A Pilot Study of Early Treatment of Acute Graft Versus Host Disease With Bone Marrow- Derived Mesenchymal Stem Cells and Corticosteroids: Correlation of Disease Severity and Response With Biomarkers
Phase: PHASE1
Conditions: Graft-Versus-Host Disease
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

1 
Abbreviated Title: Early BMSC Pilot for Acute GVHD 
Version Date: 6/16/2016 
15-H-0088 
Sawa Ito, M.D. 
CLINICAL RESEARCH PROJECT 
       Protocol # 15-H-0088 
 
 
 
 
 
 
 
 
IND # 14596 
IND Holder: A. John Barrett, M.D. 
Date:    June 16, 2016 
To: 
Richard Cannon, M.D., Chair, NHLBI, IRB 
Title: A Pilot Study of Early Treatment of Acute Graft Versus Host Disease with Bone Marrow-Derived Mesenchymal 
Stem Cells and Corticosteroids: Correlation of Disease Severity and Response with Biomarkers 
Other identifying words: BMSC, corticosteroid, GVHD, immunotherapy, adoptive cellular therapy 
 
Principal Investigator: 
* Sawa Ito, M.D. HB., NHLBI (E) 
 
 
 
 
Lead Associate Investigator:  
*Minoo Battiwalla M.D., M.S., HB, NHLBI (E)  
 
 
Medically Responsible Investigator:  
*A. John Barrett M.D., Chief, BMT Unit, HB, NHLBI (E)          
 
Associate Investigators: 
Pamela Robey Ph.D., NIDCR (E) 
 
 
 
 
David Stroncek, M.D., DTM, Clinical Center (E) 
 
 
Hanh Khuu, M.D., DTM, Clinical Center (E) 
 
 
 
Eleftheria Koklanaris, R.N., Research Nurse, HB, NHLBI (E) 
 
Debbie Draper, R.N., Research Nurse, HB, NHLBI (E)  
 
Colin Wu, Ph.D., Biostatistician, DECA/OBR, NHLBI (E) 
 
John Tisdale, M.D., HB, NHLBI (E) 
 
 
 
 
Ronald Gress, M.D., ETIB, CCR, NCI (E) 
 
 
 
Steven Pavletic, M.D., ETIB, CCR, NCI (E) 
 
 
 
Dan Fowler M.D., ETIB, CCR, NCI (E)  
 
 
 
Shoba Amarnath M.D., ETIB, CCR, NCI (E) 
 
 
  
Elizabeth Kang M.D., NIAID (E) 
 
 
 
 
Terry Fry M.D., POB, NCI (E)  
 
 
 
 
Xin Tian, Ph.D., Biostatistician, OBR/NHLBI (E) 
 
 
 
 
*Investigators designated to obtain informed consent from research subjects. 
 
Subjects of Study: 
 
 
Number 
 
Sex 
 
Age range 
 
  Subjects: 
  
  
up to 25 
 
either  
age ≥ 4 years 
 
Project involves ionizing radiation?  
Yes (Medically Indicated Only) 
Off site project? 
 
 
No 
Multi-institutional project? 
 
No 
DSMB Involved? 
 
 
Yes  

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
Version Date: 6/16/2016 
15-H-0088 
Sawa Ito, M.D. 
PRECIS  
This is a pilot study evaluating the addition of bone marrow-derived mesenchymal stem cell (BMSC) infusions 
to corticosteroids for the early treatment of acute graft versus host disease (GVHD).  Acute GVHD is a major 
complication following allogeneic stem cell transplant.  GVHD occurs when T-lymphocytes in the donor graft 
respond to signals from recipient cells and cause tissue damage.  This process can lead to organ injury, 
increased risk of infection, and graft failure.  Corticosteroids have been used as the primary therapy for acute 
GVHD for decades, and guidelines currently recommend their use as front line treatment.  Recent prospective 
data from the Blood and Marrow Transplant Clinical Trials Network shows that GVHD will be cured in about 
half of patients with steroids alone.  Patients who do not respond to steroids are considered steroid-resistant, and 
this is associated with much worse survival. It is possible to predict which patients will go on to have steroid-
resistant GVHD by measuring the plasma concentration of the molecule called suppression of tumorigenicity 2 
(ST2).  BMSC infusions have been used to treat steroid-resistant acute GVHD successfully, but despite a track 
record of safety, little is known about the use of BMSC in the early treatment setting.   The main objective of 
this study is to explore the feasibility of administering BMSC within 5 days of diagnosis of acute GVHD. Our 
study will for the first time use the ST2 biomarker to more accurately assign acute GVHD to steroid refractory 
or sensitive, and explore changes in ST2 and other biological markers of BMSC function and their correlation 
with clinical response.  In the process of this study, we will assess the safety and feasibility of early treatment 
according to our regimen, obtain estimates of efficacy at important GVHD therapy time points, and determine if 
treatment with BMSC can prevent the progression of GVHD in patients with high risk of GVHD progression as 
measured by biomarkers.   
 
The Cell Processing Section of the Department of Transfusion Medicine at the Clinical Center NIH has 
developed a BMSC repository at NIH.  The NIH BMSC are a third party, early passage product based on the 
EU manufacturing approach.  The NIH BMSC cellular product was administered safely to transplant recipients 
with steroid-resistant acute GVHD in a phase I study (protocol 12-H-0010, IND #14596) conducted from March 
2012 to October 2012 at NIH.  This pilot study is a continuation of the previous study and open to allogeneic 
stem cell transplantation recipients at NIH (age ≥4 yrs) with de novo acute GVHD requiring systemic therapy, 
either directly after allogeneic transplantation or following treatment with donor lymphocyte infusion.  Subjects 
will receive BMSC infusions (target dose of 2 x 106 BMSC/kg for up to 12 doses) in addition to standard 
upfront therapy with corticosteroids.   The primary endpoint will be the proportion of patients without a 
treatment-related severe adverse event (TRSAE) at day +56. Responses will be assessed at day +28 and +56 
from the initial diagnosis.  Responses will be correlated to changes in GVHD biomarkers including ST2, Reg3a, 
TNFR1, and IL-6.   Subjects will be enrolled at first diagnosis of acute GVHD, and the first BMSC infusion 
will be given within 120 hours of the first dose of corticosteroids.   BMSC infusions will be given twice weekly 
for the first 4 weeks.  Subjects with a complete response at the end of week 4 will not receive further infusions.  
All other subjects will receive BMSC infusions weekly for four additional weeks.  Safety will be monitored 
continuously with a stopping rule for toxicity based on the treatment-related serious adverse event rate.   
Research samples will be drawn at regular intervals to explore biological correlates of response and to 
investigate the mechanism of action of BMSC. 
 
 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
Version Date: 6/16/2016 
15-H-0088 
Sawa Ito, M.D. 
TABLE OF CONTENTS  
1. INTRODUCTION .............................................................................................................................................. 5 
1.1 
Study Objectives ...................................................................................................................................... 5 
2. BACKGROUND ................................................................................................................................................ 5 
2.1  
Acute Graft-versus-Host Disease ............................................................................................................. 5 
2.2  
Primary Therapy for Acute GVHD .......................................................................................................... 6 
2.3  
Biology of Bone marrow-derived mesenchymal stem cells..................................................................... 6 
2.4 
Clinical Experience With BMSC in GVHD ............................................................................................ 7 
2.5 
NIH Phase I BMSC Trial (12-H-0010) .................................................................................................. 10 
2.6  
Safety Of BMSC Infusions .................................................................................................................... 11 
2.7 
Scientific And Clinical Justification ...................................................................................................... 12 
2.8 
Rationale for Changes from Phase I Study ............................................................................................ 12 
3. STUDY DESIGN.............................................................................................................................................. 14 
3.1  
Study Design .......................................................................................................................................... 14 
3.2 
Subject Inclusion .................................................................................................................................... 14 
3.3 
Initial Assessment .................................................................................................................................. 14 
3.4 
Treatment ............................................................................................................................................... 15 
3.5 
Toxicity .................................................................................................................................................. 17 
3.6 
Response................................................................................................................................................. 17 
3.7 
Survival .................................................................................................................................................. 17 
4. RECRUITMENT AND REGISTRATION ...................................................................................................... 17 
5. ELIGIBILITY ASSESSMENT ........................................................................................................................ 18 
5.1 
Inclusion Criteria .................................................................................................................................... 18 
6. CLINICAL EVALUATION ............................................................................................................................. 19 
6.1  
Clinical Evaluation Of The Subject ....................................................................................................... 19 
6.2 
Response Definitions.............................................................................................................................. 20 
7. SAMPLE COLLECTION, STORAGE AND TRACKING PLAN ................................................................. 20 
7.1 
Research Sample Collection................................................................................................................... 20 
7.2 
Collection, Storage And Disposition Of Samples .................................................................................. 20 
8. MANAGEMENT OF COMPLICATIONS ...................................................................................................... 21 
8.1 
Overview ................................................................................................................................................ 21 
8.2 
Immediate Transfusion Reaction ........................................................................................................... 21 
8.3 
Treatment Limiting Toxicities ............................................................................................................... 22 
8.4 
Post-Transplant Complications .............................................................................................................. 22 
9. OFF TREATMENT AND OFF STUDY CRITERIA ...................................................................................... 22 
9.1 
Off Treatment Criteria ............................................................................................................................ 22 
9.2 
Off Study Criteria ................................................................................................................................... 22 
10. DATA MANAGEMENT................................................................................................................................ 23 
10.1 Data safety ................................................................................................................................................ 23 
11. BIOSTATISTICAL CONSIDERATIONS ..................................................................................................... 24 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
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Sawa Ito, M.D. 
11.1  Primary Endpoint ................................................................................................................................... 24 
11.3  Statistical Analysis ................................................................................................................................. 24 
12. DATA AND SAFETY MONITORING ......................................................................................................... 25 
12.1 Safety Monitoring .................................................................................................................................. 25 
12.2  Adverse Events ....................................................................................................................................... 26 
12.3  NHLBI  IRB and CD Reporting ............................................................................................................. 29 
12.4  IND Sponsor Reporting Criteria ............................................................................................................ 29 
12.5  DSMB Reporting.................................................................................................................................... 30 
12.6 Protocol Monitoring ............................................................................................................................... 31 
13. HUMAN SUBJECTS PROTECTIONS ......................................................................................................... 31 
13.1 Rationale For Subject Selection ............................................................................................................. 31 
13.2  Participation of Children, Pregnant Women and Decisionally Impaired ............................................... 32 
13.3  Hazards/Discomforts .............................................................................................................................. 32 
13.4  Risks/Benefits Analysis.......................................................................................................................... 32 
13.5  Consent and Assent Process and Documentation .................................................................................. 33 
13.6  Conflict of Interest ................................................................................................................................. 35 
14. PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION ........................................ 35 
14.1  BMSC Infusion ...................................................................................................................................... 35 
Appendix 1:  Criteria for Diagnosis, Severity, Response and Eligibility for BMSC Trial ................................... 37 
Appendix 2:  Criteria for Response to BMSC ...................................................................................................... 38 
Appendix 3:  Study Calendar for Early BMSC Pilot for Acute GVHD ............................................................... 39 
Appendix 4:  IRB Approved Hematology Branch Laboratory Research Studies ................................................ 40 
REFERENCES ..................................................................................................................................................... 45 
 
 
 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
Version Date: 6/16/2016 
15-H-0088 
Sawa Ito, M.D. 
1. INTRODUCTION 
1.1 
STUDY OBJECTIVES 
1.1.1 Primary Objective 
Determine the safety and feasibility of adding BMSC infusions to corticosteroid therapy in allogeneic 
stem cell transplant recipients within 5 days of the diagnosis of de novo acute GVHD requiring 
treatment with systemic corticosteroids.  
 
1.1.2 Secondary Objectives 
Estimate the efficacy of our proposed regimen at well-established GVHD response time points and 
correlate responses to plasma GVHD biomarkers of disease severity (ST2) and response (Reg3a, 
TNFR1, IL-6). 
 
2. BACKGROUND 
2.1  
ACUTE GRAFT-VERSUS-HOST DISEASE 
Acute GVHD is an inflammatory process that occurs in the majority of patients who receive an allogeneic stem 
cell transplant. GVHD occurs when host antigen-presenting cells (APC) activate in response to signals from 
damaged tissue or pathogens.  APC present antigens to donor T-lymphocytes, leading to alloreactivity, cytokine 
release, and increasing tissue destruction, reviewed in1. Of the more than 7,000 patients who undergo allogeneic 
stem cell transplant in the US each year2, approximately 35-50% will develop grade II-IV acute GVHD despite 
prophylactic immunosuppression3.  GVHD is the second leading cause of death in patients undergoing 
transplant and it is the leading cause of non-relapse mortality2. The standard initial therapy for acute GVHD is 
high-dose intravenous corticosteroids4. The response rate to single-agent corticosteroid therapy, when analyzed 
in large retrospective reviews, is approximately 50%5.  In a recent randomized prospective study comparing 
mycophenolate mofetil plus steroids and steroids alone, the day 56 acute GVHD survival was 50.4% in the 
steroids alone arm (n=119)6.  Mortality in patients with acute GVHD is greatest among those who fail to 
achieve a complete response with initial treatment5,7. Although steroid-resistant acute GVHD can be treated 
with anti-lymphocyte globulin or a variety of monoclonal antibodies, there is no standard treatment approach 
and steroid-resistant GVHD has a one year mortality between 50-80%8. Acute GVHD usually occurs in the first 
100 days of allogeneic stem cell transplant and may involve the skin and/or gastrointestinal tract and/or liver. 
Following is a summary of the major manifestations of acute GVHD. 
 
2.1.1  Skin GVHD  
 
Skin involvement is usually the first sign of GVHD.  It varies in extent from a rash affecting extensor 
 
surfaces face and hands to a generalized erythroderma affecting the entire body. Severity varies 
 
according to the degree of epidermal damage from a mild desquamation to bullae formation and 
 
separation of the dermo-epidermal junction. Skin GVHD is usually well controlled with steroids but 
 
severe extensive skin GVHD carries mortality from secondary infection.  
 
