A Pilot Study of the Therapeutic Potential of Stem Cell Educator Therapy in Type 1 Diabetes
Phase: PHASE1
Conditions: Diabetes Mellitus Type 1
Sponsor: Hackensack Meridian Health

Page 1 of 59 
A Pilot Study of the Therapeutic Potential of Stem Cell Educator 
Therapy in Type 1 Diabetes 
 
Principal Investigator:
Michele L. Donato, MD, FACP 
Co-Investigators:
Robert Korngold, PhD 
Steven Ghanny, MD 
Scott Rowley, MD, FACP 
Ann Van Hoven, MD   
Phyllis McKiernan, APN 
Michelle Boonstra, APN 
Nancy Durning, APN 
Alfred Gillio, MD  
David Vesole, MD, PhD 
Melissa Baker, APN 
Mary DiLorenzo, APN 
Michele Simone, APN 
Jennifer Krajewski, MD 
Jeanette Haugh, APN 
Melanie Hankewycz, APN 
Study Product: 
 
Stem Cell Educator 
Protocol Number: 
Pro00006262 
IND Number: 
16802 
Sponsor:
Hackensack University Medical Center 
30 Prospect Avenue 
Hackensack, NJ 07601 
 
Initial version: [01-29-2013] 
Amended: 
[08-01-2015] 
Amended: 
[11-12-2015] 
Amended:          [12-03-2015] 
Amended:          [12-16-2015] 
Amended:          [02-23-2016] 
Amended:          [04-12-2016] 
Amended:          [06-12-2016]   
Amended:          [01-10-2017]  
Amended:          [06-05-2017] 
Amended:          [07-05-2017] 
Amended:          [04-27-2018] 
Amended:          [11-21-2018] 
Amended:          [03-06-2019] 
 
 

 
Page 2 of 59 
The Detailed Table of Contents for Clinical Protocol
Study Summary ......................................................................................................................................................... 4 
6
Background ....................................................................................................................................................... 5 
6.1 
Introduction .............................................................................................................................................. 5 
6.2 
SCE Device ............................................................................................................................................... 6 
6.3 
Preclinical Data ......................................................................................................................................... 6 
6.3.1 
Introduction of cord blood-derived multipotent stem cells (CB-SCs) ..................................................... 6 
6.3.2 
Human Cord Blood Stem Cell-Modulated Regulatory T Lymphocytes Reverse the Autoimmune-
Caused Type 1 Diabetes in Nonobese Diabetic (NOD) Mice ............................................................................. 8 
6.3.3 
Safety of using CB-SCs
No teratoma formation. .............................................................................. 12 
6.4 
Clinical Data to Date............................................................................................................................... 12 
6.4.1 
SCE Therapy for the treatment of type 1 diabetes in a Chinese Population ....................................... 13 
6.4.2 
SCE Therapy for the treatment of type 1 diabetes in a Caucasian Population ................................... 16 
6.4.3 
SCE Therapy for the treatment of type 2 diabetes .............................................................................. 21 
6.4.4 
SCE Therapy for the treatment of Alopecia Areata ............................................................................. 26 
6.4.5 
Molecular Mechanisms underlying the SCE Therapy .......................................................................... 30 
6.5 
Dose Rationale and Risk/Benefits ........................................................................................................ 31 
7
Study Objectives ............................................................................................................................................. 32 
7.1 
Primary Objective ................................................................................................................................... 32 
7.2 
Secondary Objectives ............................................................................................................................ 32 
7.2.1 
To assess the feasibility of SCE therapy in patients with T1D ............................................................ 32 
7.2.2 
To evaluate preliminary efficacy of SCE therapy for improving islet  cell function in patients with T1D 
through 12 months. ........................................................................................................................................... 32 
7.2.3 
To evaluate markers of immune function in T1D patients after SCE therapy. .................................... 32 
8
Study Design ................................................................................................................................................... 32 
8.1 
General Design ....................................................................................................................................... 32 
8.2 
Primary Study Endpoint ........................................................................................................................ 33 
8.3 
Secondary Study Endpoints ................................................................................................................. 33 
8.3.1 
Feasibility endpoints: ........................................................................................................................... 33 
8.3.2 
Efficacy endpoints ................................................................................................................................ 33 
8.3.3 
Exploratory endpoints .......................................................................................................................... 33 
9
Subject Selection and Withdrawal ................................................................................................................ 36 
9.1 
Inclusion Criteria .................................................................................................................................... 36 
9.2 
Exclusion Criteria ................................................................................................................................... 36 
9.3 
Subject Recruitment and Screening .................................................................................................... 37 
9.4 
Early Withdrawal of Subjects ................................................................................................................ 37 
9.4.1 
When and How to Withdraw Subjects ................................................................................................. 37 
9.4.2 
Data Collection and Follow-up for Withdrawn Subjects ....................................................................... 37 
10
Study Device.................................................................................................................................................... 37 
10.1 
Description .............................................................................................................................................. 37 
10.2 
Treatment Regimen ................................................................................................................................ 38 
10.3 
Method for Assigning Subjects to Treatment Group ......................................................................... 38 
10.4 
Preparation and Use of Study Device .................................................................................................. 38 
10.5 
Subject Compliance Monitoring ........................................................................................................... 43 
10.6 
Prior and Concomitant Therapy ........................................................................................................... 43 
10.7 
Packaging ............................................................................................................................................... 43 
10.8 
Blinding ................................................................................................................................................... 43 
10.9 
Receiving, Storage, Dispensing and Return ....................................................................................... 43 
10.9.1 
Receipt of Devices ........................................................................................................................... 43 
10.9.2 
Storage ............................................................................................................................................ 43 
10.9.3 
Dispensing of Study Device ............................................................................................................. 43 
10.9.4 
Return or Destruction of Study Device ............................................................................................ 44 

 
Page 3 of 59 
11
Study Procedures ........................................................................................................................................... 44 
11.1 
Visit 1 ....................................................................................................................................................... 44 
11.2 
Visit 2 ....................................................................................................................................................... 45 
11.3 
Visit 3 ....................................................................................................................................................... 45 
11.4 
Visit 4 ....................................................................................................................................................... 45 
11.5 
Visit 5 ....................................................................................................................................................... 46 
11.6 
Visit 6 ....................................................................................................................................................... 46 
11.7 
Visit 7 ....................................................................................................................................................... 46 
11.8 
Visit 8 ....................................................................................................................................................... 46 
11.9 
Visit 9 ....................................................................................................................................................... 46 
11.10 
Visit 10................................................................................................................................................. 46 
11.11 
Visit 11................................................................................................................................................. 47 
11.12 
Visit 12................................................................................................................................................. 47 
12
Statistical Plan ................................................................................................................................................ 47 
12.1 
Statistical Methods ................................................................................................................................. 47 
12.2 
Subject Population(s) for Analysis ....................................................................................................... 48 
13
Safety and Adverse Events ............................................................................................................................ 48 
13.1 
Definitions ............................................................................................................................................... 48 
13.2 
Recording of Adverse Events ............................................................................................................... 50 
13.3 
Reporting of Serious Adverse Events and Unanticipated Problems ................................................ 50 
13.3.1 
Investigator reporting: notifying the study sponsor .......................................................................... 50 
13.3.2 
Investigator reporting: ...................................................................................................................... 51 
13.3.3 
Sponsor reporting: Notifying the FDA .............................................................................................. 51 
13.3.4 
Sponsor reporting: Notifying participating investigators .................................................................. 52 
13.4 
Stopping Rules ....................................................................................................................................... 52 
13.5 
Medical Monitoring ................................................................................................................................. 52 
13.5.1 
Internal Data and Safety Monitoring Board or DSMP...................................................................... 53 
14
Data Handling and Record Keeping .............................................................................................................. 53 
14.1 
Confidentiality ........................................................................................................................................ 53 
14.2 
Source Documents ................................................................................................................................. 53 
14.3 
Case Report Forms ................................................................................................................................ 54 
14.4 
Records Retention ................................................................................................................................. 54 
15
Study Monitoring, Auditing, and Inspecting ................................................................................................ 54 
15.1 
Study Monitoring Plan ........................................................................................................................... 54 
15.2 
Auditing and Inspecting ........................................................................................................................ 54 
16
Ethical Considerations ................................................................................................................................... 54 
17
Study Finances ............................................................................................................................................... 55 
17.1 
Funding Source ...................................................................................................................................... 55 
17.2 
Conflict of Interest .................................................................................................................................. 55 
18
Publication Plan .............................................................................................................................................. 55 
 
 
 

SCE therapy in diabetes
Version: 06Mar2019 
 
 
 
Page 4 of 59 
Study Summary 
Title 
A Pilot Study of Therapeutic Potential of Stem Cell Educator Therapy in Type 1 
Diabetes 
Short Title 
SCE therapy in type 1 diabetes 
Protocol Number 
N/A 
Phase 
Pilot study 
Methodology 
Single arm, open-label 
Study Duration 
2 year 
Study Center(s) 
Hackensack University Medical Center  
Objectives 
Primary Objective: 
1) To assess the safety of SCE therapy in subjects with T1D.   
Secondary Objectives: 
1) To assess the feasibility of SCE therapy in patients with T1D 
2) To evaluate preliminary efficacy of SCE therapy for improving  cell function 
in patients with T1D through 12 months. 
3) To evaluate markers of immune function in T1D patients after SCE therapy. 
Endpoints 
Primary Endpoint
1) The occurrence of treatment-related adverse events 
Secondary Endpoints 
1) Feasibility as determined by:  
 The number of patients who were unable to complete SCE Therapy 
 The number of patients who are lost to follow-up prior to the 12-month 
follow-up visit. 
2) Preliminary efficacy as measured by  
 Area under the C-peptide curve (AUC) over the first 2 hours of a 3-hour 
mixed meal tolerance test (MMTT) 
 Peak C-peptide levels over a 3 MMTT.   Basal C-peptide levels Daily 
insulin requirements 
 Change in HbA1C levels over time 
 Daily insulin requirements 
 Changes in auto-antibody levels over time 
3) Measurements of immune markers at baseline, 1, 3, 6, 9,  12, and 24 months
Number of Subjects 
10 subjects projected for the entire study 
Diagnosis and Main 
Inclusion Criteria 
Patients with T1D who meet the 2015 diagnosis standards of the American 
Diabetes Association and a blood test confirmed the presence of at least one 
 
Study Product, Dose, 
Route, Regimen 
SCE-treated MNCs, single treatment, infused intravenously, one time infusion.  
Duration of 
administration 
3  5 hours 
Statistical Methodology
All study data will be entered into a Microsoft Office Excel 2007 (Microsoft 
Corporation, Redmond, WA) and imported into SAS 9.2. The database will be 
validated in SAS to ensure any out range values are queried with the multiple 
sources and all data issues are resolved. Any P-value < 0.05 will be considered 
statistically significant. All data analysis will be performed using SAS 9.2 (SAS 
Institute Inc, Cary, NC). 
 

 
Page 5 of 59 
6
Background 
 
6.1 
Introduction 
regulate blood glucose, ultimately resulting in poor blood circulation, heart disease, stroke, infection, renal failure, 
and often premature death. Each day, millions of patients with T1D receive insulin injections to survive, but these 
injections do nothing to address the underlying T cell-mediated autoimmune dysfunction. Ideally, therapeutic 
approaches for treating or curing T1D should address multiple or all of the underlying causes of autoimmunity in 
the disease. Unfortunately, the etiology of T1D in humans remains largely unknown. Possible contributors to the 
initiation or potentiation of autoimmunity in T1D include genetic, epigenetic, physical, social, and environmental 
factors [9,12]. As is expected in conditions with multiple contributing factors, T1D-related immune system 
dysfunction has been traced to dysfunctions in multiple cell types and targets including T cells, B cells, regulatory 
T cells (Tregs), monocytes/macrophages (Mo/M s), dendritic cells (DCs), natural killer (NK) cells, and natural 
killer T (NKT) cells [13]. Due to the polyclonal nature of T1D-related autoimmune responses and the global 
challenges of immune regulation in T1D patients, therapies and trials that target only one or a few components of 
the autoimmune response are likely to fail. A true cure has proven elusive despite intensive research pressure 
using conventional approaches over the past 30 years [14]. Several recent clinical trials [15,16] highlight the 
challenges in conquering T1D, but their failures provide some valuable lessons about the limitations of 
conventional immune therapy and the future direction of the quest. Specifically, they point to the need for an 
approach that produces comprehensive immune modulation at both the local pancreatic and systemic levels rather 
than targeting the pancreatic effects of one or a few components of the immune system. Stem Cell Educator 
(SCE) therapy takes this broader approach [1,5,10], which was developed by Dr.Yong Zhao.  
    While traditional stem cell therapy is not likely to be effective for long-term treatment of T1D, recent studies 
suggest alternative approaches using stem cells may overcome the autoimmune component of the disease. 
Human cord blood-derived multipotent stem cells (CB-SCs) [1,2] and mesenchymal stem cells (MSCs) [3-5] have 
been shown to modulate immune activity in vitro. Subsequent studies have demonstrated that CB-SCs can be 
used to alter immune function and improve markers of T1D in nonobese diabetic (NOD) mice  [6], and they can 
modulate the immune function of T1D patient-derived islet  cell-specific pathogenic T cell clones in co-culture 
[9]. Studies in an animal model also suggest that CB-SC treatment may allow the patient to regenerate the native 
population of islet  cells without stem cell transplantation [6,9,24]. 
    To translate these findings into a clinically feasible therapy, Dr.Zhao (currently working at Hackensack 
University Medical Center) developed the procedure of SCE  
was circulated 
through a closed-loop system that separated lymphocytes from the whole blood and briefly co-cultured them with 
adherent CB-
 [1]. In an open-label, phase1/phase 2 study 
conducted in China, patients (n = 15) with T1D received one treatment with the SCE. Findings from this study 
demonstrated the feasibility and safety of SCE therapy and demonstrated that T1D patients could achieve 
improved metabolic control and reduced autoimmunity that lasted months following a single treatment. Notably, 
our initial clinical data provided powerful evidence that reversal of autoimmunity could lead to regeneration of islet 
cells and improvement of metabolic control in long-standing T1D subjects [1]. 
    The SCE device is essentially a stack of specially-designed Petri dishes with tightly adherent CB-SCs cultured 
to confluency. In the closed-loop system, 
slowly pass through the device at a constant 
flow rate and can interact with the CB-SCs in vitro, and the cells returned to the patients are thus autologous 
lymphocytes that have been treated (or educated) by the CB-SCs. The SCE therapy required only two 
venipunctures, carried a lower risk of infection than a typical blood transfusion, and did not introduce stem cells 
or reagents into the patients. In addition, CB-SCs have very low immunogenicity, and the CB-SCs cultured in the 
device are a highly restricted population and contain no CD3+ T cells or other lymphocyte subsets, eliminating 
the need for human leukocyte antigen (HLA) matching prior to treatment [8,9,11]. This innovative approach has 
the potential to provide CB-SC-mediated immune modulation therapy for multiple autoimmune diseases while 
mitigating the safety and ethical concerns associated with other approaches [2,3,5]. The relative simplicity of the 
approach may also provide cost and time savings relative to other approaches.  
    Results from this initial Chinese trial confirmed prior studies indicating that the adherence of CB-SCs could be 
exploited to develop therapies that do not introduce the CB-SCs into the patient [1,6,9]. The improvement of islet 
cell function in T1D patients with residual islet  cells is impressive, but the recovery of islet  cell function in 
T1D patients without evident  cell function prior to treatment suggested that SCE therapy could address the 
underlying challenge of autoimmunity and could control the immune response sufficiently to allow regeneration of 
the native  cell population. Thus, this trial provided initial 
-

 
Page 6 of 59 
SCs can achieve the two essential outcomes required to cure T1D: reversal of autoimmunity and regeneration of 
islet 
cells [1,5]. 
 
6.2 SCE Device 
The SCE is made of a hydrophobic material from FDA-approved (USP Class VI) Petri dishes that tightly binds 
CB-SCs without interfering with their immune modulating capability [1]. We originally designed a chamber for co-
culture of lymphocytes and CB-SCs that included nine discs of the material with a flow pathway and adherent CB-
SCs sandwiched between a top cover plate and a bottom collecting plate. In this trial, we are going to use the 12-
layer SCE device (Figure 3). The device was manufactured in a Class 100K clean room and gamma- irradiated 
prior to introducing CB-SCs  [8]. The SCE device has been registered as a Class I medical device with the FDA 
(Registered Establishment Number 3010031995). This innovative device and related technology have been 
patented (United States Patent 
and Trademark Office Patent # 
13/514, 694). 
    In the initial SCE Chinese trial, 
lymphocytes separated from a 
were 
slowly 
passed through the stacked discs 
of material with adherent CB-SCs, 
and lymphocytes finally collected 
through a hole in the bottom plate 
were returned to the patient. The 
materials used to produce the 
device are approved for in vivo 
use 
per 
the 
United 
States 
Pharmacopeia (i.e., Grade Class 
VI Plastic).  
 
6.3 Preclinical Data 
6.3.1 Introduction of cord blood-derived multipotent stem cells (CB-SCs)  
Figure 13. CB-SCs give rise to multiple lineages in response 
to different physiological growth factors and inducers. CB-
SCs are the only cells that tightly adhere to hydrophobic plastic 
(non-tissue culture treated Petri dishes) and after lymphocytes 
and monocytes die off, the CB-SCs form a single cell-derived 
colony positive for leukocyte common antigen CD45 (left panel)
and expressing embryonic markers SSEA-3 and SSEA-4. These 
CB-SCs were capable of: (A) differentiation to neuronal cell and 
oligodendrocyte when treated with 100 ng/ml NGF for 10 14days 
and characterized with lineage markers including -aminobutyric
acid 
(GABA), 
microtubule 
associated 
protein 
(MAP)1B, 
Synaptophysin (Synap), sulfatide O4, myelin basic protein (MBP), 
galactocerebroside(Galc); (B) differentiation to cardiomyocytes 
when treated with a chemical 
-aza-
24h, followed by testing with cardiomyocyte markers including 
-
actin and troponin I; (C) differentiation to macrophages when
treated with5 0 ng/ml M-CSF for 7 10 days and then 
characterized with phagocytosis 
surface 
marker 
CD11b/Mac-1; 
(D) 
differentiation 
to 
megakaryocytes when treated with 10 ng/ml TPO for 10 14 days 
polyploidy nuclear (red arrow); (E) differentiation to endothelial
cells when treated with 50 ng/ml VEGF for 10 14 days, and then 
the acetylated low density lipoprotein(Ac-LDL); and (F)
differentiation to insulin-producing cells when treated with10 nM 
exendin-4+25 mM glucose for 5 8days and characterized with 
-matched IgG 
served as negative controls for every experiment. 
 
Single cell-derived colony
(positive for CD45)
Ectoderm cells
Mesoderm cells
Endoderm cells
Oligodendrocyte
Neuronal cell
O4
MBP
GABA
Insulin-producing cell
Blood cells
CD41b
DAPI Merged
Megakaryocyte
Macrophage
Cardiomyocyte
Glut-2
Endothelial cell
CD146
Ac-LDL
Merged
Beads 
CD11b 
-actin
Troponin I
NKX2.5
Galc
MAP-1b
Synap
A
B
C
D
E
F
Maf A
Insulin
DAPI
Figure 3. Overview of SCE Device. 
CB-SCs are initially cultured inside 
the SCE device for 10-20 days at a 
cGMP facility. One allogeneic cord 
blood unit generates one Educator 
for 
one-patient 
treatment. 
Lymphocytes are isolated from the 
T1D participant by a Blood Cell 
Separator and transferred into the 
device, where they come in contact 
with CB-SCs attached to the interior 
surfaces of the device. Educated 
lymphocytes are collected from the 
bottom 
chamber 
and 
infused 
intravenously back to the patient. 

 
Page 7 of 59 
Human cord blood contains several types of stem cells including hematopoietic stem cells (HSCs), multipotent 
stem cells that have been designated cord blood-derived stem cells (CB-SCs) [8], mesenchymal stem cells 
(MSCs), endothelial progenitor cells (EPCs), and monocyte-derived stem cells [25]. Here, we focus on the novel 
type of stem cells--CB-SCs (Figure 13). We identified a unique type of stem cell CB-SC by virtue of their  
capability 
to 
attach 
to 
a 
hydrophobic plastic surface of 
non-tissue culture-treated Petri 
dishes [6,8] (Figure 14A); other 
mononuclear 
cells 
such 
as 
lymphocytes 
and 
monocytes 
were apoptotic and necrotic 
(Figure 14 B and C).  
Phenotypic characterization of 
CB-SCs indicated that they are 
different 
from 
mesenchymal 
stem 
cells 
(MSCs) 
and 
monocytes/ 
macrophages 
(Mo/M ) (Table 2). CB-SCs 
display embryonic cell markers 
(e.g., 
transcription 
factors 
OCT3/4 
and 
Nanog, 
stage-
specific 
embryonic 
antigen 
(SSEA-3, and SSEA-4) and 
leukocyte 
common 
antigen 
CD45, but are negative for blood 
cell lineage markers (e.g., CD1a, 
CD3, CD4, CD8, CD11b, CD13, 
CD14, CD19, CD20, 
CD34, 
CD41a, CD41b, CD83, CD105, 
CD131, 
and 
CD133). 
Flow 
cytometry 
revealed 
the 
expression 
of 
HVEM 
(herpesvirus-entry mediator, CD270) and PD-L1 (programmed death ligand 1) molecules on their surface, which 
may contribute to their immune modulation capacity [3,11] (Figure 14 D and E). Additionally, CB-SCs displayed 
very low immunogenicity as indicated by expression of a very low level of MHC antigens and failure to stimulate 
the proliferation of allogeneic lymphocytes [8,11]. They can give rise to three embryonic layer-derived cells in the 
presence of different inducers (Figure 13). In comparison with using bone marrow-derived mesenchymal stem 
cells (MSCs), application of CB-SCs possess several unique advantages including large resources of cord blood 
worldwide, no risk to the donor, ease to culture and expand in vitro, and possession of embryonic characteristics 
[8]. More specifically, CB-SCs tightly adhere to culture dishes with a large rounded morphology and are resistant 
to regular detaching methods (trypsin/EDTA), making it easy to collect only the lymphocytes in suspension after 
co-culture [6,8,11]. Next, we summarize our recent findings in preclinical models that shed light on the therapeutic 
potential of these cord blood stem cells in T1D.  
 
 
B
Annexin V 
C
Table 5. The phenotype of CB-SCs is different from that of Mo/M  and 
MSCs. 
Cellular markers
CB multipotent stem cells
CB-SCs
Monocytes/macrophages 
Mo/M
Mesenchymal stem cells 
MSCs
Leukocyte common 
antigen CD45
Strongly positive
100%
Strongly positive
100%
Negative
Mo/M markers
CD11b and CD14
Negative
Positive
Negative
MSC markers
CD90 and CD105
Negative
Negative
Positive
MHC class II antigens
HLA-DR, DQ
Negative
Positive
Negative
MHC class I antigens
HLA-ABC
Very low
High
High
Embryonic stem (ES) 
markers: Oct3/4, 
Nanog, Sox2
Positive
Negative
Negative
Cord blood-derived mononuclear cells
(e.g.,lymphocytes,granulocytes,Mo/M ,HSCs,CB-SCs,MSCs
Mo/M
MSCs
CB-SCs
Non-tissue culture-
treated Petri dish
Tissue culture-treated dish

 
Page 8 of 59 
 
Figure 14. Characterization of CB-SC culture by flow cytometry. (A) Morphology of CB-SC culture under phase-
contrast microscope. All floating cells and debris were washed away. (B) The apoptosis and necrosis of floating cells in the 
Petri dish. Human cord blood-derived mononuclear cells were plated in the Bioreactor in serum-free X-Vivo 15 culture 
medium (Lonza, Walkersville, MD) and incubated at 37°C, in 8% CO2. After overnight culture (17 hours), flow cytometry 
demonstrated that 92.98% of floating cells became Annexin V+7-ADD+ double positive cells, with only 3.23% of viable cells. 
The results represented from seven independent experiments with the similar results. (C) The apoptosis of monocytes.
Human cord blood-derived mononuclear cells were planted in non-treated Petri Dishes in regular cell culture medium. (B) 
After 20 hours, flow cytometry demonstrated that 99% of CD14+ monocytes became Annexin V-positive apoptotic cells. 
The data confirmed the apoptosis of monocytes; monocytes could not grow on the surface of non-treated Petri dishes. The 
results represented from five independent experiments with the similar results. (D) Expression of HVEM on the gated 
CD45+OCT3/4+ CB-SCs. CD45, a leukocyte common antigen; OCT3/4, a transcription factor marker of embryonic stem 
(ES) stem cells. (E) The gated CD45+ Oct-4+ CB-SCs display PD-L1 molecule. The isotype-matched IgGs served as 
controls for all flow cytometry.  
 
