A Phase II Study of Intrathecal Autologous Adipose-derived Mesenchymal Stromal Cells for Amyotrophic Lateral Sclerosis
Phase: PHASE2
Conditions: ALS, Amyotrophic Lateral Sclerosis
Sponsor: Mayo Clinic

A Phase II Study of Intrathecal Autologous 
Adipose-derived Mesenchymal Stromal Cells for 
Amyotrophic Lateral Sclerosis 
NCT#03268603 
19December2023 

 
 
Mayo Clinic  
 
 
A Phase II Study of Intrathecal Autologous Adipose-derived Mesenchymal Stromal Cells for 
Amyotrophic Lateral Sclerosis 
 
 
 
Study Chair: 
Nathan P. Staff, M.D., Ph.D. 
Mayo Clinic 
Department of Neurology 
200 First Street, SW 
Rochester, MN 55905 
 
 
 
Study Co-chairs: 
Bjorn Oskarsson, M.D. (Mayo Clinic – Jacksonville) 
 
 
Iryna Muzyka, M.D. (Mayo Clinic – Arizona) 
 
 
  
 
 
Anthony Windebank, M.D. 
 
 
  
 
Statistician: 
  
 
  
 
 
Investigational Agent:  adipose-derived mesenchymal stromal cells (MSCs), FDA IND #14788, FDA Orphan 
Drug #14-4270 
 
Document History 
Date 
Major Changes 
Addendum 
July 24, 2017 
Addition of fluoroscopically-guided lumbar 
puncture 
Addendum 
January 24, 2018 
Visit 4 Edinbourgh Cognitive ALS Screen (ECAS) 
was removed from the Schedule of Events 
Addendum 
March 6, 2018 
Addition of Mayo Clinic Arizona and Mayo Clinic 
Florida as enrolling sites (biopsies and stem cell 
injections done in Rochester, all other visits done at 
enrolling site). 
Addendum 
November 15, 2019 
Section 3.2: Updated exclusion criteria to include  
use of other investigational products and nutritional 
supplements; 
Added ALSSQOL-R questionnaire;  
Section 8: Expected adverse events added , 
stopping points added/clarified, definition of SAE 
clarified; 
Section 9: Added table of expected SAEs that do 
not have to be expeditedly reported; CTCAE 
version changed from v4 to v5; data collection 
method changed to RAVE. 
 
 
 
 

 
 
Mayo ALS MSC Phase II – Page 2
 
 
Document History 
Date 
Major Changes 
Addendum 
January 14, 2020 
Section 3.2: Addition of edaravone use to the 
exclusion criteria;  
Section 4.0 (Test Schedule): Moved ALSSQOL-R 
from Visit 15 to Visit 16 (final study visit); 
Section 6.2: Removed requirement for brain MRI 
Addendum 
February 26, 2020 
Section 4.0 (Test Schedule): Moved the “research” 
blood collection from V1 to V2, and clarified that 
this and “research” CSF collection are optional. 
Section 6.2 (Study Procedures Delineated by Visit): 
added optional research blood and CSF collection 
to visits to align with test schedule. 
Addendum 
August 28, 2020 
Title Page: Arizona Study Co-chair change. 
Section 3.2 (Exclusion Criteria): Changed exclusion 
of prior stem cell therapy to prior stem cell therapy 
for a neurological disease. 
Modification 
December 10, 2020 
Sections 2.1, 5.2, 7.2:  Starting aaMSC dose 
changing from 1 x 10(8) to 5 x 10(7):  Due to 
increased recognition of back/leg pain after 
intrathecal autologous aaMSC treatment that is 
impacting study feasibility, the DSMB 
recommended developing mitigation strategies to 
lessen the impact of this Adverse Event.   
Sections 5.2, 6.0, 6.2: Dose Modification Schema 
alteration to allow for dose escalation: Due to the 
continued need to determine the most efficacious 
and best tolerated dose, the Dose Modification 
Schema has been modified to allow for increasing 
doses (7.5 x 10(7) and 1 x 10(8)), if minimal pain 
(0-1/10) exists at 6 weeks post preceding injection. 
Sections 5.2, 6.2, 7.2: Dose Modification Schema 
alteration to drop threshold for dose reduction: 
Due to increased recognition of back/leg pain after 
intrathecal autologous aaMSC treatment that is 
impacting study feasibility, we have dropped the 
threshold for dose reduction from pain 1-2/10 at 2 
weeks prior to injection to pain1-2/10 at 6 weeks 
prior to injection. 
Sections 2.1, 3.2: Addition of chronic back pain 
or lumbosacral spine surgery to the exclusion 
criteria:  It has become apparent that subjects with 
history of back pain or spine surgery have increased 
likelihood of developing post aaMSC injection 
back/leg pain.  Therefore, we have made this an 
exclusion criterion. 
 
Modification 
January 11, 2022 
Sections 2.3.2, 6.2, 10.0, Table 4.0: Removal of 
VC at all visits except enrollment: In the original 
clinical protocol, pulmonary spirometry to quantify vital 
capacity (VC) was included at all visits.  In the setting of 

 
 
Mayo ALS MSC Phase II – Page 3
 
 
COVID-19 pandemic, COVID-19 testing is required 
prior to pulmonary spirometry, which requires subjects 
to come the day before spirometry.  We have required 
that spirometry be performed at enrollment because a VC 
> 65% predicted for age/BMI is part of inclusion criteria.  
Due to the undue burden on subjects to travel an extra 
day, we have not required spirometry for other visits 
since the pandemic began.  We thought this would be a 
temporary deviation, but it appears to be more 
permanent.  We are going to remove VC as a 
measurement in all visits except the enrollment visit.  
Furthermore, VC will no longer be a trial endpoint. 
Sections 6.2 and Table 4.0: Addition of window of 
timepoint to take vitals and pain assessment post-
injection: After the first two hours post-injection, a +/-
15 minutes window of time added to take vitals and pain 
assessment. 
Modification 
January 28, 2022 
Increased enrollment to 69 patients. 
Modification 
June 9, 2022 
Section 3.1 Inclusion criteria to include use of oral 
edaravone is acceptable if on stable dose for 30 
days or starts 6 weeks after second MSC injection . 
Section 3.2 Exclusion criteria clarified that 
intravenous edaravone is excluded. 
Modification  
October 11, 2022 
Enrollment completed. 
Addition of Section 8.2  Use of of RELYVRIO™ 
(sodium phenylbutyrate and taurursodiol; also 
known as AMX0035) during clinical trial. On 
9/29/22, the U.S. FDA approved the use of 
RELYVRIO™ (sodium phenylbutyrate and taurursodiol; 
also known as AMX0035) in people with ALS.  Relyvrio 
has modest efficacy with demonstrated slowing of 
disease as measured by the slope of the Revised ALS 
Functional Rating Scale. 
Patients with ALS, including subjects in the ALS-MSC 
Phase 2 clinical trial are eager to start this new FDA-
approved medication.  We have already received 
multiple requests from subjects in the trial to be able to 
take Relyvrio.   It is an oral medication that can be taken 
by mouth or via feeding tube.  Overall, the medication is 
well-tolerated with some gastrointestinal complaints 
(diarrhea, abdominal pain, and nausea being the most 
commonly seen).   
We do not anticipate any safety issues regarding the 
concomitant use of Relyvrio and participation in this 
clinical trial. 
Update to Table 4.0 Schedule of Assessments to 
accurately reflect that the HHD assessment is 
conducted at visits described in Section 6.2 Study 
Procedures Delineated by Visit. 
 
Modification 
May 2023 
Addition of treatment extension of four additional 
intrathecal injection treatments for subjects that 
completed the trial and meet inclusion and 
exclusion criteria. 

 
 
Mayo ALS MSC Phase II – Page 4
 
 
 
 
 
 
 
Index 
 
Schema 
  1.0 
Background 
  2.0 
Goals 
  3.0 
Patient Eligibility 
  4.0 
Test Schedule 
  5.0 
Protocol Treatment 
  6.0 
Study Procedures 
  7.0 
Dosage Modification Based on Adverse Events  
  8.0 
Ancillary Treatment/Supportive Care 
  9.0 
Adverse Event (AE) Reporting and Monitoring  
10.0 
Statistical Plan   
11.0 
Treatment Extension Protocol 
12.0 
References 
 
Appendix I  
Guidelines for Clinically Significant Laboratory Values, Vital Signs, and Clinical  
 
Assessments 
 
Appendix II  The ALS Specific Quality of Life-Revised (ALSSQOL-R) User’s Guide  
 
 
 

 
 
Mayo ALS MSC Phase II – Page 5
 
 
Protocol Schema:  
 
Study Schema:  FB: Fat Biopsy, IT-MSC: intrathecal MSC treatment; Arrows: clinic visits; Purple Arrows: clinic 
visit includes MRI lumbosacral spine 
 
 
 
 
1.0 
Background   
 
1.1. Overall Approach of Phase II Clinical Trial:  Amyotrophic lateral sclerosis (ALS) is a 
neurodegenerative disease of motor neurons that causes progressive, fatal paralysis. The incidence of 
ALS is 2-4 cases per 100,000 persons per year with an average life span of 2-3 years following 
diagnosis1. There are no satisfactory treatments to slow progression of the disease. In this project, we 
propose to complete a Phase II open-label multi-site clinical trial to further confirm safety and to explore 
the efficacy of intrathecal autologous adipose-derived mesenchymal stromal (stem) cell (aaMSC) therapy 
for ALS. In the context of this phase II clinical trial, our objectives are to: 
1) Confirm the safety of repeated intrathecal delivery of aaMSCs in ALS 
2) Investigate the efficacy of intrathecal aaMSCs in ALS using the Revised ALS Functional 
Rating Scale (ALSFRS-R) as the primary clinical endpoint. 
3) Validate and verify proposed biomarkers of ALS by taking advantage of the study’s 
comprehensive sampling of blood and CSF. Candidate biomarkers include markers of 
neurodegeneration (neurofilaments), and immune dysregulation (peripheral blood 
immunophenotyping (PB-IP) and CSF miRNA). This new data will provide insights into 
ALS biology and inform the design of a subsequent pivotal phase III clinical trial. 
4) Be poised to elucidate the mechanisms of action for intrathecal aaMSC therapy by 
establishing biomarkers of MSC potency via in vivo (lumbosacral root MRI, CSF miRNA, 
PB-IP) and ex vivo (proteomics, extracellular vesicles, miRNA) analyses, and exploring how 
these biomarkers correlate with efficacy of intrathecal MSC therapy in ALS.  
 