2.1.2  Gastrointestinal GVHD 
 
Acute gastrointestinal GVHD can affect the entire digestive tract leading to diarrhea, blood loss, 
 
vomiting, nutritional failure, weight loss and secondary infection. Lymphocyte infiltration in the 
 
submucosa is associated with crypt damage and loss with characteristic single-cell apoptosis in the 
 
columnar epithelium leading to complete loss of the epithelium.   

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
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Sawa Ito, M.D. 
2.1.3  Hepatic GVHD   
 
Hepatic GVHD primarily affects the bile canaliculi with peri-canalicular lymphocyte infiltration, and 
 
damage to biliary endothelium. Parenchymal tissue is secondarily affected giving rise to the typical 
 
mixed obstructive/hepatitis pattern of LFTs. While hepatic GVHD is usually mild to moderate and 
 
responds to immunosuppression, some patients develop worsening, life-threatening hepatic GVHD with 
 
a “vanishing bile duct” histological picture of rising bilirubin and worsening liver function. This usually 
 
but not always occurs in association with severe gut GVHD.  
2.2  
PRIMARY THERAPY FOR ACUTE GVHD 
Corticosteroids have been used as the first line treatment for acute GVHD for decades. Several agents 
have been added to upfront steroids in clinical trials in order to improve response rates, most commonly: 
ATG9-11, daclizumab12,13, denileukin difitox14, and etanercept15.  No agent or combination of agents has 
been proven to be more effective than steroids alone. Recently the Blood and Marrow Transplant 
Clinical Trials Network (BMT-CTN) conducted two randomized trials (BMT CTN 0302 and BMT CTN 
0802) focused on improving the initial response rates of de novo acute GVHD to therapy. 
 
2.2.1 BMT CTN 0302 
The first trial was a randomized, four-arm, phase II trial designed to identify the most promising agent to 
combine with steroids in a phase III trial for upfront treatment of acute GVHD16.  Patients (n=180) were 
accrued from August 2005 to March 2008 and randomized to receive either etanercept, mycophenolate 
mofetil (MMF), denileukin difitox, or pentostatin in combination with standard doses of corticosteroids 
for initial therapy. The proportion of complete responses at day +28 were etanercept (26%), MMF 
(60%), denileukin difitox (53%), and pentostatin (38%).  Day 56 ORR rates were 59%, 78%, 68%, 71% 
respectively, and OS at 6 months was 59%, 71%, 63%, and 55% respectively.  Infection rates and 
overall toxicities were lower in the etanercept and MMF arms.   
 
 
2.2.2 BMT CTN 0802 
Based on the results of BMT CTN 0302, a phase III, multi-center randomized trial (BMT CTN 0802) 
was conducted comparing corticosteroids/placebo to corticosteroids/MMF6,17.  Patients received 
prednisone at 2mg/kg/day with either MMF (1000mg po/IV q8h) or placebo.  MMF was continued until 
day +56 or until steroid discontinuation if sooner. The proportion of GVHD free survival at day +56 
after randomization was MMF 59.5% and placebo 50.4% (p=0.16).  Overall survival, non-relapse 
mortality, and incidence of infection were similar in both arms.  The study was stopped at a 
predetermined interim analysis when it was shown that the addition of MMF was unlikely to improve 
clinical outcomes when compared to steroids alone.   
 
Based on the findings of these recent well-designed multicenter studies, corticosteroid therapy remains 
the only standard treatment approach for de novo acute GVHD requiring systemic therapy. 
2.3  
BIOLOGY OF BONE MARROW-DERIVED MESENCHYMAL STEM CELLS 
Bone marrow derived mesenchymal stem cells (BMSC) are multi-potent bone marrow cells able to  
differentiate in vitro and in vivo into tissues of mesenchymal origin18. BMSC support growth and 
differentiation of hematopoietic progenitor cells in bone marrow microenvironments and in animal 
models, promote engraftment of hematopoietic cells19,20.  Infused BMSC home to sites of tissue injury in 
mice and non-human primates and secrete bioactive molecules21-23. BMSC are immune-modulatory and 
anti-inflammatory24,25.   BMSC do not induce proliferation or interferon-γ production, or upregulation of 
activation markers on allogeneic lymphocytes26. BMSC suppress proliferation of activated lymphocytes 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
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Sawa Ito, M.D. 
in vitro in a dose-dependent, non-HLA-restricted manner27. In a baboon skin-graft model infusion of ex-
vivo expanded BMSC prolonged the time to rejection of histoincompatible skin grafts. Suppression of 
skin graft rejection was effective whether BMSC came from the same donor as the stimulator 
lymphocytes, the same donor as the responder lymphocytes, or from an unrelated (third-party) donor21. 
Furthermore, BMSC also block established lymphocyte responses.  Addition of BMSC four days after 
the initial mixing and stimulation of lymphocytes led to a suppression of proliferation comparable to that 
seen when BMSC were present from the onset of the mixed lymphocyte reaction culture28. Infused 
BMSC do not induce immunological memory in the recipient even when they are completely 
mismatched with the recipient28. Thus there is specific tolerance to BMSC infusions which allows 
BMSC of any source to be given to recipients without being rapidly rejected. BMSC promote tissue 
repair.  Infused BMSC improve the outcome of acute renal, neural, and lung injury, possibly by 
promoting a shift from production of pro-inflammatory cytokines to anti-inflammatory cytokines at the 
site of injury. BMSC promote healing after radiation injury in experimental animals29-31.  
  
In man, BMSC can be isolated from bone marrow, cultured ex vivo, and expanded many fold32. Culture 
expanded BMSC represent a homogeneous population by flow-cytometric measures of cell-surface 
markers whose minimal criteria have been defined as positive for CD146, CD90, HLA class I and 
negative for hematopoietic cell markers33,34.  The ease with which BMSC can be cultured from human 
material, their unique properties of immunomodulation and tissue repair, and their safety profile to date 
have made the therapeutic infusion BMSC a rapidly developing area of adoptive cellular therapy35,36.  
Because of this, NIH established a repository for BMSC in the Department of Transfusion Medicine 
Cell Processing Service37,38  and conducted a phase I trial exploring the safety and efficacy of the NIH 
BMSC product in patients with steroid-resistant acute GVHD and tissue injury post-allogeneic stem cell 
transplant39. 
2.4 
CLINICAL EXPERIENCE WITH BMSC IN GVHD 
BMSC infusions have been used extensively in early phase studies for the prevention and treatment of 
GVHD.   
 
2.4.1 GVHD Prophylaxis 
The administration of BMSC for GVHD prophylaxis at the time of stem cell infusion or at the time of 
engraftment has been studied in several early stage trials in both pediatric and adult populations.   
Altogether just over one hundred patients in more than 10 cohorts have been given BMSC early in the 
course of allogeneic stem cell transplant40-50.  Patients commonly received doses of 1-2x106 BMSC/kg, 
but as much as 5x106 BMSC/kg, in one infusion.  In the majority of these studies treatment with BMSC 
was considered safe and GVHD rates compared favorably to historical control groups.  One study46 
reported an increase in the risk of leukemic relapse in a cohort of patients treated with BMSC at the time 
of stem cell infusion to aid engraftment. In this trial conducted by Ning et al. in China, patients with 
hematologic malignancy (n=10) were co-transplanted with BMSC, and a non-BMSC group was 
followed for comparison (n=15). Grades II-IV acute graft-versus-host disease (GVHD) was observed 
respectively, in one patient (11.1%) in the BMSC group, and eight (53.3%) patients in the non-BMSC 
group. The number of patients who relapsed were six (60.0%) and three (20.0%), and the 3-year disease-
free survivals were 30.0 and 66.7%, respectively. This study has been criticized for methodological 
flaws51 and no link has been established between BMSC treatment and leukemic relapse in a long term 
follow up of a larger cohort52 and in one meta-analysis inclusive of 8 randomized trials (including the 
aforementioned study by Ning et al.)53. 
 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
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2.4.2 De novo GVHD 
Kebriaei et al. conducted the first prospective trial of third-party BMSC for the treatment of de novo 
acute GVHD using a pre-manufactured, universal donor formulation of BMSC (Prochymal®)54. This 
industry sponsored, randomized, multicenter trial compared two different dose levels of BMSC (2 or 
8x106 BMSC/kg) combined with standard corticosteroid therapy.  Patients were infused with BMSC 
within 72 hours of acute GVHD diagnosis and they received one additional dose 3 days later.  Of 31 
adult patients, 71% had a CR at day 28 after therapy (primary endpoint), and the initial ORR was 94%.  
No evidence of a dose response relationship was seen. These promising results led to the use of 
Prochymal® in a large industry-sponsored phase III trial for de novo acute GVHD (Protocol 265, Osiris 
Therapeutics, Inc.).  The primary endpoint was CR within 28 days of treatment administration, and 
treatment failures included patients without CR at 28 days and those requiring increased doses of 
corticosteroids or additional immunosuppressive therapy. This study failed to reach significance in the 
primary endpoint, and the results have not been published55.  The higher than historically reported 
response to steroid therapy, the use of late passage BMSC, the predominance of skin GVHD, and the 
choice of primary endpoint at day 28 are widely attributed to have been the cause for failure to meet the 
primary endpoint56,57. 
 
2.4.3 Refractory GVHD 
By far the largest number of patients have treated with BMSC infusions have had steroid-resistant 
GVHD.   Below is a summary of over one decade of trials for BMSC in steroid-resistant GVHD.  
Response rates are promising for a disease with high morbidity and mortality, but it is difficult to draw 
comparisons between studies due to their small size, heterogeneous design, and use of different outcome 
measures. 
 
 
Year 
published 
First 
author, 
country 
Patient 
numbers 
Donor, BMSC 
source 
GVHD 
Grade 
Passage 
BMSC dose (/kg), dose 
range, number of 
infusions, number range 
Response 
Comments 
2004 
Le Blanc et 
al.58, 
Sweden. 
1 
Haplo, BM 
IV 
Early 
Two infusions- first 2x10e6, 
second 1x10e6 at relapse 
1 CR (100%) 
First use of BMSC in acute GVHD, 
patient age 9, CR with first infusion 
then relapse, CR with second 
infusion 
2006 
Ringden et 
al.59,  
Sweden 
8 
MRD, haplo, third 
party, BM 
III-IV 
Early 
1x10e6, 0.7-9x10e6, 1-2 doses, 
dosed weekly 
6 CR (75%) 
ORR 75%., no side effects or 
infusion toxicity.   
2007 
Fang et al.60 
and 61, China 
6 
Haplo, third party, 
adipose-derived 
BMSC 
III-IV 
Early 
1x10e6 x 1 dose 
5 CR (83%) 
First clinical study of adipose-
derived BMSC for the treatment of 
GVHD. 
2008 
Le Blanc et 
al.62, 
Sweden, 
Netherlands, 
Italy, 
Australia 
55 
MRD, haplo, third 
party, BM 
II-IV 
Early 
1.4x10e6, 0.4-9x10e6, 1 
(n=27), 2 (n=22), 3-5 (n=6) 
infusions, dosed weekly 
30 CR (55%) 
9 PR (16%) 
Responders had improved 2-year OS 
(52 v. 16%, p=0.018) and decreased 
TRM (13 v. 60%, p=0.002) 
2008 
Muller et al. 
63, Germany 
2 
Haplo, BM 
III-IV 
Early 
0.4-3x10e6, 1 dose 
1 CR (50%) 
Pediatric study.  Both patients had 
received a haploidentical bone 
marrow transplant 
2008 
von Bonin et 
al.64,  
Germany 
13 
Third party, BM 
III-IV 
Early 
0.9x10e6, 0.6x10e6 to 
1.1x10e6 ,1-5 doses, dosed 
weekly 
1 CR (8%) 
1 PR (8%) 
First study of platelet lysate 
expanded BMSC, disappointing 
response rate, all but 2 patients 
required secondary immune 
suppression 