6.3.2 Human Cord Blood Stem Cell-Modulated Regulatory T Lymphocytes Reverse the Autoimmune-
Caused Type 1 Diabetes in Nonobese Diabetic (NOD) Mice  
    
We reported that treatment of established autoimmune-induced diabetes in NOD mice with purified 
autologous CD4+CD62L+ Tregs co-cultured with human CB-SCs can eliminate hyperglycemia, promote islet -
cell regeneration to increase 
-cell mass and insulin production, and reconstitute islet architecture. 
Correspondingly, treatment with CB-SCs modulated CD4+CD62L+ Tregs (mCD4CD62L Tregs), resulted in a 
marked reduction of insulitis, restored Th1/Th2 cytokine balance in blood, and induced apoptosis of infiltrated 
leukocytes in pancreatic islets. These data demonstrated that treatment with mCD4CD62L Tregs can reverse 
overt diabetes, providing a novel strategy for the treatment of type 1 diabetes as well as other autoimmune 
diseases. 
 
6.3.2.1 
CB-SC modulation of NOD mouse regulatory T lymphocytes.  
To investigate the therapeutic potential of Tregs in T1D, we employed an experimental nonobese diabetic (NOD) 
mouse model. Initially, we tested the co-culture of CB-SC and NOD mouse spleen-derived lymphocytes and found 
that co-culture with CB-SC did not significantly stimulate the proliferation of mouse lymphocytes at different ratios 
of CB-SC : lymphocytes  (1:5, 1:10, and 1:20) (p = 0.25, p = 0.15, p = 0.16 respectively), which is similar to the 
co-culture of CB-SC and human lymphocytes  [11]. Next, we analyzed co-cultures of CB-SC and mouse 
lymphocytes for the presence of Tregs including conventional CD4+CD25+ Treg and CD4+Foxp3+ Treg, and the 
CD4+CD62L+ Treg. We found no significant differences in CD4+CD25+ Treg and CD4+Foxp3+ Treg in total mouse 
spleen lymphocytes that were either cultured alone or with CB-SC. In contrast, the percentage of CD4+CD62L+ 
Treg was increased about 5-fold after co-culture with CB-SC (Figure 9A). Further flow cytometry revealed that 
only a very small proportion of these CD4+CD62L+ Tregs was CD4+CD25+CD62L+Foxp3+ positive (Figure 9B), 
and this percentage was not different between lymphocytes co-cultured with or without CB-SC (0.11  0.04 % vs 
0.10   0.03 %, P = 0.44). We subsequently focused on CD4+CD62L+ Tregs, which were primarily affected by co-
culture with CB-SC (designated CB-SC-modulated CD4+CD62L+ Tregs, mCD4CD62L Tregs).  
 
D
OCT3/4
Gate Y
OCT3/4
E

 
Page 9 of 59 
 
Figure 9. Flow analysis of 
CD4+CD62L+ Tregs following 
in 
vitro 
co-culture 
with 
human cord blood stem cells 
CB-SC. 
Mouse 
spleen 
lymphocytes were isolated from 
female NOD mice (aged 6-8 
weeks) and then co-cultured 
with CB-SC for 2-4 days at a 
ratio 
1:10 
of 
CB-SC: 
lymphocytes. 
Subsequently, 
the total lymphocytes were 
harvested for flow analyses. 
Lymphocytes 
cultured 
in 
absence of CB-SC served as 
control. 
(A) 
Percentage 
of 
CD4+CD25+ Treg, CD4+Foxp3+
Treg, and CD4+CD62L+ Treg 
after in vitro co-culture with CB-
SC. (B) Flow analysis of CD25 
and 
Foxp3 
expressions 
in 
CD4+CD62L+ Tregs after in vitro
co-culture with CB-SC. (C) Flow 
analysis on CD4+CD62L+ Tregs 
after 
intra-cellular 
cytokine 
staining. Isotype-matched IgG 
served as control. Data were 
representative of three to five 
experiments.  
 
 
 
A
CD4
13.1
5.2
64.0
17.7
60.8
25.4
10.8
3.0
CD4
0.36
1.92
72.56
25.16
CD4
0.47
1.72
75.92
21.89
29.22
70.6
0.09
0.08
0.06
0.07
74.56
25.31
Control 
lymphocytes
CB-SC-cocultured 
lymphocytes
B
CD25
0.04
0.12
92.4
7.44
CD4
CB-SC-cocultured lymphocytes:
0
0.5
1
1.5
2
2.5
3
IL-4
IL-10
IL-12
Control CD4CD62L Tregs
mCD4CD62L Tregs
IFN-
TGF- 1
P=0.004
P=0.001
P=0.0001
P=0.5
C
P=0.006
 
Figure 10 CB-SC-modulated CD4+CD62L+ Tregs (mCD4CD62L Tregs) reverse hyperglycemia in overt diabetic NOD 
mice. (A) The mCD4CD62L Tregs correct hyperglycemia in diabetic NOD mice. Overt diabetic NOD mice were treated with 
mCD4CD62L Tregs (total 5 million cells/mouse, i.p., red line; representative data are from 6 diabetic mice sensitive to 
mCD4CD62L Treg treatment with euglycemia, n = 8 mice). Purified control CD4CD62L Tregs served as control (total 5 million 
cells/mouse, i.p., blue line, n = 5 mice). PBS served as an additional control (black line, n = 5 mice). (B) Intraperitoneal glucose 
tolerance testing (IPGTT) 3 weeks following the 1st treatment with mCD4CD62L Tregs. Seven-week old NOD mice served as 
normal control. (C) Determination of blood insulin levels by ELISA. (D) Morphometric analysis of pancreatic -cell mass. 
Pancreatic 
-cell mass was determined by point-counting morphometry on insulin-positive islet 
 cells followed by 
immunostaining with guinea pig anti-insulin Ab (Dako) and counter-staining with hematoxylin. (E) Quantification of Ki67-
positive cells in pancreatic islets after double immunostaining with Ki67 and insulin Abs. Isotype-matched rabbit IgG served as 
control for rabbit anti-Ki67 mAb. (F) Confocal microscopy shows double-immunostaining for insulin (red) and a cell proliferation 
nuclear marker Ki67 (green) (scale bar, 50 m), with a high magnification (two bottom rows, scale bar 10 
m). Control 
0
100
200
300
400
500
600
700
Time (day) 
Diabetic mice treated with mCD4CD62L Tregs
Diabetic mice treated with control CD4CD62L Tregs
Diabetic mice treated with PBS
1
5
6
8
10
15
20
30
45
Treatments
-
A
0
100
200
300
400
500
600
700
Time (minute) 
Non-diabetic mice (7 weeks old)
Diabetic mice treated with mCD4CD62L Tregs
Diabetic mice treated with control CD4CD62L Tregs
Diabetic mice treated with PBS
0
10
20
30
45
60
90
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
PBS-treated
diabetic mice
Control CD4CD62L
Treg-treated
diabetic mice
mCD4CD62L Treg-
treated diabetic
mice
Non-diabetic mice
(7 weeks old)
P = 0.0025
C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
mCD4CD62L Treg-
treated diabetic mice
Control CD4CD62L Treg-
treated diabetic mice
P = 0.0026
D
0
5
10
15
20
25
30
Control CD4CD62L Treg-
treated diabetic mice
mCD4CD62L Treg-
treated diabetic mice
P = 0.0014
E
Insulin
DAPI
Ki67
Merged
mCD4CD62L Treg-
treated diabetic mice
F
Control CD4CD62L
Treg-treated 
diabetic mice

 
Page 10 of 59 
To document modulation of CD4+CD62L+ Tregs by CB-SC after in vitro co-culture, intracellular cytokines related 
to helper T (Th)1 and Th2 immune responses were measured using flow analysis (Figure 9C). Results 
demonstrated that the IL-4 level was significantly down-regulated (p = 0.004), whereas IL-10, IL-12 and TGF- 1 
levels were up-regulated in mCD4CD62L Tregs compared with control CD4CD62L Tregs (p = 0.001, p = 0.0001, 
and p = 0.006 respectively). In contrast, the IFN-  expression did not change following co-culture with CB-SC 
(Figure 9C, p = 0.5). Next, we investigated expression of Th1-Th2-Th3 cell-related genes by using quantitative 
real time PCR array in the purified CD4+CD62L+ Tregs following co-culture with CB-SC. Results demonstrated 
that mCD4CD62L Tregs displayed marked down-regulation of Th cell-related genes including multiple cytokines 
and their receptors, chemokines and their receptors, cell surface molecules, along with signaling pathway 
molecules and transcription factors [6]. These data clearly indicate that in vitro co-culture with CB-SC causes 
substantial modifications of gene expression in mouse CD4+CD62L+ Tregs, specifically for function-related 
cytokine and chemokine genes.   
6.3.2.2 
CB-SC-modulated CD4+CD62L+ Tregs (mCD4CD62L Tregs) correct hyperglycemia in overt 
diabetic NOD mice.  
Next, overt diabetic NOD mice (female, at 24-28 weeks of age) were treated with mCD4CD62L Tregs (total 5 
million cells/mouse, i.p., n = 8 mice) for 5-20 days after the diagnosis of T1D to determine their therapeutic 
potential. The control CD4CD62L Tregs at the same cell amount (i.p., n = 5 mice) and vehicle PBS (total 200 
l/mouse, i.p., n = 5 mice) served as controls. Notably, we found that treatment with mCD4CD62L Tregs restored 
euglycemia in these overt diabetic mice (6/8 mice) (Figure 10A). However, treatment with control CD4CD62L 
Tregs or PBS failed to reduce hyperglycemia in diabetic mice (5/5, 5/5 mice respectively) (Figure 10A). Diabetic 
mice that had been rendered euglycemic after treatment with mCD4CD62L Tregs also showed an improved 
glucose tolerance test (IPGTT), similar to that of non-diabetic NOD mice at 7 weeks (Figure 5B). However, 
diabetic mice treated with PBS or control CD4CD62L Tregs maintained high glucose levels (>500 mg/dL) without 
any observable down-regulation (Figure 10B). Moreover, we monitored blood insulin levels 6 weeks after 
treatment with mCD4CD62L Tregs. Results showed that insulin in diabetic mice treated with control CD4CD62L 
Tregs or PBS vehicle was undetectable by ELISA (0.019 ng/ml sensitivity for the ELISA kit, Figure 10C). These 
mice had to be sacrificed because of severe hyperglycemia (BG> 600 mg/dL) and loss of body weight (>20%) 
according to the protocol approved by the Animal Care Committee. In contrast, blood insulin levels in diabetic 
NOD mice treated with mCD4CD62L Tregs were significantly increased (Figure 10C, p = 0.0025). 
At 45 days after treatment, we subjected pancreata to histological analysis and evaluated total -cell mass 
followed by immunostaining with insulin Ab on serial pancreatic sections. Morphometric analysis demonstrated 
that treatment with mCD4CD62L Tregs significantly increased total -cell mass (Figure 10E, p = 0.0026). In 
contrast, -cell mass was markedly lower after vehicle PBS treatment or control CD4CD62L Treg treatment 
(Figure 10E). To understand the mechanism of the increase in total -cell mass, we determined the expression 
of a cell proliferation nuclear marker Ki67 [18] in pancreatic islets. Double immunostaining with insulin and Ki67 
Abs revealed that 20  8  cells/islet expressed Ki67 in pancreatic islets of mCD4CD62L Treg-treated mice (Figure 
10E and F), which was much higher than that in pancreatic islets of mice treated with control CD4CD62L Tregs 
(1  0.4) (p = 0.0014). It suggests that de novo proliferation of  cells accounts for the noted increase in total  
cell mass. Moreover, double immunostaining with -cell-marker insulin and -cell-marker glucagon revealed that 
pancreatic islets in diabetic mice treated with mCD4CD62L Tregs displayed a similar pattern of 
- and -cell 
distribution as that noted in normal islets of non-diabetic NOD mice. However, islet architecture was completely 
destroyed with almost complete disappearance of  cells in the diabetic mice treated with control CD4CD62L 
Tregs [6]. Thus, treatment with mCD4CD62L Tregs can correct hyperglycemia of T1D mice by promoting -cell 
regeneration and reconstitution of islet cell architecture. 
6.3.2.3 
Treatment with mCD4CD62L Tregs reverses insulitis and immune dysfunction in NOD mice.  
    To establish whether mCD4CD62L Tregs exert an immunosuppressive influence on autoreactive effector T 
cells, we performed pancreatic histological analysis and scored insulitis at 45 days after treatment. Histological 
evaluations showed that approximately 80% of islet  cells (profound insulitis) were destroyed in diabetic NOD 
mice prior to treatment. Six weeks post treatment, we found that in diabetic mice receiving mCD4CD62L Tregs, 
36% of islets had no or few signs of infiltration of inflammatory cells; 20% of islets displayed mild insulitis; 15% of 
islets exhibited moderate insulitis; 18% of islets had severe insulitis and only 11% of islets showed profound 
insulitis (Figure 11A and B). The insulitis-free islets were of smaller size and positive for the proliferation marker 
Ki67 (data not shown), suggesting that these islets may have been newly generated. In contrast, all pancreatic 
CD4CD62L Treg-treated diabetic mice (top panels) showed the Ki67-positive cells distributed in the infiltrated inflammatory 
cells (blue, with high density), not in -cell area (dashed pink circle), with almost complete disappearance of 
cells (red). 

 
Page 11 of 59 
islets in diabetic mice receiving control CD4CD62L Tregs showed massive infiltration of inflammatory cells and 
severe destruction of pancreatic architecture (Figure 11A), and had few or no insulin-positive cells present. 
Similarly, pancreatic histological examination demonstrated that those two mice (2/8 mice) that were resistant to 
mCD4CD62L Tregs treatment also displayed profound insulitis (data not shown) after 45 days observation. 
To understand the molecular mechanism underlying reduction of insulitis, we measured plasma Th1/Th2 
cytokine levels by ELISA. We found that Th1 cytokine IFN-  and Th2 cytokine IL-4 were considerably reduced in 
the plasma of mCD4CD62L Treg-treated diabetic mice relative to control CD4CD62L Treg-treated diabetic mice 
(P = 0.017, Figure 11C; P = 0.018, Figure 11D respectively). In contrast, diabetic mice receiving mCD4CD62L 
Tregs showed a marked increase in plasma IL-10 level compared with those treated with  control CD4CD62L 
Tregs (P = 0.016; Figure 11E) and non-diabetic NOD mice at age of 6 weeks (P = 0.014; Figure 11E). Additionally, 
plasma TGF- 1 level was significantly elevated in mCD4CD62L Treg-treated diabetic mice compared with control 
CD4CD62L Treg-treated diabetic mice (P = 0.041; Figure 11F). These data suggest that both IL-10 and TGF- 1 
may contribute to an induction of immune tolerance after treatment with mCD4CD62L Tregs [19-21]. These data 
demonstrate that exposure to CB-SC induced profound changes in mCD4CD62L Tregs that helped restore 
-cell function resulting in the suppression of diabetes. 
 
Figure 11 Treatment with mCD4CD62L 
Tregs reverses insulitis and immune 
dysfunction in diabetic NOD mice.  Overt 
diabetic NOD mice treated with mCD4CD62L 
Tregs 
were 
sacrificed 
for 
pancreatic 
histological analysis and evaluation of blood 
cytokine levels after observation for 45 days. 
(A) Scoring of insulitis. Pancreatic islets were 
scored for % mononuclear cell infiltration after 
immunostaining for insulin and counter-
staining with hematoxylin.   
(B) Representative images for different type 
of 
insulitis. 
Data 
were 
collected 
from 
mCD4CD62L Treg-treated diabetic NOD 
mice. Scale bar, 50 m. (C) Determination of 
plasma IFN-  level by ELISA. Non-diabetic 
NOD mice at age of 6 weeks served as 
normal control. (D) Determination of plasma 
IL-10 
level 
measured 
by 
ELISA. 
(E) 
Determination of plasma TGF- 1 level 
measured by ELISA. Data are shown as 
mean  s.d. of mouse plasma cytokine levels 
from three experiments. 
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
No insulitis 
(0% infiltration)
Mild insulitis
(<25% infiltration)
Moderate insulitis 
(25%~50% infiltration)
Severe insulitis
(50%~75% infiltration)
Profound insulitis
(>75% infiltration)
1
2
3
4
5
A
mCD4CD62L
Tregs
Control CD4
CD62L Tregs
PBS
No insulitis
Moderate insulitis
Profound insulitis
Mild insulitis
Severe insulitis
1
2
3
4
5
B
0
20
40
60
80
100
120
140
160
Control CD4CD62L
Treg-treated DM
mCD4CD62L Treg-
treated DM
6-week non-diabetic
mice
C
P=0.017
P=0.06
0
10
20
30
40
50
60
70
Control CD4CD62L
Treg-treated DM
mCD4CD62L Treg-
treated DM
6-week non-diabetic
mice
P=0.018
P=0.0018
D
0
10
20
30
40
50
60
70
80
90
100
Control CD4CD62L
Treg-treated DM
mCD4CD62L Treg-
treated DM
6-week non-diabetic
mice
P=0.016
P=0.014
E
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Control CD4CD62L
Treg-treated DM
mCD4CD62L Treg-
treated DM
6-week non-diabetic
mice
F
P=0.041
P=0.36
TGF- 1
Insulin
Merged
Mouse IgG
Insulin
Merged
G mCD4CD62L Treg-treated diabetic mice:
H
TGF- 1
DAPI
Merged
Insulin
TUNEL
Control CD4CD62L Treg-
treated diabetic mice
mCD4CD62L Treg-
treated diabetic mice
0
5
10
15
20
25
Control CD4CD62L Treg-treated DM
mCD4CD62L Treg-treated DM
CD4+Tunel+
T cells
CD8+Tunel+
T cells
B220+Tunel+
B cells
F4/80+Tunel+
M
cells
P=0.034
P=0.024
P=0.041
P=0.032
I

 
Page 12 of 59 
TGF- 1 is one of the best characterized cytokines contributing to the induction of immune suppression and 
maintaining of self-tolerance [19].  To elucidate de novo molecular mechanism underlying the protection of islet  
cells following treatment with mCD4CD62L Tregs, we determined TGF- 1 expression in pancreatic islets by 
immunohistochemistry in addition to plasma TGF- 1 measurement (Figure 11F). Results demonstrated that TGF-
1 was presented at higher level in pancreatic islets of mCD4CD62L Treg-treated diabetic mice compared with 
control CD4CD62L Treg-treated diabetic mice (Figure 11G and H). Staining of TGF- 1-positive cells showed two 
patterns: one was distributed among islet  cells, with average positive cell number of 14  9 cells/islet, and 
another was located around islet  cells. Importantly, we found that these surrounding TGF- 1-positive cells 
(negative for macrophage marker F4/80, but positive for dendritic cell marker CD11c [22], data not shown), along 
with their released TGF- 1 in the matrix (faint staining), formed a ring surrounding pancreatic islets (Figure 11G). 
This ring may protect newly-generated islets against attack by inducing apoptosis of auto-aggressive effector 
lymphocytes, as determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining 
(Figure 11H, 58  23 TUNEL+ infiltrated leukocytes in mCD4CD62L Treg-treated group vs. 9  3 TUNEL+ infiltrated 
leukocytes in control CD4CD62L Treg-treated group, p = 0.02). To clarify which cell type became apoptotic, we 
performed double staining with different cell markers including CD4 for CD4+ T cells, CD8 for CD8+ T cells, B220 
for B cells, and F4/80 for macrophages respectively in combination TUNEL staining. We found that treatment with 
mCD4CD62L Tregs increased the apoptosis of infiltrated T cells, B cells, and macrophages compared with control 
CD4CD62L Treg treatment  (p = 0.0034, p = 0.024, p = 0.041, and p = 0.032 respectively). In comparison with the 
other three cell types however, CD4+ T cells showed a much higher percentage of apoptotic cells (Figure 11I).  
Thus, these data suggest that treatment with mCD4CD62L Tregs enhances expression of TGF- 1 in pancreatic 
islets that may contribute to local protection of newly-generated pancreatic islets from the re-destruction of 
autoreactive immune cells. 
 
6.3.3 Safety of using CB-SCs
No teratoma formation.  
 
To demonstrate the safety of 
application of CB-SCs, CB-SCs 
were 
inoculated 
into 
the 
muscles of NOD-scid mice (n = 
8, the muscle of single rear leg) 
at 2 107 cells/mouse to test the 
tumor formation  [23]. Mice 
were 
monitored 
for 
tumor 
formation twice a week for 12 
weeks. After injection for 3 
months, 
histological 
examinations failed to show 
evidence of tumor (or teratoma) 
formations in the muscles, 
lungs, 
livers, 
and 
kidneys 
(Figure 12). The data prove 
that application of CB-SCs is 
safe.    
 
 
6.4 
Clinical Data to Date 
We recently developed a novel therapy designated SCE  therapy (Figure 15), based on our results in the NOD 
mouse model and other preclinical evidence that CB-SCs can control autoimmune responses by altering Tregs 
and human islet  cell-specific T cell clones [6,9,24]. To date, several international clinical trials have confirmed 
the safety and therapeutic efficacy of SCE therapy for the treatment of: T1D in adults (in China and Spain, 
respectively) (NCT01350219) [1,5]; T1D in children (NCT01996228); type 2 diabetes (NCT01415726) [2]; and 
alopecia areata (NCT01673789) [3]. Briefly, a 16-gauge IV needle was placed in the median cubital vein to isolate 
were transferred 
into the device for exposure to CB-SCs, and other blood components were automatically returned to the patient 
[1]. In these trials, the SCE functioned as part of a closed-loop system that circulated 
the blood cell separator, briefly co-cultured 
-SCs in vitro as they slowly flowed 
through the Educator, and returned the educated  
 [1,26]. CB-SCs tightly 
attached to interior surfaces in the device, and only the CB-SC-educated autologous lymphocytes were returned 
Figure 
12. 
Histological 
examination of teratoma 
formation. 
CB-SCs 
were 
inoculated into the muscles 
of single rear leg of NOD-scid 
mice at 2 107 cells/mouse in 
100 l PBS. After injection for 
3 
months, 
mice 
were 
sacrificed for testing tumor 
formation 
by 
histological 
analysis. The isolated tissues 
were 
fixed 
in 
10% 
formaldehyde 
at 
room 
temperature for 10 minutes, 
paraffinized, and processed 
for hematoxylin and eosin 
(H&E) staining.  Original 
magnification, 400.  

 
Page 13 of 59 
to the subjects. The SCE therapy required only two venipunctures with minimal pain, and did not introduce stem 
cells or reagents into the patients in comparison with other stem cell-based therapies (e.g., MSCs and HSCs) [1]. 
Additionally, CB-SCs displayed very low immunogenicity and no presence of CD3+ T cells or other lymphocyte 
subsets derived from the cord blood, eliminating the need for human leukocyte antigen (HLA) matching prior to 
treatment [1,8,9,11]. Thus, SCE therapy proved to be advantageous in providing CB-SC-mediated immune 
modulation therapy while mitigating the safety and ethical concerns associated with other stem cell-based 
approaches and conventional immune therapies.  
 