 
1.2. MSCs are a promising therapy for ALS: Multiple lines of evidence have suggested that MSCs may 
function as a paracrine neuroprotective agent in ALS and other neurodegenerative diseases, acting via 
neurotrophic growth factor secretion and immune modulation2. We have completed treatment in our  
 

 
 
Mayo ALS MSC Phase II – Page 6
 
 
Phase I, 27-patient dose-escalation safety study using intrathecal aaMSC therapy in ALS. At this stage 
(August 2016), it is clear that there are no significant safety issues3.  
 
MSCs naturally reside in either the bone marrow or adipose tissue, whence they migrate to areas of 
injury, modulate inflammation and can differentiate into bone, cartilage, connective tissue, or fat2. In the 
context of ALS treatment, intrathecal aaMSCs are expected to be an excellent therapeutic for the 
following reasons: 
1) MSCs secrete growth factors. MSCs secrete neurotrophic factors, such as GNDF, VEGF, and 
BDNF4,5, all of which are known to be neuroprotective for motor neurons6-9. In addition to 
secreting neurotrophic factors themselves, MSCs have been shown to induce expression of 
neurotrophic factors in astrocytes10. Neurotrophic growth factors are critical for motor neuron 
health. While each of the above neurotrophic factors has been tested in ALS (systemically 
delivered), they have never been tested when given together, as will be the case here.  
2) MSCs modulate the immune system. MSCs have a strong influence on the immune system and 
modulate microglia toward a protective M2 (versus a proinflammatory M1) phenotype2,11. 
Neuroinflammation has become a clear pharmaceutical target in treating ALS pathoprogression. 
While the precise mechanism that MSCs modulate the immune system is still being elucidated, it 
is likely that they work in a different way than the modalities of immunosuppression tried 
previously in ALS12-16, thereby representing a novel approach to ALS therapy. Our repeated 
PB-IP and CSF miRNA analyses in this clinical trial will allow us to better delineate how MSCs 
alter the immune system. These results may be correlated with patient response and the potency 
of MSCs to modulate T cell proliferation in vitro aiming to evaluate MSC potency as an 
additional possible predictor of treatment response. 
3) Intrathecal injections bypass the blood brain barrier. The blood-brain barrier has limited the 
ability to allow penetration of therapeutics (especially neurotrophic growth factors) in ALS. 
Intrathecal injections enable neurotrophic growth factors produced by MSCs to enter the CNS 
directly. Furthermore, sustained secretion of MSC-derived extracellular vesicles (MSC-EVs) may 
enable a continuous shuttle of factors supporting neuroregeneration or neuroprotection. Given 
that MSCs reside within the CNS after injection, we expect that MSCs may continue to deliver 
neurotrophic growth factors for months. 
4) MSCs are easy to harvest and expand ex vivo for therapeutic indications. Adipose biopsies 
are easy to perform and are well tolerated by patients. MSCs derived from autologous sources 
eliminate issues of rejection and minimize risks of contamination.  
5) MSCs are safe and efficacious in animal models. Animal studies of MSCs in 
neurodegenerative diseases, including ALS, have been promising. MSCs can be safely infused 
into the intrathecal space of animals and survive for up to 3-6 months after injection17,18. We 
completed our own pre-clinical studies of intrathecal administration of autologous MSCs with a 
favorable safety profile19. Animal models of neurological disease have revealed MSCs 
therapeutic potential. There are data showing MSC efficacy in animal models not only of ALS2,20, 
but also in the other neurological diseases such as multiple sclerosis21, Parkinson’s disease22, and 
stroke23. These animal data suggest that MSCs may be efficacious in many neurological diseases, 
and therefore our data from this clinical trial will be useful in future human MSC trials for 
other neurological diseases. 
6) MSCs are safe and possibly efficacious in human neurodegenerative diseases. There is now 
growing evidence in human studies that MSCs can be safely administered intrathecally. Our 
Phase I dose-escalation safety trial using autologous adipose-derived intrathecal MSCs in ALS 
has had an excellent safety profile3. Our safety findings corroborate studies from other groups24-27. 
Of note, none of these studies has treated patients with more than two doses, which would likely 
be necessary in ALS. MSCs are also being used in other neurological diseases, and there are now 
some signs of success in stroke28, multiple systems atrophy29, and multiple sclerosis30. We expect 
that this treatment modality will continue to be pursued in other neurological diseases in the 

 
 
Mayo ALS MSC Phase II – Page 7
 
 
future. The Phase II clinical trial proposed here represents the first time that a large clinical 
trial will test the efficacy of intrathecal MSCs in a neurodegenerative disease. This study 
will also provide new data on the safety of repeated MSC injections over one year, while 
previous studies have not treated for over two months. 
 
7) Precise mechanisms of action for MSCs are unclear. Data regarding the mechanisms of action 
for MSC in the nervous system have exclusively come from animal models. This clinical trial 
provides a unique opportunity to better understand how aaMSCs affect the human nervous 
system. If clinical efficacy is observed in this clinical trial, we will have collected an enormous 
diverse dataset of ALS patients treated with intrathecal aaMSCs that will help us dissect how 
intrathecal MSCs improve ALS. This dataset includes: 
 
Clinical Data: All patients will be classified in detail at the time of enrolment and 
longitudinally for >12 months that they are in the trial via our primary and secondary clinical 
endpoints.  
 
ALS Biomarkers: As described above, there will be serial collection of blood and CSF 
throughout the clinical trial to assess peripheral blood immune phenotyping, CSF/serum 
neurofilaments, and CSF miRNA. We will have the ability to observe how these ALS 
biomarkers change in response to intrathecal MSC therapy. Additionally, stored blood 
samples may be used to determine ALS genotypes (SOD1, C9ORF72, etc.) in patients that 
may correlate with treatment response. 
 
MSC in vivo direct effects: We will be able to correlate lumbosacral root nodularity with 
clinical response. Collected CSF will also be assayed for neurotrophic growth factor secretion 
from MSCs. 
 
MSC ex vivo assays: MSCs will be stored from all patients in the study in order to correlate 
characteristics of each patient’s MSC with a treatment response. If clinical efficacy is 
demonstrated with intrathecal MSCs in ALS we will perform detailed analyses of subjects’ 
MSCs to better understand that response by correlating the following tests with clinical 
response to MSCs. First, we will assay the MSC culture supernatant and investigate 
extracellular vesicles31, which may provide cellular signaling via miRNA and other factors. 
Second, we will quantify the ability of each subject’s MSCs to suppress the immune 
response32. Third, the proteome and mRNA profiles33 of MSCs will be compared between 
responders and non-responders to MSC treatment.  
 
Autopsies will be requested from all patients in the clinical trial to directly investigate 
aaMSCs in the CNS and their consequences. 
 
1.3. Results from our Phase I Dose-Escalation Study of Intrathecal aaMSCs in ALS. We have treated 
all patients in our 27-patient, Phase I dose-escalation safety trial using intrathecal aaMSCs in ALS. 
Patients have been treated with either 1 x 107, 5 x 107 (one or two monthly doses), or 1 x 108 (one or two 
monthly doses)3. Mean age at injection was 56 (range 36-75) and the male:female ratio was 15:12.  The 
mean follow-up post-injection has been 345 days (range 27-759 days).  There have been 16 deaths, all 
attributed to ALS, occurring with a mean time of 391 days post-injection (range 27-892 days).  One 
autopsy has been completed and three are pending.   
 
The safety profile thus far has been excellent and neither patients nor investigators have felt that 
treatment worsened the course of disease.  Apart from single cases of self-limited headache and tinnitus, 
the most common side-effect has been temporary low back pain and position-dependent radicular pain, 
which has occurred exclusively in patients receiving 1 x 108 cells (8 of 10 patients).  In all but one patient, 
this side effect has either resolved (n=5, pain duration 21-77 days), or was improving and tolerable on last 
follow-up.  In one patient that was in the group receiving 1 x 108 cells x 2 injections, the pain after the 
first injection was severe enough to postpone the second injection for 2 months, at which point the pain 

 
 
Mayo ALS MSC Phase II – Page 8
 
 
was 0/10.  Following the second injection, the pain returned within one week and persisted at a moderate 
level 5/10 with the use of analgesics until his death 4 months following the second injection.  In no  
patient were there bowel/bladder sphincter problems, continuous numbness/paresthesia, or noted 
unexpected progression of weakness. Signs of modest CSF inflammation were noted in patients receiving 
either 5 x 107 or 1 x 108 aaMSCs (Figure 1).  Nodular thickening of lumbosacral nerve roots was observed 
on MRI in 1 patient receiving 1 x 107, 9 patients receiving 5 x 107 and 9 patients receiving 1 x 108 
aaMSCs, which we hypothesize represents engraftment of the injected MSCs (Figure 1 – autopsies on 3 
patients pending).  While our Phase I study was not designed to provide efficacy data, 17 of the 27 
patients treated thus far report that they have noted changes in their ALS impairments for up to one month 
following injection, including (with overlap) as improved bulbar function (n=8), increased limb strength 
(n=5), decreased fasciculations (n=4), decreased stiffness (n=4), and improved energy (n=4).   
 
Our safety findings corroborate studies from other groups using intrathecal administration MSCs24-27. 
Again, it is notable that none of these studies has treated patients with more than two doses, which would 
likely be necessary in a chronic disease such as ALS.  
 
