IDF-ADA TRANSLATIONAL SYMPOSIUM

Diabetes Care 1

Jinquan Cai,1 Zhixian Wu,1 Xiumin Xu,2,3,4,5

Umbilical Cord Mesenchymal Lianming Liao,1 Jin Chen,1
Lianghu Huang,1 Weizhen Wu,1 Fang Luo,1
Stromal Cell With Autologous Chenguang Wu,1 Alberto Pugliese,2,6
Antonello Pileggi,2,3,4,5
Bone Marrow Cell Transplantation Camillo Ricordi,2,3,4,5,6 and
Jianming Tan1,3,4
in Established Type 1 Diabetes:
A Pilot Randomized Controlled
Open-Label Clinical Study to
Assess Safety and Impact on
Insulin Secretion
DOI: 10.2337/dc15-0171

OBJECTIVE
To determine the safety and effects on insulin secretion of umbilical cord (UC)
mesenchymal stromal cells (MSCs) plus autologous bone marrow mononuclear
cell (aBM-MNC) stem cell transplantation (SCT) without immunotherapy in estab-
lished type 1 diabetes (T1D).

RESEARCH DESIGN AND METHODS

Between January 2009 and December 2010, 42 patients with T1D were random-
ized (n = 21/group) to either SCT (1.1 3 106/kg UC-MSC, 106.8 3 106/kg aBM-MNC
through supraselective pancreatic artery cannulation) or standard care (control).
Patients were followed for 1 year at 3-month intervals. The primary end point was
C-peptide area under the curve (AUCC-Pep) during an oral glucose tolerance test at 1
Organ Transplant Institute, Fuzhou General
1 year. Additional end points were safety and tolerability of the procedure, met- Hospital, Xiamen University, Fuzhou, China
2
abolic control, and quality of life. Diabetes Research Institute, Cell Transplant
Center, University of Miami, Miami, FL
3
RESULTS Diabetes Research Institute Federation, Holly-
wood, FL
The treatment was well tolerated. At 1 year, metabolic measures improved in treated 4
The Cure Alliance, Miami, FL
patients: AUCC-Pep increased 105.7% (6.6 6 6.1 to 13.6 6 8.1 pmol/mL/180 min, 5
Department of Surgery, University of Miami
P = 0.00012) in 20 of 21 responders, whereas it decreased 7.7% in control subjects Miller School of Medicine, Miami, FL
6
Department of Medicine, University of Miami
(8.4 6 6.8 to 7.7 6 4.5 pmol/mL/180 min, P = 0.013 vs. SCT); insulin area under the Miller School of Medicine, Miami, FL
curve increased 49.3% (1,477.8 6 1,012.8 to 2,205.5 6 1,194.0 mmol/mL/180 min, Corresponding author: Jianming Tan, tanjm156@
P = 0.01), whereas it decreased 5.7% in control subjects (1,517.7 6 630.2 to 1,431.7 6 xmu.edu.cn.
441.6 mmol/mL/180 min, P = 0.027 vs. SCT). HbA1c decreased 12.6% (8.6 6 0.81% Received 23 January 2015 and accepted 22 June
[70.0 6 7.1 mmol/mol] to 7.5 6 1.0% [58.0 6 8.6 mmol/mol], P < 0.01) in the treated 2015.
group, whereas it increased 1.2% in the control group (8.7 6 0.9% [72.0 6 Clinical trial reg. no. NCT01374854, clinicaltrials
7.5 mmol/mol] to 8.8 6 0.9% [73 6 7.5 mmol/mol], P < 0.01 vs. SCT). Fasting .gov.
glycemia decreased 24.4% (200.0 6 51.1 to 151.2 6 22.1 mg/dL, P < 0.002) and This article contains Supplementary Data online
4.3% in control subjects (192.4 6 35.3 to 184.2 6 34.3 mg/dL, P < 0.042). Daily at http://care.diabetesjournals.org/lookup/
suppl/doi:10.2337/dc15-0171/-/DC1.
insulin requirements decreased 29.2% in only the treated group (0.9 6 0.2 to
J.Ca. and Z.W. contributed equally to this work.
0.6 6 0.2 IU/day/kg, P = 0.001), with no change found in control subjects (0.9 6
This article was prepared while A.Pi. was em-
0.2 to 0.9 6 0.2 IU/day/kg, P < 0.01 vs. SCT). ployed at the University of Miami.
CONCLUSIONS © 2016 by the American Diabetes Association.
Readers may use this article as long as the work
Transplantation of UC-MSC and aBM-MNC was safe and associated with moderate is properly cited, the use is educational and not
improvement of metabolic measures in patients with established T1D. for profit, and the work is not altered.
Diabetes Care Publish Ahead of Print, published online December 1, 2015
2 UC-MSC and Autologous BM-MNC Transplant for T1D
Type 1 diabetes (T1D) remains a thera-
peutic challenge because of its elusive
etiology (1,2). Intensive insulin treat-
ment can lead to tight metabolic con-
trol, and it reduces the incidence and
delays the progression of long-term di-
abetes complications; however, main-
taining normal glycemic levels is often
difficult and associated with increased
frequency of hypoglycemic episodes
(3,4). Therapeutic interventions aimed
at preserving b-cell mass at the time of
diabetes onset thus far have shown
transient and limited efficacy (2), mostly
consisting of a less decline in insulin se-
cretion but to improvement. Promising
results in the experimental and clinical
settings support the use of stem cell
transplantation (SCT) or bone marrow
(BM)–derived hematopoietic stem cells
(HSCs) for the treatment of autoimmune
diabetes (5–10).
Mesenchymal stromal cells (MSCs)
are considered multipotent stem cells
that can be isolated from BM, umbilical
cord (UC), adipose tissue, and placenta,
among other tissues. The ability of MSCs
to modulate immune responses and tis-
sue repair through paracrine mecha-
nisms is well documented (11) and
appealing for the treatment of T1D (9).
Urbán et al. (12) showed that transplan-
tation of BM cells (BMCs) and MSCs in
sublethally irradiated diabetic mice im-
proved glycemic and serum insulin lev-
els along with tissue regeneration and
repair; in their study, combined BMC
and MSC infusion appeared to be syn-
ergistic. Recently, Thakkar et al. (13)
reported that coinfusion of insulin-
secreting adipose-derived MSCs and
BM-HSCs is a clinically safe and viable
treatment option for T1D.
Increasing evidence supports the per-
sistence of residual b-cell mass in pan-
creatic specimens obtained from
patients with T1D and the persistence
of C-peptide production years after di-
agnosis (14–16). These observations