6.4.1 SCE Therapy for the treatment of type 1 diabetes in a Chinese Population 
6.4.1.1 
Feasibility and Safety of SCE Therapy 
    Fifteen T1D patients were enrolled (baseline characteristics presented in Table 6). Median age was 29 years 
(range, 15 to 41), and median diabetic history was 8 years (range, 1 to 21). Participants were randomly assigned 
to receive SCE therapy (n=12) or sham therapy (n=3). Each participant received one treatment. Based on fasting 
C-peptide levels (a by-product of insulin biosynthesis, as an indicator for islet  cell function), participants in the 
treatment group were characterized as having moderate T1D with some residual  cell function (n = 6, Group A) 
or severe T1D with no residual pancreatic islet  cell function (n = 6, Group B) (Table 6). All Control Group 
participants had moderate T1D. 
    No participants experienced any significant adverse events during the course of treatment. Most patients 
experienced mild discomfort during venipuncture and some soreness of the arm during aphaeresis, but discomfort 
and soreness resolved quickly following the conclusion of the procedure. Twenty-four hours post treatment, no 
significant difference was noted in white blood cell counts relative to baseline (Total white blood cell count: 6.95  
109/L  1.98 vs 6.39   109/L  1.72, p = 0.38; Granulocytes: 3.79  109/L  1.43 vs 3.66  109/L  1.05, p = 0.77; 
Lymphocytes: 2.31  109/L  0.9 vs 2.08  109/L  0.67, p = 0.40; Monocytes: 0.49  109/L  0.13 vs 0.46  109/L 
0.10, p = 
-day post-
treatment observation (36.44 oC  0.24 vs 36.5 oC  0.22, n = 15, p = 0.35). No changes were observed in blood 
cell count or temperature at the 12-week follow-up.  
CB-SCs are tightly adherent [8,9] and not expected to escape the device. To confirm CB-SCs are completely 
retained in the Educator and not transferred to the patient, we examined cells leaving the device to check for stage 
specific embryonic antigen 3 (SSEA-3), a CB-SC-specific marker. Flow cytometry confirmed the absence of 
SSEA-3 in cells leaving the Educator [1]. These data indicate that the cells returned to the patients are autologous. 
Additionally, HLA matching is not required prior to SCE therapy because CB-SCs are not transferred to the patient 
and because CB-SCs have very low immunogenicity [8,9,11]. Thus, SCE therapy is a very safe approach. 
Figure 15. Scheme of the SCE Therapy Approach. CB-SCs are initially cultured inside the SCE device for 10-14 days 
at cGMP facility and shipped to clinical sites for bedside treatment. One allogeneic cord blood unit generates one Educator 
for one-
s blood is passed through a Blood Cell Separator that isolates the lymphocytes 
from peripheral blood, the collected lymphocytes are transferred into the SCE device where they encounter CB-SCs as they 
slowly transit through the chambers from the top to bottom layers; after that, 
 will 
return back to the patient via a dorsal vein in the hand (note: CB-SCs remain tightly attached inside the Educator). This is 
a closed-loop system and blood is continually processed for a total of about 8-9 hrs. 

 
Page 14 of 59 
 
6.4.1.2 
Clinical Efficacy Outcomes in Improving Islet Beta Cell Function [1] 
Participants in Group A (i.e., those with moderate T1D and some residual  cell function) exhibited improved 
fasting C-peptide levels at 12 and 24 weeks post treatment (Figure 16A and B), and participants in Group B (i.e., 
those with severe T1D and no residual pancreatic islet  cell function) exhibited successive improvement in fasting 
C-peptide levels at each follow-up (Figure 16A and 9C). C-peptide response following a 75-g oral glucose 
tolerance test (OGTT) improved among Group A participants at 4 and 12 weeks (Figure 16B). Notably, 
participants in Group B exhibited essentially no C-peptide production following glucose challenge at baseline (i.e., 
< the minimum sensitivity of 0.01 ng/ml at all time points) but demonstrated marked improvement at 12 weeks 
(Figure 16C). Improvement was maintained through the final follow-up (i.e., 40 weeks post treatment; p = 0.026) 
(Figure 16C).  Participants in the Control Group did not exhibit significant change at any follow-up (Figure 16A). 
    Consistent with improved  cell function, the median daily dose of insulin was reduced 38% at 12 weeks post 
treatment in Group A (36  13.2 units/day at baseline vs. 22  1.8 units/day 12 weeks post treatment) and 25% in 
Group B (48  7.4 units/day at baseline vs. 36  4.4 units/day 12 weeks post treatment), but no change was 
observed in the Control Group. The reduced daily dose of insulin in Group A and B was maintained through the 
last follow-up for this measure (24 weeks). The median glycated hemoglobin (HbA1C) in Group A was significantly 
lowered from 8.73%  2.49 at baseline to 7.67%  1.03 at 4 weeks post treatment (p = 0.036), and to 6.82%  
0.49 at 12 weeks post treatment (p = 0.019). The median HbA1C in Group B was reduced 1.68%  0.42 at 12 
weeks post treatment, but no change was observed in the Control Group (9.0%  2.3 at baseline vs. 8.7%  1.9 
at 12 weeks post treatment, p = 0.86). Thus, the ex vivo immune education by exposure to CB-SCs leads to 
regeneration of islet  cells and improvement of  cell function in long-standing T1D subjects. 
 
 
* The level of 0.01 ng/ml is the minimum detectable level (sensitivity) of C-peptide by radioimmunoassay (RIA). To convert 
C-peptide value to nmol/L, multiply the ng/ml by 0.331.  
Table 6. Characteristics of the T1D subjects before treatment.
Patient 
No. 
Age
Gender
Marriage
History
(year) 
Height
(cm) 
Body weight
(kg) 
C-peptide
(ng/ml)* 
Auto-Antibodies 
HbA1C
(%) 
Insulin dose
(unit/day) 
IA-2A
GAD
ICA 
IAA
Group A: Long-standing patients having some residual islet  cell function,  received Stem Cell Educator therapy with CB-SC 
1 
17 
F 
No 
5 
145 
35 
0.30 
- 
+ 
- 
- 
12.3 
52 
2 
23 
F 
No 
5 
167 
59 
0.56 
- 
+ 
- 
- 
6.6 
50 
3 
38 
F 
Yes 
11 
160 
60 
0.12 
- 
+ 
- 
- 
7.3 
23 
4 
39 
F 
Yes 
1 
156 
49 
0.636 
+ 
+ 
+ 
- 
11.3 
22 
5 
31 
M 
Yes 
14 
170 
70 
0.18 
+ 
+ 
- 
- 
6.5 
30 
6 
30 
M 
Yes 
2 
182 
70 
0.18 
- 
+ 
- 
- 
8.4 
40 
Mean  
(SD) 
30  
(9) 
 
 
6 
(5) 
163 
(12.7) 
57 
(13.4) 
0.33 
(0.22) 
 
 
 
 
8.7 
(2.5) 
36.2 
(13.2) 
Group B: Long-standing severe patients with no residual islet  cell function,  received Stem Cell Educator therapy with CB-SC 
7 
40 
M 
Yes 
17 
170 
72.5 
0.01 
- 
+ 
- 
- 
8.8 
48 
8 
15 
F 
No 
5 
163 
65 
0.01 
- 
+ 
- 
- 
15.5 
50 
9 
21 
F 
No 
4 
168 
65 
0.01 
+ 
- 
- 
- 
9.9 
46 
10 
23 
F 
No 
12 
162 
80 
0.01 
+ 
+ 
+ 
- 
16.5 
60 
11 
40 
F 
Yes 
21 
160 
67 
0.01 
+ 
- 
- 
- 
8.6 
37 
12 
21 
F 
No 
5 
157 
56 
0.01 
- 
+ 
- 
- 
13.6 
50 
Mean  
(SD) 
27 
(11)
 
 
11 
(7) 
163 
(4.9) 
67.6 
(8.1) 
0.01 
(0) 
 
 
 
 
12.2 
(3.5) 
48.5 
(7.4) 
Control group:  Long-standing patients having some residual islet  cell function, received sham therapy 
13 
35 
M 
Yes 
1 
178 
73.5 
0.37 
+ 
- 
+ 
- 
9.8 
30 
14 
41 
M 
Yes 
14 
165 
65 
0.55 
- 
+ 
- 
- 
6.4 
48 
15 
24 
M 
No 
3 
175 
58 
0.3 
- 
+ 
- 
- 
10.7 
48 
Mean  
(SD) 
33 
(9) 
 
 
6 
(7) 
173 
(6.8) 
65.5 
(7.8) 
0.41 
(0.13) 
 
 
 
 
9.0 
(2.3) 
42 
(10.4) 

 
Page 15 of 59 
Figure 16. Improvement of -cell Function by SCE
Therapy. (A) Fasting C-peptide levels of T1D 
participants over 24 weeks. Group A and Group B 
participants (n = 6 per group) received one SCE
treatment. Control group participants (n = 3) received 
sham therapy (no CB-SCs in the SCE). (B) 12-week 
follow up C-peptide levels after OGTT at 2 hours in 
Group A T1D subjects with some residual  cells. (C) 
Comparison of C-peptide levels at glucose challenge 
after 40-week follow up in Group B T1D subjects. The 
dashed red line indicates the lower limit for normal C-
peptide levels in Chinese populations. The dashed 
purple line indicates the minimum detectable level 
(sensitivity) of C-peptide by radioimmunoassay (RIA). 
 
 
6.4.1.3 
Efficacy Outcomes in Autoimmune Control [1] 
Next, we explored mechanisms underlying CB-SC-mediated immune modulation. Regulatory T lymphocytes 
(Tregs) play a crucial role in maintaining homeostasis and self-tolerance by inhibiting the action of autoreactive 
effector T cells [6,27,28], but previous attempts to manipulate Tregs for clinical applications have been problematic 
[29]. We measured changes in the percentage of CD4+CD25+Foxp3+ Tregs in peripheral blood of participants 
following SCE treatment. The percentage of Tregs in peripheral blood of participants was significantly increased 
4 weeks after SCE therapy (Figure 17A), whereas the percentage of Tregs in peripheral blood of participants 
receiving sham therapy was unchanged from baseline (Figure 17A). TGF- 1 has also been implicated in Treg-
mediated immune suppression [30] as well as in maintenance of self-tolerance in T1D animal models subjected 
to stem cell-mediated immune modulation [9,19,24]. We examined TGF- 1 and IL-10 expression to explore 
whether these pathways are activated following SCE therapy. Participants in the treatment group exhibited 
significant increases in plasma level of TGF- 1 at the 4-week follow-up (P = 0.001, Figure 17B) but did not exhibit 
changes in the plasma level of IL-10 (P = 0.44, Figure 17B). Both TGF- 1 and IL-10 failed to show changes in 
the control group.  
0
0.2
0.4
0.6
0.8
1
1.2
Group A
Group B
Control group
Before treatment
Post treatment (weeks)
4 
12 
0 
24 
Lower limit for normal 
fasting C-peptide 
A
0
0.5
1
1.5
2
2.5
3
Pre-OGTT
Post-OGTT: 2 hrs
Before treatment
Post treatment (weeks)
4 
12 
0 
P = 0.009
P = 0.031
P = 0.078
B
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Pre-OGTT
Post-OGTT: 2 hrs
C
Before treatment
Post treatment (weeks)
4 
12 
0 
P = 0.009
40
P = 0.026

 
Page 16 of 59 
    We also examined levels of CD28 [31-35] and inducible costimulator (ICOS) [36,37], which are essential for the 
establishment, maintenance, and efficacy of Tregs [31-39]. Flow cytometry revealed an increase in CD28 and 
ICOS in lymphocytes 4 weeks after SCE therapy (Figure 17C), but levels of both molecules were unchanged in 
participants receiving sham therapy [1]. We also noted other changes at the 4-week follow-up consistent with 
improved helper T cell 1 (Th1) and Th2-mediated immune function (Figure 17D). Expression of IL-4 and IL-12 
was significantly increased (p = 0.016 and p = 0.0093, respectively), and expression of IL-5 and IL-13 was 
decreased (p = 0.00039 and p = 0.00206, respectively). The production of pro-inflammatory IL-17A was also 
decreased 4 weeks after treatment (Figure 17D, p = 0.0043). No changes were observed in levels of these 
cytokines in participants who received sham therapy [1].  
 
6.4.2 SCE Therapy for the treatment of type 1 diabetes in a Caucasian Population 
6.4.2.1 
Safety profile and feasibility of two treatments with SCE therapy in Caucasian T1D subjects 
In previous clinical trials, all subjects received one treatment with the SCE therapy [1-3]. Due to the likelihood that 
significant numbers of pathogenic autoimmune cells may have remained in lymph nodes and other tissues, failing 
to enter into the bloodstream during the procedure, and thus may have escaped the exposure to CB-SCs, we 
added a second treatment three months following the initial session in these T1D subjects (n = 15) (Figure 18A). 
No participants experienced any significant adverse events during the course of the two treatments with SCE 
Figure 17. Markers of immune function in T1D patients after SCE therapy. Patient lymphocytes were isolated from 
peripheral blood by Ficoll-Hypaque (  = 1.077) for flow cytometry analyses in T1D patients at baseline and 4 weeks after
SCE therapy. Isotype-matched IgG served as control. (A) Flow Analysis of CD4+CD25+Foxp3+ Tregs demonstrating an 
increase in the percentage of Tregs at 4 weeks post treatment. (B) Cytokine ELISAs demonstrating an increase in TGF- 1 
but not IL-10 at 4 weeks post treatment. (C) Flow cytometry on co-stimulating molecules indicating increases in CD28 and 
ICOS at 4 weeks post treatment with SCE therapy (top panels). Control group failed to show increases (bottom panels). (D)
Flow analysis of intra-cellular cytokines demonstrating differential effects on key interleukins at 4 weeks post treatment. 
Data are representative of preparations from all T1D participants (n = 12) that received SCE therapy. 
0
2
4
6
8
10
12
14
16
Control group
Treated group
Before treatment
Post treatment
P < 0.0001
A
P = 0.87
0
5
10
15
20
25
30
35
IL-10
TGF-beta1
Before treatment
Post treatment
P = 0.001
P = 0.44
B
ICOS
CD28
IgG control
Before treatment
After treatment
100
101
102
103
104
100
101
102
103
104
ICOS
IgG control
Before treatment
After treatment
CD28
Control
Group
Treated
Group
C
0
10
20
30
40
50
60
70
IL-4
IL-5
IL-12
IL-13
IL-17A
Before treatment
Post teatment
P = 0.016
P = 0.0093
P = 0.0004
P = 0.0021
P = 0.0043
D

 
Page 17 of 59 
therapy or during 56-week follow-up. During the procedure, only mild discomfort at the site of venipuncture (the 
median cubital vein) and some soreness of the arm were noted for some participants. No fever or rejection was 
noted during follow-up studies. 
    Additionally, our ongoing children (NCT01996228) also confirmed the safety in Caucasian children (Figure 18 
B and C). The procedure of treatment was well tolerated in all children without any significant adverse events.      
 
Figure 18. SCE therapy in Caucasian T1D subjects. (A) Diagram of SCE therapy for the treatment and follow-up 
studies in Spanish T1D trial. All the participants received two treatments with the SCE therapy. Human cord blood 
units were derived from healthy allogeneic donors. The preparation of CB-SC cultures SCE devices were cultured in 
serum-free culture medium and incubated at 37°C, in 8% CO2.  After 2-3 weeks, CB-SCs growing at 90% confluence 
were prepared for clinical trial. One Educator device was generated from one cord blood unit, and used for one subject 
at one treatment. Follow-up visits were scheduled 2, 8, 12, 26, 40 and 56 weeks after treatment for clinical assessments 
and laboratory tests. Previous work demonstrated that participants receiving sham therapy failed to show changes in 
immune modulation [1]. (B and C) A 3-year old boy and 4-year old girl were receiving SCE therapy respectively. Pictures 
were taken from the children clinical trial (NCT01996228) performed in the Second Xiangya Hospital of Central South 
University (Changsha, China).  
6.4.2.2 
Clinical efficacy of SCE therapy in the modulation of memory T cell compartment of Caucasian 
subjects [10] 
To evaluate the immune modulating effects of the SCE therapy, we used flow cytometry to examine immune 
markers in 15 participants following SCE therapy. Clinical data indicated no changes in total cell numbers of each 
cell population during one-year follow-up, including leukocyte common antigen CD45+ nuclearized cells, CD3+ T 
cells, CD4+ T cells, CD8+ T cells, CD56+ NK cells, and CD20+ B cells (Figure 19A). Quantification of the 
percentages of total CD4+ and CD8+ T cells in peripheral blood remained very stable over a year (Figure 19B). 
Notably, the percentage of naïve CD4+ T (CD45RA+ CCR7+) cells was significantly increased at 26 weeks after 
the treatment with SCE therapy (P = 0.0042), and maintained through the final follow-up (at 56 weeks post-
treatment, P = 0.0021) (Figure 19C). The percentage of naïve CD8+ T cells did not exhibit significant changes at 
Time (week): 
1st
Prepare SCE
in GMP lab
Follow-up Studies
2nd
8
12
26
2
40
Hospitalize
Baseline tests
Recruit subjects
into clinical trial
Sign Consent Form
Receive SCE therapy
56
A
B
C

 
Page 18 of 59 
any follow-ups (Figure 19C). These findings suggested that the SCE therapy restored the regeneration of naïve 
CD4+ T cells, an essential part of normal immune capacity.        
Figure 19. Changes in immune markers in Caucasian T1D patients after SCE therapy. All subjects received two 
treatments with SCE therapy. After 3 months, all subjects received a 2nd treatment with SCE therapy. Follow-up visits were 
scheduled 2, 8, 12, 26, 40 and 56 weeks after treatment for clinical assessments and laboratory tests. Patient lymphocytes 
were isolated from peripheral blood by Ficoll-Hypaque (  = 1.077) for flow cytometry analyses in T1D patients at baseline 
and different time points after SCE therapy. Isotype-matched IgG served as control. (A) Immune cell quantification in 
peripheral blood. (B) Percentage of CD4+ and CD8+ T cells in peripheral blood. (C) Flow Analysis of naïve CD4+ and CD8+
T cells in peripheral blood, demonstrating an increase in the percentage of naïve CD4+ T cells at 26 weeks post treatment. 
(D) Flow Analysis of CD4+ TCM and CD8+ TCM cells in peripheral blood, demonstrating an increase in the percentage of 
CD4+ TCM cells at 18 weeks post treatment. (E) Flow Analysis of CD4+ TEM and CD8+ TEM cells in peripheral blood, 
demonstrating a decline in the percentage of CD4+ TEM and CD8+ TEM cells at 18 weeks and 26 weeks respectively post 
0
10
20
30
40
50
60
Naïve CD4+ T cells
Naïve CD8+ T cells
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.0021
P = 0.0042
P = 0.22
P = 0.27
P = 0.23
*
*
P = 0.35
P = 0.23
P = 0.51
P = 0.76
P = 0.62
C
0
10
20
30
40
50
60
CM CD4+ T cells
CM CD8+ T cells
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.0012
P = 0.0039
P = 0.018
P = 0.55
P = 0.204
*
*
P = 0.26
P = 0.11
P = 0.034
P = 0.19
P = 0.46
*
*
D
0
10
20
30
40
50
60
EM CD4+ T cells
EM CD8+ T cells
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.0013
P = 0.0024
P = 0.03
*
*
P = 0.003
P = 0.015
P = 0.063
*
*
*
E
0
2
4
6
Baseline
Post treatment
(26 weeks)
P = 0.002
12.5
10
7.5
5.0
2.5
0
Baseline
Post treatment
(26 weeks)
P = 0.006
F
G

 
Page 19 of 59 
treatment. (F) Flow Analysis of CD4+HLA-DR+ in peripheral blood, demonstrating a decline in their percentages at 26 weeks 
post treatment. (G) Flow Analysis of CD8+HLA-DR+ T cells in peripheral blood, demonstrating a decline in their percentages 
at 26 weeks post treatment. Data are shown as mean ± SD for all statistical analyses (A-E), pAIRE
-F). 
                                                    
Naive T cells constantly recirculate between secondary lymphoid tissue (SLTs) using the blood and lymph as 
conduits. Naive T cells become activated only after recognizing cognate antigens presented by antigen-presenting 
cells (APCs) within lymphoid organs. After the initial immune response, 90-95% of antigen-specific T cells undergo 
apoptosis; the remaining cells mainly give rise to long-term memory T cells, including central memory T cells (TCM) 
and effector memory T cells (TEM) [40,41]. These memory T cells are meant to protect the human body from the 
re-
specifically the TEM, may also play a critical role in the persistence of disease in autoimmune conditions [42]. To 
explore the effects of the SCE therapy on these memory T cells, TCM and TEM were examined by flow cytometry. 
Overall analysis in these subjects demonstrated that the percentage of CD4+ TCM (CD45RA- CCR7+) cells was 
markedly and constantly increased after receiving SCE therapy at 18 weeks (P = 0.018) (Figure 19D). In contrast, 
the percentage of CD8+ TCM cells was only temporarily improved at 18 weeks (P = 0.034), but return to baseline 
levels during continued follow-ups (Figure 19D). In comparison with Group B subjects (4/9, 44%), Group A 
subjects (4/6, 67%) were more efficiently increasing the percentage of CD4+ TCM cells over 30% of positive cells 
at 18 weeks follow-up (data not shown). Notably, overall analysis of TEM (CD45RA+ CCR7-) cells revealed that 
both CD4+ TEM cells and CD8+ TEM cells were considerably decreased at 18 weeks (P = 0.03) and 26 weeks (P = 
0.0024) respectively (Figure 19E). Group A subjects (6/6, 100%) were more efficiently decreasing the percentage 
of CD8+ TEM cells over 15% of positive cells than that in Group B subjects (7/9, 78%) at 26 weeks follow-up; 5/6 
(83%) of Group A subjects vs 7/9 (78%) of Group B subjects for the reduction of CD4+ TEM cells at 26 weeks 
follow-up (data not shown).  
In addition, using HLA-DR as an activation marker for T cells [43], clinical data demonstrated that the 
percentage of CD4+HLA-DR+ T cells  and CD8+HLA-DR+ T cells  were markedly declined at 26 weeks follow-up 
relative to the baseline levels (P = 0.002 and P = 0.006 respectively) (Figure 19F and G).  
6.4.2.3 
Up-regulation of CCR7 expression on T cells after receiving SCE therapy in Caucasian T1D 
subjects [10] 
C-C chemokine receptor 7 (CCR7) plays important roles in lymph node homing of T cells via high endothelial 
venules and mediating the T-cell homeostasis [44,45]. To further explore the modulation of SCE therapy, we 
analyzed the level of CCR7 expression on naïve T, TCM, and TEM cells by flow cytometry. Clinical data revealed 
that both Group A and B subjects significantly increased the expression of CCR7 on Naïve CD4+ T cells (Figure 
20A), naïve CD8+ T cells (Figure 20B), and CD4+ TCM cells (Figure 20C). The marked responses of Naïve CD4+ 
T cells in Group A subjects happened as early as at 8 weeks post SCE therapy, comparable to that of delayed 
responses in Group B subjects at 26 weeks (Figure 20A). The up-regulation of CCR7 expression on naïve CD8+ 
T cells was shown simultaneously at 8 weeks follow-up in both Group A and B subjects (Figure 20B). The 
expression of CCR7 on CD8+ TCM cells in Group A subjects was also improved and started at 18 weeks follow-up 
(Figure 20D), but with a postponed response in Group B subjects at 56 weeks follow-up (P = 0.046, Figure 20D). 
The levels of CCR7 expression on both CD4+ TEM and CD8+ TEM cells were markedly enhanced at 56 weeks 
follow-up after receiving SCE therapy in both groups (Figure 20E). The data suggest that the up-regulation of 
CCR7 expression on CD4+ and CD8+ T cells may lead to the re-distribution of T cells in T1D subjects after the 
treatment with SCE therapy.  
 

 
Page 20 of 59 
Figure 20. Up-regulation of CCR7 expression on T cells 
in Caucasian T1D patients after SCE therapy. Patient 
lymphocytes were isolated from peripheral blood by Ficoll-
Hypaque (  = 1.077) for flow cytometry analyses in T1D 
patients at baseline and different time points after SCE 
therapy. Isotype-matched IgG served as control. The levels 
of CCR7 expression were analyzed by Kaluza Flow 
Cytometry Analysis Software and present as arbitrary unit 
(a.u.). (A) Up-regulation of CCR7 expression on Naïve CD4+
T cells. (B) Up-regulation of CCR7 expression on Naïve 
CD8+ T cells. (C) Up-regulation of CCR7 expression on 
CD4+ TCM cells. (D) Up-regulation of CCR7 expression on 
CD8+ TCM cells. (E) Up-regulation of CCR7 expression on 
CD4+ and CD8+ TEM cells. Data are shown as mean ± SD for 
all statistical analyses (A-E), pAIRE
-E). 
 