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Abbreviated Title: Early BMSC Pilot for Acute GVHD 
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Year 
published 
First 
author, 
country 
Patient 
numbers 
Donor, BMSC 
source 
GVHD 
Grade 
Passage 
BMSC dose (/kg), dose 
range, number of 
infusions, number range 
Response 
Comments 
2009 
Martin et 
al.55 
(abstract), 
USA, 
Canada, 
Australia 
163 
Prochymal®, BM 
II-IV 
Late 
2x10e6, 8 infusions, twice 
weekly, 4 additional infusion if 
not in CR 
ORR 82% 
BMSC v. 
73% placebo 
(p=0.12) 
Prochymal® BMSC performed no 
better than placebo in this large 
randomized study.  No increase in 
adverse events compared to steroid 
alone arm.  Better responses in 
liver/gut.  Majority of patients had 
received second line IS.  Results not 
published. 
2010 
Lim et al.65, 
China 
1 
Third party, BM 
III-IV 
Early 
2x10e6, 2 infusions weeks 
apart 
1 CR (100%) 
Explored use of clonal BMSC 
through subfractionation culture 
method. 
2010 
Arima et al. 
66, Japan 
3 
MRD, haplo, BM 
III 
Early 
0.5x10e6, 1 intra-arterial 
infusion 
2 PR (66%) 
Intra-arterial injection through 
hepatic artery in liver GVHD not 
shown to be effective. 
2010 
Luccini et al. 
67, Italy 
8 
Third party, BM 
I-IV 
Early 
1.2x10e6, 1 infusion, with 2-5 
infusions given to non-
responders 
4 CR (50%) 
2 PR (25%) 
Pediatric study of acute and chronic 
GVHD.  Demonstrates safety of early 
passage BMSC in pediatric patients.   
2011 
Prasad et al. 
68, USA 
12 
Prochymal®, BM 
III-IV 
Late 
2x10e6 (n=10) or 8x10e6 
(n=2) twice weekly for 4 
weeks. 4 weekly infusion if not 
in CR at d+28, median 10 
doses 
7 CR (58%) 
2 PR (17%) 
First study of Prochymal® in 
pediatric patients.  Refractory to 
median 3 other immunosuppressive 
therapies.  No dose response 
relationship. 
2011 
Perez-Simon 
et al 69, 
Spain 
10 
MRD, haplo, third 
party, BM 
II-IV 
Early 
1x10e6, 1-4 infusions based on 
response 
1 CR (10%) 
6 PR (60%) 
Platelet lysate expanded.  Refractory 
to median 3 other 
immunosuppressive therapies. 
2011 
Wernicke et 
al.70, 
Germany 
2 
Third party, BM 
IV 
Early 
0.9-1.9x10e6. 1 infusion each 
2 CR (100%) 
Two pediatric patients, severe life 
threatening GVHD, multiple 
concurrent IS therapies. 
2011 
Wu et al 71. 
Taiwan 
2 
Third party, UCB 
IV 
Early 
3.3-4.1x10e6, 1 infusion each 
2 CR (100%) 
Two pediatric patients, severe life 
threatening GVHD, multiple 
concurrent IS therapies.  First study 
using umbilical cord blood. 
2011 
Herrmann et 
al. 72.  
Australia 
12 
MRD, haplo, third 
party, BM 
I-IV 
Early 
1.7x10e6, median 2 infusions, 
1-19 infusions  
7 CR (58%) 
4 PR (33%) 
6 of 7 patients with CR had long 
term survival.  One hepatic CR after 
19 infusions. 
2012 
Dander et 
al.73, Italy 
6 
Third party, BM 
II-IV 
Early 
0.9-1.9x10e6, 2-3 infusions  
1 CR (17%) 
4 PR (66%) 
Showed IL2Ra and TNF-R decreased 
with BMSC therapy. 
2012 
Chen et al. 
74, China 
19 
Third party, UCB 
II-III 
Early 
0.6-7.2x10e6, 1-3+ infusions 
11 CR (58%) 
4 PR (21%) 
Largest group of patients treated with 
UCB derived BMSC. 
2013 
Muroi et a.l 
75, Japan 
14 
Third party, 
Prochymal® 
collaboration, BM 
II-III 
Late 
2x10e6, 8 infusions, twice 
weekly, 4 additional infusion if 
not in CR 
8 CR (57%) 
5 PR (36%) 
ORR 92.9% 
at day +28. 
Early administration.  BMSC given 
at progression within 3 days or no 
response within 5 days of initial 
corticosteroids, impressive overall 
response. 
2013 
Resnick et 
al. 76  Israel 
50 
MRD, haplo, third 
party, BM 
II-IV 
Early 
1x10e6, 0.3-4.3x10e6 1-4 
infusions 
17 CR (34%) 
ORR 66%  
Steroid resistant grade IV GVHD in 
84% of patients.  Better response and 
OS seen in GVHD < grade IV, 
pediatric group.  BMSC responders 
had higher OS.  Two cases intra-
hepatic artery injection not effective.    
2013 
Ball et al. 77, 
Sweden, 
Netherlands, 
Italy, Canada 
37 
Third party, BM 
III-IV 
Early 
2x10e6 for median 2 doses, 
range 1-13 
24 CR (65%) 
8 PR (22%) 
The children treated between 5 and 
12 days after steroid initiation (as 
opposed to later) showed a trend 
towards better OS (56% v. 25%) and 
lower TRM (17% v 53%).  12 
children received 3-5 doses. 
2013 
Introna et al 
78, Italy 
40 
Third party, BM 
II-IV 
Early 
1.5x10e6, median 3 infusions 
CR 11 
(27.5%) 
PR 16 (40%) 
ORR 67.5 % 
Pediatric and adult.  Platelet lysate 
expanded.  Less than half of patients 
receiving secondary IS.  Plasma 
levels of IL2R lower in responders. 

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Year 
published 
First 
author, 
country 
Patient 
numbers 
Donor, BMSC 
source 
GVHD 
Grade 
Passage 
BMSC dose (/kg), dose 
range, number of 
infusions, number range 
Response 
Comments 
at day +28 
2014 
Sanchez-
Guijo et 
al.79,Spain 
25 
Third party, BM 
II-IV 
Early 
1.1x10e6 day 1, 4, 11, 18, 
additional doses given for P 
11 CR (44%) 
6 PR (27%) 
ORR (71%) 
Platelet lysate expanded.  Median 
time to response 28 days (range 9 to 
58).  Only 33% ORR for liver 
GVHD   
2014 
Yin and 
Battiwalla et 
al. 80, NIH 
7 
Third party, BM 
II-IV 
Early 
2x10e6, 3 infusions day 0, 7, 
14 
5 CR (71%) 
 
Showed Reg3a, elafin, CK18, and 
cytokines decreased in responders.   
2014 
Kurtzberg et 
al. 81, USA, 
Canada 
75 
Prochymal®, BM 
II-IV 
Late 
2x10e6, 8 infusions, twice 
weekly, 4 additional infusion if 
not in CR 
ORR 61.3 % 
at day +28 
Pediatric study.  Starting treatment 
88% had grade III (28.0%) or grade 
IV (60.0%) acute GVHD.  60% of 
patients had received two or more 
additional IS therapy.  40 of 75 
patients (53.3%) received more than 
8 infusions and were included in the 
continuing therapy analysis. More 
than one-half of those patients 
(57.5%) demonstrated additional 
improvement, with 16 patients 
achieving complete resolution. Same 
dosing regimen proposed in T-H-
0325. 
2014 
Zhao et al.82, 
China 
28 
Third party, BM 
II-IV 
Early 
1x10e6 weekly until complete 
response or 8 total doses 
ORR 75% at 
day  
CR 17 (61%) 
PR 4 (14%) 
Compared prospectively to a 19 
patient control arm.  ORR in 
intervention group 75% and 42.1% in 
control arm.  No increase in 
CMV/EBV reactivation.  Noted 
increase in Tregs and TRECs. 
Table 2 - Summary of reported BMSC use for refractory GVHD 
2.5 
NIH PHASE I BMSC TRIAL (12-H-0010) 
2.5.1 NIH Phase I Clinical Trial Design 
Results from the Phase I trial of BMSC at NIH were recently published39 and reviewed83.  At the NIH 
Clinical Center, we developed a clinical grade BMSC cell bank from third party donors37,38.  We 
approached the development of BMSC by selecting the EU method of using early passage BMSC. The 
infusion schedule of doses of 2x106 BMSC/kg weekly followed previously used schedules effective for 
controlling GVHD84.  We conducted the phase I trial using our third party, early passage BMSC for 
patients with steroid-resistant liver or gastrointestinal GVHD, tissue injury or marrow failure following 
allogeneic stem cell transplant.  The study schema is depicted in Figure 1.  
 
Figure 1 - 12-H-0010 study schema 
The BMSC were prepared from marrow aspirates from healthy volunteers with the expansion of 3 
passages38.  Ten subjects were infused a fixed dose of 2e106 BMSC/kg intravenously weekly for three 
doses.  Nine of the subjects had acute GVHD as the primary indication.  There was no treatment related 
toxicity (primary endpoint).  In addition to the demonstration of safety and exploration of efficacy, the 
major objective was to understand the biology of BMSC in post-transplant complications.  The infused 

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BMSC were fully characterized in terms of viability, freeze-thaw characteristics, and gene expression 
profiles with the goal of identifying markers for potency.  We examined the impact of BMSC in terms of 
their immunogenicity by HLA-alloimmunization, their homing, and fate (by chimerism analysis).  
Advances in new diagnostic tools using GVHD-relevant biomarkers and markers of tissue injury 
provided the opportunity to more clearly define responses to BMSC85.  Patients were monitored for 
validated plasma GVHD biomarkers, cytokines, growth factors, and lymphocyte phenotype before and 
after BMSC infusion to identify mechanisms of BMSC immunomodulation and tissue repair. 
 
2.5.2  NIH Phase I Clinical Trial Results 
Eight of nine subjects with steroid-resistant acute GVHD were evaluable for response assessment at 4 
weeks after the last infusion.  There were 5 complete responses, 2 partial responses, and one patient who 
did not respond.  Rapid reductions in markers of tissue destruction and inflammatory cytokines occurred 
after the first BMSC infusion in several patients, example shown in Figure 2.  
 
 
 
Figure 2 – Patient with a complete response after BMSC treatment (left), and corresponding fall in cytokines (right) 
 
Clinical responses correlated with a fall in biomarkers (Reg 3a, CK18, and Elafin) relevant for the site of 
GVHD, or CK18 for tissue injury.  Response to BMSC therapy and changes in cytokines and GVHD 
biomarkers segregated with survival.   
2.6  
SAFETY OF BMSC INFUSIONS 
Safety is a significant concern when translating any new therapeutic into clinical practice. Issues specific 
to BMSC therapy include the risk of infusion toxicity; an increased susceptibility to infection given their 
immune-modulatory effects; the dissemination of zoonoses associated with cell culture reagents; an 
increased neoplastic potential due to the proliferative capacity of BMSC; the potential for embolism of 
cells; and, the immunogenicity of the cells themselves86. BMSC have been used safely in clinical 
practice for over 10 years; neither acute nor long-term adverse events have been reported after infusion 
of autologous, donor-derived, or third party BMSC83. Safety of intravascular BMSC infusions in humans 
was reviewed systematically in October, 201253. In a meta-analysis of 36 studies that were deemed 
eligible, including eight RCTs, the authors were unable to detect an association between BMSC 
treatment and infusion toxicity, infection, malignancy, acute organ dysfunction, and death.  In fact, six 
of seven RCTs and all non-RCTs described equal or fewer deaths with BMSC treatment compared to 
control treatment. The only significant association with BMSC treatment was transient fever. Our 

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chosen dosing schedule of 2x106cells/kg/dose twice weekly for four weeks with additional dosing for 
partial responders is more treatment than in many earlier trials of early passage BMSC, but this same 
regimen has been shown to be safe and effective in studies using Prochymal®, the universal donor, late 
passage BMSC product and another similar BMSC product that is being developed in Japan75,81. 
 
2.6.1 Safety of the NIH BMSC Product 
The phase I study met the primary objective of safety39. Protocol defined treatment-related SAE was 
encountered by none of the subjects.  Infusion reactions were seen in one subject (grade 1 pruritus). 
There was one death while on study (gram-negative bacterial sepsis). There were seven serious adverse 
events:  bacterial infection (n=5), gastrointestinal bleeding (n=1), and a mucus plug in the respiratory 
tract (n=1). In addition, there were 13 non-SAEs: grade 1 (n=7), grade 2 (n=4), and grade 3 or 4 (n=2). 
None of these adverse events were attributable to the BMSC product. The HLA antibody screen 
remained negative prior to and immediately after completing the BMSC trial. New CMV reactivation 
without the development of CMV organ diseases occurred in two patients, controlled by pre-emptive 
antiviral therapy. 
 
2.6.2 Safety in Pediatric Patients 
Several studies have shown significant clinical benefit of BMSC for refractory acute GVHD in the 
pediatric setting, which are at least comparable to their adult counterparts, and there have been no 
reports of serious adverse events attributable to BMSC infusion (see Section 3.8.4).  Published pediatric 
studies with BMSCs with doses as high as 8x106 cells per kilogram body weight have demonstrated 
safety and efficacy for steroid refractory GVHD and to enhance engraftment43,63,67,81,87.  A pre-
manufactured, universal donor formulation of BMSC (Prochymal®) has been approved as the first stem 
cell product for refractory acute GVHD in children in Canada and New Zealand88. 
2.7 
SCIENTIFIC AND CLINICAL JUSTIFICATION  
The scientific rationale for expanding the use of BMSC to the initial treatment of GVHD is based on 
preliminary efficacy in the settings of both GVHD prophylaxis and refractory GVHD, the known 
properties of BMSC to assist in tissue repair and suppress alloimmune responses causing GVHD, and a 
track record of safety in human subjects. Importantly, treatment with BMSC offers an opportunity to 
study changes in GVHD biomarkers in the blood before and after BMSC infusion and before, during, 
and after the onset of GVHD. One of the major obstacles in GVHD research is that GVHD is a 
heterogeneous illness and responses to therapy can vary widely from study to study and BMSC 
preparation to BMSC preparation. One of the keys to understanding responses in GVHD may be the 
discovery of ST2 as a predictive biomarker of GVHD severity and subsequent mortality related to 
GVHD89.  Plasma collected at regular intervals after BMSC infusion will be used to study changes in 
these cytokines.  It has been shown recently that BMSC recipients have increased serum CD73-
expressing BMSC exosomes in the blood (submitted for publication), and BMSC exosomes are 
immunologically active90.     
2.8 
RATIONALE FOR CHANGES FROM PHASE I STUDY 
The previous phase 1 study used a dosing schedule of 2x10e6 BMSC/kg once weekly for three weeks in 
refractory GVHD, whereas the proposed pilot study will use a dosing schedule of 2x10e6 BMSC/kg 
twice weekly for four weeks for early GVHD, followed by once weekly for 4 weeks in patients who 
have a suboptimal response.  Below is justification for the change in schedule. 

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2.8.1  Upfront treatment 
Acute GVHD is caused by a cascade of events that ultimately lead to alloreactivity and tissue damage.  
Mechanistically, reversing the processes underlying GVHD early may break the cycle of alternating 
tissue damage and immune activation, thereby preventing progression to refractory GVHD.  In a 
pediatric study by Ball et al.77, of 37 children, those treated with BMSC between 5 and 12 days after 
steroid initiation showed a trend towards better OS (56%) and lower TRM (17%) as compared with 
patients receiving BMSC 13–85 days after initiation of steroids (25% and 53%, respectively; P = 0.22 
and 0.06, respectively).  More recent studies have prioritized early treatment with BMSC in refractory 
GVHD75,79,81 (see Table 2).   
 