have important repercussions on the ra-
tionale for developing new interventions
aimed at the recovery of function in pa-
tients with established diabetes. More-
over, exploring the impact of immune
interventions in this patient population
may provide invaluable insight into their
safety, mechanistic impact, and, to a cer-
tain extent, efficacy, which could help to
better tailor future T1D prevention and
intervention trials. On the basis of these
premises, we conducted a pilot ran-
domized controlled open-label trial to
investigate the potential benefits on
metabolic control and safety of com-
bined UC-MSC and autologous bone
marrow mononuclear cell (aBM-MNC)
transplantation without immunother-
apy in patients with established T1D.
RESEARCH DESIGN AND METHODS
Patients
This single-center trial was conducted
from January 2009 to December 2010.
The study protocol was approved by
the Fuzhou General Hospital (FGH) in-
stitutional review board affiliated with
Xiamen University. Written informed
consent was signed by all participants.
Inclusion criteria were both sexes, age
18–40 years, history of T1D $2 and
#16 years (a time frame selected to al-
low confirmation of T1D diagnosis and
to avoid the potentially confounding ef-
fects of long-standing diabetes compli-
cations), HbA1c $7.5% (58 mmol/mol)
and #10.5% (91 mmol/mol), fasting se-
rum C-peptide ,0.1 pmol/mL, and daily
insulin requirements ,100 IU. Patients
with chronic renal dysfunction, prolifer-
ative retinopathy, chronic liver dysfunc-
tion, pancreatitis, abdominal aortic
aneurysm, and chronic virus infections
were excluded. The diagnosis of T1D
was confirmed by measurement of se-
rum levels of GAD antibodies (GADA) at
the time of onset and measured again at
trial enrollment (17). HLA alleles A, B,
and DR were determined by PCR (18).
Between January 2009 and July 2009,
92 patients (with any HbA1c level) (Fig. 1A)
were counseled for 3 months by an endo-
crinologist on intensive insulin treatment,
self-monitoring of blood glucose, exercise
(2–3 km three times a week), and healthy
diet. At the end of this run-in phase, all
patients were individually interviewed,
and 75 were entered into the screening
phase based on the inclusion criteria.
Forty-two patients were finally enrolled
and randomized into an SCT group (n =
21 receiving UC-MSC + BM-MNC trans-
plantation and standard clinical treat-
ment) or a continued standard clinical
treatment (control) group (n = 21) be-
tween July and December 2009 and
were observed until December 2010 at
3-month intervals (Fig. 1A). Because of
the nature of the therapeutic procedures,
the control group did not receive placebo
Diabetes Care
treatment; thus, patients were not blinded
to group assignment.
Umbilical Cord Mesenchymal Stem
Cells
To ensure cellular homogeneity through-
out the study, UC-MSCs used in the trial
were all obtained from a single human
donor UC. Written consent for the use
of the UC was obtained from the donor.
Briefly, a piece of UC (;5 cm) from a full-
term newborn (blood type O) was har-
vested at the time of delivery in the FGH
Department of Obstetrics and Gynecol-
ogy. The mesenchymal tissue in Wharton’s
jelly was diced into cubes of ;0.5 cm3 and
centrifuged at 250g for 5 min. The pellet
(mesenchymal tissue) was washed with
serum-free DMEM (HyClone) and then di-
gested with collagenase type IV (1 mg/mL;
Life Technologies) at 378C for 16–18 h, di-
luted with an equal volume of DMEM, and
further digested with 0.005% trypsin
(HyClone) at 378C for 60 min. To neutralize
the excess trypsin, 25% human albumin
(Alpha Therapeutic Corporation, Los An-
geles, CA) was added to the mesenchymal
tissue followed by two washes in DMEM.
Cells were plated in DMEM supplemented
with 5% platelet lysate (Tagene Biotech,
Xiamen, China), 100 units/mL penicillin,
and 100 mg/mL streptomycin at a density
of 1 3 106 cells/mL in a 378C humidified
5% CO2 incubator as previously described
(19). The medium was renewed every 2–3
days, and nonadherent cells were dis-
carded. After reaching 80% confluence,
UC-MSCs were harvested with 0.25% tryp-
sin and 0.02% EDTA, replated at a density
of 0.5–1 3 106 cells in a 175-cm2 flask, and
incubated for 5–7 days. UC-MSCs were fro-
zen at passage 2. Ten to 14 days before
transplantation, cells were thawed and
grown again until passage 4 or 5. On the
day of transplantation, UC-MSCs were in-
cubated with M199 medium (HyClone) for
1 h at 378C humidified 5% CO2. Cells were
harvested with trypsin and washed twice
with PBS. UC-MSCs were resuspended in
PBS for transplantation. As per standard
practice at our center (19) and in accor-
dance with 2006 International Society for
Cellular Therapy criteria (20), we perform
cell surface marker analysis (Coulter EPICS
XL Flow Cytometer acquisition report) and
showed that UC-MSCs were positive for
CD29, CD73, CD90, and CD105 and nega-
tive for CD34 and CD45. The differentia-
tion potential was evaluated by culturing
UC-MSCs in differentiation medium for
care.diabetesjournals.org
Figure 1—Study chart and HLA proportions. A: Screening, randomization, and completion of
1-year evaluations. B: Representation of the distribution of patients in the two study groups on
the basis of the expression of HLA (either A or DR) known to be associated with diabetes risk
(none or one, two, or three or more risk alleles present).
21 days and staining them with alizarin
red S and oil red O for osteocytes and
adipocytes, respectively (data not shown).
Batch testing for bacteria, mycoplasma,
fungi, and endotoxin were performed be-
fore release for transplantation.
Autologous Bone Marrow
Mononuclear Cells
Cell processing was performed at the