6.4.2.4 
Clinical efficacy of SCE thera
Caucasian subjects [10] 
demonstrated that treatment with SCE therapy provided lasting reversal of autoimmunity that allowed the 
regeneration of islet  cells and improved metabolic control in Chinese subjects with long-standing T1D [1,5]. To 
test the therapeutic potential of SCE therapy in the metabolic control of Caucasian T1D subjects, 
-cell 
function was examined through the measurement of fasting plasma C-peptide and glucagon-stimulated C-peptide 
0
50
100
150
200
250
300
350
400
Group A: Naïve CD4+ T cells
Group B: Naïve CD4+ T cells
A
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.001
P = 0.035
P = 0.002
P = 0.002
P = 0.06
P = 0.002
P = 0.004
P = 0.06
0
50
100
150
200
250
300
350
400
Group A: Naïve CD8+ T cells
Group B: Naïve CD8+ T cells
B
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.018
P = 0.02
P = 0.005
P = 0.015
P = 0.04
P = 0.007
P = 0.044
P = 0.02
P = 0.019
0
10
20
30
40
50
60
70
80
90
100
Group A: CD4+ Tcm cells
Group B: CD4+ Tcm cells
C
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.012
P = 0.0006 P = 0.003
P = 0.01
P = 0.009
0
10
20
30
40
50
60
70
80
90
Group A: CD8+ Tcm cells
Group B: CD8+ Tcm cells
D
Before treatment
Post treatment (weeks)
2 
18 
0 
56 
8
26 
P = 0.001
P = 0.004
P = 0.01
P = 0.1
P = 0.046
0
1
2
3
4
5
6
7
8
9
CD8+ TEM
CD4+ TEM
CD8+ TEM
CD4+ TEM
Group A subjects
Group B subjects
P = 0.009
P = 0.004
P = 0.017
P = 0.04
E

 
Page 21 of 59 
levels. In Group A subjects, clinical results demonstrated up-regulation of both fasting and glucagon-stimulated 
C-peptide levels at 12 weeks in two recent-
populations) (Figure 21A and B). Recovered fasting and glucagon-stimulated C-peptide levels were retained in 
subject 1 through the final follow-up at 56 weeks post-treatments (Figure 21A). Glucagon-stimulated C-peptide 
levels in subject 2 were stable during one-year follow-up, while fasting C-peptide levels declined slightly (Figure 
21B). Subject 3 who had T1D 10 years at the time of study, still achieved modest improvements including an 
increase in fasting C-peptide from 0.25 ng/ml at basal to 0.36 ng/ml at 56 weeks and an increase in glucagon-
stimulated C-peptide level from 0.4 ng/ml at basal to 0.52 ng/ml at 26 weeks (Figure 21C). Subject 4 who had 
T1D 3 years at the 
-cell function with no significant change over time in fasting 
C-peptide levels from 1.05 ng/ml at baseline to 0.88 ng/ml at 40 weeks and in glucagon-stimulated C-peptide 
levels from 2.18 ng/ml at baseline to 2.01 ng/ml at 40 weeks (Figure 21D). 
 
Figure 21
-cell function in Caucasian T1D subjects. All subjects received two treatments 
with SCE therapy. T1D subjects received two treatments with SCE therapy at the beginning and 3rd month respectively. 
Fasting (blue) and glucagon-stimulated C-peptide levels (brown) were examined at different time points according to the 
protocol. For glucagon-stimulated C-peptide production, glucagon (1mg, i.v.) was administrated within 30 seconds, and six 
minutes later, plasma samples were collected for the C-peptide test by Ultrasensitive C-peptide ELISA kit. These data were 
-cell function (Group A). (A-D) Recovered fasting and glucagon-stimulated 
C-peptide levels were retained in subject 1 through the final follow-up at 56 weeks post-treatments in subject 1 -4 
respectively. 
 
Additionally, no changes were observed in fasting C-peptide levels of severe long-standing Group B patients 
with no residual 
were strikingly different from that reported in long-standing severe Chinese T1D subjects [1,5]. The potential 
mechanisms underlying this difference need to be explored.   
 
6.4.3 SCE Therapy for the treatment of type 2 diabetes 
The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health 
systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune 
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Fasting C-peptide
Glucagon-stimulated C-peptide
A
Post treatment (weeks)
12 
0 
56 
Subject 1: 1 year T1D
Basal
0
0.2
0.4
0.6
0.8
1
1.2
Fasting C-peptide
Glucagon-stimulated C-peptide
B
Basal
Post treatment (weeks)
12 
0 
56 
Subject 2: 1 year T1D
26 
40 
0
0.1
0.2
0.3
0.4
0.5
0.6
Fasting C-peptide
Glucagon-stimulated C-peptide
C
Post treatment (weeks)
26 
0 
56 
Subject 3: 10 years T1D
Basal
0
0.5
1
1.5
2
2.5
Fasting C-peptide
Glucagon-stimulated C-peptide
D
Subject 4: 3 years T1D
Post treatment (weeks)
26 
0 
40 
Basal
12 

 
Page 22 of 59 
dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune 
modulation may be a useful tool in treating the disease. In an open-label, phase1/phase 2 study, patients (N = 36) 
with long-standing T2D were divided into three groups (Group A, oral medications, n = 18; Group B, oral 
medications + insulin injections, n = 11; Group C having impaired -cell function with oral medications + insulin 
injections, n = 7). All patients received one treatment with the SCE therapy in which 
s circulated 
through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them 
with adherent CB-
  
Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation 
markers after receiving SCE therapy. Median HbA1C in Group A and B was significantly reduced from 8.61%  
1.12 at baseline to 7.25%  0.58 at 12 weeks (p = 2.62E-06), and 7.33%   1.02 at one year post treatment (p = 
0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin 
sensitivity was improved post treatment. Notably, the islet beta-cell function in Group C subjects was markedly 
recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that SCE therapy 
reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine 
production. Clinical data from current phase 1/2 study demonstrate that SCE therapy is a safe approach that 
produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a 
single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated 
with conventional stem cell-based approaches. 
6.4.3.1 
Feasibility and safety of SCE therapy in T2D 
Baseline characteristics of participants with T2D are provided in the Table [2]. Thirty six patients with T2D have 
received SCE therapy in a safety study, and their results are similar to the safety evaluation with T1D participants 
[1]. No participants experienced any significant adverse events during the course of treatment and post treatment 
for over a year. Patient complaints were limited to mild discomfort during venipunctures at the site of median 
cubital vein and some soreness of the arm that resolved quickly following aphaeresis. 
6.4.3.2 
Efficacy outcomes in improving metabolic control 
After receiving SCE therapy and being discharged from the hospital, patients continued their regular medications. 
Follow-up studies demonstrated that the median glycated hemoglobin (HbA1C) in Group A (n = 18) and Group B 
(n = 11) was significantly lowered from 8.61%  1.12 at baseline to 7.9%  1.22 at 4 weeks post treatment (p = 
0.026), 7.25%  0.58 at 12 weeks post treatment (p = 2.62E-06) (Figure 22A), and 7.33%  1.02 at one-year post 
treatment (p = 0.0002). According to the A1C goal (<7%) recommended by the American Diabetes Association 
(ADA) for the treatment of adult diabetics, 28% (5/18) of subjects in Group A, 36% (4/11) of subjects in Group B, 
and 29% (2/7) of subjects in Group C achieved this goal at 12 weeks post treatment. More than 31% of total 
subjects achieved and maintained the <7% standard for over a year. Additionally, based on the efficacy criteria, 
11 of 18 (61.1%) subjects in Group A, 8 of 11 (72.7%) subjects in Group B, and 4 of 7 (57.1%) subjects in Group 
C with reduction of A1C value (> 0.5%) at 4 weeks post treatment. Thirteen of 18 (72.2%) subjects in Group A, 9 
of 11 (81.8%) subjects in Group B, and 6 of 7 (85.7%) subjects in Group C with reduction of A1C value (> 0.5%). 
Twenty eight of 36 (78%) of total subjects reduced by 1.28 
 0.66 at 12 weeks post treatment. The data 
demonstrate that glycemic control was improved in T2D patients after SCE therapy.  
To explore the change in insulin sensitivity, we analyzed homeostasis model assessment (HOMA) of insulin 
resistance (HOMA-IR) by the product of fasting plasma glucose and C-peptide (instead of insulin due to subjects 
receiving insulin injections) in Group A and B. The data revealed that levels of HOMA-IR c-pep were markedly 
reduced at 4 weeks follow-up (Figure 22B). It suggests that insulin sensitivity has been improved post treatment. 
Consistent with their improved  cell function, the median daily dose of metformin was reduced 33% ~ 67%, and 
insulin was reduced 35% at 12 weeks post treatment. 

 
Page 23 of 59 
Figure 22. Improvement of metabolic control by SCE therapy. (A) 12-week follow-up HbA1C levels in T2D subjects.  
(B) Analysis of insulin sensitivity by HOMA-IR C-peptide at 4 weeks post treatment with SCE therapy. (C) 56-week follow-
up C-peptide levels in Group C T2D subjects with impaired islet  cell function.  (D) Analysis of islet  cell function by HOMA-
B C-peptide at 12-week follow-up post treatment with SCE therapy. 
    Notably, we found that levels of fasting C-peptide were markedly increased in the long-standing T2D subjects 
with impaired islet  cell function (Group C, diabetic duration 14  6 years, n = 7, P = 0.0073) (Figure 22C). Twelve 
weeks after receiving the SCE therapy, fasting C-peptide levels reached normal physiological levels and were 
maintained through the last follow-up for this measure (56 weeks) (0.36 ± 0.19 ng/ml at baseline vs 1.12 ± 0.33 
ng/ml at one year post treatment, p = 0.00045, Figure 22C). The -cell functional analysis by using HOMA-B C-
peptide demonstrates that the function of islet  cells was markedly enhanced in group C subjects after receiving 
SCE therapy (Figure 22D). The data suggest that the restoration of C-peptide may be associated with the 
regeneration of islet  cells as we demonstrated in our previous work in T1D [1,6].   
6.4.3.3 
Efficacy outcomes in correcting the immune dysfunction 
To determine the molecular and cellular mechanisms underlying the improvement of metabolic control, we 
examined the effects of anti-inflammation and immune modulation of SCE therapy in T2D. We used ELISA to 
examine proinflammatory cytokines IL-1, IL-6, and TNF  in the plasma, which are primarily involved in the insulin 
resistance and T2D [46,47]. We found that IL-1, IL-6, and TNF  were all at background levels in these long-
standing T2D subjects and failed to show changes after SCE therapy (p = 0.557, p = 0.316, p = 0.603 respectively), 
probably because metabolic inflammation is a chronic sub-degree inflammation [47] and the serum samples which 
were directly collected from the blood of T2D patients, not from the lipopolysaccharide (LPS)-activated monocytes 
of T2D subjects [48]. Importantly, we found that anti-inflammatory and immune suppressive cytokine TGF- 1 was 
markedly increased in the plasma of T2D subjects post treatment at 4 weeks relative to the baseline levels (Figure 
23A). However, IL-10 was unchanged in all participants (p = 0.497). These findings suggest up-regulation of TGF-
1 may be one of potential mechanisms contributing to the reversal of insulin resistance by SCE therapy. 
0
2
4
6
8
10
12
14
Group A
Group B
Group C
Before treatment
Post treatment
P = 0.0004
A
P = 0.003
P = 0.036
0
50
100
150
200
250
300
350
Group A
Group B
Before treatment
Post treatment
P = 0.04
P = 0.015
B
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
P = 0.0073
C
Before treatment
Post treatment (weeks)
4 
12 
0 
Lower limit for normal 
fasting C-peptide 
24 
56 
0
200
400
600
800
1000
1200
1400
1600
1800
Before treatment
Post treatment
P = 0.027
D

 
Page 24 of 59 
    Next, using a more sensitive intra-cellular flow cytometry analysis, we examined interleukin-17 (IL-17, also 
known as IL-17A) and Th1/Th2 immune response-associated cytokines in the peripheral blood of T2D subjects. 
IL-17A is a well-known proinflammatory cytokine involved in the autoimmune diseases. Importantly, mounting 
evidence collected over the past decade indicates that the etiology of T2D includes an autoimmune component 
that initiates an inflammation affecting pancreatic islet  cells  [47,49-53], which provides new insight into the 
mechanism and potential treatment of insulin resistance through immune modulation. Recent clinical studies 
showed the increasing of circulating Th17 cells and IL-17 production in T2D patients [54] and obese patients [55]. 
Additionally, recent studies showed that the level of Th1-associated cytokine IL-12 is increased in T2D subjects 
[56,57].  We found that the production of IL-17, IL-12, and Th2-associated cytokine IL-4 and IL-5 were all markedly 
decreased after SCE therapy (Figure 23B). 
    To explore the cellular mechanism underlying the modulation on the Th1/Th2 immune responses, we focused 
on the changes of co-stimulating molecules CD80/CD86 expressed on the monocytes/macrophages, the 
professional antigen-presenting cells that play a key role in the onset of chronic inflammation and obese-
associated insulin resistance of T2D [58-62]. Flow results demonstrated that the percentage of CD86+CD14+ 
monocytes was markedly decreased 4 weeks after treatment (Figure 23C, P = 0.0212). There was no significant 
change in the level of CD80+CD14+ monocytes (P = 0.13). The ratio of CD86+CD14+ monocytes / CD80+CD14+ 
monocytes was reduced from 3.86  2.56 to 1.22  0.48 (P = 0.01). Further flow analysis of the ligands of 
CD80/CD86, CD28/CTLA-4 expressed on lymphocytes revealed that the expression of CTLA-4 was markedly 
increased 4 weeks after receiving SCE therapy (0.51%  0.5 before treatment vs 1.98%  0.51 post treatment, P 
= 9.02E-05). However, flow analysis failed to show differences in the expression of co-stimulating molecule CD28 
(69.98%  14.17 before treatment vs 61.5%  10.89 post treatment, P = 0.225). Additionally, we examined 
changes in the CD4+CD25+Foxp3+ Tregs population after receiving SCE therapy. Flow analysis did not identify 
any differences between baseline and 4- or 12-weeks post treatment (Figure 23D, P = 0.689). Therefore, these 
data suggest that SCE therapy may modulate the Th1/Th2 immune responses through the action of antigen-
presenting cells monocytes rather than Tregs.  
 
0
5
10
15
20
25
30
35
40
45
IL-10
TGF-beta 1
Before treatment
Post treatment
P = 0.00059 
P = 0.497 
A
0
5
10
15
20
25
30
35
40
45
IL-4
IL-5
IL-12
IL-13
IL-17A
Before treatment
Post treatment
P = 0.00041 
P = 0.024 
P = 0.0015 
P = 0.0045 
P = 0.35 
B
0
2
4
6
8
10
12
14
16
Before treatment
Post treatment
P = 0.0212 
P = 0.13 
CD80+CD14+
Monocytes
CD86+CD14+
Monocytes
C
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
Baseline
Post treatment
P = 0.689 
D

 
Page 25 of 59 
Figure 23. Anti-inflammatory effects of SCE therapy. (A) Up-regulation of plasma level of TGF- 1 in T2D patients at 
baseline and 4 weeks after SCE therapy. (B) Flow analysis of intra-cellular cytokines demonstrating differential effects on 
key interleukins at 4 weeks post treatment. (C) Down-regulation percentage of CD86+CD14+monocytes in T2D patients at 
baseline and 4 weeks after SCE therapy. (D) Flow Analysis of CD4+CD25+Foxp3+ Tregs demonstrating no change in the 
percentage of Tregs at 4 weeks post treatment. 
6.4.3.4 
In vitro mechanistic studies of the immune modulation of CB-SCs on monocytes. 
To better understand the immune modulation of CB-SC on monocytes, we performed in vitro co-culture 
experiments by using CD14+ monocytes purified from human peripheral blood. The purified CD14+ monocytes 
were co-cultured with CB-SCs at different ratios. We found that there were strong reactions after adding the CD14+ 
monocytes to CB-SCs (Figure 24A, bottom left panel). Flow analysis demonstrated that co-culture with CB-SCs 
for 18 hrs resulted in the significant apoptosis of monocytes at the ratio 1 : 5 of CB-SC : monocytes (Figure 24B). 
Correspondingly, both the cell viability and attachment of CB-SCs were also affected in the presence of apoptotic 
monocytes (Figure 24A, bottom left panel). The cellular processes of CB-SCs were reduced in length, but most 
were still attached to the bottom (Figure 24A, bottom left panel). Interestingly, these impaired CB-SCs were 
restored after co-culture for 2-3 days; they continually expanded and became 90 ~ 100% confluence after 7-10 
days (Figure 24A, bottom right panel). Mechanistic studies revealed that CB-SCs displayed the cellular inhibitor 
of apoptosis protein (cIAP) 1 [63] that protects CB-SCs against the cytotoxic effects of monocytes, allowing them 
to survive and proliferate (Figure 24C). To further explore the molecular mechanisms underlying the cytotoxic 
effects of monocytes on CB-SCs, we found that CB-SCs expressed tumor necrosis factor receptor II (TNF R II) 
but not TNF R I (Figure 24D). Recombinant TNF showed cytotoxicity to CB-SCs at different doses (Figure 24E). 
Notably, CB-SCs pre-treated with TNF RII mAb (20 g/ml) at ratio of 1 : 10  could markedly block the toxic action 
of monocytes and protect 50% of CB-SCs with good cell viability and morphology.  
Figure 24. In vitro study of the immune modulation of CB-SCs on monocytes. (A) Phase contrast microscopy shows the 
co-culture of CB-SC with monocytes (bottom left panel) for 18 hrs. CB-SCs co-culture with lymphocytes (top right panel) served 
as control. The impaired CB-SCs after co-culture with monocytes were restored to expansion and became 90 ~ 100% 
confluence after 7-10 days (bottom right). Original magnification,  100. (B) Apoptotic analysis of floating cells from the co-
culture of CB-SCs with monocytes for 18 hrs. (C) Western blotting show the expression of the cellular inhibitor of apoptosis 
protein (cIAP) 1, not cIAP2, in four preparations of CB-SCs. (D) Western blotting show the expression of tumor necrosis factor 
receptor II (TNF R II), not TNF-RI, in four preparations of CB-SCs. (E) TNF  suppress the proliferation of CB-SCs in a dose-
CB-SC control
CB-SC + lymphocytes
CB-SC + monocytes
A
CB-SC + monocytes on 10 days
5.62%
39.94%
50.92%
3.51%
Annexin V
B
cIAP1
cIAP2
-actin
M.W. (kD)
70
72
42
P2
P3
P4
P1
C
TNF RI
TNF RII
-actin
M.W. (kD)
51
48
42
P2
P3
P4
P1
D
0
5
10
15
20
25
30
35
TNF alpha dose (ng/ml)
100
50
25
12.5
0
E
0
50
100
150
200
250
300
350
400
450
500
LPS-stimulated monocytes versus control
LPS-stimulated monocytes + CB-SCs versus control
LPS-stimulated monocytes + CB-SCs + 1400W versus control
F
Chemokines
Cytokines
Metallo-
peptidase

 
Page 26 of 59 
response manner. Cell proliferation was evaluated using CyQUANTR Cell Proliferation Assay Kit (Millipore, OR) [25]. (F)
Blocking experiment with iNOS inhibitor 1400W demonstrates that CB-SC-derived nitric oxide (NO) contributes to the immune 
modulation of CB-SCs on monocytes. Monocytes were initially stimulated with lipopolysaccharide (LPS, 10 g/ml) for 8 hrs, 
and then co-cultured with CB-SCs at ratio 1 : 5 of CB-SCs : monocytes for 48 hrs in the presence or absence of 1400W 
(100nM), followed by real time PCR analysis by using Human Th17 for Autoimmunity & Inflammation PCR Array kit 
(SABiosciences, Valencia, CA).  
    
  To further explore the immune modulation of CB-SCs on monocytes, lipopolyssacharide (LPS)-stimulated 
purified CD14+ monocytes were co-cultured with CB-SCs. Real time PCR array showed that co-culture with CB-
SC could significantly down-regulate numbers of LPS-stimulated, inflammation-related genes including 
chemokines, multiple cytokines, and matrix metallopeptidase, along with signaling pathway molecule NF- B 
(Figure 24F). These data clearly indicate that in vitro co-culture with CB-SCs causes substantial down-regulation 
of inflammation-associated gene expressions in monocytes. Previous work showed that CB-SCs function as 
immune modulators on lymphocytes via nitric oxide (NO) production [11]. To confirm the action of NO involved in 
the immune modulation of CB-SCs on monocytes, the specific inducible nitric oxide synthase (iNOS) inhibitor 
1400W was applied to the co-culture system. The data demonstrated that the inhibitory effects of CB-SC on LPS-
stimulated monocytes could be significantly reversed in the presence of iNOS inhibitor 1400W (Figure 24F). 
Interestingly, we found that blocking NO production in CB-SCs could markedly increase the expressions of 
chemokine CCL20 and cytokines (e.g., IL-1 , IL-6, IL-8, IL-23, and TNF ) in monocytes. Thus, it indicates that 
CB-SC-derived NO plays an essential role in the immune modulating and anti-inflammatory effects of CB-SCs on 
monocytes. 
6.4.4 SCE Therapy for the treatment of Alopecia Areata 
    Alopecia areata (AA) is one of the most common autoimmune diseases and targets the hair follicles, with high 
impact on the quality of life and self-esteem of patients due to hair loss. Clinical management and outcomes are 
challenged by current limited immunosuppressive and immunomodulating regimens. In an open-label, phase 
1/phase 2 study, patients (N = 9) with severe AA received one treatment with the SCE therapy. The median age 
was 20 years (median alopecic duration, 5 years). Clinical data demonstrated that patients with severe AA 
achieved improved hair regrowth and quality of life after receiving SCE therapy. Flow cytometry revealed the up-
regulation of Th2 cytokines and restoration of balancing Th1/Th2/Th3 cytokine production in the peripheral blood 
-
newly generated hair follicles against autoimmune destruction. Mechanistic studies revealed that co-culture with 
CB-SC may up-regulate the expression of coinhibitory molecules B and T lymphocyte attenuator (BTLA) and 
programmed death-1 receptor (PD-
+NKG2D+ effector T cells and suppress their proliferation via 
herpesvirus entry mediator (HVEM) ligands and programmed death-1 ligand (PD-L1) on CB-SCs. Current clinical 
data demonstrated the safety and efficacy of the SCE therapy for the treatment of AA. This innovative approach 
produced lasting improvement in hair regrowth in subjects with moderate or severe AA. 
6.4.4.1 Suppressed Proliferation of Antigen-Specific T cells by co-culture with CB-SC  
The expansion of antigen-specific autoreactive T cells is the critical step leading to the destruction of tissues in 
autoimmune diseases. Recently, mouse and human data have demonstrated that CD8+NKG2D+ effector T cells 
function as a key mediator in the pathogenesis of AA [27]. To explore the therapeutic potential of CB-SC in AA, 
CD8+NKG2D+ effector T cells from human peripheral blood mononuclear cells (PBMC) were activated and 
expanded with Dynabeads coupled with anti-CD3, anti-CD28, and anti-CD137 mAb in the presence of IL-2 and 
IL-7. After ex vivo expansion with this mAb combination for 5 days, there were large numbers of cell clusters with 
different sizes floating in the supernatant (Figure 25A, left panel), suggestive of significant cell proliferation. 
However, this phenomenon was not evident in the presence of CB-SCs (Figure 2A, right panel). Flow cytometry 
revealed that 52% of lymphocytes proliferated in response to costimulation with this combination of mAb 
molecules and growth factors (Figure 25B, middle panel). By contrast, there were only 13% of lymphocytes 
proliferating after co-culture with CB-SCs (Figure 25B, right panel). Triple color staining demonstrated that 25% 
of CD8+NKG2D+ T cells were proliferated upon costimulation in the absence of CB-SCs. Notably, the percentage 
of proliferating CD8+NKG2D+ T cells was reduced to 5% following co-culture with CB-SCs. Further multi-color flow 
cytometry indicated that the percentage of CD8+NKG2D+ T cells was decreased from 25.6% ± 0.43% to 13.87% 
± 3.43% in the presence of CB-SCs (Figure 2C, left panels) (P = 0.04). Additionally, we examined the expression 
of coinhibitory molecules on CD8+NKG2D+ T cells, such as BTLA (B and T lymphocyte attenuator) and PD-1 
(programmed death-1 receptor). Results confirmed that co-culture with CB-SCs increased the percentage of 
CD8+NKG2D+BTLA+PD-1+ T cells from 69% to 91%. Their mean fluorescence intensity (MFI) also increased after 
co-culture with CB-SCs (Figure 25C, right panels). These data demonstrated that CB-SCs could markedly 

 
Page 27 of 59 
suppress the proliferation of CD8+NKG2D+ T cells and up-regulate the expression of coinhibitory molecules on 
those cells. This finding supports the clinical-translational potential of CB-SCs in AA subjects. 
6.4.4.2 
Hair Regrowth in Alopecia Areata Subjects 
Nine AA subjects (the median age was 20 years; median alopecic duration was 5 years) received one treatment 
with SCE therapy and completed the study. All patients tolerated the procedure well, without any significant 
adverse events during the course of treatment. Their baseline clinical characteristics are described in Table 1. No 
participants experienced any significant adverse events during the course of treatment and the 2-year follow-up 
period. At 4 weeks post-treatment with SCE therapy, there was hair regrowth in subjects with patchy AA and 
Alopecia totalis (Figure 22). There were short vellus hairs found over the scalp of AA universalis patients (3/4) at 
the 12-week follow-up. Two participants (one having Alopecia totalis and another having multiple patches of AA) 
achieved complete hair regrowth at 12 weeks and 16 weeks, respectively, post-treatment, and remained 
completely recovered with no relapse after 2 years (Figure 22). Patients (3/4) with Alopecia universalis exhibited 
regrowth of eyebrows and eyelashes at the 12-week follow-up. Notably, the regrowth of eyebrows and a mustache 
were found in a 17-year-old boy affected by severe Alopecia universalis since he was 1-year-old.  Additionally, 
one of nine subjects with nail pitting was also improved, as indicated by the reduction of the number and the cavity 
of nail pitting at 4 weeks after receiving SCE therapy. All of these improvements were maintained throughout the 
final follow-up at 2 years. Of nine AA subjects, only one participant with Alopecia universalis failed to show a 
response to the SCE therapy, possibly due to the previous long-term therapy with oral prednisone. Overall, the 
proof-of-concept data demonstrated the therapeutic potential of SCE therapy for the treatment of AA subjects.  
 
