Umbilical Cord Mesenchymal Stromal Cell With Autologous Bone Marrow Cell Transplantation in Established Type 1 Diabetes: A Pilot Randomized Controlled Open-Label Clinical Study to Assess Safety and Impact on Insulin Secretion

Type 1 diabetes (T1D) remains a therapeutic challenge because of its elusive etiology (1,2). Intensive insulin treatment can lead to tight metabolic control, and it reduces the incidence and delays the progression of long-term diabetes complications; however, maintaining normal glycemic levels is often difficult and associated with increased frequency of hypoglycemic episodes (3,4). Therapeutic interventions aimed at preserving β-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 secretion 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 tissue repair through paracrine mechanisms is well documented (11) and appealing for the treatment of T1D (9). Urbán et al. (12) showed that transplantation of BM cells (BMCs) and MSCs in sublethally irradiated diabetic mice improved glycemic and serum insulin levels along with tissue regeneration and repair; in their study, combined BMC and MSC infusion appeared to be synergistic. 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 persistence of residual β-cell mass in pancreatic specimens obtained from patients with T1D and the persistence of C-peptide production years after diagnosis (14–16). These observations have important repercussions on the rationale for developing new interventions aimed at the recovery of function in patients with established diabetes. Moreover, exploring the impact of immune interventions in this patient population may provide invaluable insight into their safety, mechanistic impact, and, to a certain extent, efficacy, which could help to better tailor future T1D prevention and intervention trials. On the basis of these premises, we conducted a pilot randomized controlled open-label trial to investigate the potential benefits on metabolic control and safety of combined UC-MSC and autologous bone marrow mononuclear cell (aBM-MNC) transplantation without immunotherapy in patients with established T1D.

This single-center trial was conducted from January 2009 to December 2010. The study protocol was approved by the Fuzhou General Hospital (FGH) institutional 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 allow confirmation of T1D diagnosis and to avoid the potentially confounding effects of long-standing diabetes complications), HbA_1c ≥7.5% (58 mmol/mol) and ≤10.5% (91 mmol/mol), fasting serum C-peptide <0.1 pmol/mL, and daily insulin requirements <100 IU. Patients with chronic renal dysfunction, proliferative retinopathy, chronic liver dysfunction, pancreatitis, abdominal aortic aneurysm, and chronic virus infections were excluded. The diagnosis of T1D was confirmed by measurement of serum 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 HbA_1c level) (Fig. 1A) were counseled for 3 months by an endocrinologist 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 transplantation and standard clinical treatment) or a continued standard clinical treatment (control) group (n = 21) between 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 treatment; thus, patients were not blinded to group assignment.

To ensure cellular homogeneity throughout 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 harvested at the time of delivery in the FGH Department of Obstetrics and Gynecology. 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 digested with collagenase type IV (1 mg/mL; Life Technologies) at 37°C for 16–18 h, diluted with an equal volume of DMEM, and further digested with 0.005% trypsin (HyClone) at 37°C for 60 min. To neutralize the excess trypsin, 25% human albumin (Alpha Therapeutic Corporation, Los Angeles, 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 μg/mL streptomycin at a density of 1 × 10⁶ cells/mL in a 37°C humidified 5% CO₂ incubator as previously described (19). The medium was renewed every 2–3 days, and nonadherent cells were discarded. After reaching 80% confluence, UC-MSCs were harvested with 0.25% trypsin and 0.02% EDTA, replated at a density of 0.5–1 × 10⁶ cells in a 175-cm² flask, and incubated for 5–7 days. UC-MSCs were frozen 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 incubated with M199 medium (HyClone) for 1 h at 37°C 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 accordance 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 negative for CD34 and CD45. The differentiation potential was evaluated by culturing UC-MSCs in differentiation medium for 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 before release for transplantation.

Cell processing was performed at the FGH current good manufacturing practices 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 resuspended in ∼500 mL isotonic saline solution, and then aBM-MNCs in normal saline solution were transported for immediate transplantation along with UC-MSCs.

Before transplantation, patients were fasted and received prophylactic octreotide. 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 × 10⁶/kg) plus 30–50 mL UC-MSCs (1 × 10⁶/kg) were sequentially infused within 30 min. Amylase levels were tested at day 1 postinoculum to monitor for the occurrence of pancreatitis.