FGH current good manufacturing prac-
tices facility. Under local anesthesia with
2% lidocaine, BM was aspirated from both
iliac crests to obtain 300–375 mL; the
aspirate was mixed with 20,000 units
heparin, separated using a quadruple
blood collection bag (Terumo Medical
Corporation, Changchun, China), and
centrifuged (J-26XP; Beckman Coulter)
at 2,000g for 15 min. Red blood cells,
plasma, and fat layers were discarded.
The buffy coat was washed and resus-
pended in ;500 mL isotonic saline solu-
tion, and then aBM-MNCs in normal saline
solution were transported for immediate
transplantation along with UC-MSCs.
Transplantation Procedures
Before transplantation, patients were
fasted and received prophylactic octreo-
tide. As reported by Wu et al. (21), the
catheterization procedure was carried
out under angiography guidance in all
subjects. The dorsal pancreatic artery
or its substitute was identified, and
60–80 mL BM-MNCs (106.8 3 106/kg)
plus 30–50 mL UC-MSCs (1 3 106/kg)
were sequentially infused within 30
min. Amylase levels were tested at day
1 postinoculum to monitor for the oc-
currence of pancreatitis.
End Points
The primary end point was C-peptide area
under the curve (AUCC-Pep) during a 3-h
oral glucose tolerance test (OGTT) per-
formed after .12 h fasting since the
last insulin injection at 1 year after SCT.
Blood samples for C-peptide and serum
insulin levels were collected at OGTT time
points 210, 25, 30, 60, 90, 120, and 180
min. The AUCC-Pep and insulin area under
the curve (AUCIns) calculations were per-
formed using the trapezoidal method
with subtraction of the baseline (22).
Secondary end points were safety,
HbA1c, exogenous insulin requirement
(daily dose), fasting blood glucose
(FBG), fasting C-peptide, and serum
AUCIns of OGTT. Blood samples were col-
lected after overnight fasting before
and every 3 months post-SCT for FBG
Cai and Associates 3
(hexokinase method, AU2700; Olym-
pus), HbA1c (high performance liquid
chromatography assay, Variant II; Bio-
Rad), and C-peptide (chemiluminescent
immunoassay, ADVIA Centaur XP; Sie-
mens) analysis. Safety parameters in-
cluded close observation at 3-month
intervals for infectious diseases (e.g.,
upper respiratory tract infection) and
monitoring of white blood cell counts
as well as levels of C-reactive protein,
hemoglobin, serum creatinine (sCr),
and alanine aminotransferase. Immune
parameters analyzed were qualitative
determination of GADA by ELISA (23),
levels of T-cell activation and regulatory
T-cell (Treg)–related cytokines (inter-
feron-g [IFN-g] and IL-10) measured by
ELISA (R&D Systems), and cellular im-
mune status index based on CD4 T-cell
ATP released after mitogenic stimula-
tion in vitro (ImmuKnow; Cylex) (24).
Serum was collected at baseline and 1
year after treatment.
Clinical Management
During hospitalization and home care,
fingertip glycemic monitoring was per-
formed before meals, 2 h after meals or
at bedtime by turns, one to two times a
day. It was similar to a whole-day in-
tense monitoring when those values in
$1 week were pooled together. Insulin
dosing was based on FBG before meals
and 2 h postprandially, with target levels
of ,110 mg/dL (6.1 mmol/L) and ,140
mg/dL (7.8 mmol/L), respectively. If the
patient presented clinical symptoms of
hypoglycemia or blood glucose ,90
mg/dL (5.0 mmol/L), the insulin dose
would be decreased, even when blood
glucose levels before meals or 2 h post-
prandially were .110 mg/dL (6.1 mmol/L)
or 140 mg/dL (7.8 mmol/L). Endocrinolo-
gists periodically counseled patients on
healthy diet and exercise to avoid clinical
care discrepancies or nonadherence. In-
sulin doses were managed by the study’s
endocrinologist (Z.W.).
Quality-of-Life Measures
Global anxiety and depression status was
assessed separately at baseline and 1 year
after SCT by the participants and the
study physician, who was unaware of
the group assignment, using the Self-
Rating Anxiety Scale (range 20–80, with
higher scores indicating greater anxiety),
the Self-Rating Depression Scale (range
20–80, with higher scores indicating more
severe depression), and the summary
4 UC-MSC and Autologous BM-MNC Transplant for T1D
scores for the physical and mental quality-
of-life (QOL) components of the Medical
Outcomes Study 36-Item Short-Form Sur-
vey (range 0–100, with higher scores in-
dicating better health status).
Statistical Analysis
A computer-generated block randomiza-
tion was used to assign each subject to
one of the experimental groups. Statistical
analysis was performed using SPSS ver-
sion 10.1, GraphPad Prism 6, and Micro-
soft Excel software. Data are presented
as mean 6 SD. The x2 test, independent
t test, two-factor repeated-measures
ANOVA, and mixed-effects linear model
were used for two-group numeration
data comparison, two-group measure-
ment data comparison, repeated-measures
comparison (normal distribution), and
repeated-measures comparison (nonnor-
mal distribution), respectively. Tests yield-
ing P , 0.05 were considered statistically
significant. Power and sample size consid-
erations assume a 50% increase of AUCC-Pep
at 1 year after treatment from an average
6.67 pmol/mL/180 min of Chinese patients
with T1D. Student t test of independence
considered two independent groups of
21 patients each as having adequate power
to detect this assumed difference (type I
error = 0.05, 90% power).
RESULTS
Patient Characteristics
Forty-two patients (22 female and 20 male)
with established T1D were enrolled
and randomized to receive SCT or stan-
dard treatment (Fig. 1A). Both groups
were well matched in terms of baseline
characteristics (Table 1 and Fig. 1B),
with no statistically significant dif-