 

 
Page 28 of 59 
6.4.4.3 Systematic immune modulation after receiving SCE therapy To explore the immune modulation of 
SCE therapy in AA subjects, we examined changes in the percentage of regulatory T cells (Treg) in their peripheral 
blood by using the specific intracellular marker, FoxP3. Flow cytometry revealed that the percentage of FoxP3 + 
Treg at 4 weeks was unchanged from the baseline (1.69% ± 1.02 versus 1.38% ± 0.85, P = 0.49). This result 
suggested that the immune modulation via SCE therapy in AA subjects may act through a different mechanism 
than induction of Treg. TGF-
-characterized cytokines contributing to the induction of peripheral 
immune tolerance [29], also plays a crucial role in modulating the normal cycling of hair follicles [30]. Flow 
cytometry demonstrated a marked increase in TGF-
receiving SCE therapy (P = 0.015, Figure 23A). Additionally, participants exhibited significant up-regulation of 
Th2 cytokines IL-4 and IL-5 expression at the 4-week follow-up (P = 0.012 and P = 0.022, respectively, Figure 
5A). Expression of IL-13 was also significantly increased in 6/9 participants (3.69 ± 3.27 versus 15.55 ± 7.48, P = 
0.005). No changes were observed in the level of Th1 cytokine IL-12 (P = 0.24, Figure 5A). Thus, these data 
suggested that the up-regulation of Th2 cell responses may suppress the Th1 cell-mediated autoimmune 
Figure 25. Ex vivo studies of the immune modulation 
of CB-SCs on T cells. (A)Phase contrast microscopy 
shows the formation of cell clusters in human peripheral 
blood-derived lymphocytes that were activated with 
Dynabeads coupled with anti-CD3, anti-CD28 and anti-
CD137 antibodies, 50 U/ml rIL-2 and 5 ng/ml rIL-7 for 5 
days, in absence (left panel) and presence (right panel) 
of CB-SCs.  co-culture with lymphocytes (top right panel) 
served as control. Original magnification,  100. (B) Cell 
proliferation was analyzed with C
Proliferation kit. Untreated lymphocytes (left panel) 
served as control. Flow cytometry dot plots and the 
percentage of each population were representative of 
three independent experiments with similar results.  
Figure 22. Regrowth of hair following SCE therapy. A 
patient with alopecia totalis received SCE therapy and 
followed up for two years, without relapse.  
CD3/CD28/CD137Abs/IL2/IL7-treated 
lymphocytes
CD3/CD28/CD137Abs/IL2/IL7-treated 
lymphocytes + CBSCs
A
Before treatment
Post treatment
4 weeks
8 weeks
12 weeks
40 weeks
2 years
CD3/CD28/CD137Abs/
IL2/IL7-treated 
lymphocytes
Untreated 
Lymphocytes
CD3/CD28/CD137Abs/
IL2/IL7-treated 
lymphocytes + CBSCs
Fluorescence Intensity
B

 
Page 29 of 59 
response in AA subjects [1,4,31] via associated cytokines [32]. The SCE therapy may shift the balance towards 
Th2-mediated immune responses, leading to the clinical efficacy in AA subjects.   
    The costimulatory molecule, CD28, functions as a key signal leading to Th2 cell differentiation and activation 
[33-36]. To determine changes in expression of costimulatory molecules, we examined lymphocytes for their levels 
of CD28 and inducible costimulator (ICOS). Flow cytometry demonstrated that the expression of CD28 was 
markedly increased in 8/9 participants 4 weeks after SCE therapy (P = 0.013, Figure 23B), but levels of ICOS 
were unchanged in all participants (P = 0.84, Figure 23B). Therefore, the up-regulation of CD28 expression, 
together with the increase of IL-4 production in AA subjects, can provide critical signals that shift the differentiation 
of human CD4+ T cells into Th2 cells [36] and attenuate Th1 cell responses. 
Figure 23. Immune modulation of SCE
therapy. Patient lymphocytes were isolated from 
peripheral blood by Ficoll-
= 1.077) for flow cytometric analyses in AA 
patients at baseline and 4 weeks after SCE
therapy. Isotype-matched IgG served as control. 
Histologic examination of alopecic skin (C 
and D). (A) Flow analysis of intracellular 
cytokines demonstrating differential effects on 
key interleukins at 4 weeks post-treatment. (B)
Flow 
analysis 
of 
costimulating 
molecules 
demonstrating an increase of CD28 expression 
at 
4 
weeks 
post-treatment. 
Data 
are 
representative of preparations from all AA 
participants (n = 9) who received SCE therapy. 
(C) Fresh skin tissues were collected from the 
scalps via biopsy for immunohistochemistry 
testing in participants with alopecia totalis before 
treatment and 12 weeks after receiving SCE
therapy. TGF-
of AA participants after receiving SCE therapy, 
with vertical section of hair follicle (top panels) 
and horizontal section of hair follicle. Isotype-
matched mouse IgG1 served as a negative 
control. Representative images were obtained 
(D) H&E 
 
6.4.4.4 Formation of a 
-
privilege of hair follicles after SCE therapy To clarify the molecular and cellular mechanism underlying the 
regrowth of hairs and the immune modulation, we performed immunohistochemistry on fresh tissues via the biopsy 
of alopecic lesions from subjects after receiving SCE therapy. The histology of the alopecic lesions demonstrated 
a dense, perifollicular lymphocytic infiltration around anagen hair follicles. Twelve weeks after receiving SCE 
therapy, histological examination revealed the restoration of hair follicle architecture and the disappearance of the 
substantial perifollicular infiltration of lymphocytes. Additionally, horizontal sections confirmed an increase in the 
density of total hair follicles in these participants with alopecia totalis.  
    TGF-
[29], but also in the regulation of cycling of hair follicles via the inhibition of keratinocyte proliferation and the 
induction of apoptosis [30,37,38]. Previous works demonstrated that TGF-
efficacy of SCE therapy in both autoimmune-caused diabetic NOD mice [23] and diabetic patients [24,25]. Notably, 
in addition to the up-regulation of TGF-
he peripheral blood mononuclear cells (Figure 15A), 
immunohistochemistry demonstrated that the level of TGF-
-treatment (21.78 ± 0.27) was much 
higher than that of baseline before treatment (5.14 ± 0.01, P = 3.14337E-14), specifically at the proximal anagen 
hair follicle (Figure 15C, middle panel). The expression of TGF-
und the hair follicles (Figure 
15C
-
this unique feature of TGF-
and protect the regenerated hair follicles against further destruction by autoimmune cells. HE staining further 
demonstrated the reduction of immune cell infiltration and restoration of normal architecture of the hair follicles 
(Figure 15D). 
To elucidate the major origin of the TGF-
-
performed double immunostaining in biopsied tissues by using the markers of monocyte/macrophage (CD14 and 
CD11b), Langerhans/dendritic cells (CD83 and DEC205), myeloid dendritic cells (CD1c), and Treg FoxP3, 
respectively. There were a few CD14+ monocytes/CD11b+ macrophages, Langerhans/dendritic cells, and FoxP3+ 
0
5
10
15
20
25
30
35
40
45
50
IL-4
IL-5
IL-12
TGF-beta 1
Before treatment
Post treatment
P = 0.012
P = 0.24
P = 0.022
P = 0.015
TGF- 1
DAPI
Merged 
Before 
treatment
Post 
treatment
A
C
0
10
20
30
40
50
60
70
Before treatment
Post treatment
P = 0.013
P = 0.84
CD28                                ICOS
B
D

 
Page 30 of 59 
Treg cells distributed in the dermal tissues. Double immunostaining showed weak staining for TGF-
immune cells, indicating that these immune cells were not the major source of TGF-
there was a strong expression of TGF-
SCE therapy (Figure 15C, bottom panels) relative to the baseline level (Figure 15C, top panel). These data 
suggested that keratinocytes were recovered after receiving SCE therapy and became the major TGF-
-
producing cells leading to the restoration of immune privilege and the induction of immune tolerance in hair 
follicles. The molecular mechanisms underlying the up-regulation of TGF-
rified 
in current studies. 
6.4.5 Molecular Mechanisms underlying the SCE Therapy  
In comparison with conventional immune therapies (e.g., monoclonal antibodies, vaccines, Treg therapy, and 
dendritic cell therapy) for T1D, the ex vivo immune modulation by CB-SCs inside a device can be controlled and 
monitored during the treatment. The lymphocytes (including Naïve T cells, TCM, and TEM) purified by apheresis 
can be intensively educated by directly contact with CB-SCs, with minimum interference from red blood cells, 
granulocytes, and other blood components. This approach also reduces side effects associated with conventional 
immune therapies. Based on our combined preclinical [1,5,6] and clinical studies [8,12] to date, immune 
modulation by CB-SCs seems to be mediated by a variety of molecular and cellular mechanisms including: 1) 
Expression of autoimmune regulator (AIRE) in CB-SCs plays an essential role (Figure 24A-C) [8]; 2) Functioning 
via cell-cell contact mechanisms involving the surface molecule programmed death ligand 1 (PD-L1) [5] and 
CD270 on CB-SCs, and their ligands PD-1 and BTLA on variety of immune cells (e.g., T cells, B cells, monocytes, 
dendritic cells, and granulocytes (Figure 24D and E) [10]; 3)  Acting through soluble factors released by CB-SCs 
(e.g., nitric oxide, TGF- 1) (Figure 24F) [5]; and 4) Adjusting the cell-cell interaction between antigen-presenting 
cells monocytes/macrophages and T cells through co-stimulating molecules and their ligands [12]. Thus, during 
the ex-vivo brief exposure to CB-SCs, T1D-
microenvironment created by CB-SCs through cell to cell contact and soluble factors. Additionally, previous work 
demonstrated that the proliferation of the T cell clone stimulated with antigen-presenting cells (APC) and different 
doses of GAD peptide were markedly and specifically decreased in the presence of CB-SCs compared to control 
group in the absence of CB-SCs [1]. Thus, it indicates that CB-SCs have a potential to eliminate the pathogenic 
T cells.  
    Human CB-SCs mediate immune modulation through the release of soluble factors (for example, nitric oxide 
and TGF-
-L1 (programmed death-1 ligand) [19,20,28]. 
To investigate additional potential molecular mechanisms underlying the immune modulation, we also found that 
CB-SCs strongly displayed the surface molecule HVEM (Figure 24D), the ligand of BTLA. Triple color staining 
confirmed the co-expression of HVEM on CB-SCs positive with molecular markers of leukocyte common antigen 
CD45+ and embryonic transcription factor OCT3/4+ (Figure 24D).To further substantiate that HVEM may be a 
mechanism of immune modulation by CB-SCs, flow cytometry also demonstrated the expression of BTLA and 
PD-1 on 
myeloid dendritic cells (mDCs), CD123+ plasmacytoid dendritic cells (pDCs), and CD66b+ granulocytes (Figure 
24E). There were about 40-80% BTLA+PD-1+ cells in each subpopulation of immune cells [10]. These data 
suggest that CB-SCs may display a broad spectrum of modulatory capacity on immune cells via HVEM/BTLA and 
PD-L1/PD-1 signaling pathways.     
 
100
200
300
400
500
Marker (bp)
AIRE
(166 bp)
-actin
(191 bp)
1
2
3
1
2
3
CB-SC preparations:
A
Real Time PCR Analysis
B
Immunostaining
AIRE
IgG
C Western blot: 

 
Page 31 of 59 
Figure 24. Characterization of AIRE and molecular mechanisms in CB-SC. (A) Expression of AIRE mRNA in CB-SCs. 
Real time PCR analysis for AIRE mRNA expression followed by electrophoresis in 2% agarose gel. Data are representative of 
three CB-SC preparations. (B) Immunocytochemistry for AIRE. Isotype- matched IgG served as control (left) for AIRE staining 
(right) with magnification 200. (C) Western blot showed the dose-dependent knockdown response of AIRE following AIRE
siRNA treatment, and decreased expression of PD-L1 and carboxypeptidase M (CPM) in CB-SCs. (D) Expression of HVEM 
on the gated CD45+OCT3/4+ CB-SCs. CD45, a leukocyte common antigen; OCT3/4, a transcription factor marker of embryonic 
stem (ES) stem cells. (E) Expression of BTLA and PD-1 on peripheral blood-derived immune cells. The percentage of BTLA+
PD-1+ cells in each subpopulation that was gated using the specific surface markers. (F) Outline of the molecular mechanisms 
for the immune modulation by CB-SC. AIRE, autoimmune regulator; Arg
arginine; B1R, kinin  B1 receptor; B2R, kinin B2 
receptor; BTLA, B and T lymphocyte attenuator; CRH, corticotropin-releasing hormone; CRHR, CRH receptor; CTLA4, 
cytotoxic T lymphocyte-associated molecule-4; iNOS, inducible nitric oxide synthase; NO, nitric oxide; PD-L1, programmed 
death ligand 1; PD-1, programmed death 1; TGF- 1, transforming growth factor.  
6.5 Dose Rationale and Risk/Benefits 
To date, a true cure for T1D has proven elusive despite intensive research pressure using conventional 
approaches over the past 30 years [64]. The situation highlights the challenges that we face in conquering this 
disease and the need for novel alternative approaches for the treatment of T1D. Our previous work demonstrated 
that CB-SCs are a unique type of stem cell identified from human cord blood [8,9], distinct from other stem cell 
types including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and endothelial progenitor 
cells (EPCs) [25]. We characterized these stem cells and harnessed some of their unique properties with SCE 
therapy by using CB-SCs in a closed-
briefly co-
-SCs in vitro
lymphocytes (but not the CB-SCs) to t
Figure 15) [1,2,5,6,8,9,11,24,65]. This treatment 
leads to global immune modulations and immune balance as demonstrated by clinical data and animal studies 
[1,3,6,9,11]. The SCE therapy may revolutionize the clinical treatment of diabetes and other immune-related 
diseases through CB-
concerns associated with conventional stem cell-based approaches. 
    Ideally, therapeutic approaches to treating or curing T1D should address multiple or all of the underlying causes 
of autoimmunity in T1D. Unfortunately, the etiology of T1D remains largely unknown in humans. Possible triggers 
for autoimmunity in T1D include genetic, epigenetic, physical, social, and environmental factors. These factors 
may act independently or jointly to initiate or potentiate the development of autoimmunity. As is expected in 
conditions with multiple contributing factors, T1D-related dysfunction in the immune system has been traced to 
dysfunctions in multiple cell types and targets including T cells, B cells, regulatory T cells (Tregs), 
monocytes/macrophages, dendritic cells (DCs), natural killer (NK) cells, and natural killer T (NKT) cells [13]. Due 
to the polyclonal nature of T1D-related autoimmune responses and the global challenges of immune regulation in 
T1D patients, therapies and trials that only target one or a few components of the autoimmune response are likely 
to fail just as recent trials involving anti-CD3 Ab for T cells, anti-CD19 Ab for B cells, and GAD 65 vaccination have 
failed [15,16,66]. Successful therapies will likely restore immune balance and peripheral tolerance by addressing 
changes in multiple targets within the immune system. Specifically, they point to the need for an approach that 
produces comprehensive immune modulation at both the local pancreatic and systematic levels rather than 
targeting the pancreatic effects of one or a few components of the immune system. Our SCE therapy takes this 
broader approach. 
In the initial use of this therapy, a novel device (the SCE) functioned as part of a closed-loop system that 
circulated 
-cultured 
adherent CB-SCs in vitro, and returned the educated lymphocytes (but not the CB-
 
It takes 8 -9 hours for the whole process. CB-SCs attached to interior surfaces in the device present secreted and 
cell-surface signaling molecules to passing lymphocytes, and only the autologous lymphocytes are returned to 
the subjects. Findings from ongoing clinical trials provide powerful evidence that a single treatment with the SCE 
provides lasting reversal of autoimmunity that allows regeneration of islet  cells and improvement of metabolic 
OCT3/4
Gate Y
D
0
10
20
30
40
50
60
70
80
90
E
CB-SC
PD-L1/PD-1
iNOS
NO
AIRE
CD86 /CD80
CD28 /CTLA4
T cell
Mo/M
TGF- 1
CPM
B1R/B2R
Arg
Notch1/2
Jagged1
CRHR1/2
CRH
CRH
CRH
HVEM/BTLA
F

 
Page 32 of 59 
control in individuals with long-standing T1D [1,5]. Findings from these trials indicated that CB-SC-mediated 
reversal of autoimmunity results from modulation of the immune response in multiple immune cell types, thereby 
meeting the expectation that successful therapies will likely address multiple sources of the autoimmune response.  
Additionally, the clinical data from the Spanish trial (NCT01350219) demonstrated that SCE therapy was well 
tolerated in all Caucasian subjects. The percentage of naïve CD4+ T cells was significantly increased at 26 weeks 
and maintained through the final follow-up at 56 weeks. The percentage of CD4+ central memory T cells (TCM) 
was markedly and constantly increased at 18 weeks. Both CD4+ effector memory T cells (TEM) and CD8+ TEM cells 
were considerably decreased at 18 weeks and 26 weeks respectively. Additional clinical data demonstrated the 
up-regulation of C-C chemokine receptor 7 (CCR7) expression on naïve T, TCM, and TEM cells. Following two 
treatments with SCE therapy, i
-
-cell 
function. Thus, SCE therapy provided lasting reversal of autoimmune memory that could improve islet -cell 
function in Caucasian subjects. These findings provide a much-needed boost for T1D therapeutic research. If 
further shown to be safe and effective in subjects, immune modulation by CB-SCs has the potential to address 
T1D and other autoimmune diseases [9]. 
7
Study Objectives 
7.1 Primary Objective 
         To assess the safety of SCE therapy in patients with T1D.   
7.2 Secondary Objectives 
7.2.1 
To assess the feasibility of SCE therapy in patients with T1D 
7.2.2 
To evaluate preliminary efficacy of SCE therapy for improving islet  cell function in patients with T1D 
through 12 months. 
7.2.3 
To evaluate markers of immune function in T1D patients after SCE therapy.   
8
Study Design 
8.1 General Design 
This is a prospective, single arm, open-label, single-center pilot study to assess the safety, feasibility, and efficacy 
of SCE therapy for the treatment of patients with T1D.  Up to 10 patients meeting eligibility criteria will be enrolled. 
Patients with T1D will be evaluated by the study principal investigator or co-investigators.  Informed consent will 
be obtained at the initial screening visit (Visit 1).  The initial screening visit will occur within 30 days of initiation of 
SCE therapy (Table 3).  The second screening visit (Visit 2) will occur within 7 days of therapy. Subjects who 
meet all criteria will be scheduled for treatment.  All enrolled subjects will receive treatment with the SCE system 
consisting of a single session of MNC collection by apheresis where 10 L (expected collection of ~1010 
mononuclear cells) of blood will be processed on day -1. The MNC product will then be exposed over a 17-hour 
period of time to the SCE and on day 0 the product will be infused intravenously back to the patient. The infusion 
will take place in the Outpatient Transplant Facility at John Theurer Cancer Center (JTCC) or Inpatient at 
Hackensack University Medical Center (HUMC) on the stem cell transplantation or cellular therapy unit. The 
Name of Procedure: Infusion of 
Non-Frozen Hematopoietic Stem Cell (HSC) Products and Cytotoxic T Lymphocytes . After finishing the infusion, 
the patients will be hospitalized at the main hospital of Hackensack University Medical Center for a 24-hours post-
infusion observation. If the patients show the stable blood glucose, blood pressure, temperature, and normal vital 
signs, they can be discharged from the hospital after 24 hours. Subjects will be treated sequentially, with a 
minimum of one week between subjects for the first three subjects. .  All treated subjects will attend a safety visit 
on Day +7 ± 1 day. Follow-up visits will occur on 1st, 3rd, 6th, 9th, 12th, and 24th month (± 5 days) post-therapy.  
Subjects will be instructed to record daily insulin doses, sugar levels, and physical activities. After receiving the 
SCE th
their own endocrinologist.                                           

 
Page 33 of 59 
Figure 25. Outline the SCE therapy and follow-up. Ten participants will receive a single treatment with the SCE and 
follow-up studies, as described in above diagram. The preparation of CB-SC cultures and SCEs will be performed as 
previously described [1]. Briefly, human cord blood units derived from healthy allogeneic donors will be obtained from FDA-
registered CORD : USE Cord Blood Bank (Orlando, FL). All cord blood samples will be screened for HIV ½, HBsAg, HBcAg, 
HCV, HIVNAT, STS, HBVNAT, HCVNAT, AbScr, HTLV I/II, West Nile, Chagas, and CMV, and only pathogen-free cord 
blood units will be used for clinical treatment. Human CB-SC will be generated as previously described [6,8]. Cord blood 
mononuclear cells will be plated in serum-free culture medium (Lonza, Walkersville, MD) and incubated at 37°C, in 8% CO2. 
After 2-3 weeks, CB-SC growing at 80% confluence will be prepared for clinical trial. One Educator device will be generated 
from one cord blood unit, and used for one subject. After overnight treatment through the co-culture of MNC product with 
CB-SCs, the SCE (SCE)-treated MNC will be collected and infused intravenously (i.v.) back to the subject in 3 - 5 hours.  
8.2 Primary Study Endpoint                                      
The primary study endpoint will be the occurrence of treatment-related adverse effects.  Adverse events that occur 
during therapy (especially those that necessitate temporary or permanent discontinuation of therapy) and over the 
12-month follow-up period will be assessed.   
 