1.4 Biomarker Discovery and Validation are critical in ALS: Clinical trials in ALS are hampered by 
the lack of biomarkers for diagnosis, or that track with disease severity and respond to therapies. Recent 
evidence suggests that neurofilaments and peripheral blood immune phenotyping (PB-IP) are excellent 
candidates for biomarkers of ALS. Currently there are no validated biomarkers in ALS for diagnosis, nor  
 
FIGURE 1: Results from Phase I dose-escalation safety trial of intrathecal aaMSCs in ALS.  Nodularity of lumbosacral nerve roots 
(red arrows) was observed in patients 3 weeks following treatment with 5 x 107 aaMSCs (40%) and 1 x 108 aaMSCs (100%),  as 
shown on MRI images (A: axial T2-weighted sequence, B: sagittal T2-w2eighted sequence and C: sagittal gadolinium-enhanced T1-
weighted sequence).  D: ALSFRS-R decreased over time in all patients. E: Elevations in CSF protein and nucleated cells were seen in 
a dose-dependent fashion.  
 

 
 
Mayo ALS MSC Phase II – Page 9
 
 
 
that track with disease severity or rate of progression. Furthermore, there are no pharmacodynamics 
biomarkers that are altered by the one ALS treatment, riluzole.  
 
Cerebrospinal Fluid (CSF) neurofilament, reflecting motor neuron axonal breakdown, has emerged as 
a promising surrogate of disease progression34 and we will incorporate this biomarker into the present 
study. Measured by ELISA or chemiluminescent immunoassay, reactivity for the phosphorylated heavy 
subunit of neurofilament (pNF-H) and neurofilament light subunit (NF-L) is increased in CSF and plasma 
in ALS compared to healthy subjects and disease controls, and is stable over 6 months. This supports the 
hypothesis that elevated pNF-H and NF-L concentrations may be useful biomarkers in ALS34-36. pNF-H 
and NF-L levels in CSF are reported to correlate with rate of disease progression and survival in ALS, 
with elevated levels consistently shown to be associated with more rapidly progressive disease and shorter 
survival35,36.  
Published and unpublished data from longitudinal studies in patients with ALS have shown that 
pNF-H in CSF measured serially remain relatively stable over intervals of at least 6 months35. These 
observations are consistent with studies demonstrating that a single baseline measurement of pNF-H or 
NF-L may predict subsequent progression rate and survival. However, there are no data available 
regarding the half-life of CSF pNF-H or NF-L in chronic neurologic disorders that have persistent 
elevation of neurofilament levels (such as ALS). In the absence of effective 
neuroprotective/neuroregenerative therapy in ALS, there has been no means to readily investigate 
anticipated reductions in CSF or plasma pNF-H or NF-L levels in response to treatment. Evaluation of 
pNF-H and NF-L as putative pharmacodynamic biomarkers as such requires further study and is an 
appropriate aim in ALS therapeutic trials in which pNF-H and NF-L can be collected pre- and post-
treatment. 
 
Immune markers: Likewise, alterations of the immune system have been associated with ALS37,38. A 
recent study has demonstrated that drugs that modulate macrophage phenotype may positively impact 
ALS39. Our preliminary data using 10-color flow cytometry demonstrate differences in the peripheral 
blood immune phenotyping (PB-IP) between patients with ALS and healthy volunteers. We are in the 
process of correlating these findings with clinical data to determine how they may be used in clinical 
practice and clinical trials.  
 
MicroRNAs (miRNAs) are non-coding RNA molecules that are involved in post-transcriptional 
regulation and have recently gained attention as biomarkers associated with ALS. Alterations of specific 
miRNAs have been reported in ALS patient samples of leukocytes37,40,41, CSF37,40, and CNS tissue40,42,43. 
Further studies will be necessary to validate these findings and interpret their meaning. Importantly, it 
will be necessary to establish if these miRNA change over time and whether they are correlated with 
disease progression and/or in response to therapies. We have established the ability to detect miRNA in 
patient CSF and will carry this further into this trial. 
 
2.0 
Goals  
 
The Goal of the Proposed Study is to perform an open label, 69 subject, Phase II multi-site clinical trial to 
investigate the safety and efficacy of intrathecal treatment of aaMSCs in ALS.  Patients will be treated 
with up to 1 x 108 aaMSCs every 3 months for a total of 4 intrathecal injections over 12 months. Reduced 
dose treatments will be allowed based on specific adverse events described below.  Multiple biomarkers 
will be tracked throughout the clinical trial and correlated with response to treatment. Patients will be 
enrolled at Mayo Clinic Rochester, Arizona or Florida.  This study will initially have all fat biopsies and 
MSC administrations performed at Mayo Clinic in Rochester.  Subsequently, these procedures will 
expand to the two other Mayo Clinic sites in Arizona and Florida as the manufacturing capabilities are 
finalized. 

 
 
Mayo ALS MSC Phase II – Page 10
 
 
 
2.1 Primary Endpoint (Safety): Our Phase I dose-escalation study of this treatment demonstrated safety 
at the maximum dose (1 x 108 aaMSCs x 2 monthly injections).  It is likely that any stem cell therapy will 
require multiple treatments and this Phase II proposal extends the treatment period to 4 injections every 3 
months in order to establish safety with this extended regimen. The primary safety endpoint will be the 
safety of repeated intrathecal aaMSCs. The overall number and frequency of adverse events and 
unexpected severe adverse events during the 12-month treatment period among study subjects will be 
documented. Number and frequency of adverse events will be recorded from the time of enrollment until 
the end of the follow-up period or, in the case of early withdrawal, to the time of study withdrawal. 
Neurological examination, MRI lumbosacral spinal cord, blood and urine samples will be analyzed at 
specified times throughout the study for safety.  At the time of each intrathecal injection, CSF will be 
sampled for routine safety parameters.   
 
Summary of Recommendations from November 19, 2020 DSMB Meeting: 
At the November 19, 2020 DSMB meeting, it was recommended that the trial protocol be updated to 
address the increased recognition that subjects appear to be having increased back/leg pain following ad-
MSC intrathecal injection and that this AE is threatening long-term feasibility of the study and the 
therapy.  In response to this, the investigators have decided to change the starting dose from 1 x 108 
aaMSCs to 5 x 107 aaMSCs.  For those that tolerate this dose, there is opportunity to increase the dose by 
2.5 x 107 aaMSCs at subsequent doses to maximum dose of 1 x 108 aaMSCs.  For those that do not 
tolerate a given dose, there continues to be option to decrease subsequent doses (by 2.5 x 107 aaMSCs to a 
minimum dose of 1 x 107 aaMSCs) or skip dose if there is moderate pain.  Furthermore, the DSMB 
recommended that subjects with preexisting chronic back pain and treatment (epidural injections or 
surgeries) should be excluded from study. 
 
As part of this study, all future patients will start at 5 x 107 aaMSCs.  Dose-Modification Rules (Section 
7.2) are in place to allow for lower doses in patients that have a significant Expected Adverse Event of 
back and leg pain.  The Dose-Modification Rules also allow for increasing doses for those who tolerate a 
given dose (to a maximum dose of 1 x 107 aaMSCs). This will provide further safety information that can 
be beneficial in future studies. 
 
2.2 Secondary Endpoints (Efficacy):  In order to investigate efficacy in this study, we will use both 
conventional ALS clinical endpoints and perform a “responder” analysis that can be correlated with the 
extensive proposed biomarker studies. 
 
All 69 patients enrolled in this Phase II study will have a 3 month lead-in observation period to determine 
baseline slope of progression, and subsequently will receive 4 intrathecal aaMSC treatments every 3 
months.  Throughout the study, we will collect measures of function with the ALS Functional Rating 
Scale – Revised (ALSFRS-R), which is the current standard clinical endpoint in ALS clinical trials.     
 
Primary statistical analyses for secondary efficacy endpoints will be: 
1) Average slope of ALSFRS-R progression in the study group will be compared with a cohort of 
subjects from the PRO-ACT ALS clinical trial database44 that will be matched for age, gender, 
and riluzole use (5 PRO-ACT subjects for every study subject). 
2) Average slope of ALSFRS-R progression in the 3 month lead-in period will be compared to the 
average slope of ALSFRS-R progression in the 12 month treatment period for study subjects. 
 
2.3 Exploratory Endpoints:  
 
2.3.1 Responder Analysis of Biomarkers:  

Mayo ALS MSC Phase II – Page 11
At the maximum dose in our Phase I study, 60% of patients reported subjective improvements in speech, 
dexterity, and spasticity domains. Other patients; however, reported no changes in their ALS symptoms.  
It is critical to understand this “responder effect” and whether it is a treatment effect or whether it is 
placebo-related.   We define “responder” as those who exhibit a >25% improvement in the slope of 
ALSFRS-R between the lead-in and treatment periods.  The following biomarkers will then be 
compared in the “responders” and “non-responders” (no improvement or worsening in ALFRS-R slope): 
1)
Peripheral blood immunophenotyping: we have evidence that a proportion of patients
with ALS have a unique peripheral blood immune system signature.  Furthermore, we
have observed specific changes in peripheral blood immune system in patients receiving
intrathecal aaMSCs in our Phase I study.
2)
Neurofilaments: CSF and serum neurofilaments have emerged as a promising marker of
neurodegeneration in ALS.  Samples will be collected throughout the study to be used for
markers of “responders” and progression in ALS.
3)
CSF microRNA: Several lines of evidence suggest that miRNA may serve as biomarkers
of ALS. Additionally, aaMSC potency may relate to miRNA secretion from extracellular
vesicles.  We will evaluate miRNA from CSF in study patients.
4)
aaMSC Biomarkers: If there are clear responders to aaMSC therapy, stored MSCs will
be evaluated ex vivo (via proteomics, extracellular vesicles, miRNA and immune
suppression assays) to help determine whether response may reflect the aaMSC or the
patient response to aaMSC therapy.
2.3.2 Exploratory Clinical Endpoints: 
1)
Change in the slope of hand-held dynamometry (HHD):  We will assess global
strength using a composite of 9 standard motions, performed bilaterally (shoulder flexion,
elbow flexion/extension, wrist extension, finger spread, hip flexion, knee
flexion/extension, and ankle dorsiflexion). Comparisons will be made between the
treatment period in study subjects and PRO-ACT controls.  Comparisons will also be
made between the 3-month lead-in and the 12-month observation period in study
subjects.
2)
Modified Ashworth Spasticity Scale: The muscle tone of bilateral elbows and ankles
will be quantified using the Modified Ashworth Spasticity Scale45. Comparisons will be
made between the 3-month lead-in and the 12-month observation period in study
subjects.
3)
Edinbourgh Cognitive ALS Screen (ECAS): This is a 136-point test of cognitive
function designed for patients with ALS46, which assesses executive, language, memory,
visuospatial functions. Comparisons will be made between the 3-month lead-in and the
12-month observation period in study subjects.
4)
ALS Specific Quality of Life-Revised (ALSSQOL-R):  This is a 50 item instrument
that measures quality of life (QOL) for subjects with ALS47. Comparisons will be made
between the 3-month lead-in (V3) and the 12-month observation period (V16) in study
subjects.
The data gathered in this Phase II study will be used to decide whether to proceed with a pivotal Phase III 
study.  The current sample size is powered to detect a major change (50%) in ALSFRS-R slope.  A key 
goal is to identify correlations of “responders” and biomarkers in order to enrich their enrollment for a 
Phase III study.   
3.0 
Patient Eligibility   
3.1 Inclusion Criteria 

 
 
Mayo ALS MSC Phase II – Page 12
 
 
 
All patients will have ALS diagnosed as possible, laboratory-supported probable, probable, or 
definite as defined by the World Federation of Neurology criteria for the diagnosis of ALS. 
  