ferences between the SCT and control
conditions in terms of mean age at the
time of T1D onset (18.29 [range 5–28]
and 20.38 [13–27] years), mean dura-
tion of diabetes (9.2 [2–16] and 7.0
[2–13] years), body weight (59.50 6 8.42
and 60.33 6 10.76 kg), BMI (21.99 6 1.78
and 22.06 6 2.46 kg/m2), HbA1c (8.56 6
0.81% [70.0 6 6.5 mmol/mol] and 8.68 6
0.87% [71.0 6 7.1 mmol/mol]), FBG
(200.06 6 51.09 and 192.43 6 35.318
mg/dL), insulin dose (0.91 6 0.23 and
0.90 6 0.20 IU/day/kg), and sCr
(68.95 6 14.79 and 73.90 6 13.26
mmol/L). At the time of enrollment,
66.67% of patients (14 of 21) in the SCT
group and 55.14% (12 of 21) in the control
group were GADA positive (Table 1 and
Supplementary Table 2), which did not
appear to correlate with disease duration
(data not shown). The frequency of HLA
alleles associated with T1D risk were com-
parable among the study subjects in both
groups (Fig. 1B and Supplementary Tables
1 and 2); 95% of patients (n = 20) in the
SCT group and 86% (n = 18) in the control
group had at least one predisposing al-
lele, 62% and 52% of SCT and control
group patients had at least the HLA-DR
allele associated with T1D (DR9, DR4,
and/or DR3) (25). Of these, 43% (n = 9)
in the SCT group and 38% (n = 8) in the
control group had at least one HLA-DR
plus at least one HLA-A (A11 and/or A24)
T1D risk alleles present (25). Only 5% (n =
1) in the SCT group and 14% (n = 3) in the
control group had none of the risk alleles.
Therapeutic Efficacy of SCT
At 1 year, metabolic measures improved
in SCT recipients. The AUCC-Pep increased
105.7% from basal (6.6 6 6.1 to 13.6 6
8.1 pmol/mL/180 min, P = 0.00012),
with 15 of 21 patients (71.4%) showing
increased levels at 1 year. In contrast,
AUC C-Pep decreased 7.7% in con-
trol subjects (8.4 6 6.8 to 7.7 6
4.5 pmol/mL/180 min, P = 0.013 vs. SCT),
with 8 of 21 patients (38.0%) showing
improved values at 1 year (Fig. 2A and
Supplementary Fig. 1A). The AUCIns in-
creased in SCT recipients 49.3% from
basal (1,477.8 6 1,012.8 to 2,205.5 6
1,194.0 mmol/mL/180 min, P = 0.01),
whereas it decreased 5.7% in control sub-
jects (1,517.7 6 630.2 to 1,431.7 6
441.6 mmol/mL/180 min, P = 0.027 vs.
SCT) (Fig. 2B and Supplementary Fig. 1B).
After SCT, HbA1c levels decreased signif-
icantly at 3, 6, 9, and 12 months (repeated-
measures ANOVA P , 0.01), whereas they
remained stable in the control group during
the follow-up period (SCT vs. control P ,
0.01 for all time points) (Fig. 2C and Sup-
plementary Fig. 1C). HbA1c decreased
12.6% in the SCT group from 8.6 6 0.81%
(70.0 6 7.1 mmol/mol) to 7.5 6 1.0%
(58.0 6 8.6 mmol/mol) (P , 0.01), whereas
it increased 1.2% in control subjects from
8.7 6 0.9% (72.0 6 7.5 mmol/mol) to 8.8 6
0.9% (73 6 7.5 mmol/mol) (P , 0.01 vs.
SCT) (Fig. 2C and Supplementary Fig. 1C).
FBG was unchanged during the follow-
up period in the control group, whereas it
decreased significantly in the SCT group
at 3, 6, 9, and 12 months (P , 0.002 vs.
baseline for all time points in SCT) and
was significantly lower than in the control
group (P , 0.042 at 3 months, P , 0.01
Diabetes Care
thereafter) (Fig. 2D and Supplementary
Fig. 1D). At 12 months, FBG decreased
24.4% in SCT recipients (200.0 6 51.1 to
151.2 6 22.1 mg/dL) and 4.3% in con-
trol subjects (192.4 6 35.3 to 184.2 6
34.3 mg/dL) (Fig. 2D and Supplementary
Fig. 1D).
Progressive and significant reductions
in insulin dose requirements after trans-
plantation were observed in the SCT
group at 3, 6, 9, and 12 months (P ,
0.002), whereas they were unchanged
in the control group, which was signifi-
cantly different from SCT (P , 0.01 at 6,
9, and 12 months) (Fig. 2E and Supple-
mentary Fig. 1E). The insulin require-
ment was reduced 29.2% in the SCT
group (0.9 6 0.2 to 0.6 6 0.2 IU/day/kg,
P = 0.001) and was unchanged (;0%) in
the control group (0.9 6 0.2 to 0.9 6
0.2 IU/day/kg, P , 0.01 vs. SCT) (Fig. 2E
and Supplementary Fig. 1E).
Fasting C-peptide levels were mainly
unchanged in the control group (Fig. 2F
and Supplementary Fig. 1F), whereas
they markedly increased in the SCT
group at 9 and 12 months (compared
with baseline, P , 0.01) (Fig. 2F and
Supplementary Fig. 1F). Fasting C-peptide
in the SCT group was significantly higher
than in the control group at 9 and
12 months (P , 0.01 and P = 0.00001,
respectively). Comparing baseline with
12-month data, an increase was ob-
served in the SCT group (0.03 6 0.02
to 0.06 6 0.03 pmol/mL, P , 0.01),
with 20 of 21 patients (95.2%) showing
improvement, whereas no change was
found in the control group (0.02 6 0.02
to 0.03 6 0.02 pmol/mL, P not signifi-
cant), with only 9 of 21 patients (42.9%)
showing improvement (Fig. 2F and Sup-
plementary Fig. 1F).
QOL Measures
At baseline, patients in both groups
demonstrated similar symptoms of anx-
iety and depression and QOL scores
(Table 2). At 12 months, patients in the
SCT group showed decreased anxiety
and depression symptoms and improved
QOL score, whereas these measures did
not change markedly in the control group
(Table 2).
Safety
Patient-reported severe hypoglycemic
events were lower in the SCT group
than in the control group (0.43 [0–2] vs.
20.048 [21 to 1], P = 0.02). In the SCT
group, transient abdominal pain was
Table 1—Baseline characteristics of patients
Infusion HLA allele
Age at T1D AUCC-Pep Insulin
Study group onset duration Body GADA pos. (pmol/mL/ dose (IU/ sCr MSC BM-MNC
and pt. no.* Sex (years) (years) wt (kg) BMI (kg/m2) at enroll. 180 min) HbA1c (%) FBG (mg/dL) day/kg) (mmol/L) (3 106/kg) (3 106/kg) DR A B
SCT
1 F 14 7 54 20.58 Yes 21.24 7.5 306.0 0.74 76 1.05 151.85 1\14 2\3 7\35
2 F 20 11 55 19.26 No 12.70 7.8 201.6 1.18 84 1.12 136.36 12\– 3\24 7\51
care.diabetesjournals.org