8.3 
Secondary Study Endpoints 
8.3.1 Feasibility endpoints:  
8.3.1.1 
The number of patients who were unable to complete SCE Therapy.  Any non-adverse event related 
reasons for discontinuation of therapy will be recorded (eg: technical problems with the SCE system). 
8.3.1.2 
The number of patients who are lost to follow-up prior to the 12-month follow-up visit.   
8.3.2 Efficacy endpoints  
8.3.2.1 
Area under the C-peptide curve (AUC) over the first 2 hours of a 3-hour mixed meal tolerance test 
(MMTT).  The AUC at 12-months and changes over time will be assessed. 
8.3.2.2 
Peak C-peptide levels over a 3-hour MMTT.   The peak C-peptide level at 12-months and changes over 
time will be assessed. 
8.3.2.3 
Basal C-peptide levels at 12-months and changes over time will be assessed. 
8.3.2.4 
Daily insulin requirements 
8.3.2.5 
Change in HbA1C levels over time 
8.3.2.6 
Changes in auto-antibody levels over time 
 
8.3.3 Exploratory endpoints 
8.3.3.1 
Measurements of regular immune cell markers at baseline, 1, 3, 6, 9, 12, and 24 months 
8.3.3.2 
Flow cytometry of memory T cell markers at baseline, 1, 3, 6, 9, 12, and 24 months 
 
 
Time: 
Day:
Prepare Educators
in GMP lab
Follow-up Studies
1
2
Month: 1st
3rd
6th
9th
12th
Baseline tests
Recruit subjects
into clinical trial
Sign Consent Form
Send to
GMP lab
Infused (i.v.)
Follow-up
assessment
-1
0
MNC collection
by Apheresis
Treatment
overnight
-30
24th

SCE therapy in diabetes 
Version: 06Mar2019
Page 34 of 59 
 
Table 3. Schedule of Events   
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 
Visit 10 
Visit 11 
Visit 12
PROCEDURE 
Within 30 
Days of 
Cord 
Blood 
Order 
Day -7  
to -1 
Day -7  
to -1 
Day 0 
Day +1 
to +2 
Day +7 
+1 day 
Month 1 
+ 5 days 
Month 3 
+ 5 days 
Month 6 
+ 5 days 
Month 9 
+ 5 days 
Month 12 
+ 5 days 
Month 24
+ 5 days 
Informed Consent 
x 
 
 
 
 
 
 
 
 
 
 
 
Verify Inclusion/Exclusion Criteria 
x 
 
 
 
 
 
 
 
 
 
 
 
Vital Signs (Ht/Wt/Temp/BP/HR/RR) 
x 
 
 
x 
 
x 
 
x 
x 
x 
x 
x
Medical History 
x 
 
 
x 
 
x 
 
x 
x 
x 
x 
x
Physical Exam  
x 
 
 
x 
 
x 
 
x 
x 
x 
x 
x
Venous assessment for apheresis 
x 
 
 
 
 
 
 
 
 
 
 
 
LABS 
 
 
 
 
 
 
 
 
 
 
 
 
CBC 
x 
 
 
x 
 
x 
x 
x 
x 
x 
x 
x
CMP 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x
ABO Blood Typing Test 
x 
 
 
 
 
 
 
 
 
 
 
 
Infectious disease testing (HIV, HTLV, 
Hep B, cAB, Hep C ab. 
x 
 
 
 
 
 
 
 
 
 
 
 
HbA1C 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x
Serum Pregnancy Test 
x 
x 
 
 
 
 
x 
x 
x 
 
 
 
Auto-antibodies (IAA, IA2, GAD, ZnT8 ) 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x
Fasting C-peptide level 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x
Fasting Blood Glucose Level 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x 
x
Fasting plasma pro-insulin level 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
Insulin dose recording 
x 
 
 
 
 
 
x 
x 
x 
x 
x 
x
EKG 
x 
 
 
 
 
 
 
 
 
 
 
 
METABOLIC STATUS 
 
 
 
 
 
 
 
 
 
 
 
 
Mixed meal tolerance test 
 
x 
 
 
 
 
 
x 
x 
x 
x 
x
Pro-Insulin level 
 
x 
 
 
 
 
 
x 
x 
x 
x 
x
Glucagon levels 
 
x 
 
 
 
 
 
x 
x 
x 
x 
x
Daily Insulin Doses: Patient Diary 
 
Continuous 
Daily Glucose Levels: Patient Diary 
 
Continuous 
IMMUNE MARKERS 
 
 
 
 
 
 
 
 
 
 
 
 
CD4+CD25+CD127low/-Tregs 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
CD4+CD45RO+CCR7+ TCM cells 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x

 
Page 35 of 59 
Table 3. Schedule of Events   
 
Visit 1 
Visit 2 
Visit 3 
Visit 4 
Visit 5 
Visit 6 
Visit 7 
Visit 8 
Visit 9 
Visit 10 
Visit 11 
Visit 12
PROCEDURE 
Within 30 
Days of 
Cord 
Blood 
Order 
Day -7  
to -1 
Day -7  
to -1 
Day 0 
Day +1 
to +2 
Day +7 
+1 day 
Month 1 
+ 5 days 
Month 3 
+ 5 days 
Month 6 
+ 5 days 
Month 9 
+ 5 days 
Month 12 
+ 5 days 
Month 24
+ 5 days 
CD4+ICOS+ and CD8+ICOS+ T cells 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
IL-
-17 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
IL-10 and TGF  
 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
Th1/Th2 cytokines (IL4, IL5, IL12, IL13) 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
PLATELET MARKERS 
 
 
 
 
 
 
 
 
 
 
 
 
Platelet function testing 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
CD41+CD42+CD270+CD274+Galectin9+ 
 
x 
 
 
 
 
x 
x 
x 
x 
x 
x
TREATMENT 
 
 
 
 
 
 
 
 
 
 
 
 
Apheresis 
 
 
day -1 
x 
cell 
infusion 
 
 
 
 
 
 
 
 
SAFETY ASSESSMENTS 
 
 
 
 
 
 
 
 
 
 
 
 
Phone call for safety follow-up 
 
 
 
 
x 
x 
 
 
 
 
 
 
Concomitant med monitoring 
 
 
 
Continuous 
Adverse Event monitoring 
 
 
 
Continuous 
 
 

SCE therapy in diabetes
Version: 06Mar2019 
 
 
 
Page 36 of 59 
9 Subject Selection and Withdrawal 
This study will enroll a total of 10 subjects who meet the following eligibility criteria.  
9.1 Inclusion Criteria 
1) Adult patients (  18 years)  
2) Must have a diagnosis of type 1 diabetes mellitus based on the 2015 American Diabetes 
Association criteria for the Clarification and Diagnosis of diabetes. 
3) Must have a blood test confirming the presence of at least one autoantibody to pancreatic islet  
Cells (IAA, IA2, GAD 65, ZnT8). 
4) Fasting C-peptide level > 0.3 ng/ml 
5) HbA1C < 10% at enrollment 
6)  Recent diagnosis (within two years of enrollment) 
7) Adequate venous access for apheresis 
8) Must be equipped with a continuous glucose monitoring system (CGMS) 
9) Ability to provide informed consent 
10) For 
female 
patients 
only, 
willingness 
to 
use 
FDA-recommended 
birth 
control 
(http://www.fda.gov/downloads/ForConsumers/ByAudience/ForWomen/FreePublications/UCM3
56451.pdf) until 6 months post treatment. 
11) Must agree to comply with all study requirements and be willing to complete all study visits 
9.2 Exclusion Criteria 
Potential subjects meeting any of the following criteria will be excluded from participation: 
 
1) AST or ALT 2 > x upper limit of normal. 
2) Abnormal bilirubin (total bilirubin > 1.2 mg/dL, direct bilirubin > 0.4 mg/dL) 
3) Creatinine > 2.0 mg/dl. 
4) Known coronary artery disease or EKG suggestive of coronary artery disease unless cardiac 
clearance for apheresis is obtained from a cardiologist. 
5) Known active infection such as Hepatitis B, Hepatitis C, or Human Immunodeficiency Virus (HIV) 
6) Pregnancy assessed by a positive serum pregnancy test or breastfeeding mothers 
7) Use of immunosuppressive medication within one month of enrollment including but not limited 
to prednisone, cyclosporine, tacrolimus, sirolimus, and chemotherapy. 
8) Presence of any other autoimmune diseases (lupus, rheumatoid arthritis, scleroderma, etc.) 
9) Anticoagulation other than ASA. 
10) Hemoglobin < 10 g/dl or platelets < 100 k/ml 
11) Is unable or unwilling to provide informed consent 
12) Presence of any other physical or psychological medical condition that, in the opinion of the 
investigator, would preclude participation 

 
Page 37 of 59 
9.3 Subject Recruitment and Screening 
Subjects will be recruited for the study by any of the investigators, co-investigators, endocrinologists, other 
medical professionals who treat Type 1 Diabetes. Patients may also be self-referred.  If subjects consent 
to participate in the trial, diagnostic testing necessary to meet above inclusion or exclusion criteria will be 
performed. The list of screening requirements will include a regular blood count analysis, a blood test to 
confirm the presence of at least one autoantibody to pancreatic islet  cells, and other associated testing 
to exclude pregnancy, clinically significant liver, kidney, or heart disease.  Patients will be referred to the 
Principal Investigator, Dr. Donato, for the apheresis and re-infusion therapy portion of the protocol only.  
There will be a follow-up with a safety phone call on Day 1 or 2 after re-infusion and on Day 7 +1. This will 
be performed by the research nurses.  
Copies medical records from all visits to the JTCC will be sent to the treating physicians.  
The rescreening of subjects who previously failed eligibility is allowable. 
9.4 Early Withdrawal of Subjects 
9.4.1 When and How to Withdraw Subjects 
Based on our current published [16] and unpublished clinical data, there were no subjects who needed to 
be withdrawn from the study prior to completion.  The risks to the patients are expected to be minimal and 
akin to a standard apheresis procedure.  The cells returned to the patients are autologous cells that will be 
treated (or educated) by CB-SCs.  
    
    In this study a patient may be withdrawn from this protocol at any points: 
1) If they suffer some type of allergic or hypersensitivity reaction to the SCE treated cells precluding 
the infusion of the product. 
2) If technical difficulties preclude the completion of apheresis. 
3) If they do not want to complete the required post treatment follow-up testing.  
Participation in this trial is VOLUNTARY for all subjects. If they choose not to participate, this will not affect 
their relationships with Hackensack University Medical Center or their rights to health care or other services 
to which they are otherwise entitled.  If they decide to participate, patients are free to withdraw their consents 
and discontinue participation at any time without affecting their future care at Hackensack University 
Medical Center. In the event that a patient is withdrawn for above or any other unexpected reasons they 
will be allowed to continue to receive medical care for treatment associated problems.   
9.4.2 Data Collection and Follow-up for Withdrawn Subjects 
Even though subjects may be withdrawn prematurely from the study, it is imperative to collect toxicity data 
for 12 weeks after.  We will use all possible methods (e.g. number of phone calls to subject, phone calls to 
health care proxy, if possible, certified letters, etc.) to confirm that the subject is truly lost for follow-up. If a 
patient withdraws from the study prior to their 12 month follow-up, enrollment of a replacement patient will 
occur.   
 
10 Study Device 
 
10.1 Description 
To build upon previous clinical trials abroad [1-3] using a closed-loop system, we will investigate the use of 
the SCE as a static device, which will allow increased interaction time (14-17 hours) between CB-SCs and 
patient lymphocytes/monocytes under a controlled good manufacturing process (GMP) environment before 
reintroducing the lymphocytes back into the patient. The longer interaction between CB-SCs and 
lymphocytes/monocytes will improve treatment efficacy using this optimized discontinuous system. 
Through secreted and cell-surface signaling molecules, the CB-SCs educate the lymphocytes [1,5]. The 
cells returned to the patients are autologous cells that have been treated (or educated) by CB-SCs. CB-
SCs remain inside of the device due to their unique attaching ability [1-3,5]. Additionally, in this clinical 
trial, we design a chamber (160 160 200 cm) for co-culture of lymphocytes and CB-SCs that includes 
twelve discs of the material with adherent CB-SCs sandwiched between a top cover plate and a bottom 

 
Page 38 of 59 
collecting plate (Figure 1). The device is manufactured, assembled and packaged in a Class 100K clean 
room. After being sterilized by gamma-irradiation (Cesium-137), the devices are stored at the room 
temperature in dark cabinets of Class 100K clean room, which is FDA-approved facility for cell isolation and 
cultures. The materials used to produce the device are FDA-approved for in vivo use per the United States 
Pharmacopeia (i.e., Grade Class VI Plastic). The sterilized device is a single use, which CB-SCs are 
generated from one cord blood unit for one subject application.  
 
10.2 Treatment Regimen 
Subjects who meet all criteria will be scheduled for treatment.  All enrolled subjects will receive treatment 
with the SCE system consisting of a single session of MNC collection by apheresis approximately 10 L 
(~1010 mononuclear cells) of blood will be processed on day -1. The MNC product will then be exposed 
over a 17-hour period of time to the SCE and on day 0 the product will be infused intravenously back to the 
patient. All treated subjects will receive a telephone follow-up assessment on Day +1 to monitor for acute 
adverse events.  All treated subjects will attend a safety visit on Day +7 ± 1 day. Follow-up visits will occur 
on 1st, 3rd, 6th, 9th, 12th, and 24th month (± 5 days) post-therapy.  Subjects will be instructed to record daily 
insulin doses, sugar levels, and physical activities. After receiving the SCE 
doses will be monitored and adjusted by their own physician.  
 
10.3 Method for Assigning Subjects to Treatment Group 
There is only one treatment group.  Based on fasting C-peptide levels (a by-product of insulin biosynthesis, 
with normal reference range 0.8  3.1 ng/mL), as an indicator for islet  cell function, all participants will be 
characterized and assigned as one group having moderate T1D with some residual  cell function (fasting 
C-peptide level  0.3 ng/mL, n = 10). Each participant will receive one treatment. 
 
10.4  Preparation and Use of Study Device 
To build upon previous clinical trials abroad [1-3] using a closed-loop system, we will investigate the use of 
the SCE as a static device, which will allow increased interaction time (at least 17 hours) between CB-SCs 
and patient lymphocytes/monocytes under a controlled good manufacturing process (GMP) environment 
before reintroducing the lymphocytes back into the patient. The longer interaction between CB-SCs and 
lymphocytes/monocytes will improve treatment efficacy using this optimized discontinuous system (Figures 
13 A and B). Please see the Appendix 4 Master Batch Record (MBR) for the detailed information on the 
Preparation and Use of Study Device. Briefly, the whole procedure is outlined in the diagram (Figure 4C): 
1) Preparation of SCE: A fresh donated human umbilical cord blood unit is received, and mononuclear cells 
are isolated using a Ficoll gradient separation followed by removal of remaining Red Blood Cells (RBCs) 
using lysis buffer. The entire mononuclear cell suspension is seeded in the SCE, and cultured for 10-20 
days. Once the culture is  80% confluent, the SCE is ready to use. 2) Preparation of SCE-treated MNC: 
The day after the culture is  80% confluent, t
 collected on day -1 and 
shipped to the GMP facility; the SCE is washed and the CB-SCs in the SCE are cocultured overnight with 
 monocytes. After incubation, the 
patient cells are drained into a product collection bag. After final QC testing and product release, the product 
is sent to the recipient for administration. 
 

 
Page 39 of 59 
QC Test:  cell number, 
viability, endotoxin 
Prepare 
Device: 
Y
Y
Insert Y 
Connector 
and Tubing
Insert Y 
Connector
Wash 2 
with NS
Pass !
Receive
Cord Blood
Isolate CBMCs
Cell count
CBMCs
Pass QA test
Receive
MNC Apheresis
Premix MNC with 
saline in a 500 ml tube
Load MNC
Into Educator
Save a sample for QC test
Y
Y
Y
Overnight
Co-culture
Harvest
QC Test
Labeling 
Packing 
shipping
Plant CBMCs
into Educator
A. Produce Stem Cell Educator           B. Ex Vivo Treatment
Cell culture

 
Page 40 of 59 
Figure 4. Outline the preparation and use of Educator device. (A) Show the procedure how to produce SCE in 
GMP facility. (B) Display the protocols on ex vivo treatment of MNC by overnight co-culture with CB-SCs in GMP 
facility. (C) The detailed process overview.  
 
 
Following steps will be applied for the Preparation and Use of Study Device. The production of 
clinical-grade SCEs and associated protocol have been mirrored by two Performance Qualification 
(PQ) runs at the FDA-registered cGMP facility, as demonstrated by PQ Reports from NYBC (see 
Appendix 2).   
 
Step 1: Collection of human cord blood units.  
Human cord blood units derived from healthy donors will be provided by FDA-registered CORD:USE Cord 
Blood Bank Inc.  All cord blood samples are screened for communicable disease as requested by regulatory 
agencies.    
Accession
Cord Blood Unit
Isolation of 
Mononuclear cells
Using Ficoll
RBC Lysis and 
washing of 
Mononuclear cells
Culture Cells in SCE
Once culture is 
80% confluent
Sampling of SCE
Addition of MNC 
Apheresis Product 
to SCE
Harvest Cells from 
SCE
Final Product
Ship Final Product
to Clinical Site
Overnight Incubation
Cell Count
Gram Stain
Endotoxin
Pre MNC Retain
Oct3/4/CD45/ CBC
Pre MNC ABO
Gram Stain
Endotoxin
Viability
Mycoplasma
Sterility
Product Identity
Oct3/4/CD45 purity
Accession MNC
Apheresis Product
Pre CBU Retain
Pre CBU ABO
Cell Concentration
Post Retain
Stability
Potency
C

 
Page 41 of 59 
 
Step 2: Preparation of CB-SC in the device. 
Prior to introducing CB-SCs into the device, cord blood mononuclear cells are isolated from fresh cord blood 
unit (at least 100 ml/unit) according to the following protocol:  
1) Take 50ml tubes and put in holders: normally use 4 tubes; 
2) Add  HISTOPAQUE®-1077 to each tube at 20ml/tube to isolate the mononuclear cells; 
3) Plant mononuclear cells in the SCE Device (Tianhe Stem Cell Biotechnologis Inc.) at 1 106cells/ml, 
25-30ml/dish in serum-free culture medium; if > 20x109 CD3 positive cells are collected by the 
apheresis procedure, the collection may be divided between two SCE Devices for one treatment. 
4) Incubate cells at 37°C, 8% CO2 conditions for 10~20 days. 
5) Cell observation: CB-SC are round and attach on the bottom of dishes. If cell density reaches the 
80% of confluence, CB-SC can be prepared for clinical application.   
   
Step 3: Test the CB-SC culture for endotoxin and Gram stain.  
1) Testing for endotoxin. The supernatant from the culture of CB-SCs will be collected into 1.8 ml 
sterilized tubes. Endotoxin will be tested by using the Endosafe-PTS Portable Test System (Charles 
River, Charleston, SC) and Endosafe-Licensed PTS Endotoxin Cartridge (0.005 EU/mL sensitivity, 
Catalogue #: PTS20005F, Fisher Scientific). The standard endotoxin level will be < 0.5 EU/mL. 
Only the Educator apparatus that meets this standard can be used for the clinical trial.  
2) Gram stain. The supernatant from the culture of CB-SCs will be collected into 1.8 ml sterilized 
tubes. Gram staining will be performed by using the Gram Stain kit (BD Diagnostic Systems, 
Sparks, MD). Only the Educator apparatus that is negatively stained can be used for the clinical 
trial.  
 
 
Step 4: Preparation of SCE.  
 
1) Sterilized the hood in a GMP facility for preparation of SCE;  
2) Clean out side of device with 70% ethanol gauze and carefully remove all caps;  
3) Discard all supernatants inside of device; 
4) Add  physiological saline to each layer at 20ml/layler, washing each layer and removing all floating 
cells and debris; 
5) Additional wash with physiological saline (20ml/layler);  
6) Remove the caps from the top and bottom, replace with Plastisol Horseshoe Y Connectors; 
 
Step 5: Treatment of MNC with SCE therapy.  
1) On day -1 patients with undergo a steady state MNC collection by apheresis, with an optimal goal 
of processing approximately 10 L (~1010 mononuclear cells) of blood. Apheresis may be stopped 
before the goal is reached based on tolerability and venous access. A femoral apheresis catheter 
may be placed for the duration of the procedure if venous access is found to be insufficient on the 
day of collection, despite the initial venous assessment. The femoral apheresis catheter will be 
removed approximately 1 hour after the collection as per institutional SOP.   A minimum of 6 L 
processed will be required to continue on to the next phase. Plasma will be added to the collection 
bag at the end of apheresis to target a product volume of 250 mL. Labeling of the product will be 
done prior to disconnecting the product from the donor in accordance with the Blood and Marrow 
Transplant program labelling policy.  
2) The product will be packaged, stored and picked up by New York Blood Center (NYBC) in 
accordance with the Blood and Marrow Transplant program SOPs.  
3) Testing for stem cell markers CD45 and OCT3/4 by flow cytometry on the cellular product. To 
exclude the contamination of CB-SCs in the Final Product, we will perform the flow cytometry by 
double-staining with CB-
Figure 3D).  
4) Testing for identity markers CD4+CD25+CD127Low/- Tregs by flow cytometry on the cellular product. 
The isotype-matched IgG antibodies will be served as negative controls. The Fc Blocker (BD 
Pharmingen) will be applied to block non-specific staining.  
5) Testing 
for 
potency 
markers 
CD4+CD45RO+CCR7+ 
central 
memory 
T 
(TCM) 
cells, 
CD4+ICOS+,CD8+ICOS+ T cells by flow cytometry on the cellular product. The isotype-matched IgG 

 
Page 42 of 59 
antibodies will be served as negative controls. The Fc Blocker (BD Pharmingen) will be applied to 
block non-specific staining.  
6) Once at NYBC, the cellular product will be processed and treated by CB-SCs according to the 
SOPs in Master Batch Record (Appendix 4). 
7) On day 0, the educated cellular product will be infused to the patient in accordance with the Blood 
and Marrow Transplant program cellular infusion SOP.  
 
Step 6: Test the Final Product for cell viability, endotoxin, Gram stain, mycoplasma, and stem cell 
markers.  
1) Cell viability. The sample from the CB-SC-treated MNCs will be collected into 1.8 ml sterilized 
tubes. The cell viability will be tested by the trypan blue based exclusion assay. The mononuclear 
nuclear cell number will be tested by using the Automated Complete Blood Counts (CBC) Using 
the Beckman Coulter ® AC-
. 
2) Testing for endotoxin. The sample from the CB-SC-treated MNCs will be collected into 1.8 ml 
sterilized tubes. Endotoxin will be tested by using the Endosafe-PTS Portable Test System (Charles 
River, Charleston, SC) and Endosafe-Licensed PTS Endotoxin Cartridge (0.005 EU/mL sensitivity, 
Fisher Scientific). The standard endotoxin level 
 5 EU/kg/hr according 
to FDA guidelines. Only the Final Product that meets this standard can be used for the clinical 
infusion.  
3) Gram stain. The sample from the CB-SC-treated MNCs will be collected into 1.8 ml sterilized tubes. 
Gram staining will be performed by using the Gram Stain kit (BD Diagnostic Systems, Sparks, MD). 
Only the Educator apparatus that is negatively stained can be used for the clinical trial.  
4) Testing for mycoplasma and sterility. The Sterility and contamination of mycoplasma in the 
sample from the CB-SC-treated MNCs can be tested and excluded by following methods: 1). 
Regular cell culture: Cell viability can be monitored under phase-contrast microscope to exclude 
the contamination of bacteria. 2). Real time PCR: The sample from the CB-SC-treated MNCs will 
be collected into 1.8 ml sterilized tubes for mycoplasma and and 50 mL tubes (total 120 mL) for 
sterility tests by Eurofins (Lancaster, PA) in compliance with the requirements of the US FDA Good 
Laboratory Practice Regulations (21 CFR 58). 3). The Sterility Testing will be performed by using 
a Direct Inoculation Method at Eurofins Company. 
5) Testing for stem cell markers CD45 and OCT3/4 by flow cytometry. To exclude the 
contamination of CB-SCs in the Final Product, we will perform the flow cytometry by double-staining 
with CB-
T3/4 (Figure 3D).  
6) Testing for stem cell markers CD45 and OCT3/4 by flow cytometry. To exclude the 
contamination of CB-SCs in the Final Product, we will perform the flow cytometry by 
double-staining with CB-
Figure 3D). The isotype-matched IgG 
antibodies will be served as negative controls. The Fc Blocker (BD Pharmingen) will be applied to 
block non-specific staining.  
7) Testing for identity markers CD4+CD25+CD127Low/- Tregs by flow cytometry. The 
isotype-matched IgG antibodies will be served as negative controls. The Fc Blocker (BD 
Pharmingen) will be applied to block non-specific staining.  
8) Testing for potency markers CD4+CD45RO+CCR7+ central memory T (TCM) cells, 
CD4+ICOS+,CD8+ICOS+ T cells by flow cytometry. The isotype-matched IgG antibodies will be 
served as negative controls. The Fc Blocker (BD Pharmingen) will be applied to block non-specific 
staining.  
 