 
Examination and neurophysiological testing confirm a pure motor syndrome compatible with the 
diagnosis of ALS.  All other possible causes of weakness have been excluded by extensive 
investigations. 
 
Age greater than 18 years, if female, must be post-menopausal, had a hysterectomy, or agree to 
two forms of birth control. 
 
Permanent resident or citizen of the United States. 
 
Geographic accessibility to the study site and willingness and ability to comply with follow-up. 
 
History of a chronic onset of a progressive motor weakness of less than two years duration. 
 
Subjects must be taking a stable dose of riluzole for at least 30 days prior to enrolment or not be 
on riluzole, and not have been on it for at least 30 days prior to enrolment (riluzole-naïve subjects 
are permitted in the study).  
 
Subjects must be taking a stable dose of oral Radicava® (edaravone) for at least 30 days prior to 
enrolment or not be on oral Radicava® (edaravone), and not have been on it for at least 30 days 
prior to enrolment (edaravone-naïve subjects are permitted in the study).  
 
Able to comply with protocol requirements, including MRI testing. 
 
Can provide written informed consent. 
 
3.2 Exclusion Criteria 
 
Use of intravenous Radicava® (edaravone) within 30 days of screening or intent to use 
Intravenous Radicava® at any time during the course of the study including the follow up period. 
 
Any clinically significant medical condition (e.g., within six months of baseline, had myocardial 
infarction, angina pectoris, and/or congestive heart failure) that, in the opinion of the investigator, 
would compromise the safety of patient. 
 
Pulmonary Slow Vital Capacity (SVC) less than 65% of predicted for age, gender, and body type. 
 
Autoimmunity, including Crohn’s disease or rheumatoid arthritis 
 
Current use of immunosuppressant medication or use of such medication within 4 weeks of 
Screening visit (Visit 1). 
 
Current use of any of the following investigational medicinal products or nutritional supplements 
being used or studied  for the treatment of ALS: 
 
o Acetyl carnitine 
o Methylcobalamin (B12) 
o BASIS (nicotinamide riboside and 
pterostilbene) 
o N-acetyl-cysteine 
o Biotin 
o  
o Calogen 
o Retigabine 
o Inosine 
o Rasagiline 
o Interleukin 2(Aldesleukin, 
Proleukin®) 
o  
o L-Serine 
 
 
 
Unwilling to forgo initiating the use of any new supplements during participation in the study. 
 
Malignancy 5 years prior to enrollment, including melanoma, with the exception of localized skin 
cancers (with no evidence of metastasis, significant invasion, or re-occurrence within three years 
of baseline).  
 
Active systemic or local infection near the lumbar puncture site. 

 
 
Mayo ALS MSC Phase II – Page 13
 
 
 
Inability to lie flat for the duration of intrathecal cell transplantation, or inability to tolerate study 
procedures for any other reason. 
 
Other active systemic disease as defined by laboratory abnormalities delineated in Appendix III. 
 
Unwillingness to discontinue herbal medications or other unapproved drugs 
 
Enrolled in an investigational drug trial within 30 days of baseline visit 
 
Prior stem cell therapy for a neurological disease 
 
Kokmen Short Test of Mental Status score <32 
 
Presence of a tracheostomy 
 
Ventilator dependent 
 
Pregnancy  
 
Men or women of childbearing potential who are unwilling to employ adequate contraception 
 
Chronic low back pain requiring invasive procedures (i.e. epidural injections or lumbar spine 
surgery) 

 
 
Mayo ALS MSC Phase II – Page 16
 
 
5.0 Protocol Treatment 
 
5.1 Study Drug Description 
The investigational product consists of autologous adipose-derived MSCs, suspended in 5-10 mL 
Lactated Ringer’s.  The MSC are provided in a sterile syringe labelled with appropriate patient and 
product identifiers ready for intrathecal injection.  The MSCs uniformly express CD90, CD105, CD73 
HLA Class I, and CD44.  The MSC are negative for CD14, CD45 and HLA DR.  Additionally the cells 
must have no clonal abnormalities detected by cytogenetic testing.  
 
5.2 MSC Dose Regimen 
Cells will be administered intrathecally at a single dose in a volume of 5-10 mL, all patients will receive 5 
x 107 intrathecal aaMSCs at the first injection (Visit 4).  Subsequent doses may be reduced or increased 
by 2.5 x 107, or skipped based on Dose Modification Rules (Section 7.2). Maximum dose is 1 x 108 
aaMSCs, and minimum dose is 1 x 107 aaMSCs.  
  
5.3 Preparation of Cellular Product  
The adipose biopsy tissue will be delivered to and processed in the Mayo Clinic Rochester Human 
Cellular Therapy Lab.  Cells will be expanded ex vivo in this cGMP facility using standard operating 
procedures.  Cells will be cryopreserved during release criteria analysis. Cells will be tested for 
phenotype, mycoplasma, culture sterility, and cytogenetic analysis.  Cells not meeting release criteria will 
not be administered to the patient.  Upon meeting release criteria and upon appropriate patient scheduling, 
cells will be returned to culture and administered 3-5 days after culture.  Additional release testing will 
occur, but cells will be administered without completion of final testing.  In the event that the final 
number of MSCs is less than the scheduled amount (if scheduled amount is 2.5 x 107 – 1 x 108 aaMSC), 
MSCs will be delivered intrathecally to patient as long as the MSC number is > 2 x 107 (20-100 million 
MSCs). 
 
In the event there is no cell growth from the tissue obtained from the first biopsy, one further attempt will 
be made from a second biopsy. If the second attempt fails to grow cells, no further attempts will be made, 
and the subject will not continue in the study.  Thus far we have successfully generated cell growth from a 
single fat sample in all 28 ALS patients attempted. Thus we anticipate a very low failure rate for cellular 
manufacturing. 
 
6.0  Study Procedures 
 
This is an open-label Phase II study of intrathecal aaMSCs in patients with ALS.  The goal is to treat 69 
patients in this study.  Patients will undergo a 3-month lead in period followed by 4 intrathecal injections 
spaced 3 months apart.  The standard dose will be 5 x 107 aaMSCs, but dose modification may occur for a 
given patient as dictated by Expected Adverse Events and tolerability.   Patients will undergo systematic 
safety studies (blood, CSF, MRI) and efficacy measures (ALSFRS-R, HHD) throughout the trial. 
 
 
 

 
 
Mayo ALS MSC Phase II – Page 17
 
 
6.1 Study Schema 
 
 
 
 
6.2 Study Procedures Delineated by Visit 
 
Subjects that are enrolled at Mayo Clinic Rochester will have all Visits in Rochester.  Subjects that are 
enrolled in Mayo Clinic Arizona or Mayo Clinic Florida will have Visit 2 (fat biopsy) and Visits 4, 7, 10 
and 13 (MSC treatments) in Rochester, but all other visits will occur locally at the enrolling site. 
 
Screening Visit & Informed Consent (Visit 1 – V1; 12 weeks prior to 1st MSC treatment) 
Informed Consent will be obtained during the screening visit by a neurologist with experience in the conduct 
of clinical trials for treatment of ALS.  The draft informed consent document is attached (Appendix II).  
Patients will have access to the consent document at least 24 hours prior to meeting with the study 
neurologist.   
 
As part of the informed consent, potential subjects will be asked to volunteer for two voluntary aspects of 
the study: 
 
Genetic screening for ALS-causing genes 
 
Autopsy at the time of death 
 
After informed consent is obtained, screening tests will take place during which the patient will be assessed 
for study eligibility.  This includes the following: 
1) Review of inclusion and exclusion criteria 
2) Screening blood collection in order to screen for systemic disease, including analysis for 
complete blood counts (with WBC differential), comprehensive metabolic panel (Na, K, Cl, 
HCO3, BUN, creatinine, Ca, AST, ALT, alkaline phosphatase), Mg, Phosphorus, uric acid, 
creatine kinase, ESR, and PT/INR 
3) 12 lead ECG 
4) Urinalysis and urine pregnancy test 
5) Slow vital capacity 
6) Hand-held dynamometry (HHD) 
Study Schema:  FB: Fat Biopsy, IT-MSC: intrathecal MSC treatment; Arrows: clinic visits; Purple Arrows: 
clinic visit includes MRI lumbosacral spine 

 
 
Mayo ALS MSC Phase II – Page 18
 
 
7) ALSFRS-R 
8) Review of concomitant medications and instructions regarding the use of these medications while 
enrolled in this study. 
9) Edinbourgh Cognitive ALS Screen (ECAS) 
10) Columbia Suicide Severity Rating Scale (C-SSRS) 
11) Modified Ashworth Spasticity Scale 
12) Review activities of daily living (ADL) 
 
Within two years of MSC treatment, patients must undergo MRI of the lumbosacral spinal cord (lumbar 
level).  If MRIs were performed at an outside institution, a study investigator will review these images for 
quality and pathology.  If the MRIs have not been performed elsewhere (or are poor quality), these studies 
will be performed prior to MSC treatment.  The MRIs will serve two functions:  1) helps ensure that there 
is no alternate explanation for patients’ symptoms, 2) serves as a baseline safety study prior to MSC 
treatment.    
 