3 M 23 16 80 23.89 Yes 1.90 8.0 61.2 0.83 69 1.04 77.50 3\9 2\24 46\75
4 M 19 6 64 19.97 Yes 12.14 7.9 190.8 0.80 100 1.36 57.81 3\9 11\33 51\58
5 M 28 2 44 20.09 No 2.53 8.4 203.4 0.70 64 0.95 61.36 3\9 11\– 51\60
6 M 14 11 65 24.46 Yes 19.90 8.4 203.4 0.80 92 0.96 29.23 3\7 33\74 44\58
7 M 5 14 57 19.72 Yes 1.90 7.9 230.4 1.19 73 1.24 133.33 4\15 11\24 61\62
8 F 22 15 60 25.30 Yes 1.90 8.9 221.4 0.83 55 1.07 43.33 3\– 11\33 58\–
9 F 13 9 55 21.48 No 14.85 10.5 282.6 0.80 43 1.01 103.64 3\9 2\– 46\58
10 F 22 15 56 22.43 Yes 2.02 8.3 142.2 1.00 67 1.58 67.86 4\9 2\24 48\60
11 F 18 5 58.2 22.45 No 2.10 9.2 176.4 1.00 48 0.88 116.84 4\9 2\11 56\70
12 F 19 5 58.2 22.45 No 7.02 7.9 199.8 1.17 52 1.57 147.77 9\14 2\– 46\51
13 F 25 2 57 22.27 Yes 3.17 8.5 203.4 1.12 56 0.91 138.60 8\11 2\11 60\75
14 F 16 6 57 22.27 No 2.18 10.4 244.8 1.37 72 0.89 161.40 4\12 24\32 39\51
15 M 21 15 65 21.97 Yes 4.84 9.2 235.8 0.58 74 1.39 132.31 8\12 2\11 60\75
16 M 19 10 70 24.80 No 5.01 7.9 185.4 0.44 82 1.23 52.86 1\11 24\32 39\51
17 F 14 11 52 21.10 Yes 4.60 8.9 226.8 1.00 63 1.04 182.69 11\12 11\– 51\55
18 F 11 15 50 21.36 Yes 2.27 7.8 169.2 1.16 52 0.87 164.00 15\11 11\24 13\60
19 F 20 12 51 19.43 Yes 8.98 9.2 140.4 0.84 69 0.96 178.43 8\12 11\24 60\–
20 M 23 4 71 23.72 Yes 5.33 8.8 172.8 0.72 72 0.99 30.99 9\13 2\33 46\58
21 M 18 3 70 22.86 Yes 1.93 8.4 203.4 0.79 85 0.96 74.29 3\9 11\33 46\58
Mean 12/9 18.29 9.24 59.50 21.99 14/21 6.60 8.56 200.06 0.91 68.95 1.10 106.78 d d d
Ctrl.
22 M 20 11 62 22.77 Yes 2.62 9.3 210.6 1.00 73 d d 14\15 11\24 35\60
23 F 20 6 52 21.64 Yes 4.35 8.1 176.4 1.08 63 d d 9\14 2\11 61\62
24 M 21 7 63 19.88 Yes 8.31 7.7 237.6 0.94 86 d d 12\15 2\24 46\56
25 M 15 3 68 24.38 No 9.43 8.2 165.6 1.06 76 d d 4\8 2\24 61\–
26 F 16 8 49 19.63 Yes 1.92 8.7 187.2 0.98 54 d d 7\12 11\30 13\60
27 M 24 11 64 19.97 No 9.02 10.3 237.6 0.97 93 d d 4\13 26\30 38\70
28 F 21 13 47 20.08 No 7.81 9.4 199.8 1.23 67 d d 8\10 1\2 37\60
29 F 22 5 52 20.83 Yes 4.52 7.5 142.2 1.08 79 d d 3\13 11\33 58\60
30 F 20 4 55 21.22 Yes 3.65 8.4 136.8 0.95 81 d d 4\14 11\24 13\38
31 F 16 6 49 22.37 No 16.23 9.1 190.8 0.67 47 d d 4\9 11\24 60\61
32 F 27 10 57 22.27 Yes 30.88 8.3 241.2 1.19 54 d d 1\3 24\26 35\60
33 M 24 5 68 22.46 Yes 4.25 9.9 273.6 0.76 78 d d 12\– 2\24 61\75
34 M 24 5 76 26.30 Yes 1.90 10.2 207 0.61 86 d d 4\14 2\31 54\75
35 M 19 13 72 26.45 No 12.95 8.2 147.6 0.61 99 d d 8\13 3\– 7\46
36 M 22 10 79 26.70 Yes 3.79 8.8 176.4 0.72 68 d d 12\3 11\24 51\60
37 F 25 3 48 18.99 No 5.95 7.7 185.4 1.02 59 d d 12\– 2\24 51\58
38 M 22 2 75 22.89 Yes 5.21 9.6 207 0.71 75 d d 12\4 11\74 51\60