 
 
 

 
Page 43 of 59 
10.5 Subject Compliance Monitoring 
 
Subjects will receive one treatment with the SCE.   
 
Follow-up visits will be scheduled 1, 3, 6, 9, 12, and 24 months (± 5 days) after treatment for clinical 
assessments and laboratory tests as described in Section 4.2. A telephone follow-up will be performed the 
day after the cell infusion. 
10.6 Prior and Concomitant Therapy 
 
All T1D subjects receive their daily insulin injections prior to or during the study. Their daily insulin doses 
will be adjusted after receiving the SCE therapy by their own physicians, not the study PI. There will be no 
other new diabetes medicines or therapies will be permitted during this study.  
 
10.7 Packaging 
 
The SCEs will be packaged in box (4 devices/box) and shipped to the GMP facility NYBC, with the Good 
Manufacturing Practices (GMP) Labeling System.  
After treatment with the SCE, the Final Product will be shipped to clinical site at Hackensack University 
Medical Center in a sterilized cooler.   
 
10.8 Blinding  
 
In this single-arm study, all subjects will receive open label treatment with the SCE. 
  
10.9 Receiving, Storage, Dispensing and Return 
 
10.9.1 Receipt of Devices 
 
Upon receipt of the study treatment supplies, an inventory must be performed and a device receipt log filled 
out and signed by the person accepting the shipment.  It is important that the designated study staff counts 
and verifies that the shipment contains all the items noted in the shipment inventory.  Any damaged or 
unusable study device in a given shipment will be documented in the study files.  The investigator must 
notify study sponsor of any damaged or unusable study treatme
site. 
 
10.9.2 Storage 
 
The devices are stored at the room temperature in dark cabinets of Class 100K clean room (or GMP facility 
for cell cultures). There is no need for protection from light. Storage considerations include avoiding 
dropping of the devices or placing heavy weight on top of the devices.  
 
10.9.3 Dispensing of Study Device 
 
All empty devices (without stem cells) are in the same quality. Any single device will be assigned to any 
single subject and dispensed. The only standard will be the quality of CB-SCs cultured in the device. If cell 
density reaches the 80% of confluence, endotoxin level <0.5 EU/mL, the device with CB-SCs can be 
assigned to subject and prepared for clinical application. 
  
Regular study device reconciliation will be performed to document device assignments, devices used, 
devices remaining, and inadvertently damaged devices.  This reconciliation will be logged on the device 
accountability form, and signed and dated by the study team. 
 

 
Page 44 of 59 
10.9.4 Return or Destruction of Study Device 
 
Devices are discarded at the conclusion of the treatment as per institutional biohazard waste disposal SOP.  
At the completion of the study, there will be a final reconciliation of devices shipped, devices consumed, 
and devices remaining.  This reconciliation will be logged on the device reconciliation form, signed and 
dated.  Any discrepancies noted will be investigated, resolved, and documented prior to return or 
destruction of unused study devices.  Any devices destroyed on site will be documented in the study files. 
11 
Study Procedures 
11.1 Visit 1 
Visits to the John Theurer Cancer Center will be as outlined in this study. The investigators will not be 
medications, with the following exceptions: 
 If medically necessary while the participant is physically present at the JTCC during a scheduled 
visit.
 On the day of apheresis
 On the day of re-infusion.   
 If one of the co-investigators is the primary endocrinologist for one of the participants. 
5) In case of emergency or if the participant is unable to contact their primary treating physician. 
Screen for Enrollment: First, all consented subjects will be screened for enrollment in accordance with the 
Inclusion Criteria and Exclusion Criteria of SCE therapy. Patients will be qualified for enrollment if they meet 
the 2015 diagnosis standards of the American Diabetes Association and a blood test confirm the presence 
of at least one autoantibody to pancreatic islet beta cells. Depending on age, either the adult endocrinologist 
(Dr. Van Hoven) or pediatric endocrinologist (Dr.Ghanny, for patients aged from 18 to 22 years old) will 
review pre-procedure blood sugar logs. Respective MD would be accessible by medical team for any 
questions during visit regarding blood sugars. Patients will be provided additional blood sugar logs for the 
duration of the study. We will ask the patient to check their blood sugars at least 4 times daily and twice a 
week overnight. We will also give option to obtain a cloud-based glucometer (e.g., Accucheck Aviva 
Connect). This meter is a cloud-based meter and has a physician portal so MD can review blood sugars 
remotely as weekly. We can also recommend that the patient obtain a CGMS (continuous glucose 
monitoring system) unit to help monitor blood sugars more closely. The visit must be completed within 30 
days prior to the cord blood order from cord blood bank.  
 
1) 
CBC with different immune cell markers 
2) 
Complete chemistry 
3) 
EKG 
4) 
Venous assessment done by an apheresis nurse.  Patients without sufficient venous access will be 
deferred.  Patients with acceptable venous access at the time of screening but who fail to collect peripherally 

 
Page 45 of 59 
on the day of apheresis may have an apheresis catheter placed femorally by a surgeon for the purpose of 
the collection. 
5) 
Autoantibody testing (see Table 3). 
6) 
Infectious disease testing (HIV, HTLV, Hep B, cAB, Hep C ab.) 
11.2 Visit 2 
Women of childbearing potential will require a serum pregnancy test between day -7 and day -1. 
The Mixed Meal Tolerance Test (MMTT): MMTT testing should be done in the morning while the subject is 
fasting. Subjects will be nothing by mouth (NPO) after midnight except water. Liquids that contain only 
simple sugars can be used to treat or prevent hypoglycemia during the night and morning prior to study. 
The target glucose level at the start of the test should be between 70 and 200 mg/dL for subjects with 
T1DM.  
Insulin Application: Subjects with T1DM should take their usual dose of insulin on the evening before the 
test. Subjects on Lantus or Levemir should take their usual injection the evening before and/or on the 
morning of the test, as per their normal routine. Subjects on CSII should continue with their usual basal 
settings on night prior to test. Usual doses of rapid-acting insulin analog will be used until midnight the 
evening before the test. Small correction doses of rapid-acting insulin analog may also be used up to 4 
hours before the test if needed to achieve a blood glucose level <150 mg/dl at the start of the study. Usual 
doses of NPH insulin and rapid-acting insulin analogs are not given on the morning of the test. 
Blood Sampling: Blood will be obtained for measurement of plasma glucose, c-peptide and glucagon at -
10, 0, 15, 30, 60, 90, 120, 150 and 180 minutes. A split-duplicate sample for quality control will also be 
collected. IV lines will be inserted in one arm for blood sampling. 
Boost Dose: The mixed meal used will be Boost High Protein Gluten Free Drink with 20g of sugar per bottle. 
The MMTT should take 180 minutes to perform. The dose of Boost High Protein Gluten Free Drink with 20g 
of sugar per bottle, given at time 0 minutes. The dose should be consumed in no more than 5 minutes. 
 
11.3 Visit 3 
Patients will undergo leukapheresis. Adult endocrinologist or Pediatric Endocrinologist to be on-call for 
questions.   
11.4 Visit 4 
Infusion of the cellular product after exposure to the SCE. Adult endocrinologist or Pediatric 
Endocrinologist to be on-call for questions.  
The cellular product will be released based on the results of a Gram stain and endotoxin test, with results 
of sterility available after the 14 day sterility test. If the tests reveal a sterility failure and there are any signs 
(e.g., fever, vomiting, and increased CBC count) of infection in subjects after infusion, the management and 
Management and Handling of Cellular Products with Positive Micro
. Antibiotics need to be 
administered in the event that microbial contamination is present in the product that the subject already 
received. The route (oral or i.v.) and dosage of administering antibiotics will be determined according to the 
type of infection. To identify the microorganism(s), anti-microbial agent sensitivity testing needs to be 
performed during the sterility test. If the subject experiences any serious and unexpected adverse reaction 
that could be from administration of a non-sterile cellular product, then the endocrinologist must report this 
information to the FDA in an IND safety report no more than 15 calendar days after the initial receipt of the 
information. The endocrinologist must also report the sterility failure, results of investigation of cause and 
any corrective actions, in an information amendment submitted to the IND within 30 calendar days after 

 
Page 46 of 59 
initial receipt of the positive culture test result. All these reports will be handled according to our SOPs at 
 
Errors, Accidents, Deviations, Variances and Complai
. 
To determine which step caused contamination and to prevent sterility lapses in the future, all procedures 
will be fully examined from the collection of the cord blood unit, all steps of Operations, the isolation of 
mononuclear cells (MNCs) by apheresis, and to the packing and shipping of final product between the 
clinical site and the GMP facility. If the reagents and materials are contaminated, they will be discarded. 
New reagents and materials will be used for the production of Stem Cell Educators (SCE) and SCE-treated 
MNCs.     
11.5 Visit 5 
Safety Phone Call Day + 1 to + 2 after the re-infusion.   
 
11.6 Visit 6 
Safety visit 7 days + 1 day after the re-infusion.   
 
11.7 Visit 7 
Follow-up visit 1: It is scheduled 1 month (± 5 days) after treatment for clinical assessments and 
laboratory tests as per Section 3.2, Table 3. To be reviewed by respective Endocrinologist.   
 
11.8 Visit 8 
Follow-up visit 2: It is scheduled 3 months (± 5 days) after treatment for clinical assessment (e.g., fasting 
blood glucose, C-peptide, and HbA1C) and laboratory tests as per Section 3.2, Table 3. To be reviewed 
by respective Endocrinologist. 
 
11.9 Visit 9 
Follow-up visit 3: It is scheduled 6 months (± 5 days) after treatment for clinical assessment (e.g., fasting 
blood glucose, C-peptide, and HbA1C) and laboratory tests as per Section 3.2, Table 3. To be reviewed 
by respective Endocrinologist. 
 
11.10 Visit 10 
Follow-up visit 4: It is scheduled 9 months (± 5 days) after treatment for clinical assessment (e.g., fasting 
blood glucose, C-peptide, and HbA1C) and laboratory tests as per Section 3.2, Table 3. To be reviewed 
by respective Endocrinologist. 
 

 
Page 47 of 59 
11.11 Visit 11 
Follow-up visit 5: It is scheduled 12 months (± 5 days) after treatment for clinical assessment (e.g., fasting 
blood glucose, C-peptide, and HbA1C) and laboratory tests as per Section 3.2, Table 3. To be reviewed 
by respective Endocrinologist. 
 
11.12 Visit 12 
Follow-up visit 6: It is scheduled 24 months (± 5 days) after treatment for clinical assessment (e.g., fasting 
blood glucose, C-peptide, and HbA1C) and laboratory tests as per Section 3.2, Table 3. To be reviewed 
by respective Endocrinologist. 
12 Statistical Plan 
12.1 Statistical Methods 
 
This is a prospective, single-arm, open-label, single-center study to assess the safety, feasibility, and 
efficacy of SCE therapy for the treatment of patients with T1D. Ten patients meeting eligibility criteria will 
be enrolled for this study, with an effect size of 0.8 ng/mL at a 0.025 significance level and 80% power. All 
patients will be included in the safety analyses. The primary safety analysis will be performed in the safety 
population. The occurrences of treatment-related adverse events will be evaluated and presented as counts 
(percentages) and binomial 95% exact binomial confidence intervals (CI). 
    The feasibility of the SCE therapy will assessing by analyzing the number of the patients unable to 
complete the therapy and the number of patients who are lost to follow-up prior to the 12-month visit. Both 
of these parameters will be analyzed as counts (percentages) and corresponding 95% CI as described in 
the safety analysis section above. 
    Statistical analyses of data will be performed by t-test using Power and Precision software (www.power-
analysis.com). The paired t test will be used to study the significance between baseline and follow-ups. 
Using the fasting blood glucose levels and the plasma C-peptide, as well as their levels following OGTT or 
MMTT as the parameters, we want to detect the difference after the SCE therapy with an effect size of 0.8 
ng/mL at 2.5% significance level, 80% power, and one side. According to our calculations, we will need 10 
subjects in order to find this difference.  
    Area under the C-peptide curve will be calculated with C-peptide level measured at 0, 30, 60, 90, 120, 
150 and 180 minutes of the MMTT using the trapezoid rule. The AUC for C-peptide  over 120 minutes of 
the MMTT will be evaluated and summarized using descriptive stats mean (SD) or median (IQR), as 
appropriate. This calculation will be obtained for each of the time points, baseline, 3, 6, 9 12, and 24 months 
and longitudinal analysis will be performed to evaluate the changes over time. This analysis of changes 
over the time points will be achieved using mixed effect models for repeated measures (MMRM) 
implemented using PROC MIXED in SAS version 9.4. As this is a secondary analysis no adjustment for 
multiple test will be employed for this analysis. 
    Mean AUC for C-peptide over 3 hour MMTT, which is defined as the AUC divided by the duration of the 
test i.e., 180 minutes, will be calculated at baseline, and all subsequent post-baseline points, and changes 
over time will be analyzed using MMRM method as defined in the AUC for C-peptide subsection above. 
    HbA1C levels assessed at baseline, 3, 6, 9, 12, and 24 months will be summarized using descriptive 
statistics at each time point. To evaluate changes in HbA1C levels assessed at baseline and post-baseline 
time points, the MMRM analysis defined in AUC for C-peptide will be performed. 
    All study data will be entered into a Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, WA) 
and imported into SAS 9.4. The database will be validated in SAS to ensure any out of range values are 
queried for all multiple source until all data issues are resolved. Any p <0.05 will be considered statistically 
significant. All data analysis will be performed using SAS version 9.4 (SAS Institute Inc. Cary, NC). 
 

 
Page 48 of 59 
Stopping rule: If a grade 3 adverse event, as per the Common Terminology Criteria for Adverse Events 
(CTCAE) Version 4.0, related to the study procedure occurs in 2 patients, the study will be terminated.      
12.2 Subject Population(s) for Analysis 
 
All subjects who received treatment will be included in the safety analyses.  The primary efficacy end point 
will be the change in C-peptide secretion between baseline and follow-up. 
 
13 Safety and Adverse Events 
13.1 Definitions 
13.1.1  Unanticipated Problems Involving Risk to Subjects or Others 
Any incident, experience, or outcome that meets all of the following criteria:  
 Unexpected in nature, severity, or frequency  (i.e. not described in study-related documents such 
as the IRB-approved protocol or consent form, the investigators brochure, etc) 
 Related or possibly related to participation in the research (i.e. possibly related means there is a 
reasonable possibility that the incident experience, or outcome may have been caused by the 
procedures involved in the research, 
 Apheresis-related side effects such as citrate toxicity (symptomatic hypocalcemia), hypotension, 
vasovagal reaction, and access site thrombophlebitis.  If central venous catheter (CVC) is used, 
additional adverse events include bleeding at the cvc site, deep-vein thrombosis (DVT), 
thrombophlebitis, and central line infections. 
 
that applies a grade to the AE. 
 
13.1.2  Adverse Event 
An adverse event (AE) is any symptom, sign, illness or experience that develops or worsens in severity 
during the course of the study.  Concurrent illnesses or injuries should be regarded as adverse events.  
Abnormal results of diagnostic procedures are considered to be adverse events if the abnormality: 
 results in study withdrawal 
 is associated with a serious adverse event 
 is associated with clinical signs or symptoms 
 leads to additional treatment or to further diagnostic tests 
 is considered by the investigator to be of clinical significance 
 
13.1.3  Serious Adverse Event 
Adverse events are classified as serious or non-serious.  A serious adverse event is any AE that is:  
 fatal 
 life-threatening 
 requires or prolongs hospital stay 
 results in persistent or significant disability or incapacity 
 a congenital anomaly or birth defect 
 an important medical event 
 
Important medical events are those that may not be immediately life threatening, but are clearly of major 
clinical significance.   They may jeopardize the subject, and may require intervention to prevent one of the 
Patient logs in in-patient hospitalization, or intensive treatment of bronchospasm in an emergency 
department would typically be considered serious.  
 
All adverse events that do not meet any of the criteria for serious should be regarded as non-serious 
adverse events.  

 
Page 49 of 59 
 
13.1.4  Adverse Event Reporting Period 
The study period during which adverse events must be reported is normally defined as the period from the 
initiation of any study procedures to the end of the study treatment follow-up.   
 
For this study, the study treatment follow-up is defined as 30 days following the administration of study 
treatment. Serious adverse events should be reported to the local IRB and FDA within 7-10 days from the 
time of the event. Minor events can be reported in an annual report 
 
13.1.5  Preexisting Condition 
A preexisting condition is one that is present at the start of the study.  A preexisting condition should be 
recorded as an adverse event if the frequency, intensity, or the character of the condition worsens during 
the study period. 
 
13.1.6  General Physical Examination Findings 
At screening, any clinically significant abnormality should be recorded as a preexisting condition.  At the 
end of the study, any new clinically significant findings/abnormalities that meet the definition of an adverse 
event must also be recorded and documented as an adverse event.  
 
13.1.7  Post-study Adverse Event 
All unresolved adverse events should be followed by the investigator until the events are resolved, the 
subject is lost to follow-up, or the adverse event is otherwise explained.  At the last scheduled visit, the 
investigator should instruct each subject t
personal physician, believes might reasonably be related to participation in this study.  The investigator 
should notify the study sponsor of any death or adverse event occurring at any time (during the 6-month 
follow-up) after a subject has discontinued or terminated study participation that may reasonably be related 
to this study.  The sponsor should also be notified if the investigator should become aware of the 
development of cancer or of a congenital anomaly in a subsequently conceived offspring of a subject that 
has participated in this study.  
 
13.1.8 Abnormal Laboratory Values 
A clinical laboratory abnormality should be documented as an adverse event if any one of the following 
conditions is met:  
 The laboratory abnormality is not otherwise refuted by a repeat test to confirm the abnormality 
 The abnormality suggests a disease and/or organ toxicity 
 The abnormality is of a degree that requires active management; e.g. change of dose, 
discontinuation of the drug, more frequent follow-up assessments, further diagnostic investigation, 
etc. 
 
13.1.9 Hospitalization, Prolonged Hospitalization or Surgery 
Any adverse event that results in hospitalization or prolonged hospitalization should be documented and 
reported as a serious adverse event unless specifically instructed otherwise in this protocol.  Any condition 
responsible for surgery should be documented as an adverse event if the condition meets the criteria for 
and adverse event.  
 
Neither the condition, hospitalization, prolonged hospitalization, nor surgery are reported as an adverse 
event in the following circumstances: 
 Hospitalization or prolonged hospitalization for diagnostic or elective surgical procedures for a 
preexisting condition.  Surgery should not be reported as an outcome of an adverse event if the 
purpose of the surgery was elective or diagnostic and the outcome was uneventful. 
 Hospitalization or prolonged hospitalization required to allow efficacy measurement for the study. 
 Hospitalization or prolonged hospitalization for therapy of the target disease of the study, unless it 
is a worsening or increase in frequency of hospital admissions as judged by the clinical investigator. 

 
Page 50 of 59 
13.2 Recording of Adverse Events 
Based on our current clinical data in 200 subjects including Chinese and Caucasian patients, we did not 
see any significant adverse events during the treatment with SCE therapy, and follow-up studies after 
treatment for 4 years. No participants experienced any significant adverse events during the course of 
treatment. Most patients experienced mild discomfort during venipuncture and some soreness of the arm 
during aphaeresis, but discomfort and soreness resolved quickly following the conclusion of the procedure. 
No tumor formation and other safety concerns in all subjects after receiving SCE for 4 years.   
 
At each contact with the subject, the investigator must seek information on adverse events by specific 
questioning and, as appropriate, by examination.  Information on all adverse events should be recorded 
immediately in the source document, and also in the appropriate adverse event module of the case report 
form (CRF).  All clearly related signs, symptoms, and abnormal diagnostic procedures results should 
recorded in the source document, though should be grouped under one diagnosis. 
 
All adverse events occurring during the study period must be recorded.  The clinical course of each event 
should be followed until resolution, stabilization, or until it has been determined that the study treatment or 
participation is not the cause.  Serious adverse events that are still ongoing at the end of the study period 
must be followed up to determine the final outcome.  Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study participation should be 
recorded and reported immediately. 
13.3 Reporting of Serious Adverse Events and Unanticipated Problems 
Investigators and the protocol sponsor must conform to the adverse event reporting timelines, formats and 
requirements of the various entities to which they are responsible, but at a minimum those events that must 
be reported are those that are: 
 related to study participation, 
 unexpected, and  
 serious or involve risks to subjects or others  
(see definitions, section 8.1).   
If the report is supplied as a narrative, the minimum necessary information to be provided at the time of the 
initial report includes: 
 
Study identifier 
 
Study Center 
 
Subject number 
 
A description of the event 
 
Date of onset 
 
Current status 
 
Whether study treatment was discontinued 
 
The reason why the event is classified as serious
 
Investigator assessment of the association 
between the event and study treatment 
 
13.3.1 Investigator reporting: notifying the study sponsor 
Any study-related unanticipated problem posing risk of harm to subjects or others, and any type of serious 
adverse event, must be reported to the study sponsor by telephone within 24 hours of the event.  To report 
such events, a Serious Adverse Event (SAE) form must be completed by the investigator and faxed to the 
study sponsor within 24 hours.  The investigator will keep a copy of this SAE form on file at the study site.  
Report serious adverse events by phone and facsimile to: 
 
Dr.Michele Donato, 551 996 5900 (Phone) 
Dr.Steven Ghanny, 551 996 5329 (phone) 
Dr. Anne Van Hoven, 551 996 3500 (Phone) 
 
Within the following 48 hours, the investigator must provide further information on the serious adverse event 
or the unanticipated problem in the form of a written narrative.  This should include a copy of the completed 
Serious Adverse Event form, and any other diagnostic information that will assist the understanding of the 

 
Page 51 of 59 
event.  Significant new information on ongoing serious adverse events should be provided promptly to the 
study sponsor 
13.3.2 Investigator reporting:   
For reportable deaths, the initial submission to the Hackensack UMC IRB may be made by contacting the 
IRB Director or Associate Director. The AE/Unanticipated Problem Form is required as a follow up to the 
initial submission. 
 
Other Reportable events: 
 
 Any adverse experience that, even without detailed analysis, represents a serious unexpected adverse 
event that is rare in the absence of drug exposure (such as agranulocytosis, hepatic necrosis, Stevens-
Johnson syndrome). 
 Any adverse event that would cause the sponsor to modify the investigators brochure, protocol or 
informed consent form, or would prompt other action by the IRB to assure protection of human subjects. 
 Information that indicates a change to the risks or potential benefits of the research, in terms of severity 
or frequency. For example:  
 An interim analysis indicates that participants have a lower rate of response to treatment than 
initially expected. 
 Safety monitoring indicates that a particular side effect is more severe, or more frequent than 
initially expected. 
 A paper is published from another study that shows that an arm of your research study is of no 
therapeutic value. 
 Change in FDA safety labeling or withdrawal from marketing of a drug, device, or biologic used in a 
research protocol. 
 Breach of confidentiality 
 Change to the protocol taken without prior IRB review to eliminate apparent immediate hazard to a 
research participant. 
 Incarceration of a participant when the research was not previously approved under Subpart C and 
the investigator believes it is in the best interest of the subject to remain on the study. 
 Complaint of a participant when the complaint indicates unexpected risks or the complaint cannot 
be resolved by the research team. 
 Protocol violation (meaning an accidental or unintentional deviation from the IRB approved protocol) 
that in the opinion of the investigator placed one or more participants at increased risk, or affects 
the rights or welfare of subjects. 
13.3.3 Sponsor reporting: Notifying the FDA 
If this protocol is being conducted under an FDA IND, it is the responsibility of the study regulatory sponsor 
(Hackensack University Medical Center./Dr.Michele Donato responsible for the initiation, management, of 
the clinical trial --- i.e. the IND holder) to report certain adverse events or unanticipated problems to the 
FDA.   
 