Subcutaneous Fat Biopsy (V2; 8 weeks prior to 1st MSC treatment) 
 
A subcutaneous fat biopsy will be taken from either the abdomen or thigh of the study subject. The actual 
site of the biopsy will be determined by the general surgeon or trained health professional, at the time of 
the procedure.  The subject will be given a local anesthetic (usually 1% lidocaine) at the site of the biopsy.  
The fat biopsy will be done through a small (1-2 inch) incision where approximately 15 cc of subcutaneous 
fat is removed from under the skin.  The biopsy site will be sutured with an intradermal absorbable suture 
that will not require suture removal. The suture will be reabsorbed in 1-3 weeks.  This type of biopsy is 
usually well tolerated and offers minimal risk to the subject.  These risks can include discomfort and 
bruising at the biopsy site.  The subject will be given instructions on after care of the biopsy site following 
the procedure.   
 
Lead-in Period (V1-3) 
Patients will have monthly study visits prior to MSC infusion, for a total of a 3-month lead-in period.  At 
each lead-in visit, patients will undergo the following:  
1) Physical Examination 
2) Neurological Examination 
3) Vital Signs 
4) Medication Review 
5) ALSFRS-R 
6) C-SSRS 
7) HHD 
8) Review Adverse Events (V2 and V3 only) 
9) Review Activities of Daily Living (ADL) (V1 only) 
10) ALSSQOL-R (V3 only) 
11) Optional research blood collection (V2 only; only if patient has consented to this optional 
collection/testing) 
 
 
Phone Calls 
Two weeks prior to each intrathecal administration of aaMSCs, the study coordinator will contact the 
subject by phone, review adverse events, ADL, ALSFRS-R, pain assessment and review patient perceived 
changes.   
 
 
 

 
 
Mayo ALS MSC Phase II – Page 19
 
 
Intrathecal administration of aaMSCs and acute monitoring (V4, V7, V10, V13; 12 weeks apart) 
Study subjects will be admitted to Hospital the day of MSC infusion, and will stay there for at least 24 
hours after treatment.  During the day of hospital admission, the patients will undergo the following: 
1) Physical Examination 
2) Neurological Examination 
3) Safety blood collection to serve as baseline prior to MSC injection, including analysis for 
complete blood counts (with WBC differential), comprehensive metabolic panel (Na, K, Cl, 
HCO3, BUN, creatinine, CA, AST, ALT, alkaline phosphatase), Mg, Phosphorus, uric acid, 
creatine kinase, ESR, and PT/INR 
4) Optional research blood collection (to be collected at same time as safety bloods – if patient 
consents to optional collection/testing) 
5) Urinalysis and Urine Pregnancy test 
6) Modified Ashworth Spasticity Scale 
7) Vital Signs 
8) Medication Review 
9) ALSFRS-R 
10) C-SSRS 
11) Pain assessment (prior to discharge from CRTU at each injection visit) 
12) Review patient perceived  changes  (starting  at V5) 
13) HHD 
14) Safety CSF collection 
15) Optional research CSF collection (to be collected at same time as safety CSF – if patient consents 
to optional collection/testing) 
16) Intrathecal administration of MSCs 
17) Distribute Patient Diary (for review during V5, V8, V11 and V14) 
 
Prior to the intrathecal injection, the patient will have a saline lock placed. This is a safety measure should 
the subject need IV fluids or medications should they experience a medical emergency during or after the 
injection.   
 
MSCs will be delivered from the Human Cellular Therapy Lab in syringes at 4oC and administered via 
lumbar puncture within 12 hours of preparation. A lumbar spinal needle will be placed in the subarachnoid 
space by a trained health practitioner (possibly under fluoroscopic guidance), and a baseline CSF sample 
will be collected (later analyzed for protein, glucose, nucleated cells, RBCs, cytology, oligoclonal bands, 
IgG Index). Subsequently, aaMSCs will be infused into the CSF in 5-10 mL of Lactated Ringers solution 
over 1-2 minutes, followed by 1mL Lactated Ringers flush by one of the study physicians.  In the event that 
the final number of MSCs is less than the scheduled amount, MSCs will be delivered intrathecally to patient 
as long as the MSC number is > 2 x 107 (20-100 million MSCs). After cell infusion and if the patient is 
tolerant, they will be rotated every 15 minutes in a Trendelenburg position (with help from nursing staff if 
necessary) for 2 hours to maximize even distribution of cells in the CSF.  
 
During the patient’s stay in the CRTU the subject will be observed by research nurses for any Adverse 
Events during and immediately following the intrathecal injection of the MSC’s.  The patient’s vital signs 
(including pain assessment) will be monitored every 15 minutes for one hour, and then hourly for four hours 
(+/-15 minutes after the 2nd, 3rd, and 4th hour), and then every four hours (+/-15 minutes) until discharged 
from the CRU, which will be at least 24 hours after injection. 
 
If after MSC injection, release testing reveals unexpected contamination, the following procedure will be 
implemented: 
a) The Principal Investigator and study physicians and patient will be immediately notified by 
members of the Human Cell Therapy Laboratory.   

 
 
Mayo ALS MSC Phase II – Page 20
 
 
b) An emergent infectious disease specialty consultation will be made to help determine appropriate 
further testing (CSF and/or blood cultures), antibiotic therapy directed to identified pathogen, and 
continued clinical follow-up.  Continued active patient monitoring will comprise weekly patient 
contacts by study staff (clinic visits or phone calls) for four weeks (or until completion of 
antibiotics) to document symptoms. 
c) An investigation will be conducted by the Human Cell Therapy laboratory to determine the root 
cause of contamination,  
d) An action plan will be enacted for corrective/preventative actions in order to mitigate the likelihood 
for recurrence.  
 
Early Follow-up Visits (V5, V8, V11, V14; two weeks after aaMSC treatment) 
Two weeks following MSC therapy, patients will have a study visit and undergo the following:  
 
1) Physical Examination 
2) Neurological Examination 
3) Modified Ashworth Spasticity Scale 
4) Vital Signs 
5) Medication Review 
6) Adverse Events Review 
7) ALSFRS-R 
8) C-SSRS 
9) Pain assessment   
10) Review patient perceived  changes  
11) HHD 
12) Safety Bloods 
13) Optional research blood collection (V5 only; (to be collected at same time as safety bloods – if 
patient consents to optional collection/testing)) 
14) Urinalysis 
15) Review Patient Diary 
 
Late Follow-up Visits (V6, V9, V12, V15; 6 weeks after aaMSC treatment) 
Patients will have a visit 6 weeks after aaMSC treatment.  At Late Follow-up visit, patients will undergo 
the following:  
1) MRI of lumbosacral spinal cord for safety purposes (V6 only) 
2) Physical Examination 
3) Neurological Examination 
4) Vital Signs 
5) Medication Review 
6) Adverse Events Review 
7) ALSFRS-R 
8) C-SSRS 
9) Pain assessment (which will dictate subsequent aaMSC dose) 
10) Review patient perceived  changes  
11) HHD 
12) Lumbar Puncture for safety CSF collection (possibly under fluoroscopic guidance) for safety 
(protein, glucose, nucleated cells, RBCs, cytology, oligoclonal bands, IgG Index) and stored for 
research 
13) Optional CSF collection (to be collected at same time as safety CSF collection – if patient 
consents to optional collection/testing) 
 
 

 
 
Mayo ALS MSC Phase II – Page 21
 
 
Final Observation Visit (V16; 48 weeks after 1st aaMSC treatment; 6 weeks after last Late Follow-up Visit) 
 
At the Final Observation Visit, patients will undergo the following:  
1) Physical Examination 
2) Neurological Examination 
3) Vital Signs 
4) Medication Review 
5) Adverse Events Review 
6) Safety blood collection 
7) Optional research blood collection (to be collected at same time as safety bloods – if patient 
consents to optional collection/testing)  
8) Urinalysis  
9) ALSFRS-R 
10) C-SSRS 
11) Modified Ashworth Spasticity Scale 
12) Pain assessment  
13) Review patient perceived  changes  
14) ALSSQOL-R  
15) HHD 
16) MRI of lumbosacral spinal cord.   
17) ECAS 
 
6.3 Procedures for when patients are unable to attend Study Visits. 
 
ALS is a progressive paralytic disorder and it is common that patients progress to where they are 
unable to travel to a clinic visit.  When this occurs for study patients, subjects (and/or caregivers) 
will be contacted by phone to review adverse events and perform the ALSFRS-R. 
 
6.4 Voluntary testing for patients 
 
6.4.1 Genetic testing: Given the increasing number of genes that cause ALS, it has been suggested 
that patients voluntarily subject to genetic testing for clinical trials in ALS in order to have 
this information available as a post-hoc analysis. We will collect DNA for post-hoc genetic 
testing following informed consent. 
 
6.4.2  Autopsy:  A key question in stem cell therapy and ALS is: What is the fate of injected cells 
and what are their effects?  All patients in this study will be asked to volunteer for eventual 
autopsy at the time of their death in order to address this question.  
 
 

 
 
Mayo ALS MSC Phase II – Page 22
 
 
7.0 Dosage Modification Based on Adverse Events 
 
7.1 Adverse Event Monitoring 
An adverse event is defined as any noxious, pathologic or unintended change in anatomical, 
physiologic, or metabolic function as indicated by physical signs, symptoms, and/or laboratory 
changes occurring during this phase II clinical trial.  This includes an exacerbation of a pre-
existing condition. 
 