2.8.2  Increased number and frequency of doses 
Also in the study by Ball et al cited above77, the authors retrospectively analyze their results in a series 
of 37 children with steroid-resistant acute GVHD.   Multiple doses of BMSC were associated with a 
higher rate of CR.  Cumulative incidence of transplantation-related mortality (TRM) in patients who did 
or did not achieve CR was 17% and 69%, respectively.  In a more recent manuscript describing the 
Spanish experience, investigators examined a dosing schedule of 1x106 BMSC/kg on days 1, 4, 11, and 
18 for treatment refractory acute GVHD and found an ORR of 71%.  Most patients received 3 or more 
infusions, and additional infusions were allowed in patients achieving a partial response.  A maximum 
of 8 infusions were given in two patients and proved to be safe79.  In a larger study81, Kurtzberg et al. 
reported on 75 children with treatment-resistant grade II - IV acute GVHD who received the pre-
manufactured, universal donor formulation of BMSC (Prochymal®).  The BMSC dose was 2x106 
cells/kg/dose infused twice weekly for 4 weeks.  In the case of a partial or mixed response, patients were 
treated with 2x106 cells/kg/dose weekly for four more weeks.  This is the same schedule proposed in our 
study.  Patients received a median of 10 doses.  At the start of treatment, the majority of patients had 
grade III (28.0%) or grade IV (60.0%) refractory acute GVHD and patients were heavily pretreated with 
immunosuppression.  The overall response rate was 61.3% at day 28.  No infusion or other identifiable 
acute toxicity was seen in any patient.   
 
2.8.3  Continuation of therapy 
In the pediatric study previously cited by Kurtzberg et al.81, in the case of a partial or mixed response 
after 2x106 cells/kg/dose twice weekly for 4 weeks, patients were treated with 2x106 cells/kg/dose 
weekly for an additional four weeks and assessed again for response.  For a patient to be considered a 
responder between day 28 and day 100, they must have experienced additional improvement in at least 1 
organ of at least 1 stage without worsening in any other organ.  Forty (40) patients on this study received 
more than the planned 8 infusions and more than one-half of these patients (57.5%) demonstrated 
additional improvement in acute GVHD, with 16 patients achieving complete resolution of acute 
GVHD.  
 
2.8.4 Addition of pediatric patients  
Several studies have shown significant clinical benefit of BMSC for acute GVHD in the pediatric 
setting, which are at least comparable to their adult counterparts.  In the largest cohort of children to 
date68,81, Kurtzberg et al. reported on 75 children with treatment-resistant grade II - IV acute GVHD 
who received Prochymal®.  The BMSC dose was 2x106 cells/kg/dose, and infused twice weekly for 4 
weeks.  In the case of a partial or mixed response, patients were treated with 2x106 cells/kg/dose weekly 
for four more weeks. Patients received a median of 10 doses.  At the start of treatment, the majority of 

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patients (88%) had grade III (28.0%) or grade IV (60.0%) acute GVHD.  Many patients (60%) had 
received two or more immunosuppressive therapies in addition to steroids.  The overall response rate 
was 61.3% at day 28.  No infusion or other identifiable acute toxicity was seen in any patient.  
Prochymal® is approved for use in Canada and New Zealand for refractory GVHD in pediatric patients.  
Similar results have recently been reported in a smaller European pediatric study of eleven (11) patients 
with acute or chronic GVHD (grade I to IV) who received HLA-disparate donor’s bone marrow-derived 
BMSC, expanded in platelet-lysate containing medium on compassionate use studies67.  The median 
dose administered to patients (aged 4-15 years) was 1.2 x 106 cells/kg/dose (range: 0.7-3.7x106 
cells/kg/dose).  No acute or late side effects were reported with a median follow up of 8 months.  The 
overall response rate was 71.4% (CR = 23.8%).  Müller et al. reported on the administration of ex vivo-
expanded BMSC in seven pediatric patients in escalating doses (0.4x 106 to 3.0x106 cells/kg/dose)63.  No 
adverse effects were observed in a follow up of 29 months.   
 
3. STUDY DESIGN 
3.1  
STUDY DESIGN 
This is a pilot trial designed to explore the feasibility of using BMSC combined with standard therapy for the 
initial treatment of acute GVHD requiring systemic therapy  
3.1.1  Accrual Objective  
 
Twenty-five (25) patients will be accrued with the goal of attaining twenty-three (23) evaluable patients. 
3.1.2  Accrual Period 
 
The estimated accrual period is 18 months. 
3.1.3  Study Duration 
The expected maximum duration of treatment according to the study protocol will be 11 weeks total as 
outlined in Section 3.4.  
3.1.4  Rationale for Design 
Twenty five patients over 12-18 months is a realistic target for accrual given the incidence of acute 
GVHD in the clinical center. 
3.2 
SUBJECT INCLUSION 
Provided subjects satisfy inclusion criteria in Section 5.1, the protocol is open to all subjects 
transplanted at NIH who are diagnosed with acute GVHD, irrespective of conditioning regimen, primary 
disease, or transplant type.  The protocol will include subjects at any time after allogeneic stem cell 
transplantation.   
3.3 
INITIAL ASSESSMENT 
Subjects will be assessed by clinical laboratory criteria and also where appropriate, by biopsy for degree 
and severity of GVHD of the liver and/or gastrointestinal tract according to criteria in Appendix 1.   
Patients with confirmed acute GVHD will be enrolled within 120 hours of receiving systemic 
corticosteroids for the treatment of acute GVHD.    

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3.4 
TREATMENT 
3.4.1 Schema and Treatment Summary 
 
Figure 3 - Study schema 
This is a pilot trial where eligible subjects may be entered into this study if they meet the diagnostic 
criteria for acute GVHD requiring systemic corticosteroid therapy.  Subjects will receive the first 
infusion of BMSC within 120 hours of the first dose of systemic corticosteroids.  Each subject will 
receive 2x106 cells/kg of BMSC given twice weekly, no sooner than 48 hours apart, for 8 doses.  Four 
(4) additional weekly doses will be given in the absence of a complete response. BMSC will be given 
for a maximum of 12 doses. We will endeavor to administer BMSC on Tuesdays and Fridays in order to 
facilitate planning with CPS DTM.  Each BMSC infusion will contain a target dose of 2x106 BMSC/kg 
(+/- 10%) viable BMSC (30-150 mL).  Donor sample identifiers will be tracked for each infusion. Since 
there are no specific or antidotal therapies for AEs arising from BMSC, any toxicity that arises during 
the subject’s infusion or post infusion assessments will be managed with standard supportive care as 
outlined below. Patients will also be treated with systemic corticosteroids per established treatment 
guidelines (Section 3.4.3).   
 
3.4.2  Treatment with BMSCs 
3.4.2.1 Pre-infusion 
Pre-infusion steps will be followed according to the NIH institutional standard operating procedure: 
Administration of Products of Cellular Therapy. Emergency equipment will be made available in the 
patient room including: oxygen, suction machine, vital signs monitor, and 0.9% sodium chloride 
solution and administration. All adult subjects will receive diphenhydramine 25-50 mg IV or PO over 
10-15 minutes and/or acetaminophen 325-650 mg PO 30-60 minutes prior to infusion; all pediatric 
subjects will receive diphenhydramine 0.5-1 mg/kg IV or PO over 10-15 minutes (max dose 50 mg) 
and/or acetaminophen 10-15 mg/kg PO (max dose 650 mg) 30-60 minutes prior to infusion. Subjects 
will remain in the NIH Clinical Center (inpatient ward or day hospital) to be evaluated for toxicity as 
specified by the standard operating procedure.  Pre-medications may be substituted.   Acetaminophen 
can be held for patients with abnormal liver function studies. 
3.4.2.2 Infusion 
BMSC infusions will be scheduled with the CPS DTM in advance.  BMSC will be thawed rapidly in a 
37˚C water bath.   BMSC bags will be hand carried by CPS personnel to the patient care unit. BMSC 
will be administered according to NIH institutional standard operating procedure: Administration of 
Products of Cellular Therapy. In brief, subjects will be present either in the inpatient ward or the day 
hospital during the entire time of the infusion. It is recommended to use a central venous catheter for 
infusion of BMSC, but a patent peripheral access is acceptable. Infusion rate shall not exceed 4-5 
mL/min (approximately 10-30 minutes) by gravity. The product should not be given by an infusion 

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pump. The total dose of the product given with each infusion is 2x106 BMSC/ kg (+/- 10%) viable 
BMSC (30-150 mL). 
3.4.2.3 Post-infusion 
Patients will be monitored post-infusion according to the NIH institutional standard operating procedure: 
Administration of Products of Cellular Therapy. Outpatients may be released thirty minutes to one hour 
after completion of infusion if patient condition and vital signs are stable, as specified in the standard 
operating procedure above. 
3.4.2.4 Missed infusions 
Unanticipated logistical challenges including, but not limited to, adverse weather, availability of 
transportation, or equipment-related delays may occur.  Subjects can miss up to four (4) BMSC 
infusions in total over the course of the treatment protocol and remain enrolled and evaluable for 
response.  If an infusion or series of infusions are missed, the infusion will not be rescheduled and the 
subject will go on to receive the next scheduled infusion in the treatment course.  Subjects missing more 
than four (>4) BMSC infusions will be removed from the treatment protocol and will not be evaluable 
for the primary endpoint. 
 
3.4.3  Treatment with corticosteroids 
The optimal dose of steroid and duration of therapy for GVHD is unknown and there is considerable 
variation in practice, particularly in early stage GVHD, but in general steroid dose should be sufficient 
to treat GVHD but limited to avoid any side effects of long-term high dose therapy.  The preferred rate 
to taper steroids is also not well-established91, but general guidelines have been provided in the design of 
a recent randomized, prospective, multi-center trial (BMT CTN 0802) comparing steroids/placebo vs. 
steroids/MMF for acute GVHD6,17.   It is suggested that patients receive an initial steroid dose by IV 
route (usually methylprednisolone) that is equivalent to prednisone at a dose 2 mg/kg/day at diagnosis of 
acute GVHD.  Steroids can be tapered as tolerated according to individual practice.  It is suggested that 
steroids are tapered if signs of clinical improvement defined as any clinically recognizable lessening of 
skin rash, redness, or extent; lessening of diarrhea or lowered bilirubin (though it does not have to be 
greater than or equal to one stage improvement in any involved organ), without worsening in any organ. 
 
Suggested steroid taper for responders (round to nearest 5mg of prednisone) 
Day 1-5 
Prednisone 2 mg/kg/day (use IV methylprednisolone equivalent in single or divided dose(s)) 
Day 6-10 
Prednisone 1.5 mg/kg/day PO (or IV methylprednisolone equivalent) 
Day 11-15 
Prednisone 1 mg/kg/day PO (or IV methylprednisolone equivalent) 
Day 16-20 
Prednisone 0.5 mg/kg/day PO (or IV methylprednisolone equivalent) 
Day 21-28 
Prednisone 0.25 mg/kg/day (or equivalent) PO (or IV methylprednisolone equivalent) 
Day >28 
Continue taper with a goal of <0.2 mg/kg/day of prednisone PO once daily (or equivalent). 
Table 3 – Suggested steroid taper 
3.4.4  Additional immunosuppressive therapy 
3.4.4.1 Standard GVHD prophylaxis 
Most patients will receive medications that are considered GVHD prophylaxis as part of their transplant 
regimen.  Patients developing acute GVHD during GVHD prophylaxis therapy (e.g. calcineurin 
inhibitor, sirolimus, MMF, etc.) should have this medication continued during the study if possible. 
Patients who have been tapered off of or completed standard calcineurin inhibitor based prophylaxis 
(e.g. cyclosporine, tacrolimus) can be restarted on this medication concurrent to treatment on this 

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protocol. Concurrent or addition of topical steroid therapy (skin creams, oral budesonide, or other 
locally acting steroid preparation) is allowed.   
3.4.4.2 Progression of GVHD 
If there is no response to therapy or acute GVHD progresses, then the patient can be treated by adding 
one of many alternative systemic secondary GVHD therapies.  BMSC therapy will continue per protocol 
design, given the prior established efficacy of BMSC in refractory GVHD and the poor outcomes 
associated with primary refractory and progressive acute GVHD. 
3.4.4.3 Flare of GVHD 
If acute GVHD flares during taper of steroids or GVHD prophylaxis medications, steroid or GVHD 
prophylaxis dosing may be re-escalated or secondary therapy added at the discretion of the treating 
physician.  Re-escalation of steroid or GVHD prophylaxis medications for GVHD flare alone will not be 
considered as need for secondary therapy. 
 
3.4.5 Retreatment with BMSC 
There will be no retreatment with BMSC infusions after BMSC treatments are stopped. 
 
3.4.6  Concurrent Treatments 
Subjects may receive any concurrent treatment except other investigational agents for treatment of acute 
GVHD (See PBSCT Supportive Care Guidelines) 
3.5 
TOXICITY 
BMSC infusion toxicity will be defined as alteration in vital organ function within the 11 weeks of the 
study that cannot be explained by other complications; for example, post-stem cell transplant, infections, 
underlying transplant indication, or other therapy.  See Section 8 for definition of Treatment Limiting 
Toxicity. 
3.6 
RESPONSE 
Subjects will be assessed for response for the primary indication according to criteria listed in Appendix 
2.  The critical time points for response are at day +28 and day +56.  Subjects are considered off 
protocol after 11 weeks of total enrollment.  Subjects will receive additional infusions after day +28 
based upon whether or not there is a complete response at the day +28 response assessment (Section 
3.4.1). After coming off protocol, subjects will be followed for best response for up to 1 year on their 
parent transplant protocols. 
3.7 
SURVIVAL 
Survival assessment will be done at day +28 and day +56.  After coming off protocol, subjects will be 
followed for survival for up to 1 year on their parent transplant protocols. 
 
4. RECRUITMENT AND REGISTRATION 
The study will be listed on clinicaltrials.gov, clinical center research studies, PDQ, Aplastic Anemia 
Foundation, and the NHLBI patient recruitment websites.  If recruitment goals are not met, a recruitment 
plan will be developed by the Clinical Center Office of Patient Recruitment to extend the protocol to 
subjects outside NIH. 
 