The study sponsor is required to report certain study events in an expedited fashion to the FDA.  These 
written notifications of adverse events are referred to as IND safety reports. The following describes the 
safety reporting requirements by timeline for reporting and associated type of event: 
 
 Within 7 calendar days 
Any study event that is: 
 associated with the use of the study drug 
 unexpected,  
 fatal or life-threatening, and  
 

 
Page 52 of 59 
 Within 15 calendar days 
Any study event that is: 
 associated with the use of the study drug, 
 unexpected, and 
 serious, but not fatal or life-threatening 
-or- 
 a previous adverse event that was not initially deemed reportable but is later found to fit the 
criteria for reporting (reporting within 15 calendar days from when event was deemed 
reportable). 
Any finding from tests in laboratory animals that:  
 suggest a significant risk for human subjects including reports of mutagenicity, teratogenicity, 
or carcinogenicity. 
 
Additional reporting requirements 
Sponsors are also required to identify in IND safety reports all previous reports concerning similar adverse 
events and to analyze the significance of the current event in light of the previous reports. 
 
Reporting Process 
Adverse events may be submitted on FDA Form 3500A or in a narrative format. If supplied as in a narrative 
format, the minimum information to be supplied is noted above at the beginning of section 8.3. The contact 
information for submitting IND safety reports is noted below: 
 
FDA Emergency Operations Branch 
Office of Regional Operations, HFC-162 
Phone: 1-866-300-4374 or 301-796-8240 
FAX: 301-443-3757 
13.3.4 Sponsor reporting: Notifying participating investigators 
It is the responsibility of the study sponsor (Hackensack University Medical Center) to notify all participating 
investigators, in a written IND safety report, of any adverse event associated with the use of the drug that 
is both serious and unexpected, as well as any finding from tests in laboratory animals that suggest a 
significant risk for human subjects.  Additionally, sponsors are also required to identify in IND safety reports 
all previous reports concerning similar adverse events and to analyze the significance of the current event 
in light of the previous reports. 
 
13.4 Stopping Rules  
This single-arm study, in which all patients will receive the same experimental treatment at one dose, 
assess treatment safety and efficacy. The conduct of this trial may thus be stopped by a stopping rule 
setting out the circumstances under which the trial will end and the action that will then be taken, if the 
primary safety endpoint (Section 3.3.1) and efficacy endpoints (Section 3.3.2) have been achieved, and/or 
the confirmation of the treatment is safe and efficacious.  
If a grade 3 adverse event, as per the Common Terminology Criteria for Adverse Events (CTCAE) Version 
4.0, related to the study procedure occurs in 2 patients, the study will be terminated. This is defined in the 
statistical section of the protocol (Section 7.1). 
13.5 Medical Monitoring 
Michele Donato, MD, FACP, Division of Stem Cell Transplantation, HUMC is assigned as the medical 
monitor and will review all unanticipated problems involving risks to subjects or others, serious adverse 
events associated with the protocol.   
It is the responsibility of the study sponsor (Hackensack University Medical Center) to notify all participating 
investigators, in a written IND safety report of any adverse event associated with the use of the drug that is 
both serious and unexpected, as well as any finding from tests in laboratory animals that suggest a 
significant risk for human subjects.  Additionally, sponsors are also required to identify in IND safety reports 

 
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all previous reports concerning similar adverse events and to analyze the significant of the current event in 
light of the previous reports.   
13.5.1 Internal Data and Safety Monitoring Board or DSMP 
Hackensack University Medical Center (HUMC) places the highest priority on ensuring the safety of patients 
participating in clinical trials. The initial review of a new protocol lies with the Institutional Review Board 
(IRB) in reviewing new trials to ensure each trial, regardless of sponsorship or support, contains adequate 
plans for data and safety monitoring. Our Data and Safety Monitoring Board (DSMB) is responsible for 
monitoring all investigator-initiated trials (IITs) authored by HUMC investigators, regardless of federal, 
institutional or industry support. This includes single site IITs, as well as multicenter IITs coordinated by 
HUMC investigators with data management and site coordination by the principal investigator and includes 
all phases of clinical trials conducted at the HUMC. The members of DSMB at HUMC have clinicians, 
biostatisticians, 
bioethicists, 
and 
research 
scientists. 
Where 
appropriate, 
the 
use 
of 
an 
external/independent DSMB for a trial (e.g., high risk, multicenter) will be recommended.  
    Once a trial is determined as appropriate for monitoring by the internal DSMB, the DSMB has the 
written in to the IRB-approved protocol for such trials. The DSMB provides oversight of study progress and 
safety by review of the following information by un-blinded analyses: 1) Rates of accrual and accrual 
retention. 2) Frequency and severity of adverse events (AEs) and serious adverse events (SAEs). 3) 
Response rates, where appropriate. 4) New information related to the trial, i.e., published scientific reports 
or other developments that may affect subject safety or ethical concerns. 5) Any changes to the anticipated 
risk/benefit ratio of the study that would affect its continuation. 6) Protocol deviations and violations. 7) 
Matters that pertain to serious errors or potential misconduct by any of the investigators or research staff, 
i.e., breaches in confidentiality, research fraud. 8) Subject complaints. 9) Conflict of interest. 
    The timeline for review of trials by the DSMB is determined at the outset of the study with approval by 
the IRB. The frequency of DSMB review required for a protocol (i.e., 6 month, yearly) is recorded by the 
PRMS Administrator and tracked in order that the appropriate DSMB submission documentation is 
requested from the study team in advance of a scheduled DSMB meeting. The PI and clinical research 
coordinator (CRC) are provided forms for submission of required documentation to the DSMB.   
14 Data Handling and Record Keeping 
14.1 Confidentiality 
Information about study subjects will be kept confidential and managed according to the requirements of 
the Health Insurance Portability and Accountability Act of 1996 (HIPAA).  Those regulations require a signed 
subject authorization informing the subject of the following:  
 What protected health information (PHI) will be collected from subjects in this study 
 Who will have access to that information and why 
 Who will use or disclose that information 
 The rights of a research subject to revoke their authorization for use of their PHI.  
 
In the event that a subject revokes authorization to collect or use PHI, the investigator, by regulation, retains 
the ability to use all information collected prior to the revocation of subject authorization.  For subjects that 
have revoked authorization to collect or use PHI, attempts should be made to obtain permission to collect 
at least vital status (i.e. that the subject is alive) at the end of their scheduled study period. 
14.2 Source Documents 
Source data is all information, original records of clinical findings, observations, or other activities in a 
clinical trial necessary for the reconstruction and evaluation of the trial.  Source data are contained in source 
documents.  Examples of these original documents, and data records include: hospital records, clinical and 
ensing 
records, recorded data from automated instruments, copies or transcriptions certified after verification as 
being accurate and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, 

 
Page 54 of 59 
subject files, and records kept at the pharmacy, at the laboratories, and at medico-technical departments 
involved in the clinical trial. 
14.3 Case Report Forms 
The study case report form (CRF) is the primary data collection instrument for the study.  All data requested 
on the CRF must be recorded.  All missing data must be explained.  If a space on the CRF is left blank 
ibly in black ink.  If any entry error has 
been made, to correct such an error, draw a single straight line through the incorrect entry and enter the 
correct data above it.  All such changes must be initialed and dated.  DO NOT ERASE OR WHITE OUT 
ERRORS.  For clarification of illegible or uncertain entries, print the clarification above the item, then initial 
and date it. 
14.4 Records Retention 
approval of a marketing application in their country and until there are no pending or contemplated 
marketing applications in their country or at least 2 years have elapsed since the formal discontinuation of 
clinical development of the investigational product.  These documents should be retained for a longer period 
if required by an agreement with the sponsor.  In such an instance, it is the responsibility of the sponsor to 
inform the investigator/institution as to when these documents no longer need to be retained.  
 
15 Study Monitoring, Auditing, and Inspecting 
15.1 Study Monitoring Plan 
This study will be monitored according to the monitoring plan.  The investigator will allocate adequate time 
for such monitoring activities.  The Investigator will also ensure that the monitor or other compliance or 
quality assurance reviewer is given access to all the above noted study-related documents and study 
related facilities (e.g. pharmacy, diagnostic laboratory, etc.), and has adequate space to conduct the 
monitoring visit. The following issues will be addressed at this visit: all CRFs have been completed and 
appropriately filed, a copy of the monitoring Patient Log is obtained, maintenance and retention of study 
records.   
    The close-
se report forms have been completed, study has 
been closed with reviewing IRB/IEC and all regulatory issues have been addressed.  
    In summary the Monitor will serve an important role in the successful conduct of the study. The 
relationship between the Monitor and the site staff is strengthened by open effective communication with 
the Monitor providing training and support to ensure 
and compliance with all applicable regulations of the regulatory authorities. 
15.2 Auditing and Inspecting 
The investigator will permit study-related monitoring, audits, and inspections by the IRB, the sponsor, 
government regulatory bodies, and University compliance and quality assurance groups of all study related 
documents (e.g. source documents, regulatory documents, data collection instruments, study data etc.).  
The investigator will ensure the capability for inspections of applicable study-related facilities (e.g. 
pharmacy, diagnostic laboratory, etc.). 
 
Participation as an investigator in this study implies acceptance of potential inspection by government 
regulatory authorities and applicable University compliance and quality assurance offices. 
16 Ethical Considerations 
This study is to be conducted according to US and international standards of Good Clinical Practice (FDA 
Title 21 part 312 and International Conference on Harmonization guidelines), applicable government 
regulations and Institutional research policies and procedures. 

 
Page 55 of 59 
 
This protocol and any amendments will be submitted to a properly constituted independent Ethics 
Committee (EC) or Institutional Review Board (IRB), in agreement with local legal prescriptions, for formal 
approval of the study conduct.  The decision of the EC/IRB concerning the conduct of the study will be 
made in writing to the investigator and a copy of this decision will be provided to the sponsor before 
commencement of this study.  The investigator should provide a list of EC/IRB members and their affiliate 
to the sponsor. 
 
All subjects for this study will be provided a consent form describing this study and providing sufficient 
information for subjects to make an informed decision about their participation in this study. This consent 
form will be submitted with the protocol for review and approval by the EC/IRB for the study.  The formal 
consent of a subject, using the EC/IRB-approved consent form, must be obtained before that subject 
undergoes any study procedure.  The consent form must be signed by the subject or legally acceptable 
surrogate, and the investigator-designated research professional obtaining the consent.  
17 Study Finances 
17.1 Funding Source 
This study is financed through a funding from Hackensack University Medical Center Foundation.   
17.2 Conflict of Interest 
No investigators have conflict of interests that may be relevant to the submitted clinical trial. All Hackensack 
UMC investigators will follow the HUMC conflict of interest policy. 
18 Publication Plan 
Neither the complete nor any part of the results of the study carried out under this protocol, nor any of the 
information provided by the sponsor for the purposes of performing the study, will be published or passed 
on to any third party without the consent of the study sponsor.  Any investigator involved with this study is 
obligated to provide the sponsor with complete test results and all data derived from the study. 
 
 
 
 
 
 
 
 
 
 
 

 
Page 56 of 59 
Bibliography 
 
 
1.  Zhao Y, Jiang Z, Zhao T, Ye M, Hu C, Yin Z et al.: Reversal of type 1 diabetes via islet beta 
cell regeneration following immune modulation by cord blood-derived multipotent stem 
cells. BMC Med 2012, 10: 3. 
 
2.  Zhao Y, Jiang Z, Zhao T, Ye M, Hu C, Zhou H et al.: Targeting insulin resistance in type 2 
diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in 
stem cell educator therapy: phase I/II clinical trial. BMC Med 2013, 11: 160. 
 
3.  Li Y, Yan B, Wang H, Li H, Li Q, Zhao D et al.: Hair regrowth in alopecia areata patients 
following Stem Cell Educator therapy. BMC Med 2015, 13: 87. 
 
4.  Delgado E, Perez-Basterrechea M, Suarez-Alvarez B, Zhou H, Revuelta EM, Garcia-Gala JM et 
al.: Modulation of Autoimmune T-Cell Memory by Stem Cell Educator Therapy: Phase 1/2 
Clinical Trial. EBioMedicine 2015, 2: 2024-2036. 
 
5.  Zhao Y: Stem Cell Educator Therapy and Induction of Immune Balance. Curr Diab Rep 2012, 
12: 517-523. 
 
6.  Zhao Y, Lin B, Darflinger R, Zhang Y, Holterman MJ, Skidgel RA: Human cord blood stem cell-
modulated regulatory T lymphocytes reverse the autoimmune-caused type 1 diabetes in 
nonobese diabetic (NOD) mice. PLoS ONE 2009, 4: e4226. 
 
7.  Zhao Y, Huang Z, Lazzarini P, Wang Y, Di A, Chen M: A unique human blood-derived cell 
population displays high potential for producing insulin. Biochem Biophys Res Commun 
2007, 360: 205-211. 
 
8.  Zhao Y, Wang H, Mazzone T: Identification of stem cells from human umbilical cord blood 
with embryonic and hematopoietic characteristics. Exp Cell Res 2006, 312: 2454-2464. 
 
9.  Zhao Y, Mazzone T: Human cord blood stem cells and the journey to a cure for type 1 
diabetes. Autoimmun Rev 2010, 10: 103-107. 
 
10.  Delgado E, Perez-Basterrechea M, Suarez-Alvarez B, Zhou H, Martinez Revuelta E, Garcia-Gala 
JM et al.: Modulation of autoimmune T-Cell memory by Stem Cell Educator therapy: phase 
1/2 clinical trial. EBioMedicine 2015, 2: 2024-2036. 
 
11.  Zhao Y, Huang Z, Qi M, Lazzarini P, Mazzone T: Immune regulation of T lymphocyte by a 
newly characterized human umbilical cord blood stem cell. Immunol Lett 2007, 108: 78-87. 
 
12.  Bluestone JA, Herold K, Eisenbarth G: Genetics, pathogenesis and clinical interventions in 
type 1 diabetes. Nature 2010, 464: 1293-1300. 
 
13.  Lehuen A, Diana J, Zaccone P, Cooke A: Immune cell crosstalk in type 1 diabetes. Nat Rev 
Immunol 2010, 10: 501-513. 
 
14.  Couzin-Frankel J: Trying to reset the clock on type 1 diabetes. Science 2011, 333: 819-821. 
 
15.  Bach JF: Anti-CD3 antibodies for type 1 diabetes: beyond expectations. Lancet 2011, 378: 
459-460. 
 
16.  Wherrett DK, Bundy B, Becker DJ, DiMeglio LA, Gitelman SE, Goland R et al.: Antigen-based 
therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 
1 diabetes: a randomised double-blind trial. Lancet 2011, 378: 319-327. 
 
17.  Zhao Y, Glesne D, Huberman E: A human peripheral blood monocyte-derived subset acts as 
pluripotent stem cells. Proc Natl Acad Sci U S A 2003, 100: 2426-2431. 

 
Page 57 of 59 
 
18.  Meier JJ, Lin JC, Butler AE, Galasso R, Martinez DS, Butler PC: Direct evidence of attempted 
beta cell regeneration in an 89-year-old patient with recent-onset type 1 diabetes. 
Diabetologia 2006, 49: 1838-1844. 
 
19.  Li MO, Flavell RA: TGF-beta: a master of all T cell trades. Cell 2008, 134: 392-404. 
 
20.  Roncarolo MG, Battaglia M, Gregori S: The role of interleukin 10 in the control of 
autoimmunity. J Autoimmun 2003, 20: 269-272. 
 
21.  Wan YY, Flavell RA: The roles for cytokines in the generation and maintenance of 
regulatory T cells. Immunol Rev 2006, 212: 114-130. 
 
22.  Perruche S, Zhang P, Liu Y, Saas P, Bluestone JA, Chen W: CD3-specific antibody-induced 
immune tolerance involves transforming growth factor-beta from phagocytes digesting 
apoptotic T cells. Nat Med 2008, 14: 528-535. 
 
23.  Wesselschmidt RL: The teratoma assay: an in vivo assessment of pluripotency. Methods Mol 
Biol 2011, 767: 231-241. 
 
24.  Zhao Y, Lin B, Dingeldein M, Guo C, Hwang D, Holterman MJ: New type of human blood stem 
cell: a double-edged sword for the treatment of type 1 diabetes. Transl Res 2010, 155: 211-
216. 
 
25.  Zhao Y, Mazzone T: Human umbilical cord blood-derived f-macrophages retain 
pluripotentiality after thrombopoietin expansion. Exp Cell Res 2005, 310: 311-318. 
 
26.  Zhao Y, Jiang Z, Guo C: New hope for type 2 diabetics: Targeting insulin resistance through 
the immune modulation of stem cells. Autoimmun Rev 2011, 11: 137-142. 
 
27.  Bluestone JA, Tang Q, Sedwick CE: T Regulatory Cells in Autoimmune Diabetes: Past 
Challenges, Future Prospects. J Clin Immunol 2008, 28: 677-684. 
 
28.  Roncarolo MG, Battaglia M: Regulatory T-cell immunotherapy for tolerance to self antigens 
and alloantigens in humans. Nat Rev Immunol 2007, 7: 585-598. 
 
29.  Leslie M: Immunology. Regulatory T cells get their chance to shine. Science 2011, 332: 
1020-1021. 
 
30.  Shevach EM: Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 
2009, 30: 636-645. 
 
31.  Bour-Jordan H, Esensten JH, Martinez-Llordella M, Penaranda C, Stumpf M, Bluestone JA: 
Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of 
the CD28/ B7 family. Immunol Rev 2011, 241: 180-205. 
 
32.  Hombach AA, Kofler D, Hombach A, Rappl G, Abken H: Effective proliferation of human 
regulatory T cells requires a strong costimulatory CD28 signal that cannot be substituted 
by IL-2. J Immunol 2007, 179: 7924-7931. 
 
33.  Hori S: c-Rel: a pioneer in directing regulatory T-cell lineage commitment? Eur J Immunol 
2010, 40: 664-667. 
 
34.  Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK et al.: Cutting edge: CD28 
controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol 2003, 171: 
3348-3352. 

 
Page 58 of 59 
 
35.  Vang KB, Yang J, Pagan AJ, Li LX, Wang J, Green JM et al.: Cutting edge: CD28 and c-Rel-
dependent pathways initiate regulatory T cell development. J Immunol 2010, 184: 4074-
4077. 
 
36.  Herman AE, Freeman GJ, Mathis D, Benoist C: CD4+CD25+ T regulatory cells dependent on 
ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004, 199: 
1479-1489. 
 
37.  Gotsman I, Grabie N, Gupta R, DaCosta R, MacConmara M, Lederer J et al.: Impaired 
regulatory T-cell response and enhanced atherosclerosis in the absence of inducible 
costimulatory molecule. Circulation 2006, 114: 2047-2055. 
 
38.  Nurieva RI, Liu X, Dong C: Molecular mechanisms of T-cell tolerance. Immunol Rev 2011, 
241: 133-144. 
 
39.  Rudensky AY: Regulatory T cells and Foxp3. Immunol Rev 2011, 241: 260-268. 
 
40.  Masopust D, Picker LJ: Hidden memories: frontline memory T cells and early pathogen 
interception. J Immunol 2012, 188: 5811-5817. 
 
41.  Mueller SN, Gebhardt T, Carbone FR, Heath WR: Memory T cell subsets, migration patterns, 
and tissue residence. Annu Rev Immunol 2013, 31: 137-161. 
 
42.  Devarajan P, Chen Z: Autoimmune effector memory T cells: the bad and the good. Immunol 
Res 2013, 57: 12-22. 
 
43.  Maecker HT, McCoy JP, Nussenblatt R: Standardizing immunophenotyping for the Human 
Immunology Project. Nat Rev Immunol 2012, 12: 191-200. 
 
44.  Forster R, Davalos-Misslitz AC, Rot A: CCR7 and its ligands: balancing immunity and 
tolerance. Nat Rev Immunol 2008, 8: 362-371. 
 
45.  Moschovakis GL, Forster R: Multifaceted activities of CCR7 regulate T-cell homeostasis in 
health and disease. Eur J Immunol 2012, 42: 1949-1955. 
 
46.  Devaraj S, Dasu MR, Jialal I: Diabetes is a proinflammatory state: a translational 
perspective. Expert Rev Endocrinol Metab 2010, 5: 19-28. 
 
47.  Shoelson SE, Lee J, Goldfine AB: Inflammation and insulin resistance. J Clin Invest 2006, 
116: 1793-1801. 
 
48.  Devaraj S, Jialal I: Low-density lipoprotein postsecretory modification, monocyte function, 
and circulating adhesion molecules in type 2 diabetic patients with and without 
macrovascular complications: the effect of alpha-tocopherol supplementation. Circulation 
2000, 102: 191-196. 
 
49.  Brooks-Worrell B, Palmer JP: Is diabetes mellitus a continuous spectrum? Clin Chem 2011, 
57: 158-161. 
 
50.  Brooks-Worrell BM, Reichow JL, Goel A, Ismail H, Palmer JP: Identification of autoantibody-
negative autoimmune type 2 diabetic patients. Diabetes Care 2011, 34: 168-173. 
 
51.  Goldfine AB, Fonseca V, Shoelson SE: Therapeutic approaches to target inflammation in 
type 2 diabetes. Clin Chem 2011, 57: 162-167. 

 
Page 59 of 59 
 
52.  Mathis D, Shoelson SE: Immunometabolism: an emerging frontier. Nat Rev Immunol 2011, 
11: 81. 
 
53.  Naik RG, Palmer JP: Latent autoimmune diabetes in adults (LADA). Rev Endocr Metab Disord 
2003, 4: 233-241. 
 
54.  Jagannathan-Bogdan M, McDonnell ME, Shin H, Rehman Q, Hasturk H, Apovian CM et al.: 
Elevated proinflammatory cytokine production by a skewed T cell compartment requires 
monocytes and promotes inflammation in type 2 diabetes. J Immunol 2011, 186: 1162-1172. 
 
55.  Sumarac-Dumanovic M, Stevanovic D, Ljubic A, Jorga J, Simic M, Stamenkovic-Pejkovic D et al.: 
Increased activity of interleukin-23/interleukin-17 proinflammatory axis in obese women. 
Int J Obes (Lond) 2009, 33: 151-156. 
 
56.  Wu HP, Kuo SF, Wu SY, Chuang DY: High interleukin-12 production from stimulated 
peripheral blood mononuclear cells of type 2 diabetes patients. Cytokine 2010, 51: 298-304. 
 
57.  Mishra M, Kumar H, Bajpai S, Singh RK, Tripathi K: Level of serum IL-12 and its correlation 
with endothelial dysfunction, insulin resistance, proinflammatory cytokines and lipid 
profile in newly diagnosed type 2 diabetes. Diabetes Res Clin Pract 2011, 94: 255-261. 
 
58.  Osborn O, Olefsky JM: The cellular and signaling networks linking the immune system and 
metabolism in disease. Nat Med 2012, 18: 363-374. 
 
59.  Chatzigeorgiou A, Karalis KP, Bornstein SR, Chavakis T: Lymphocytes in obesity-related 
adipose tissue inflammation. Diabetologia 2012, 55: 2583-2592. 
 
60.  Glass CK, Olefsky JM: Inflammation and lipid signaling in the etiology of insulin resistance. 
Cell Metab 2012, 15: 635-645. 
 
61.  Olefsky JM, Glass CK: Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 
2010, 72: 219-246. 
 
62.  Sell H, Habich C, Eckel J: Adaptive immunity in obesity and insulin resistance. Nat Rev 
Endocrinol 2012. 
 
63.  Beug ST, Cheung HH, Lacasse EC, Korneluk RG: Modulation of immune signalling by 
inhibitors of apoptosis. Trends Immunol 2012, 33: 535-545. 
 
64.  Herold KC, Vignali DA, Cooke A, Bluestone JA: Type 1 diabetes: translating mechanistic 
observations into effective clinical outcomes. Nat Rev Immunol 2013, 13: 243-256. 
 
65.  Zhao Y, Guo C, Hwang D, Lin B, Dingeldein M, Mihailescu D et al.: Selective destruction of 
mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 
diabetic model. Biochem Biophys Res Commun 2010, 399: 629-636. 
 
66.  Mathieu C, Gillard P: Arresting type 1 diabetes after diagnosis: GAD is not enough. Lancet 
2011, 378: 291-292.