Dose limiting toxicity (DLT) criteria:  DLT will be defined as an adverse event attributed 
(definitely, probably, or possibly) to the study treatment AND meeting one of the following 
criteria: 
Toxicity 
DLT Definition (CTCAE v5) 
Hematologic 
≥ Grade 3 ANC for  7 days or PLT < 75,000 for 
≥7 days 
Infection 
≥ Grade 3 
Febrile Neutropenia 
Defined as fever ≥ 38.5oC (38 > 1 hour) with 
grade ≥ 3 neutropenia 
Dermatologic 
≥ grade 2 erythema multiforme, ulceration, or 
urticaria that does not resolve to < grade 2 within 
two weeks 
Pulmonary 
Newly identified ≥ grade 2 bronchial obstruction, 
pneumonitis, or wheezing 
Immune system 
≥ grade 2 allergic reaction or autoimmunity 
Other Non-hematologic 
 grade 2 that does not resolve to < grade 2 
within two weeks 
Neurologic or psychiatric 
≥ grade 3 that do not resolve within two weeks 
 
 
Serious Adverse Events:  Serious Adverse Events are defined as an adverse event meeting any 
one of the following criteria: 
1.  Fatal or life-threatening 
2.  Temporarily or permanently disabling 
3.  Requires or prolongs inpatient hospitalization 
4.  Requires intervention to prevent permanent impairment or damage 
5.  Is a cancer or overdose 
 
In addition, specific neurological incidents that will be considered a Serious Adverse Event 
include the following:  
 unexpected CNS infection or inflammation 
 new onset seizures 
 sudden unexplained deterioration of ALS 
 new CNS MRI abnormalities with mass effect, or  
 severe and long-lasting (> two weeks) headache with increased intracranial pressure.   
 
Furthermore, experiences that in the investigator’s opinion suggest a significant hazard, 
contraindication, side effect, or precaution that may be associated with the use of the intrathecal 
MSC therapy will be considered a Serious Adverse Event.   
 
When assessing whether an adverse event is related to a medical treatment or procedure, the 
following attribution categories are utilized: 

 
 
Mayo ALS MSC Phase II – Page 23
 
 
 
Definite - The adverse event is clearly related to the investigational agent(s). 
Probable - The adverse event is likely related to the investigational agent(s). 
Possible - The adverse event may be related to the investigational agent(s). 
Unlikely - The adverse event is doubtfully related to the investigational agent(s). 
Unrelated - The adverse event is clearly NOT related to the investigational agent(s) 
 
Expected adverse events:  In our Phase I dose-escalation safety trial of intrathecal aaMSCs for 
ALS, eight of the ten subjects receiving 1 x 108 MSC dosage developed low back and leg pain 
that began within a week of treatment, peaked in the first three weeks, and then resolved or 
became very mild (1/10 on visual analog scale).  The pain exhibited variable intensity (2-8/10 on 
visual analog scale) and duration (range 42-77 days) among the subjects. In one subject, the pain 
led to postponement of the 2nd treatment by 2 months until pain had resolved.  The pain recurred 
upon 2nd injection, again lasting two months3 (Appendix I).   
 
Additionally, previously reported MRI lumbosacral spine abnormalities will be considered 
expected adverse events.  Elevated CSF protein and cell count will also be considered expected 
adverse events.  These adverse events related to MRI and CSF will be logged as part of the 
patient’s CRF. 
 
In the study by Karussis et al.48, which used intrathecal treatment of bone-marrow derived 
MSCs, no Serious Adverse events occurred; however, adverse events occurred (without mention 
of severity).  In their 19 ALS patients, the following adverse events were noted: fever (11), 
headache (5), leg pain (2), and dyspnea (1).  Based on the available data, fever, headache, and 
back/leg pain may be regarded as expected adverse events.   
 
In summary, Expected Adverse Events for this study attributable to the treatment are: 
 Back pain developing within 2 weeks of lumbar puncture, improving or resolving within 4 
weeks of onset 
 Headache developing within 2 weeks of lumbar puncture, improving or resolving within 4 
weeks of onset 
 Leg pain developing within 2 weeks of lumbar puncture, improving or resolving within 4 
weeks of onset 
 CSF pleocytosis (<50 cells/uL) 
 CSF protein elevation 
 Abnormal MRI spine with meningeal thickening, nerve root enlargement and nodularity, 
+/- gadolinium enhancement 
 
7.2 Dose modifications and stopping rules based on adverse events 
 
Adverse events, MRI lumbosacral spine abnormalities, and subjective clinical responses 
occurred in a dose-dependent fashion.  Eight of ten patients receiving frequency 1 x 108 aaMSCs 
in our Phase I study developed low back pain and leg pain of variable intensity and duration, 
which was not observed at the 5 x 107 dosage.  MRI lumbosacral abnormalities were seen in 
9/12 receiving 5 x 107 and 9/10 receiving 1 x 108 aaMSCs.  Subjective clinical response 
frequency was similar in the 5 x 107 and 1 x 108 dosages; however, the responses were more 
compelling in the higher dosage.  The following dose modification rules were developed with 
the Phase I data in mind, and modified following the DSMB meeting of November 19, 2020 
(Figure 2).  The goal is to provide the maximum tolerated dose during the one-year study, 
starting at the middle dose in our Phase I trial (5 x 107).  In summary, if patients tolerate a given 
dose, the next dose can be increased by 2.5 x 107 (to 7.5 x 107 or 1 x 108).  Conversely, if a 
subject develops significant low back and leg pain, the offending dose can be lowered at the next 
injection (either to 7.5 x 107, 5 x 107, or 1 x 107, depending on what dose caused pain). These 
rules will provide dose finding information for subsequent studies. 

 
 
Mayo ALS MSC Phase II – Page 24
 
 
 
7.2.1  If patients tolerate a given dose (defined by 0-1/10 pain at six weeks prior to the next 
scheduled injection), the next injection dose may be increased by 2.5 x 107, up to a 
maximum dose of 1 x 108 aaMSCs. 
 
7.2.2 If patients develop an Expected Adverse Event (back and leg pain of any severity) that 
remains at 2-4/10 intensity (mild-to-moderate pain on visual analog scale) six weeks prior 
to the next scheduled injection, patients must receive the next lower dose (7.5 x 107, 5 x 
107, or 1 x 107  aaMSC) in subsequent doses.  If the patient is already receiving 1 x 107 
aaMSCs, this dose may continue. 
 
7.2.3    If patients develop an Expected Adverse Event (back and leg pain of any severity) that 
remains 5-10/10 intensity (moderate-to-severe pain on visual analog scale) at six weeks 
prior scheduled aaMSC treatment, treatments will be discontinued until pain reduces to 
<5/10.  At that point, the patient will receive the next lower dose (7.5 x 107, 5 x 107, or 1 
x 107 aaMSCs) at the next scheduled injection timepoint. If the patient is already 
receiving 1 x 107 aaMSCs, this dose may continue. 
 
7.2.4  If patients develop a Dose-Limiting Toxicity (defined in 7.1), patients must receive the 
next lower dose (7.5 x 107, 5 x 107, or 1 x 107 aaMSC) in subsequent doses.  If the patient 
is already receiving 1 x 107 aaMSCs, this dose may continue.  
 
7.2.5 Once the dose has been lowered, it may not be increased at subsequent treatments. 
 
7.2.6 If at any point a Serious Adverse Event occurs that is determined to be possible, 
probable, or definitely related to aaMSC, no further intrathecal aaMSC treatments will be 
given to that patient until thorough review by the principal investigator, Institutional 
Review Board (IRB) and FDA. 
 
7.2.7  If at any point during the course of the trial, Serious Adverse Events occur in three 
subjects that are determined to be probable or definitely related to aaMSC, no further 
intrathecal aaMSC treatments will be given to any patient until thorough review by the 
principal investigator, DSMB, Institutional Review Board (IRB) and FDA. 
 
 
 
 

 
 
Mayo ALS MSC Phase II – Page 26
 
 
for subjects already enrolled at the time of Relyvrio FDA approval, we will request that 
subjects do not start Relyvrio until 6 weeks after the 2nd MSC injection (Visit 7).  If 
subjects choose to initiate Relyvrio prior to that time it will be treated as a protocol 
deviation, but subjects may stay in the clinical trial.  Inclusion criteria did not need to be 
amended because all subjects were enrolled at the time of the amendment regarding 
Relyvrio. 
 
 
 
9.0 Adverse Event (AE) Reporting and Monitoring 
 
Throughout the study the investigators will record all adverse events on the Adverse Event Case 
Report Forms (CRF), regardless of the severity or relationship to the study treatment.   
 
Investigators will treat patients with adverse events appropriately and observe them at suitable 
intervals until the experiences resolve or stabilize.  Adverse events may be discovered through 
observation of the patient, questioning of the patient, complaint by the patient, or by abnormal 
clinical laboratory values.  
 
The subject will be observed and questioned regarding the occurrence of possible adverse events.  
The questioning should be conducted with due regard for objectivity and, in particular, the 
questioner should gather information regarding adverse events from questioning the patient, 
spontaneous report by the patient, laboratory reports (including ECG), and clinician’s observations. 
 
In addition, adverse experiences may also include laboratory values that become clinically 
significant out-of-range values (defined in Appendix IV).  In the event of an out-of-range value, the 
laboratory test should be repeated until it returns to normal, or can be explained, and the patient’s 
safety is not at risk.   
 
Worsening of a pre-existing condition that is not consistent with the patient’s usual clinical course 
will be reported as an adverse event. 
 
9.1 Adverse Event Characteristics 
 
CTCAE term (Adverse Event description) and grade:  The descriptions and grading scales 
found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 
5.0 will be used for Adverse Event reporting.  All appropriate treatment areas should have 
access to a copy of the CTCAE version 5.0.  A copy of the CTCAE version 5.0 can be 
downloaded from the CTEP web site. 
 
9.1.1 
Attempts will be made to characterize all Adverse Events.  Patients enrolled in the 
study will be followed per Schedule of Events, out to 3 months after the last 
injection therapy.  Patients who are unwilling (due to study withdrawal) or unable 
(due to ALS debility) to attend clinic appointments will be contacted by phone at the 
scheduled intervals (if the subject gives consent) for inquiry into Adverse Events. 
 
9.1.2 
Adverse event monitoring and reporting is a routine part of every clinical trial.  
First, the event will be identified and graded using the CTCAE v5.0.  Next, it will be 
determined if the adverse event is related to the medical treatment or procedure.  
With this information, it will be decided whether an adverse event should be 
reported to the IRB and FDA as an expedited report (see Section 9.2) or as part of 
the routinely reported clinical data.  All Adverse Events reported via expedited 

 
 
Mayo ALS MSC Phase II – Page 27
 
 
mechanisms will also be reported via the routine data reporting mechanisms defined 
by the protocol. 
 