Continued on p. 6
Cai and Associates
5
6 UC-MSC and Autologous BM-MNC Transplant for T1D Diabetes Care

observed in one patient during cell trans- evidence that some level of insulin pro-
plantation, which resolved without se- duction is maintained in many patients

13\39

39\60
37\38
60\–

d
B
quel. Bleeding at the puncture site was years after diagnosis, and some recent
observed in another patient (1 of 21 trials have enrolled patients within
HLA allele

24\26
2\33

24\–
1\–
[4.7%]), which resolved after applying 2 years from diagnosis. Herein, we de-

d
A

local pressure. Upper respiratory tract scribe the results of combined UC-MSC
infections were comparable between and aBM-MNC transplantation in pa-
12\15
10\4

14\–
7\–
DR

d
groups, with seven cases in the SCT group tients with established T1D.
(7 of 21 [33%]) and five in the control We show that cotransplantation of
(3 106/kg)
BM-MNC

group (5 of 21 [23.8%]); all resolved allogeneic Wharton’s jelly UC-MSC and

d
d
d
d
d

with medical therapy (Table 2). No re- aBM-MNC is followed by signs of im-
markable changes in C-reactive protein, proved insulin secretion and reduce in-
Infusion

white blood cell counts, hemoglobin, sulin requirement, as indicated by

(3 106/kg)

sCr, and alanine aminotransferase were significantly improved fasting C-peptide

MSC

d
d
d
d
d

observed (data not shown). No severe levels, AUCC-Pep (primary end point), and
adverse events, such as malignant tu- AUC Ins during OGTT performed at 1
mors, were observed during the follow- year. As well, we observed reductions
(mmol/L)

up period. in HbA1c, FBG, and insulin requirement

73.90
sCr

73
82
85
74

Immunological Parameters
At baseline, the SCT and the control
dose (IU/
compared with baseline and the control
group. Although the absolute change in
C-peptide is marginal, it is relatively sig-

groups had similar GADA-positive rates

day/kg)
Insulin

1.04
0.86
0.53
0.81
0.90

(66.7% vs. 57.1%), IL-10 levels (4.7 6 4.2 nificant in view of the long disease du-
vs. 5.3 6 4.4 pg/mL), IFN-g levels (6.0 6 ration of the study patients, many of
3.0 vs. 7.2 6 3.2 pg/mL), and ATP levels in whom had no or barely detectable fast-
FBG (mg/dL)

CD4+ T cells (378.7 6 52.8 vs. 376.0 6 ing C-peptide levels.

192.43
169.2
212.4
172.8
163.8

71.7 ng/mL) (P . 0.05 for all compari-
sons). At 1 year, patients in the SCT group
showed a 75% increase in IL-10 levels
HbA1c (%)
Clinical trials of MSC therapy for the
treatment of acute graft-versus-host
disease following allogeneic hematopoi-
etic SCT (26) and to improve outcome in

(8.2 6 7.7 vs. 4.7 6 4.2 pg/mL; one-tailed

8.10
8.68

allogeneic renal transplantation (19,27),

8.1
9.2
7.5

t test P , 0.03), a 50.7% decrease in IFN-g

levels (3.0 6 1.8 vs. 6.0 6 3.0 pg/mL; two- among other applications (28), have
tailed t test P , 0.001, 1 year vs. baseline shown encouraging results. Carlsson
(pmol/mL/

et al. (9) recently reported on the ben-

180 min)
AUCC-Pep

in SCT group; P , 0.00004, SCT vs. control

13.36

17.50
8.25
4.34

8.39

at 1 year), and a 9.7% decrease in ATP
levels in CD4+ T cells (345.3 6 43.6 vs.
378.7 6 52.8 ng/mL; one-tailed t test
GADA pos.
at enroll.
eficial effect of BM-MSC in newly diag-
nosed individuals with T1D. Hu et al. (29)
reported metabolic improvements (fast-