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5. ELIGIBILITY ASSESSMENT 
5.1 
INCLUSION CRITERIA 
5.1.1 History of any grade acute GVHD requiring systemic therapy after allogeneic stem cell transplant or 
 
DLI. 
Subjects must have received an allogeneic stem cell transplant at NIH and be diagnosed with acute 
GVHD.  Acute GVHD is defined using the NIH consensus definition inclusive of classic acute (< 100 
days after transplant or DLI, presence of acute GVHD features, absence of chronic GVHD features) 
AND persistent/recurrent/late onset acute  (>100 days after transplant or DLI, presence of acute GVHD 
features, absence of chronic GVHD features). Subjects with stage I and II skin only (overall Grade I) or 
isolated upper gastrointestinal involvement are eligible if the treating physician deems that systemic 
corticosteroid treatment is indicated.  Biopsy confirmation of GVHD is desirable, but not required for 
study entry because enrollment should not be delayed awaiting biopsy or pathology results.  Patients 
must be diagnosed with a first episode of acute GVHD requiring systemic corticosteroids and associated 
with preceding administration of a cellular therapy including stem cells and donor lymphocyte infusion.  
Patients who were treated for GVHD associated with another cellular therapy product (e.g. prior 
allogeneic transplant or DLI) will be allowed into the study.  
5.1.2 Previous immunosuppressive therapy 
The patient must have received no systemic immune suppressive therapy for treatment of new acute 
GVHD (e.g. pentostatin, etanercept, denileukin difitox, etc.), except for a maximum 120 hours prior 
corticosteroid therapy.  This does not include immune suppressive therapy for GVHD prophylaxis (e.g. 
calcineurin inhibitor, sirolimus, MMF, etc.).  It is expected that most patients will be receiving GVHD 
prophylaxis as part of their transplant regimen, thus patients developing acute GVHD while on GVHD 
prophylaxis will still be considered eligible.  Concurrent or addition of locally-acting steroid therapy 
(skin creams, oral budesonide, or any other locally-acting steroid preparation) is allowed.   
 
There is one exception to the above stipulations:  Use of the oral medication MMF (in addition to 
systemic corticosteroids) for the treatment of acute GVHD will be allowed.  MMF is commonly given 
early in the treatment of acute GVHD, but it has not been shown to improve outcomes compared to 
steroids alone in a randomized, prospective study; therefore, treatment with MMF will not exclude 
patients from BMSC treatment.  
 
5.1.3 Age 
 
Age ≥ 4 years old will be allowed. 
 
5.1.4 Birth control 
 
Subjects of childbearing or child-fathering potential must be willing to use a medically acceptable form 
 
of birth control, which includes abstinence, while they are being treated on this study. 
 
5.1.5  Informed consent 
Signed informed consent and/or assent is required.  Assent and educational materials will be provided 
to, and reviewed with, patients under the age of 18.  The informed consent process will begin at 
recognition of patient eligibility. 
 

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5.2 
Exclusion Criteria  
5.2.1 Breast feeding or pregnant females (due to unknown risk to fetus or newborn). 
 
5.2.2 Known allergy to gentamicin. 
 
6. CLINICAL EVALUATION  
6.1  
CLINICAL EVALUATION OF THE SUBJECT 
 
6.1.1  Eligibility Evaluation 
  
a) Karnofsky performance status 
  
b) Eligibility to participate in trial 
  
c) Pregnancy test in females of child-bearing potential 
  
d) Relevant GVHD organ-specific studies, confirmation of diagnosis, and severity assessment  
  
(Appendix 1) 
  
e) Acute GVHD diagnosed by clinical criteria and no more than 120 hours of prior steroid  
  
therapy for diagnosis 
 
Note that eligibility screening may be performed on a separate NIH-CC screening protocol 
 
 
6.1.2 Pre-Treatment Evaluation 
 
 
Pretreatment evaluation is to be completed within 30 days of the protocol enrollment date. 
  
a) Physical examination 
  
b) Vital signs: temperature, pulse oximetry, blood pressure, respiratory rate, height and weight 
  
c) CBC with differential + reticulocyte 
 
 
d) Chemistry panel (Na, K, Cl, CO2, Cr, glucose, BUN, albumin, Ca, Mg, Phosphorus, Alk  
 
 
Phos, ALT/AST, total and direct bilirubin, LDH, total protein, CK, uric acid) 
  
e) As per GVHD evaluation in Appendix 1 
  
  
6.1.3  
Toxicity monitoring 
 
a) Administration and monitoring of BMSC will be performed according to NIH Clinical Center 
standard operating procedure:  Administration of Products of Cellular Therapy.    
 
6.1.4  
Follow up Evaluations 
  
a) Physical exam and performance score 
  
b) Vital Signs 
 
c) Routine CBC with differential, Chemistry panel (Na, K, Cl, CO2, Cr, glucose, BUN, albumin, 
Ca, Mg, Phosphorus, Alk Phos, ALT/AST, total and direct bilirubin,  LD, total protein, CK, uric 
acid),  and reticulocyte count. 
d) Acute GVHD severity and response monitored by skin exam, stool volume, and liver function 
tests on days of BMSC infusion by the investigators while inpatient and during the day of clinic 
visit as outpatient. 

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Follow up evaluations after the last BMSC infusion will be based on subject’s condition, and may be as 
frequent as twice weekly.  All radiographic examinations at baseline and follow up would be considered 
standard of care for the evaluation and management of these protocol subjects as clinically warranted. 
6.2 
RESPONSE DEFINITIONS  
6.2.1 Response 
Response criteria are defined in Appendix 2.  Response will be recorded at day +28 and day +56.  Best 
response will also be recorded: i.e. the maximum improvement achieved following BMSC infusion in 
the follow up period between day +56 and one year post transplantation.  
 
7. SAMPLE COLLECTION, STORAGE AND TRACKING PLAN 
7.1 
RESEARCH SAMPLE COLLECTION 
All research sampling will be obtained based upon the clinical situation and may be optional at the 
discretion of the PI. Such situations may occur without violating the protocol when the PI determines 
that sample collection is precluded by the subject’s performance status, or would interfere with 
management of an acute medical situation or when logistic considerations prevent procurement or 
processing of the specimen. 
 
7.1.1  Research Blood Sampling: 
Research blood samples:  54 mL of blood will be drawn [1 (6 ml) lavender top- K2-EDTA for plasma 
and 6 (8 ml) red-green “tiger top”] on Day 0 (pre BMSC infusion), Day 28, and Day 56.  3 mL of blood  
[1 (3 ml) lavender top- K2-EDTA for plasma] will be drawn before and after each BMSC infusion (+/- 
24 hours) and at 3 weeks after the last infusion (+/- 7 days).  Testing will include for cytokines, GVHD 
biomarkers, plasma exosomes, lymphocyte subset analysis and other Stem Cell Allotransplantation 
Section research studies (Appendix 3).   For logistic considerations, a +/- 3-day window can be applied 
to sample draw dates on D0, D28, and D56. Blood will be stored at room temperature and transported to 
the laboratory of Dr. John Barrett, NHLBI, NIH for analysis. A list of IRB-approved research tests that 
will be conducted on the collected samples is included in Appendix 4. 
 
Pharmacokinetics (PKs): In selected patients, and after selected infusions (as determined by the 
investigator, clinical course, and available nursing/lab resources), up to 10 blood samples will be drawn 
for PKs to determine the kinetics of BMSC-derived exosome release. 2mL of blood in 3mL lavender 
top- K2-EDTA tubes will be drawn at intervals following infusion. The suggested intervals are 20 min, 
40 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs, 12 hrs, 18 hrs, and 24 hrs from infusion. The suggested intervals 
are not mandatory and can be modified as needed based on nursing/laboratory availability and clinical 
course.    
7.2 
COLLECTION, STORAGE AND DISPOSITION OF SAMPLES 
7.2.1 Intended use 
Any research specimen will not be read by a pathologist or used for diagnostic purposes.   These studies 
will not be used in assessing clinical endpoints but are undertaken for descriptive or exploratory 
ancillary research. 

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7.2.2 Tracking   
Samples will be ordered and tracked through the CRIS Research Screens.  Should a CRIS screen not be 
available, the NIH form 2803-1 will be completed and will accompany the specimen and be filed in the 
medical record.  Samples will not be sent outside NIH without IRB notification and an executed MTA.  
 
7.2.3 Storage 
Research samples will be stored with identifiers in the secure laboratory of Dr. John Barrett, NHLBI, 
NIH. Specimens will be entered in the NHLBI Biospecimen Inventory System (BSI). 
 
7.2.4  End of study procedures  
The study will remain open so long as sample or data analysis continues to a maximum of one year after 
the last subject completes treatment.  Samples from consenting subjects will be stored until they are no 
longer of scientific value or if a subject withdraws consent for their continued use, at which time they 
will be destroyed.  
 
7.2.5 Loss or destruction of samples 
Should we become aware that a major breech in our plan for tracking and storage of samples has 
occurred, the IRB will be notified. 
 
8. MANAGEMENT OF COMPLICATIONS 
8.1 
OVERVIEW 
Serious toxicities related to infusion of BMSC have not been reported.  Hives and nasal congestion have been 
identified infrequently.  Infusion associated fever is recognized.  As with any infusion of a cell product, 
immediate transfusion reactions are a theoretical possibility. 
8.2 
IMMEDIATE TRANSFUSION REACTION 
Administration of BMSC will be performed according to NIH Clinical Center standard operating procedure: 
Administration of Products of Cellular Therapy. If an allergic or other acute reaction occurs, studies appropriate 
for investigation of a transfusion reaction will be performed (urinalysis, CBC, Coomb's test).   Any adverse 
reactions should prompt interruption of the infusion and should be reported to the principal investigator.  
 
No further BMSC infusions will be administered to a subject in the event of: 
• recurrent infusion related toxicity despite premedication (moderate toxicity is defined as requiring 
interruption or discontinuation of BMSC administration, which is not rapidly responsive to symptomatic 
medication) 
• any CTCAE v4.0 grade 4 acute infusion reaction (defined as requiring discontinuation of infusion AND 
urgent cardiopulmonary intervention) or grade 4 hypersensitivity reaction (anaphylaxis) at least possibly 
related to BMSC infusions 
• any CTCAE v4.0 grade 3 infusion reaction that does not resolve completely within 2 hours 
• any CTCAE v4.0 grade 3 acute infusion reaction or hypersensitivity that recurs when the infusion is 
restarted. 

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8.3 
TREATMENT LIMITING TOXICITIES 
The observational period for determining Treatment Limiting Toxicities is from the initiation of the 
BMSC infusion to 3 weeks after the last infusion. A treatment related serious adverse event (TRSAE) is 
defined as an SAE which is at least probably or definitely related to the BMSC infusions. Any subject 
who encounters a TRSAE will not receive further infusions.   
8.4 
POST-TRANSPLANT COMPLICATIONS 
New post-transplant complications will be managed according to standard NIH Intramural transplant 
guidelines (See PBSCT Supportive Care Guidelines).  
 
9. OFF TREATMENT AND OFF STUDY CRITERIA 
9.1 
OFF TREATMENT CRITERIA 
Subjects will receive no further BMSC infusions if any of the following occur: 
9.1.1  Toxicity 
Subjects experiencing BMSC infusion reaction as defined in Section 8.2 will receive no further BMSC 
infusions; subjects experiencing a grade 4 acute infusion reaction or hypersensitivity reaction will 
receive no further treatments; subjects experiencing a grade 3 acute infusion reaction or hypersensitivity 
reaction will receive no further treatments if symptoms do not resolve within 2 hours; subjects 
experiencing a grade 3 acute infusion reaction or hypersensitivity reaction will receive no further 
treatments if symptoms recur when infusion is restarted.  Any subject who encounters a TRSAE will not 
receive further infusions.  Subjects will be followed until off study criteria are met.  
 
9.1.2  Completion of Infusions 
A maximum of 12 infusions is planned.  Final evaluation of outcomes and response to the BMSC will 
occur 3 weeks after the last infusion.  After planned infusions and the three week monitoring period, 
subjects will be taken off protocol. Subjects will be followed for GVHD response and survival as a 
secondary endpoint for up to 1 year on their parent transplant protocol. 
 
9.1.3  Subject Choice 
The subjects are at liberty to withdraw from further infusions at any time. 
9.2 
OFF STUDY CRITERIA 
9.2.1  Withdrawal per subject choice 
Subjects are at liberty to withdraw from the protocol at any time. They will continue to receive standard 
care for their post-transplant complication. 
 
9.2.2  Subject Death 
 
9.2.3  Withdrawal by physician decision   
Subjects may be withdrawn from the protocol at any time if the PI deems that the subject will not 
survive to be evaluable beyond 7 days from transfusion, or that BMSC infusion will complicate the 
routine management of the recipient.  In addition, subjects who are noncompliant with treatment or 

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monitoring and those who develop such severe inter-current illness that compromises treatment or 
evaluation will be taken off study. 
 
9.2.4  Completion of the study 
At the conclusion of this study, all surviving subjects will be taken off study or if appropriate enrolled 
on a follow up study to obtain survival data. 
 
10. DATA MANAGEMENT 
10.1 DATA SAFETY 
The PI will be responsible for overseeing entry of data into an in-house password protected electronic 
system and ensuring data accuracy, consistency and timeliness.  The principal investigator, associate 
investigators, Hematology Branch fellows, research nurses and/or a contracted data manager will assist 
with the data management efforts.   Some primary source data on baseline characteristics, BMSC 
infusions, adverse events, concomitant medications and clinical response will be captured directly on 
case report forms (CRFs). Data will also be abstracted from Clinical Center progress notes.  Laboratory 
data from NIH will be imported electronically from CRIS into an in-house clinical trial database.   
 
All human subjects personally identifiable information (PII) as defined in accordance to the Health 
Insurance Portability and Accountability Act,  eligibility and consent verification will  be recorded.  
Primary data obtained during the conduct of the protocol will be kept in secure network drives or in 
approved alternative sites that comply with NIH security standards.  Primary and final analyzed data will 
have identifiers so that research data can be attributed to an individual human subject participant, e.g., 
study-specific identifying number (SSPIN) or other unique code, or minimum PII required for subject 
identification.  All protocol data will be stored in the NHLBI secured network drive (P drive) with the 
following security measures: restricted access, password protection and daily off-site back-up. 
 