9.1.3  Assessment of Attribution 
 
When assessing whether an adverse event is related to a medical treatment or 
procedure, the following attribution categories are utilized: 
 
Definite - The adverse event is clearly related to the investigational agent(s). 
Probable - The adverse event is likely related to the investigational agent(s). 
Possible - The adverse event may be related to the investigational agent(s). 
 
Unlikely - The adverse event is doubtfully related to the investigational agent(s). 
Unrelated - The adverse event is clearly NOT related to the investigational agent(s) 
 
9.2   Expedited Adverse Event Reporting Requirements 
 
A report will be submitted to the FDA and IRB within7 working days in the following 
scenarios: 
 
 Unexpected Adverse events that are CTCAE v5.0 Grade 3, 4, or 5, and attributed to the 
treatment (possible, probable, or definite), that occur within 30 days of the last 
treatment 
 Any hospitalizations attributed to the treatment (possible, probable, or definite) that 
occur within 30 days of the last treatment  
 Adverse events that are CTCAE v5.0 Grade 5 (death), and attributed to the treatment 
(possible, probable, or definite), regardless of timeframe 
 Specific neurological adverse events: unexpected CNS infection or inflammation, new 
onset seizures, sudden unexplained deterioration of ALS, new CNS MRI abnormalities 
with mass effect (lumbosacral nerve roots excluded), or severe and long-lasting (> two 
weeks) headache with increased intracranial pressure, regardless of timeframe 
 
 
 

 
 
Mayo ALS MSC Phase II – Page 28
 
 
 
9.21 
Special Situations for Expedited Reporting 
EXPECTED Serious Adverse Events: Protocol Specific Exceptions 
to Expedited Reporting   
For this protocol only, the following Adverse Events/Grades are 
expected to occur within this ALS population and do not require 
Expedited Reporting, unless considered attributed to the treatment 
(possible, probable, or definite). These events must still be reported via 
Routine Reporting (see Section 10.3). * 
 
System Organ Class 
(SOC) 
Adverse event/ 
Symptoms 
CTCAE Grade at which 
the event will not be 
expeditedly reported. 
Generalized 
disorders and 
administration site 
conditions 
Gait disturbance 
<Grade 3 
Gastrointestinal 
disorders 
Dysphagia 
<Grade 3 
Investigations 
Vital Capacity Abnormal 
<Grade 3 
Weight loss 
<Grade 3 
Injury, poisoning 
and procedural 
complications 
Fall 
<Grade 3 
Musculoskeletal and 
connective tissue 
disorders 
Generalized muscle 
weakness 
<Grade 3 
Muscle Cramp 
<Grade 3 
Muscle weakness lower 
limb 
<Grade 3 
Muscle weakness trunk 
<Grade 3 
Muscle weakness upper 
limb 
<Grade 3 
Nervous system 
disorders 
Dysarthria 
<Grade 3 
Facial muscle weakness 
<Grade 3 
Muscle weakness left-
sided 
<Grade 3 
Muscle weakness right-
sided 
<Grade 3 
Spasticity 
<Grade 3 
Respiratory, thoracic 
and mediastinal 
disorders 
Dyspnea 
<Grade 4 
Hypoxia 
<Grade 3 
Laryngospasm 
<Grade 3 
 
NOTE: Suspected/actual events covered in this exception include any event that is 
considered ALS disease progression. 
 
9.3   Routine Adverse Event Reporting 

 
 
Mayo ALS MSC Phase II – Page 30
 
 
endpoints as well as secondary endpoints. For an assessment of primary efficacy endpoint, we 
will regress each patient’s ALSFRS-R score at each of the visits against time (months) to estimate 
a patient specific slope i.e. rate of decline in score per month. This will allow us to use all data 
points from a patient without a need for any imputation as long as a patient has a baseline and at 
least one post baseline measurement. This slope estimate will then be compared between two 
groups using two sample t-test or Wilcoxon rank sum test. If we identify imbalances among the 
groups at baseline or would consider accounting for any possible confounders then we will use 
regression based approach with slope as an endpoint adjusting for respective variables of interest. 
All tests will be two sided, and p values of less than 0.05 will be considered statistically 
significant. Given the smaller sample size and Phase IIB nature of the study, no adjustment for 
multiple comparisons will be made while analyzing several secondary endpoints. Analysis will be 
performed using intention to treat (ITT) data, which will include patients that were randomized 
and have received at least one intrathecal injection.  ITT approach minimizes biases and allow for 
more accurate assessment of the safety or efficacy of the treatment. Both safety and efficacy 
assessments will be considered prior to making a decision on future Phase III study.  Analysis of 
secondary endpoints such as ALSFRS-R (patient baseline as control), HHD and modified 
Ashworth Spasticity Scale will be assessed by comparing slope estimates between the lead-in 
period and the treatment period in a similar fashion as in the case of primary efficacy endpoint. A 
graphical analysis will also be done using scatter plots and/or boxplots by displaying the pattern 
of actual values as well as slopes between placebo and treatment groups.   
 
11.0 Treatment Extension Protocol 
 
Background:  During the execution of this Phase 2 trial, there have been subjective positive responses 
from patients that encouraged the researchers to pursue an extension arm of this trial. Due to limited 
resources, we have elected to offer Treatment Extension to the following sub-cohort meeting the 
following Inclusion & Exclusion Criteria within the Phase 2 clinical trial: 
 
Inclusion Criteria: 
1) Completion of the Phase 2 clinical trial, including the final visit. 
2) Improvement (or < 0.2 overall change) of the slope of the ALSFRS-R between the lead-in (V1-
V4) and treatment periods (V4-V16) 
3) If available, a decrease in the serum neurofilament light chain levels between the lead-in and 
treatment periods.  Decrease in neurofilament light chain has been accepted as a surrogate 
biomarker of reduced neurodegeneration by the FDA for the submission of Tofersen (anti-sense 
oligonucleotide) for SOD1-associated ALS. 
 
Exclusion Criteria 
1) Any clinically significant medical condition that, in the opinion of the investigator, would 
compromise the safety of patient. 
 
At the time of this protocol amendment submission, 6 patients meet these criteria (4 are yet to complete 
study).We will begin screening / inviting subjects that completed the study first and proceed to those that 
recently completed the study until the funding is exhausted. 
 
Treatment Extension Protocol 
The trial will offer the option for subjects who meet criteria as defined above to be retreated every 3 
months for one year (4 injections total). See Schedule of Events for details. In-person visits will be 
performed for the consent visit and MSC injection visits.  All other visits will be via phone or video.  
Dose-escalation rules will be used as per the main Phase 2 study (Section 7.2). Subjects will undergo 
repeat fat biopsy for manufacturing of MSCs.  
 

 
 
Mayo ALS MSC Phase II – Page 31
 
 
Patients will have post-treatment video visit follow-ups at 2 weeks and 6 weeks (in-person), which may 
be converted to in-person at the discretion of the investigator. Safety monitoring and reporting will 
continue as in the main protocol (Section 9.0) 
 
Informed Consent 
A new modified Informed Consent will be obtained by one of the investigator-neurologists with 
experience in the conduct of clinical trials for treatment of ALS.  The modified informed consent 
document is attached. 
 
Subcutaneous Fat Biopsy & MSC manufacturing 
 
A subcutaneous fat biopsy will be taken from either the abdomen or thigh of the study subject as per 
Section 6.2 (Subcutaneous Fat Biopsy V1).  MSC manufacturing will occur as per Section 5.3 
(Preparation of Cellular Product).   
 
Intrathecal administration of autologous MSCs and acute monitoring 
The protocol will follow the procedures detailed in Section 6.2 (Intrathecal administration of aaMSCs and 
acute monitoring). 
 

 
 
Mayo ALS MSC Phase II - 33
 
 
 
Test Schedule Footnotes 
 
1 CBC with differential, Comprehensive Metabolic Panel (Na, K, Cl, HCO3, BUN, Creatinine, Ca, AST, 
ALT, alkaline phosphatase), Mg, Phos, Uric Acid, Creatine Kinase, ESR, and PT/INR, Neurofilament light 
chain 
2 Urinalysis (dipstick) (Urine pregnancy test) 
3 CSF: protein, glucose, nucleated cells, RBCs, cytology 
   
4 Vital signs include blood pressure, temperature, pulse and respiratory rate. 
5 Height measured only at visit 1 if able to stand; weight measured at every visit.  
6 Activities of Daily Living (ADL): reviewed at V1, and phone calls 1, 2, 3 and 4 
7 Vital signs and pain assessment will be taken post-transplant every 15 minutes for the first hour. Vital 
signs and pain assessment will be taken hourly for four hours (+/- 15minutes after the 2nd, 3rd, and 4th 
hour) and then every four hours +/-15 minutes until discharge which is 18-24 hours after injection 
8 If there is a positive response on the C-SSRS, within 4 hours an MD-investigator will discuss with subject whether 
further evaluation and treatment is indicated. 
 