P , 0.03, baseline vs. 1 year in SCT group; ing and postprandial glycemia, HbA1c,
12/21
Yes
No

No
No

two-tailed t test P = 0.045, SCT vs. control fasting C-peptide) paralleled by reduc-
at 1 year). These changes were significant tions of exogenous insulin requirements
Ctrl., control; enroll., enrollment; pos., positive; pt. no., patient number.

compared with those in the control group following administration of UC-MSCs in

BMI (kg/m2)

(Supplementary Fig. 2). The overall newly diagnosed patients with T1D.
20.20
19.61
25.06
19.56
22.06

GADA-positive rates at 1 year were not
significantly different between the two
groups (57% in SCT and 52% in control),
wt (kg)
Moreover, Thakkar et al. (13) recently
reported the safety and efficacy of coin-
fusion of insulin-secreting adipose-
derived MSCs and BM-HSCs in patients

60.33
Body

with only two and one patients having

53
56
75
47

converted from GADA positive to GADA with T1D in which the use of autologous
negative in the SCT and control groups, inoculum appeared to confer better
duration

long-term control of hyperglycemia

(years)

respectively. None of the study subjects

7.00
T1D

8
7
5
5

showed conversion from GADA negative compared with allogenic SCT.

to GADA positive. Compared with new-onset T1D, MSCs
may be less effective in long-standing
(years)
Age at
onset

20.38

CONCLUSIONS
13
22
20
15

T1D because of the fewer inflammatory

Therapeutic strategies for T1D must ad- signals in the pancreatic microenviron-
Table 1—Continued

dress the autoreactive host immune sys- ment, which are essential for homing
10/11
Sex

M
M
F

tem as well as pancreatic b-cell repair MSCs toward the pancreas. Moreover,
and regeneration. Most of the T1D clin- MSCs infused intravenously undergo a
and pt. no.*
Study group

ical trials have been conducted in pa- pulmonary first-pass effect and are
tients soon after disease onset (7,8) likely to be sequestered in the lungs
Mean
39
40
41
42

when it is more likely to expect a clinical (30). Therefore, cells were injected
benefit. However, there is increasing through the pancreatic artery in the
care.diabetesjournals.org Cai and Associates 7