10.1.1 End of study procedures 
 
Data will be stored in locked cabinets and in a password protected database until it is no longer of 
 
scientific value.  
 
10.1.2 Loss or destruction of data  
 
Should we become aware that a major breech in our plan to protect subject confidentiality and trial data 
 
has occurred, the IRB will be notified. 
 
10.1.3 Future use of data 
Given the research mandate of the NIH, patient data including the results of testing and responses to 
treatment will be entered into an NIH-authorized and controlled research database. Any future research 
use will occur only after appropriate human subject protection institutional approval such as prospective 
NIH IRB review and approval or an exemption from the NIH Office of Human Subjects Research 
Protections (OHSRP).  No identifiable data will be sent outside NIH without IRB notification and an 
executed MTA or CTA. 
 

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11. BIOSTATISTICAL CONSIDERATIONS 
11.1  PRIMARY ENDPOINT 
The primary endpoint of this study is: 
• The proportion of subjects reaching day +56 without a TRSAE. 
 
11.2  Secondary Endpoints 
Secondary endpoints for this study are: 
• Proportions of complete response (CR), partial response (PR), mixed response (MR), no response 
(NR), and worsening disease (WD) among surviving patients at day +28 and +56 of study 
(Appendix 2).   Need for secondary immunosuppression by day +28 and +56 of study.  Steroid dose 
(prednisone equivalent) at day +28 and +56 of study.  Proportion of responses in patients with high 
and low ST2 levels. 
 
Secondary endpoints will not be used in assessing success or failure of the primary endpoint but are 
undertaken for descriptive or exploratory research and will be reported accordingly.  The duration of 
treatment limiting toxicity monitoring will be 3 weeks after the last dose of BMSC. The total duration of 
treatment limiting toxicity monitoring will be 7-11 weeks.  Total serious adverse event monitoring will 
continue until at least 3 weeks after the last dose of BMSC, and subjects experiencing adverse events 
related to BMSC will be monitored until toxicity resolution or stabilization.  
11.3  STATISTICAL ANALYSIS 
11.3.1  Methods of Statistical Analysis  
The planned analyses will include descriptive statistics on response rates, biomarkers, and the incidence 
and severity of adverse events. The proportions of treatment related adverse events and their severity 
will be estimated using the sample proportions, and their inferences including confidence intervals and 
hypotheses testing will be evaluated using Binomial distributions. The time to adverse events will be 
analyzed using appropriate tools in survival analysis such as Kaplan-Meier estimates. Graphical tools 
will be used to display the appropriate estimates. 
 
11.3.2  Sample Size 
Let p be the probability of subjects reaching day +56 without a TRSAE.  The sample size is determined 
by testing the null hypothesis H0 :  p= 75%  versus the alternative hypothesis Ha:  p> 75%  with a one-
sided significant level of 0.05.  Based upon our previous phase 1 trial (12-H-0010), the TRSAE rate was 
0 out of 10 subjects and we expect 95% subjects would reach day +56 without a TRSAE.    Thus a 
sample size of 23 subjects will achieve 89.5% power to test the above hypotheses using an exact 
binomial test.   
 
To account for the possibility of non-treatment related drop out, up to 2 additional subjects may be 
enrolled to replace those subjects that are non-evaluable for the study’s primary endpoint for a total 
accrual of 25 patients. At an accrual rate of 1-2 subjects per month, this study will be completed in 12-
18 months. 
 
11.3.3  Subject Replacement 
Subjects who do not complete at least two doses of BMSCs will be replaced in this study.  

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11.3.4  Stopping Rules 
11.3.4.1 For Mortality 
Any death occurring through the follow up period (3 weeks after last BMSC infusion) will halt further 
accrual irrespective of causality pending IRB review.  The study will continue only with IRB 
determination that the event was not related to study procedures.  
 
11.3.4.2 For Safety  
The trial will be monitored for safety based on Treatment Related Serious Adverse Events (TRSAE), 
which is also the primary endpoint of the study.  A TRSAE is defined as an SAE which is at least 
probably or definitely related to the BMSC infusions. The trial will be stopped if 3 subjects have 
developed one or more TRSAE’s.  This is based on the binomial test of the primary hypothesis (Section 
11.3.2) and at least 21 of 23 subjects without a TRSAE are needed to reject the null hypothesis.   If 3 or 
more subjects developed TRSAE’s are observed,   the null hypothesis will not be rejected and we will 
stop the study early. 
 
12. DATA AND SAFETY MONITORING  
12.1 
SAFETY MONITORING 
12.1.1  Principal Investigator  
Accrual and safety data and conduct of the trial will be monitored by the PI and research team on an 
ongoing basis. The protocol will be continuously evaluated for any unusual or unpredicted 
complications with the aim of detecting and preventing unacceptable increase in morbidity and mortality 
over and above that anticipated from standard allogeneic stem cell transplants. (See stopping rules in 
section 11.3.4).  Given that safety was established in the prior Phase 1 study, non-serious adverse events 
will not be collected (see definition Section 12.2). 
 
12.1.2 NHLBI IRB  
Prior to implementation of this study, the protocol and the proposed subject consent and assent forms 
will be reviewed and approved by the properly constituted IRB operating according to the 45 CFR 46, 
21 CFR 50 and 56.  This committee must approve all amendments to the protocol or informed consent, 
and conduct continuing annual review so long as the protocol is open to accrual or sample and/or data 
analysis continues. 
 
12.1.3  DSMB 
The NHLBI Data Safety and Monitoring Board will review the protocol annually or semiannually.  A 
progress report will be forwarded to the DSMB at this time.  The DSMB may recommend early 
termination of the study for considerations of safety and efficacy.  
 
12.1.4  FDA 
An annual progress report, any amendments to the protocol, and any change in the status of the protocol 
will be forwarded to FDA. 

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12.1.5  Data Integrity and Study Conduct   
Regularly scheduled clinical trial monitoring will be performed by experienced contracted clinical trial 
monitors according to established procedures established by the Office of Clinical Affairs, NHLBI. 
12.2  ADVERSE EVENTS 
 
12.2.1  Definitions 
Adverse Event (AE):  Any untoward or unfavorable medical occurrence in a human subject, including 
any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or disease, 
temporally associated with the subject’s participation in the research, whether or not considered related 
to the research. 
 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by 
any of the following: 
 
• Results in discontinuation from the study 
• Is associated with clinical signs or symptoms 
• Requires treatment or any other therapeutic intervention 
• Is associated with death or another serious adverse event, including hospitalization 
• Is judged by the Investigator to be of significant clinical impact 
• If any abnormal laboratory result is considered clinically significant, the investigator will provide 
details about the action taken with respect to the test drug and about the patient’s outcome. 
 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
• results in death; 
• is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
• results in in-patient hospitalization or prolongation of existing hospitalization; 
• results in a persistent or significant incapacity; 
• results in a congenital anomaly/birth defect; or 
• based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition. 
 
Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there is a 
reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 
‘reasonable possibility’ means there is evidence to suggest a causal relationship between the drug and 
the adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than 
adverse reaction, which means any adverse event caused by a drug. 
 
Serious event: An event is serious if it meets the definition of a serious adverse event (above) or if it 
requires immediate corrective action by a PI and/or IRB to protect the safety, welfare or rights of 
subjects. 
 

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Unexpected adverse reaction: An adverse event or suspected adverse reaction is considered 
“unexpected” if it is not listed in the investigator brochure or is not listed at the specificity or severity 
that has been observed; or, if an investigator brochure is not required or available, is not consistent with 
the risk information described in the general investigational plan or elsewhere in the current application. 
"Unexpected”, also refers to adverse events or suspected adverse reactions that are mentioned in the 
investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological 
properties of the drug, but are not specifically mentioned as occurring with the particular drug under 
investigation. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that meets all of the following 
criteria: 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and informed consent 
document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. related or possibly related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, economic, or 
social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that does not fit the 
definition of an adverse event, but which may, in the opinion of the investigator, involves risk to the 
subject, affect others in the research study, or significantly impact the integrity of research data.  For 
example, report occurrences of breaches of confidentiality, accidental destruction of study records, or 
unaccounted-for study drug. 
 
Protocol Deviation (PD):  Any change, divergence, or departure from the IRB approved research 
protocol. 
 
Non Compliance: The failure to comply with applicable NIH HRPP policies, IRB requirements, or 
regulatory requirements for the protection of human research. Noncompliance may be further 
characterized as: 
1. Serious non-compliance: Non-compliance that: 
a. Increases risks, or causes harm, to participants. 
b. Decreases potential benefits to participants. 
c. Compromises the integrity of the NIH HRPP. 
d. Invalidates the study data. 
2. Continuing non-compliance: Non-compliance that is recurring. An example may be a pattern of 
non-compliance that suggests a likelihood that, absent an intervention, non-compliance will 
continue. Continuing noncompliance could also include a failure to respond to IRB requests to 
resolve previous allegations of non-compliance. 
3. Minor (non-serious) non-compliance: Non-compliance that, is neither serious nor continuing. 

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12.2.2 Adverse Event Management 
The following adverse event management guidelines are intended to ensure the safety of each subject 
while on the study.  The descriptions and grading scales found in the revised NCI Common 
Terminology Criteria for Adverse Events (CTCAE) version 4.0 will be utilized for AE reporting.  All 
appropriate treatment areas should have access to a copy of the CTCAE version 4.0.  A copy of the 
CTCAE version 4.0 can be downloaded from the CTEP website. 
  
 
Attribution 
Relationship 
Attribution 
Description 
Unrelated to investigational 
agent/intervention1 
Unrelated 
The AE is clearly NOT related to the intervention 
Unlikely 
The AE is doubtfully related to the intervention 
Related to investigational 
agent/intervention1 
Possibly 
The AE may be related to the 
intervention 
Probably 
The AE is likely related to the intervention 
Definitely 
The AE is clearly related to the intervention 
1NOTE: AEs listed as ‘possibly, probably, or definitely’ related to the investigational agent/intervention are 
considered to have a suspected ‘reasonable causal relationship’ to the investigational agent/intervention (ICH 
E2A). 
 
The following are expected outcomes for the transplant recipient (not necessarily as a result of this study) 
and will be documented in the subject’s medical record but not reported to IRB unless they meet the 
criteria for an SAE or UP: 
• Renal insufficiency 
• Hepatic insufficiency 
• Transient cardiac arrhythmias 
• Transient cardiac insufficiency 
• Pulmonary insufficiency 
• Neutropenia and its complications 
• Thrombocytopenia and its complications 
• Anemia and its complications 
• Transfusion reactions 
• Treatable infections from bacteria, viruses, protozoa and fungi 
• Late effects of transplant regimens including: chronic fatigue, cataracts, infertility, growth 
 
impairment, hypothyroidism, bone complications, and dental caries 
• Headache, insomnia, psychosis, mood changes, disorientation, seizures from metabolic imbalance 
• Nausea, vomiting, diarrhea, mucositis, weight loss, dry mouth, hiccoughs, constipation 
• Well-characterized drug reactions - allergic manifestations, "red man" syndrome, steroid effects 
• Well-characterized drug side effects from drugs used routinely in transplant recipients (e.g.; preparative 
regimen chemotherapy, immunosuppressive drugs, antimicrobials)  
• Common side effects of antiemetics, analgesics, anti-inflammatory agent and known complications of 
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• Complications from intravenous catheters, thrombotic occlusion, infection, local reactions, cardiac 
arrhythmia  
• Expected adverse events related to investigational reagents and transplant drugs are listed in section 
13.3. 
 
The following are expected transplant outcomes that will be reported in summary form at the time of 
continuing review but will not be reported to IRB at each occurrence unless they meet the criteria of an 
SAE or UP: 
• Acute graft-versus-host disease 
• Chronic graft-versus-host disease 
• Graft failure / graft rejection 
• Veno-occlusive disease 
• Hemorrhagic cystitis 
• Cytomegalovirus reactivation or disease 
• Autoimmune phenomena 
• EBV reactivation or disease 
• Fungal infections 
• Disease relapse or progression 
12.3  NHLBI  IRB AND CD REPORTING 
12.3.1  Serious Events 
Reports to the IRB and CD:   
The PI must report Serious UPs and Serious PDs, to the IRB and CD as soon as possible but not more than 7 
days after the PI first learns of the event via iRIS using the NIH Problem Form. 
 
Reports to the IRB Chair and CD 
The PI must report all SAEs that do not meet the definition of UP to the IRB chair and CD not more than 14 
days after the PI first learns of the event via iRIS, using the NIH Problem form. 
 
12.3.2. Non-serious Events   
Reports to the IRB and CD: 
The PI must report all UPs that are not Serious to the IRB and CD, and PDs that are not Serious to the IRB, not 
more than 14 days after the PI first learns of the event via iRIS using the NIH Problem Form. 
 
12.3.3 Deaths 
The PI must report all deaths (that are not UPs) to the CD as soon as possible, but not more than 7 days after the 
PI first learns of the event.  
12.4  IND SPONSOR REPORTING CRITERIA 
An investigator must immediately report to the sponsor, using the mandatory MedWatch form 3500a, any 
serious adverse event, whether or not considered drug related, including those listed in the protocol or 
investigator brochure and must include an assessment of whether there is a reasonable possibility that the drug 
caused the event.  
 
 

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Events will be submitted to Dr. A John Barrett, IND Sponsor at: 
A. John Barrett, M.D. 
Rm 3-5330, HB, NHLBI, CRC,  
10 CenterDrive,  
Bethesda, MD 20892-2012. 
 
12.5  DSMB REPORTING  
Any UP observed during the clinical trial and for which there is a relationship with the use of BMSC cells and 
the conduct of the study will be reported within 24 hours to the Data and Safety Monitoring Board (DSMB). A 
summary of all SAEs and AEs will be included for review at the regularly scheduled DSMB meeting.  
 