 
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Levy, Y. S., Gilgun-Sherki, Y., Melamed, E. & Offen, D. Therapeutic potential of neurotrophic 
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Storkebaum, E. et al. Treatment of motoneuron degeneration by intracerebroventricular delivery 
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Sun, H. et al. Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium 
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Prigione, I. et al. Reciprocal interactions between human mesenchymal stem cells and 
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Appel, S. H. et al. Hematopoietic stem cell transplantation in patients with sporadic amyotrophic 
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Mayo ALS MSC Phase II - 34
 
 
 
13 
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Gordon, P. H. et al. Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a 
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Meucci, N., Nobile-Orazio, E. & Scarlato, G. Intravenous immunoglobulin therapy in 
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Jin, K. et al. Comparison of ischemia-directed migration of neural precursor cells after 
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Satake, K., Lou, J. & Lenke, L. G. Migration of mesenchymal stem cells through cerebrospinal 
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20 
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21 
Gerdoni, E. et al. Mesenchymal stem cells effectively modulate pathogenic immune response in 
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22 
Park, H. J., Lee, P. H., Bang, O. Y., Lee, G. & Ahn, Y. H. Mesenchymal stem cells therapy exerts 
neuroprotection in a progressive animal model of Parkinson's disease. J Neurochem 107, 141-
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23 
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recovery. Neurology 59, 514-523 (2002). 
24 
Karussis, D. et al. Safety and immunological effects of mesenchymal stem cell transplantation in 
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25 
Mazzini, L. et al. Mesenchymal stromal cell transplantation in amyotrophic lateral sclerosis: a 
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26 
Oh, K. W. et al. Phase I trial of repeated intrathecal autologous bone marrow-derived 
mesenchymal stromal cells in amyotrophic lateral sclerosis. Stem Cells Transl Med 4, 590-597, 
doi:10.5966/sctm.2014-0212 (2015). 
27 
Petrou, P. et al. Safety and Clinical Effects of Mesenchymal Stem Cells Secreting Neurotrophic 
Factor Transplantation in Patients With Amyotrophic Lateral Sclerosis: Results of Phase 1/2 and 
2a Clinical Trials. JAMA neurology, 1-8, doi:10.1001/jamaneurol.2015.4321 (2016). 
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Kim, S. J., Moon, G. J., Chang, W. H., Kim, Y. H. & Bang, O. Y. Intravenous transplantation of 
mesenchymal stem cells preconditioned with early phase stroke serum: current evidence and 
study protocol for a randomized trial. Trials 14, 317, doi:10.1186/1745-6215-14-317 (2013). 
29 
Lee, P. H. et al. A randomized trial of mesenchymal stem cells in multiple system atrophy. Ann 
Neurol 72, 32-40, doi:10.1002/ana.23612 (2012). 
30 
Connick, P. et al. Autologous mesenchymal stem cells for the treatment of secondary progressive 
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150-156, doi:10.1016/S1474-4422(11)70305-2 (2012). 
31 
Katsuda, T., Kosaka, N., Takeshita, F. & Ochiya, T. The therapeutic potential of mesenchymal 
stem cell-derived extracellular vesicles. Proteomics 13, 1637-1653, doi:10.1002/pmic.201200373 
(2013). 

 
 
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32 
Bloom, D. D. et al. A reproducible immunopotency assay to measure mesenchymal stromal cell-
mediated T-cell suppression. Cytotherapy 17, 140-151, doi:10.1016/j.jcyt.2014.10.002 (2015). 
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34 
Boylan, K. B. et al. Phosphorylated neurofilament heavy subunit (pNF-H) in peripheral blood and 
CSF as a potential prognostic biomarker in amyotrophic lateral sclerosis. J Neurol Neurosurg 
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ALS biomarker. J Neurochem 111, 1182-1191, doi:10.1111/j.1471-4159.2009.06386.x (2009). 
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Brettschneider, J., Petzold, A., Sussmuth, S. D., Ludolph, A. C. & Tumani, H. Axonal damage 
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Butovsky, O. et al. Modulating inflammatory monocytes with a unique microRNA gene signature 
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Zhao, W., Beers, D. R. & Appel, S. H. Immune-mediated mechanisms in the pathoprogression of 
amyotrophic lateral sclerosis. J Neuroimmune Pharmacol 8, 888-899, doi:10.1007/s11481-013-
9489-x (2013). 
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Miller, R. G. et al. NP001 regulation of macrophage activation markers in ALS: A phase I 
clinical and biomarker study. Amyotroph Lateral Scler Frontotemporal Degener 15, 601-609, 
doi:10.3109/21678421.2014.951940 (2014). 
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De Felice, B. et al. miR-338-3p is over-expressed in blood, CFS, serum and spinal cord from 
sporadic amyotrophic lateral sclerosis patients. Neurogenetics 15, 243-253, doi:10.1007/s10048-
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41 
De Felice, B., Guida, M., Coppola, C., De Mieri, G. & Cotrufo, R. A miRNA signature in 
leukocytes from sporadic amyotrophic lateral sclerosis. Gene 508, 35-40, 
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42 
Butovsky, O. et al. Targeting miR-155 restores abnormal microglia and attenuates disease in 
SOD1 mice. Ann Neurol 77, 75-99, doi:10.1002/ana.24304 (2015). 
43 
Campos-Melo, D., Droppelmann, C. A., He, Z., Volkening, K. & Strong, M. J. Altered 
microRNA expression profile in Amyotrophic Lateral Sclerosis: a role in the regulation of NFL 
mRNA levels. Mol Brain 6, 26, doi:10.1186/1756-6606-6-26 (2013). 
44 
Atassi, N. et al. The PRO-ACT database: design, initial analyses, and predictive features. 
Neurology 83, 1719-1725, doi:10.1212/WNL.0000000000000951 (2014). 
45 
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46 
Abrahams, S., Newton, J., Niven, E., Foley, J. & Bak, T. H. Screening for cognition and 
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Simmons, Z. et al. The ALSSQOL: balancing physical and nonphysical factors in assessing 
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Karussis, D. et al. Safety and immunological effects of mesenchymal stem cell transplantation in 
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doi:10.1001/archneurol.2010.248 (2010). 
 

Appendix I 
 
Mayo ALS MSC Phase II - 36
 
 
Guidelines for Clinically Significant Laboratory Values, Vital Signs, and Clinical Assessments 
 
AST and ALT 
(SGOT and SGPT) 
>3 times ULN OR pretreatment >4 times ULN 
Albumin 
<0.9 LLN OR pretreatment value <0.75 LLN 
Albumin/Globulin ratio 
<0.75 
Alkaline phosphatase 
>3 times ULN 
Anion Gap 
>25 m/Eq/L 
Beck’s Depression Inventory (BDI) 
Worsening of BDI score by 3 or more points 
Bilirubin  
Direct 
>1.5 times ULN  OR pretreatment >2 times ULN 
Total 
>2 times ULN, OR pretreatment above >4 times ULN 
Blood urea nitrogen (BUN) 
>2 times ULN OR pretreatment >3 times ULN 
BUN/Creatinine ratio 
>35 
C-reactive Peptide 
>20 mg/L 
Calcium 
<0.8 times LLN  OR>1.2 times ULN 
Chest X-ray 
Clinically significant change in chest x-ray as judged by the site 
principal investigator 
Chloride 
<0.9 LLN OR > 1.1 times ULN 
Cholesterol 
>300 mg/dl OR 2 times pretreatment value 
Creatinine 
>4.0 OR 2 times pretreatment value  
EKG 
Clinically significant change in EKG as judged by the site 
investigator 
Erythrocytes 
<0.75 times LLN OR< 0.75 times pretreatment value 
Erythrocyte Sedimentation Rate 
>60 mm/hour 
Gamma Glutamyl transpeptidase (GGT) 
1.25 times ULN 
Glucose 
< 60 or > 140 for two consecutive readings 
Hematocrit 
male <0.37, female <0.32 OR < 0.75 pretreatment 
Hemoglobin 
>3 g/dl decrease from pretreatment value OR male  
<115 g/L, female < 95g/L 
INR 
> 1.5 
Lactic Dehydrogenase 
>3 times pretreatment value 
Leukocyte count 
(WBC) 
 
<2.8 times 109 OR >16 times 109/L 
Blasts 
immature forms >0 
Basophils (%) 
>3% if 0-1% pretreatment OR >3% times pretreatment  
value if pretreatment >1% 
Eosinophils 
>2 times ULN pretreatment, and >6% if pretreatment normal;  
OR if >6% pretreatment, >3 ULN pretreatment >10% 
Lymphocytes 
< 10 % 

Appendix I 
 
Mayo ALS MSC Phase II - 37
 
 
Mammogram 
Any clinically significant change in mammogram as judged by 
the site investigator 
Neutrophils and Bands 
< 15 %  
Ophthalmological examination 
Presence of papilledema OR loss of visual acuity of 2 or more 
lines on the standard visual acuity card. 
Partial thromboplastin time 
Subject’s baseline > 1.33 ULN  
Phosphate 
< 0.75 times LLN OR > 1.25 times ULN 
Platelet count 
< 75 times 109 OR > 700 times 109 
Potassium 
< 0.9 times LLN OR  >1.1 times ULN 
Prostate Specific Antigen (PSA) 
>2 times increase in PSA level 
Protein, Total 
<0.9 times LLN OR >1.1 times ULN 
Prothrombin time 
Subject’s baseline  > 1.33 times pretreatment  
Serum Iron 
< 20 mg/Dl OR > 200 mg/dL 
SGOT and SGPT (AST and ALTT) 
> 3 times ULN OR if pretreatment > ULN, >4 times 
pretreatment value 
Sodium 
< 0.95 times LLN OR > 1.05 times ULN 
Spinal Fluid 
Protein 
> 150 mg/dL 
WBC 
> 10 cells/uL 
Triglycerides 
 
>450 mg/kl OR> 2 times pretreatment 
Uric Acid 
 
> 1.5 times ULN or then > 2 times pretreatment value if 
pretreatment > ULN 
Urinalysis 
urinary RBC 
->5 /HPF OR >4 times pretreatment value if pretreatment value 
>5/HPF 
urinary WBC 
->5 /HPF OR >4 times pretreatment value if pretreatment value 
>5/HPF 
Urine pH 
< 4 or > 9 
Urine specific gravity 
< 1.001 or > 1.050 
Vital signs 
Systolic blood pressure   
< 80 or > 200 mm/hg 
Diastolic blood pressure 
< 50 or > 115 mm/hg 
Pulse 
 
 
< 50 or > 120 beats/minute 
Temperature 
 
< 97 or > 100 degrees F 
< 35 or > 38.3 degrees C 
Respirations 
 
< 10 or > 40 breaths/minute 
 
 

Appendix II 
 
Mayo ALS MSC Phase II - 38
 
 
 
 
 
 
The ALS Specific Quality of Life-Revised (ALSSQOL-R) User’s Guide 
 
Version 1.0 June 14, 2011 
 
Stephanie H Felgoise, PhD; Susan M Walsh, RN, ACNS-BC; Helen E Stephens, MA;  
Allyson Brothers, MA; Zachary Simmons, MD 
 
 
(Separate PDF uploaded into IRBe.)