with allogeneic adjuvant cells, such as

UC-MSCs with, reportedly, hypoimmu-
nogenicity, may represent a viable strat-
egy toward retaining and recovering
stem cell properties and increasing effi-
cacy of SCT for the treatment of T1D.
Urbán et al. (12) suggested that MSCs
alone might be inadequate for tissue re-
generation and repair in experimental
models of T1D, therefore requiring a com-
plementary treatment. Moreover, BM-
MNCs comprising multiple cell fractions
of undifferentiated stem cells and differ-
entiated cells are appealing owing to their
tissue regeneration and repair potential
(35); thus, they could be used in combi-
nation to enhance MSC-mediated effects
(i.e., tissue repair, immune modulation).
The mechanisms underlying the effect
of MSC and SCT in patients with T1D are
not yet fully understood. First, the impact
of immunomodulation by stem cells
should be noted (13,36,37). In the pres-
ent study, we could investigate T-cell ac-
tivation and Treg-related cytokines
before and after SCT. We found that
SCT was associated with increased serum
levels of IL-10, a regulatory cytokine, with
decreased serum levels of IFN-g (T-helper
1 cytokine) and ATP production by CD4+ T
cells (ImmuKnow), indicating reduced
Figure 2—Metabolic function. A: The AUCC-Pep increased markedly from baseline in the SCT
group at 1 year after transplantation (two-tailed t test P = 0.0012). The change was significant T-cell activation (24). Residual b-cell
compared with the control group at 1 year (two-tailed t test P = 0.013). B: AUCIns significantly mass has been described in pancreatic
increased in the SCT group at 1 year compared with baseline levels (two-tailed t test P = 0.01) and specimens obtained from cadaveric do-
the control group at 1 year (one-tailed t test P = 0.027, SCT vs. control). C: HbA1c levels signif- nors with T1D, suggesting that interven-
icantly decreased from baseline in the SCT group (repeated-measures ANOVA P , 0.01 for all)
tions able to dampen inflammation may
and appeared to be significantly lower than those of the control group at 3, 6, 9, and 12 months
(P , 0.032 at 3 months, P , 0.006 thereafter). D: FBG levels were unchanged in the control be beneficial toward achieving recovery
group, whereas they improved over time in the SCT group compared with baseline (repeated- of function (14). Whether SCT influence
measures ANOVA P , 0.002) and respective values of the control group during follow-up (P , on T-cell and Treg function is one of the
0.042 at 3 months, P , 0.01 thereafter). E: Exogenous insulin requirements were significantly mechanisms involved in the current study
lower than baseline in the SCT group during follow-up (repeated-measures ANOVA P , 0.002),
which required significantly lower insulin than the control group at each time point assessed
remains controversial. Zhao et al. (38)
(P , 0.01, SCT vs. control from 6 to 12 months). F: The SCT group showed improved fasting showed that treatment of established T1D
C-peptide levels from baseline to 9 and 12 months (repeated-measures ANOVA P , 0.01) and with UC-MSCs provided lasting reversal of
compared with the same time points in the control group (two-tailed t test P , 0.001 at autoimmunity. In the present study, we
9 months, P = 0.00001 at 12 months). *P , 0.05 comparison of changes from baseline between could also assess GADA levels, which re-
the two groups.
mained largely unchanged; however, this
is only a partial assessment of autoimmune
present study to promote homing of antigens, it may introduce the issue of responses, and more studies are needed to
stem cells directly to the pancreas (31). diverse yields of stem cell numbers or address the effects of anti-islet responses.
Of note, we did not observe any patient failure to expand cells during culture Similarly, BMCs were shown to con-
with abnormal amylase levels, which in- (data not shown) in established T1D tribute to b-cell expansion and to de-
dicated the safety of pancreatic arterial due to impaired function of stem cells velop into functionally competent
infusion. Furthermore, the patients’ obtained from individuals with diabetes pancreatic b-cells when homed to pan-
QOL significantly improved, possibly re- (32–34). Conversely, UC stem cells ob- creatic islets after transplantation in ex-
flecting the positive impact of improved tained from healthy donor tissues have perimental animals (35). It could be
metabolic control following SCT. Protocol- the advantage of abundant yield, which speculated that the positive effects of
associated side effects were mild and self- could guarantee homogeneity and simi- SCT on insulin secretion could also result
limiting. lar quantities of infused cells. Therefore, from the regeneration of residual b-cells
Although the use of autologous SCT in designing the present trial, we rea- or from the generation of new b-cells
may prevent sensitization to allogeneic soned that combination of aBM-MNCs from BMC precursors; however, this
8 UC-MSC and Autologous BM-MNC Transplant for T1D
Table 2—QOL and adverse events
Anxiety (SAS) score
Before treatment
After treatment
Depression (SDS) score
Before treatment
After treatment
QOL (SF-36) score
Before treatment
After treatment
Adverse event type
Number of occurrences
Time after treatment (months)
Adverse event grade
Attribution
Intervention
Outcome
Data are mean 6 SD or mean (range). SAS, Self-Rating Anxiety Scale; SDS, Self-Rating Depression Scale; SF-36, Medical Outcomes Study 36-Item
Short-Form Survey; URTI, upper respiratory tract infection. *P , 0.05 compared with the occurrence rate in the control group. †Occurred in the ward
3 h after the arterial intervention therapy because the patient did not adhere to counseling. ‡Occurred during the arterial intervention therapy,
which was transient and recovered without intervention after transplantation.
hypothesis cannot be tested in the ab-
sence of biopsy data.
The limitations of this pilot study
include a relatively small sample size
and the short duration of follow-up.
Moreover, the independent contribu-
tion of each cell product (namely, UC-
MSCs and aBM-MNCs) was not assessed
separately. Although metabolic control
improved in patients receiving SCT, in-
sulin independence was not achieved.
Additionally, the lack of a placebo group
may generate bias in the QOL measure-
ments, which should be verified in a fu-
ture large-scale study. Assessment of
long-term safety is paramount, consider-
ing the potential risk of tumors generated
from unwanted MSC differentiation or
from other unknown factors related to
MSCs. Of note, we did not find abnormal-
ities in chromosome numbers in the UC-
MSCs used in the current trial. Others
have reported the stability of cultured
MSCs regarding the development of ab-
normal chromosomes after several pas-
sages well beyond that used in the
present trial (39,40). Patients are coun-
seled to have regular health checks to de-
termine early any malignancy that may
develop during follow-up.
In conclusion, we established the
safety of the approach and proof of con-
cept that SCT may lead to measurable
improvements of metabolic function in
patients with established T1D. The en-
couraging results point to a number of
issues that should be addressed in the
SCT group
38.5 6 7.1
34.6 6 5.4
38.0 6 6.6
33.3 6 4.7
78.6 6 8.4
82.4 6 5.9
URTI Bleeding Abdominal pain
7* 1* 1*
3 (1–6) 0† 0‡
Mild Mild Mild
Unrelated Definite Definite
Medical Local pressure None
Resolved Resolved Resolved
design of future large-scale trials to help
to improve clinical outcomes.
Acknowledgments. The authors thank Jinhua
Chen (Statistics Office, Fuzhou General Hospital,
Xiamen University, Fuzhou, China) for critical
contributions to the statistical work of this study.
Funding. This study was supported by the
Fujian Province (Major Research Project Fund
2009Y4001, Technology Innovation Platform
Project Fund 2008J1006 and 2010Y2006, and
Special Program for Key Science Research
2012YZ0001), the People’s Liberation Army
Clinical Innovation Major Project Fund
(2010gxjs026), and the Natural Science Founda-
tion of Fujian Province (2012J01408). Generous
support by the Diabetes Research Institute
Foundation, Hollywood, FL, is acknowledged.
Duality of Interest. No potential conflicts of
interest relevant to this article were reported.
Author Contributions. J.Ca. contributed to
performing the study and writing the manu-
script. Z.W. contributed to performing the
study, collecting and analyzing the data, and
writing the manuscript. X.X., L.L., A.Pu., A.Pi.,
and C.R. contributed to analyzing the data and
writing the manuscript. J.Ch. and L.H. prepared
the cells. W.W. and C.W. contributed to per-
forming the study. F.L. contributed to collecting
the data. J.T. designed the study. J.T. is the
guarantor of this work and, as such, had full
access to all the data in the study and takes
responsibility for the integrity of the data and
the accuracy of the data analysis.
Prior Presentation. Parts of this study were
presented at the International Diabetes Federa-
tion’s 2015 World Diabetes Congress, Vancouver,
Canada, 30 November–4 December 2015.
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