12.5.1 Serious Adverse Event Reporting on Cell Therapy Products to the NHLBI IRB and FDA 
If a cell product deviation (or any component thereof) occurs during or after its manufacture, a report of 
the event, and information relevant to the event, associated with the manufacturing will be submitted to 
the NHLBI IRB and the FDA by the PI and/or IND sponsor, in collaboration with CPS/DTM, to include 
testing, processing, packing, labeling, or storage, or holding or distribution of the product, if the event 
meets the following criteria: 
(i) Represents a deviation from current good manufacturing practice, applicable regulations, 
applicable standards, or established specifications that may affect the safety, purity, or potency of 
that product; or 
(ii) Represents an unexpected or unforeseeable event that may affect the safety, purity, or 
potency of that product; 
 
Cell product deviations will be reported as soon as possible but at a date not to exceed 45-calendar days 
from the date the PI/research team or CPS, DTM acquired information reasonably suggesting that a 
reportable event has occurred. 
 
FDA reporting: All HCT/P deviations involving 361 cell products will be reported using MedWatch 
Form FDA 3500A according to FDA publication “Guidance for Industry: MedWatch Form FDA 
3500A: Mandatory Reporting of Adverse Reactions Related to Human Cells, Tissues, and Cellular and 
Tissue-Based Products (HCT/Ps).   
 
12.5.2  Action Plan for Positive Results on Cell Product Safety Testing:  
In the unlikely event that a positive sterility test or mycoplasma test result is obtained after distribution 
of the BMSC product, or after administration of the product to the subject, the following steps will be 
initiated IMMEDIATELY: 
a)  CPS, DTM personnel will notify the principal investigator, Dr. Dunavin (240-274-3599) (and 
pager 10682) or through the page operator.  As soon as the identification and sensitivity report 
from positive sterility is available, a copy of the final report will be sent to the PI.  The DTM QS 
officer and CPS Supervisor will determine the need for quality improvement based on the nature 
and extent of the incident. 
b)  If CPS is unable to reach Dr. Battiwalla within 15 minutes, - the page operator will be 
contacted (301-496-1211) to page the Hematology Branch Fellow on-call.  NOTE: Dr. Barrett or 

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the Fellow on-call will contact the attending physician for the BMT Service, who will determine 
the extent of the work-up of a positive culture in consultation with staff from the Microbiology 
Service. 
c) Dr. Dunavin/Barrett/Battiwalla/attending physician will discuss the positive results with the 
subject/parents, and specify the clinical therapy, antibiotic regimen and/or monitoring plan in 
consultation with staff from the Microbiology Service. 
d) A contaminated sample of a product that has been administered to a subject will be handled in 
the same fashion as a serious adverse event, i.e., the PI or IND Sponsor will be responsible for 
notifying the NHLBI IRB, DSMB and the FDA.  A full written report with description of events, 
laboratory findings, clinical evaluation and treatments would be submitted to the IRB within 15 
working days.  The PI or IND Sponsor will be responsible for filing this report with the FDA 
within 15 calendar days.  
12.6 
PROTOCOL MONITORING 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50 clinical protocols are required to be adequately 
monitored by the study sponsor. The monitoring of this study will be conducted by Clinical  Research 
Associates (CRAs)/Monitors employed by an independent contract organization working under an 
agreement with NHLBI to monitor aspects of the study in accordance with the appropriate regulations 
and the approved protocol. The objectives of a monitoring visit will be: 1) to verify the existence of 
signed informed consent form (ICF) and documentation of the ICF process for each monitored subject; 
2) to verify the prompt and accurate recording of all monitored data points, and prompt reporting of all 
SAEs; 3) to compare abstracted information with individual subjects’ records and source documents 
(subject’s charts, laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and any 
other relevant original subject information); and 4) to help ensure investigators are in compliance with 
the protocol. The monitors also will inspect the clinical site regulatory files to ensure that regulatory 
requirements of the Office for Human Research Protections (OHRP), FDA and applicable guidelines 
(ICH-GCP) are being followed. During the monitoring visits, the investigator (and/or designee) and 
other study personnel will be available to discuss the study progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms and pertinent 
hospital or clinical records readily available for inspection by the local IRB, FDA, the site monitors, and 
the NHLBI staff for confirmation of the study data.  
 
13. HUMAN SUBJECTS PROTECTIONS  
13.1 
RATIONALE FOR SUBJECT SELECTION 
This protocol is open to pediatric and adult males and females from all ethnic and racial groups 
undergoing stem cell transplantation treatment protocols of NHLBI, NCI, NIAID, and NIDDK. 
 
13.1.1 Competition with other Branch transplant protocols: 
 
 
There are currently no active protocols for the treatment of subjects who do not respond to the  
 
 
standard therapies of these post-transplant complications. 
 
 
13.1.2 Reimbursement for protocol participation, travel, food, and lodging  
 
 
Compensation will not be given for participation in this study or for subjects’ time and  
 
 
 
inconvenience.  In determining reimbursement, the following factors are considered applicable to 

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this protocol:   the subjects are diagnosed with a rare disease, the subject population is sick, the  
 
 
protocol offers the potential for direct benefit, the protocol regimen is demanding, and in order to 
 
 
complete accrual in a reasonable timeframe a geographically dispersed participant population is  
 
 
required. No specific provisions for reimbursement are planned for this protocol since almost all  
 
 
subjects will be hospitalized and supported financially on existing stem cell transplant protocols.  
13.2  PARTICIPATION OF CHILDREN, PREGNANT WOMEN AND DECISIONALLY IMPAIRED 
 
13.2.1 Pediatric subjects 
Pediatric subjects will be included in this protocol.  BMSC have been tested extensively in 
children and the NIH BMSC product has been tested in adults in a Phase 1 study and has been 
shown to be safe. 
 
 
 
13.2.2 Pregnant women 
Pregnant women will not be included in this protocol as pregnancy is a contraindication to the 
underlying transplant procedure and because of the unknown risks of the BMSC product to the 
fetus. 
 
 
13.2.3 Decisionally-impaired 
Patients who are decisionally-impaired will be included provided that they have a surrogate 
decision maker willing to provide informed consent. 
13.3  HAZARDS/DISCOMFORTS 
 
13.3.1  Related to the BMSC infusion  
Serious complications or side effects from BMSC infusions have not been described.  Infusion side 
effects have been minor including fever, hives, and nasal congestion. 
 
 
13.3.2  Related to transplant-related complications. 
Subjects in this trial may experience the spectrum of post-transplant complications described in 
detail in the associated protocols. 
13.4  RISKS/BENEFITS ANALYSIS 
 
13.4.1  For Transplant Subjects 
According to previous studies using similar infusions, the risks of BMSC infusion appear to be 
low.  As described in Section 2, in-vitro expanded BMSC have been used successfully to treat de 
novo and severe steroid refractory acute GVHD.  In a recently published multicenter phase II 
study, 55 patients with steroid-resistant, severe, acute GVHD were treated with up to five 
infusions of BMSC at a median dose of 1·4x106 cells per kg bodyweight.  HLA mismatched or 
unrelated HLA mismatched donors were used in most cases.  Thirty patients had a complete 
response and nine showed improvement.  No patients had side-effects during or immediately 
after BMSC infusions.  Therefore, it is our expectation that this study presents greater than 
minimal risk but there is the potential for benefit in subjects enrolled.  Stopping rules will apply 
if unexpected complications arise in this study.   
 

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For children, our expectation is that the research involves greater than minimal risk but 
presenting the prospect of direct benefit (Children's Risk Category Assignment: 2; 45 CFR 
46.405). 
13.5  CONSENT AND ASSENT PROCESS AND DOCUMENTATION 
 
13.5.1  Adults 
The investigational nature and research objectives of this trial, the procedure and its attendant 
risks and discomforts will be carefully explained to the subject and a signed informed consent 
document will be obtained prior to entry onto this study.  Participants eligible for this study may 
be very ill and incapable of providing consent at the time in which enrollment to this study is 
warranted, in which case a relative or legal surrogate would provide consent.  All participants 
will have undergone transplant at NIH, and therefore, discussions introducing this study and 
potential participation in this study with possible need for surrogate consent will be held with 
potential candidates and their families, when warranted.   Dr. Ito or any of the investigators listed 
on the cover page with consenting privileges will lead the discussion. 
 
 
13.5.2  Informed Consent for adult participants unable to provide consent: 
If there is an unexpected enrollment of a participant that is a patient unable to provide informed 
consent, the following justification and procedures per NIH HRPP SOP 14E will be used to 
enrolled participants in the this protocol. 
 
Justification for inclusion:  This research provides the prospect of direct benefit, therefore 
inclusion is justified. 
 
Risk/Benefit Assessment: 
This research involves greater than minimal risk to subjects with the prospect of direct benefit 
(45 CFR 46.102) 
 
Consent and Assent: 
Procedures to determine capacity: If documentation of decision making capacity is not present in 
the medical record or the investigator questions the decision making capacity of the individual, 
then the Ability to Consent Assessment Team (ACAT) (301-496-9675 or 301-496-2429) will be 
contacted to make the determination. 
 
Procedures for obtaining consent for legally authorized representative (LAR) (Risk Level B per 
SOP 14E):  The following procedures will be followed starting with (1) in order to determining 
the LAR.  
(1) For adults who cannot consent and have a court appointed guardian from a jurisdiction that 
allows it or a Durable Power of Attorney (DPA) for health care and/or research participation, the 
PI/designee or ACAT confirms appropriateness of surrogate to consent to research, including 
that: 
(a)The surrogate understands that the protocol involves research; 
(b)The surrogate understands the risks, potential benefits, (if any), and alternatives to the study; 
(c)The surrogate has sufficient reason to believe participation in the study is consistent with the 
subject’s preferences and values. 
 

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(2) Adults who cannot consent and who do not have a DPA or court-appointed guardian, but who 
are capable of understanding the DPA process and can assign a DPA, then ACAT confirms 
appropriateness of surrogate to consent to research, which includes assessing criteria (a)-(c) 
above. 
 
(3) Adults who cannot consent, who do not have a DPA or court-appointed guardian, and who 
are not able to understand the DPA process to appoint a DPA, then a person at the highest level 
of the following list may serve as surrogate and authorize subject’s participation if ACAT 
confirms surrogate appropriateness (which includes assessing criteria (a)-(c) above): 
1. spouse or domestic partner; 
2. adult child; 
3. parent; 
4. sibling; 
5. other close relative 
 
If at any time there is a question about the authority of the LAR to provide consent based on the 
jurisdiction appointing the durable power of attorney or other legal question regarding the LAR 
to provide consent, the Office of the General Counsel will be consulted. 
 
Procedures to obtain assent and documentation of assent or dissent:  The informed consent 
discussion will include the individual unable to provide informed consent along with LAR.  The 
individual unable to provided informed consent will be asked if they agree to participate in the 
research and this will be documented in the medical record. 
 
 
13.5.3  Informed Consent of Non-English Speaking Research Participants. 
We anticipate the enrollment of non-English speaking research participants into our study.  The 
IRB approved full consent document will be translated into that language in accordance with the 
Clinical MAS Policy M77-2.  If there is an unexpected enrollment of a research participant for 
which there is no translated extant IRB approved consent document, the principal investigator 
and or those authorized to obtain informed consent will use the Short Form Oral Consent Process 
as described in MAS Policy M77-2, 45 CFR 46.117 ( b) (2), and 21 CFR50.27 (b) (a).  The 
summary that will be used is the English version of the extant IRB approved consent document.  
We request prospective IRB approval of the use of the short form for up to a maximum of 5 
participants in a given language and will notify the IRB at the time of continuing review of the 
frequency of the use of the Short Form.  Should we reach the threshold of 5, we will notify the 
IRB of the need for an additional use of the Short Form and that we will have that consent 
document translated into the given inherent language.  
 
13.5.4 Re-Consent for Minors when they reach the age of majority:   
When a pediatric subject reaches age 18, continued participation will require re-consenting of the 
now adult with the standard protocol consent document to ensure legally effective informed 
consent has been obtained. Should sample or data analysis continue following completion of 
active participation and the subject has reached 18 years of age, we will attempt to contact the 
subject using the last known contact information to obtain consent for continued use of data or 
samples  collected during their prior visit. Given the length of time that may have transpired for 

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some of the subjects since their last visit for this study, we request waiver of informed consent 
for those individuals who after good faith efforts to contact them, we are unable to contact. 
 
 
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d), each of which must be 
addressed in relation to the protocol: 
 
(1) 
The research involves no more than minimal risk to the subjects. 
a. Analysis of samples and data from this study  involves no additional risks to subjects. 
(2) 
The waiver or alteration will not adversely affect the rights and welfare of the subjects. 
a. This is an FDA-regulated study and as such, we are mandated to retain all samples, 
once collected, regardless of the age of the subject at the time of collection.  
Retention of these samples or data does not affect the welfare of subjects.  
(3) 
The research could not practicably be carried out without the waiver or alteration. 
a. Considering the length of time between a minor’s enrollment and their age of 
majority, it is possible that more than a few subjects may be lost to follow up.  A 
significant reduction in the number of samples analyzed could impact the quality of 
the research.  
(4) 
Whenever appropriate, the subjects will be provided with additional pertinent information 
after participation. 
a. We only plan to request a waiver of reconsent for those subjects who have been lost 
to follow-up. 
   
 
13.5.5  New Study Related Information 
At any time during participation in the protocol that new information becomes available relating 
to risks, adverse events, or toxicities, this information will be provided orally or in writing to all 
enrolled or prospective subject participants.  Documentation will be provided to the IRB and if 
necessary the informed consent amended to reflect relevant information. 
 
13.6  CONFLICT OF INTEREST 
A Guide to Preventing Financial and Non-Financial Conflicts of Interest in Human Subjects Research at 
NIH  has been distributed to all NIH investigators who have reviewed the guide and indicated no 
conflicts of interest exist.  No investigators hold patents or patents pending, or have any financial 
conflict of interest associated with this study.  
 
14. PHARMACEUTICAL AND INVESTIGATIONAL DEVICE INFORMATION   
14.1  BMSC INFUSION 
 
14.1.1  Source 
BMSC are an adherent, fibroblast-like cell population found in the bone marrow.  Allogeneic 
BMSC for treatment can be grown from bone marrow aspirates or biopsies of normal donors, 
and are currently being used to treat a number of disorders, including graft-versus-host disease 
(GVHD), ischemic heart disease, peripheral vascular disease, and autoimmune diseases. The 
BMSC are produced in the Clinical Cell Processing Laboratory, located in the Cell Processing 
Section (CPS), Department of Transfusion Medicine (DTM), Clinical Center, NIH. Donors must 
be greater than or equal to 18 years of age, be afebrile, have normal hemodynamic parameters, 

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not be pregnant and must meet the screening requirements according to the policies and 
procedures of DTM.  The HLA-type of the donor will be determined. Bone marrow is collected 
for generation of BMSC on protocol 10-CC-0053.  Gentamicin is used in the processing of 
BMSC on 10-CC-0053 and thus allergy to gentamicin is specified as an exclusion criterion on 
this study. The collected cells are serially passaged into multilayer cell factories until 
approximately 300 to 900 million cells are generated.  The cells are packaged into units of 
approximately 100 million cells and cryopreserved.     
 
 
14.1.2  Toxicity 
 
As previously noted, serious toxicities are not expected.  Reported toxicities include fever, hives, 
 
and nasal congestion.  Theoretically, transfusion reaction and allergic reactions are possible. 
 
 
14.1.3  Formulation and preparation 
Cells will be processed and prepared as per the BB-DMF filed by CPS, DTM with the FDA.  
Thawed BMSC suspended in Plasmalyte A will be given in an infusion and may be repeated 
twice weekly for four weeks followed by weekly for four weeks based on response.  Each BMSC 
infusion will contain a target dose of 2 x 106 BMSC /kg (+/- 10%) viable BMSC (30-150 mL).  
Where possible repeat infusions will be obtained from the same donor but  BMSC from other 
donors is permitted if same donor cells are not available.   Donor sample identifiers will be 
tracked for each infusion. 
 
 
14.1.4  Stability and Storage  
The cryopreserved BMSC will be stored in the vapor phase of liquid nitrogen in the Cell Process 
Laboratory.  This is a secure laboratory and the temperature and liquid nitrogen levels in the 
storage tanks are monitored continuously.  The duration of time in which BMSCs retain their 
complete biological activity is not certain and a stability and viability testing plan is included in 
the CPS BB-DMF with the FDA.  Previously BMSC have been stored in the Laboratory of Dr. P. 
Robey (NIDCR, NIH) for up to 15 years with retention of 90% viability upon thawing and 
hematopoietic progenitor cells have been stored even longer without evidence of loss of 
engraftment, proliferation, or differentiation potential.  Data to date has demonstrated stability of 
the product for 4 hours post thaw; hence the expiration of the product will be considered, 4 hours 
post thaw. 
 
 
14.1.5  Administration procedures 
BMSC will be thawed rapidly in a 37˚C water bath and infused by IV infusion as per Section 3.4.

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Appendix 1:  Criteria for Diagnosis, Severity, Response and Eligibility for BMSC Trial 
 
Consensus Conference Criteria92 
Extent of Organ Involvement 
 
Skin 
Liver 
Gut 
Stage 
1 
Rash on <25% of skina 
Total bilirubin 2-3 mg/dLb 
Diarrhea ≥ 500 mL/dayc (child 
10-19.9 ml/kg/day), or 
persistent nausead 
2 
Rash on 25-50% of skin 
Total bilirubin 3-6 mg/dL 
Diarrhea ≥ 1000 mL/day (child 
20-30 mL/kg/day) 
3 
Rash on >50% of skin 
Total bilirubin 6-15 mg/dL 
Diarrhea 1500 mL/day (child ≥ 
30 mL/kg/day) 
4 
Generalized erythroderma 
with bullous formation 
Total bilirubin >15 mg/dL 
Severe abdominal pain with or 
without ileus 
Gradee 
I 
Stage 1 or 2 
None 
None 
II 
Stage 3 or 
Stage 1 or 
Stage 1 
III 
- 
Stage 2-3 or 
Stage 2-4 
IVf Stage 4 or 
Stage 4 
- 
(a) Use “rule of nines” or burn chart to determine extent of rash 
(b) Range given as total bilirubin.  Downgrade by one stage if an additional cause of 
elevated bilirubin has been documented. 
(c) Volume of diarrhea applies to adults.  For pediatric patients, the volume of diarrhea 
should be based on body surface area.  Downgrade by one stage if an additional cause of 
diarrhea has been documented. 
(d) Persistent nausea with histological evidence of GVHD in the stomach or duodenum. 
(e) Criteria for grading given as minimal degree of organ involvement required to confer 
that grade. 
(f) Grade IV may also include lesser degree of organ involvement but with extreme 
decrease in performance status 

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Appendix 2:  Criteria for Response to BMSC 
 
Term 
Definition 
Complete response (CR) 
Resolution of acute GVHD in all involved organs 
Partial response (PR) 
Organ improvement of at least 1 stage without worsening in any 
other organ system 
Overall response (OR) 
CR or PR 
Mixed response (MR) 
Improvement by at least 1 organ stage in at least 1 evaluable 
organ with worsening by at least 1 organ stage in at least 1 other 
organ 
Stable disease 
The absence of any clinically significant differences 
(improvement or worsening) sufficient to meet minimal criteria 
for improvement or deterioration in any evaluable organ 
Worsening disease 
Deterioration in at least 1 evaluable organ by 1 stage or more 
No response 
MR or stable disease or worsening disease 
 
 
 

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Appendix 3:  Study Calendar for Early BMSC Pilot for Acute GVHD 
 
a Tissue biopsy if indicated: See Appendix 1for GVHD staging.  aGVHD evaluation may occur in inpatient or outpatient setting and will occur at minimum on days of BMSC 
infusion (+/- 24 hours) and at any additional time that is determined necessary by the investigators.  aGVHD evaluation will be done at least once weekly for the duration for the 
first 56 days of treatment. 
b Monitoring according to NIH Clinical Center SOP: Administration of Products of Cellular Therapy 
c In females of child-bearing potential 
d Response assessment according to condition; response criteria in Appendix 2. 
e Follow up between infusions and after the last BMSC infusion will be based on the subject’s condition, as frequently as twice weekly. 
f Premedicate 30-60 minutes prior to infusion. Infusions will be scheduled with DTM and given twice weekly no sooner than 48 hours apart (typically on Tuesday and Friday).  
The first dose of BMSC will be given within 120 hours of the first dose of corticosteroids.  Infusion rate not to exceed 4-5 mL/min in subjects ≥35 kg, and infused over 
approximately 60 minutes in subjects < 35 kg (see Section 3.4).   
g 54 mL of blood will be drawn [1 (6 ml) lavender top- K2-EDTA for plasma and 6 (8 ml) red-green “tiger top”] on Day 0 (pre BMSC infusion, +/- 24 hrs), Day 28 (+/-72 hrs), and 
Day 56 (+/- 72 hrs).  3 mL of blood [1 (3 ml) lavender top- K2-EDTA for plasma] will be drawn before and after each BMSC infusion (+/- 24 hours as long as before or after, 
respectively) and at 3 weeks after the last infusion (+/- 7 days).    
Procedure 
Protocol 
Screen 
 
 
Day 
0 +/- 
24 
hrs 
Week 
1 
Week 
2 
Week 
3 
Day 
28 +/- 
72 
hrs 
Week 
5 
Week 
6 
Week 
7 
Day 
56 +/- 
72 
hrs 
Before and after 
each infusion +/- 
24 hoursb, g 
3 weeks after 
last infusion 
+/- 7 days 
Post Therapy 
Follow-upe 
History 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Physical Exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs: BP, HR, 
RR, Temp, pulse ox 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Acute GVHD 
evaluation 
Xa 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
Performance Score 
(KPS) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Labs 
 
 
 
 
 
 
 
 
 
 
 
 
 
• Pregnancy Testc 
X 
 
 
 
 
 
 
 
 
 
 
 
 
• CBC/differential 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
• Chemistry Panel 
X 
 
 
 
 
 
 
 
 
 
 
X 
X 
BMSC INFUSIONf 
 
 
 
 
 
 
 
 
 
 
 
X 
 
Correlative Research 
Studiesg 
 
X 
 
 
 
 
 
 
 
 
X 
X 
 
Response evaluation d 
 
 
 
 
 
X 
 
 
 
X 
 
X 
 
Adverse events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Concomitant 
Medications 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 

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Appendix 4:  IRB Approved Hematology Branch Laboratory Research Studies  
  NHLBI HEMATOLOGY BRANCH LABORATORY RESEARCH STUDIES v. 2.5.2013 
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION 
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 45 
CFR 46.404? 
Does this test 
pose a 
greater than 
minimal risk 
to healthy 
pediatric 
donors per 45 
CFR 46.404? 
 
 
A 
Stem Cell Allotransplantation Section (Dr. A. John Barrett) 
 
 
A.1 
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine  
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques. 
 
No 
No 
A.2 
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples. 
No 
No 
A.3 
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses. 
No 
No 
A.4 
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies. 
No 
No 
A.5 
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions. 
No 
No 
A.6 
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application. 
No 
No 
A.7 
Identification of individual T cell clones by their T cell receptor sequence. 
No 
No 
A.8 
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA,protein, or peptide expression in cells or fluids. 
No 
No 
A.9 
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes. 
No 
No 
A.10 
DNA and RNA typing of genes that control immune responses in lymphocytes. 
No 
No 
A.11 
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions. 
No 
No 
 
 
B 
Molecular Hematopoiesis  Section  (Dr. Cynthia Dunbar)  
 
 
B.1 
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells. 
No 
No 

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B.2 
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function. 
No 
No 
B.3 
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro. 
No 
No 
B.4 
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro. 
No 
No 
 
 
C 
Cell Biology Section (Dr. Neal Young)  
 
 
C.1 
Studies of blood and bone marrow hematopoietic progenitor numbers, including 
early and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion. 
No 
No 
C.2 
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests. 
No 
No 
C.3 
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins. 
No 
No 
C.4 
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow. 
No 
No 
C.5 
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained. 
No 
No 
C.6 
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations. 
No 
No 
C.7 
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations. 
No 
No 
C.8 
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells. 
No 
No 
C.9 
Studies of chromosomal instability in myelopdysplastic syndromes including BM 
cell and CD34 cell response to PAS crosslinking and examination of the cytotoxic 
effect of lymphocytes to the abnormal clone of cells. 
No 
No 
C.10 
Surface Enhanced Laser/Desorption Ionization (SELDI)  time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology). 
No 
No 
C.11 
Mitochondrial DNA (mtDNA) sequence heterogeneity. 
No 
No 
C.12 
Measurement of EBV viral load. 
No 
No 
C.13 
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1. 
No 
No 
C.14 
Outgrowth assay of EBV transformed B cells. 
No 
No 

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C.15 
Quantification of serumchemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12). 
No 
No 
C.16 
Quantification of EBV cytotoxic T cells (tetramerstaining). 
No 
No 
C.17 
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA 
No 
No 
C.18 
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10 , NHP2. 
No 
No 
C.19 
Analysis of  inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation. 
No 
No 
C.20 
Confocal microscopic imaging of bone marrow. 
No 
No 
C.21 
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots. 
No 
No 
C.22 
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques. 
No 
No 
C.23 
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection. 
No 
No 
C.24 
Quantification of gene expression with RNA-seq 
No 
No 
C.25 
Characterization of chromatin and promoter/enhancer landscapes with ATAC-seq 
No 
No 
C.26 
Measurement of protein markers with SomaLogic's SOMAscan assay 
No 
No 
 
 
D 
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS 
 
 
D.1 
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19  variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays. 
No 
N/A 
D.2 
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements. 
No 
N/A 
D.3 
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2). 
No 
N/A 
D.4 
Spectra-typing of blood cells to determine response to known or putative viral 
infections. 
No 
N/A 
D.5 
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies. 
No 
N/A 
D.6 
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity. 
No 
N/A 
 
 
E 
Solid Tumor Section (Dr. Richard Childs) 
 
 
E.1 
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells. 
No 
No 
E.2 
ELISA for IL-12  maturity of DC's made from subjects monocytes. 
No 
No 
E.3 
ELISA for IFN ã to evaluate specificity of CTL clones. 
No 
No 

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Sawa Ito, M.D. 
E.4 
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells. 
No 
No 
E.5 
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant. 
No 
No 
E.6 
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets. 
No 
No 
E.7 
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry. 
No 
No 
E.8 
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect. 
No 
No 
E.9 
Geno typing of tumor or tissue samples by high density cDNA arrays. 
No 
No 
E.10 
VHL mutation analysis on kidney cancer tissue. 
No 
No 
E.11 
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions. 
No 
No 
E.12 
Lasar capture microdisection of cells from tumor biopsies and tissue samples  to 
determine origin (donor vs patient). 
No 
No 
E.13 
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples. 
No 
No 
E.14 
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples. 
No 
No 
E.15 
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant. 
No 
No 
E.16 
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts. 
No 
No 
E.17 
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts. 
No 
No 
E.18 
Determination of etiology of membraneous nephropathy using serum from subjects. 
No 
No 
E.19 
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation. 
No 
No 
E.20 
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH. 
No 
No 
 
 
F 
Lymphoid Malignancies Section (Dr. Adrian Wiestner) 
 
 
F.1 
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions. 
No 
No 
F.2 
Generation of stable cell lines for the study of hematologic malignancies. 
No 
No 
F.3 
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions. 
 
 
F.4 
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression. 
No 
No 
F.5 
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays. 
No 
No 
F.6 
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing. 
No 
No 
F.7 
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes. 
No 
No 

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Sawa Ito, M.D. 
 
F.8 
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens. 
No 
No 
F.9 
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy. 
No 
No 
F.10 
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma. 
No 
No 

45 
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Sawa Ito, M.D. 
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