The primary end point was C-peptide area under the curve (AUC_C-Pep) during a 3-h oral glucose tolerance test (OGTT) performed 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 −10, −5, 30, 60, 90, 120, and 180 min. The AUC_C-Pep and insulin area under the curve (AUC_Ins) calculations were performed using the trapezoidal method with subtraction of the baseline (22).

Secondary end points were safety, HbA_1c, exogenous insulin requirement (daily dose), fasting blood glucose (FBG), fasting C-peptide, and serum AUC_Ins of OGTT. Blood samples were collected after overnight fasting before and every 3 months post-SCT for FBG (hexokinase method, AU2700; Olympus), HbA_1c (high performance liquid chromatography assay, Variant II; Bio-Rad), and C-peptide (chemiluminescent immunoassay, ADVIA Centaur XP; Siemens) analysis. Safety parameters included 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 (interferon-γ [IFN-γ] and IL-10) measured by ELISA (R&D Systems), and cellular immune status index based on CD4 T-cell ATP released after mitogenic stimulation in vitro (ImmuKnow; Cylex) (24). Serum was collected at baseline and 1 year after treatment.

During hospitalization and home care, fingertip glycemic monitoring was performed before meals, 2 h after meals or at bedtime by turns, one to two times a day. It was similar to a whole-day intense 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 postprandially were >110 mg/dL (6.1 mmol/L) or 140 mg/dL (7.8 mmol/L). Endocrinologists periodically counseled patients on healthy diet and exercise to avoid clinical care discrepancies or nonadherence. Insulin doses were managed by the study’s endocrinologist (Z.W.).

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 scores for the physical and mental quality-of-life (QOL) components of the Medical Outcomes Study 36-Item Short-Form Survey (range 0–100, with higher scores indicating better health status).

A computer-generated block randomization was used to assign each subject to one of the experimental groups. Statistical analysis was performed using SPSS version 10.1, GraphPad Prism 6, and Microsoft Excel software. Data are presented as mean ± SD. The χ² test, independent t test, two-factor repeated-measures ANOVA, and mixed-effects linear model were used for two-group numeration data comparison, two-group measurement data comparison, repeated-measures comparison (normal distribution), and repeated-measures comparison (nonnormal distribution), respectively. Tests yielding P < 0.05 were considered statistically significant. Power and sample size considerations assume a 50% increase of AUC_C-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).

Forty-two patients (22 female and 20 male) with established T1D were enrolled and randomized to receive SCT or standard treatment (Fig. 1A). Both groups were well matched in terms of baseline characteristics (Table 1 and Fig. 1B), with no statistically significant differences 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 duration of diabetes (9.2 [2–16] and 7.0 [2–13] years), body weight (59.50 ± 8.42 and 60.33 ± 10.76 kg), BMI (21.99 ± 1.78 and 22.06 ± 2.46 kg/m²), HbA_1c (8.56 ± 0.81% [70.0 ± 6.5 mmol/mol] and 8.68 ± 0.87% [71.0 ± 7.1 mmol/mol]), FBG (200.06 ± 51.09 and 192.43 ± 35.318 mg/dL), insulin dose (0.91 ± 0.23 and 0.90 ± 0.20 IU/day/kg), and sCr (68.95 ± 14.79 and 73.90 ± 13.26 μmol/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 comparable 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 allele, 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.

At 1 year, metabolic measures improved in SCT recipients. The AUC_C-Pep increased 105.7% from basal (6.6 ± 6.1 to 13.6 ± 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 control subjects (8.4 ± 6.8 to 7.7 ± 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 AUC_Ins increased in SCT recipients 49.3% from basal (1,477.8 ± 1,012.8 to 2,205.5 ± 1,194.0 mmol/mL/180 min, P = 0.01), whereas it decreased 5.7% in control subjects (1,517.7 ± 630.2 to 1,431.7 ± 441.6 mmol/mL/180 min, P = 0.027 vs. SCT) (Fig. 2B and Supplementary Fig. 1B).

After SCT, HbA_1c levels decreased significantly 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 Supplementary Fig. 1C). HbA_1c decreased 12.6% in the SCT group from 8.6 ± 0.81% (70.0 ± 7.1 mmol/mol) to 7.5 ± 1.0% (58.0 ± 8.6 mmol/mol) (P < 0.01), whereas it increased 1.2% in control subjects from 8.7 ± 0.9% (72.0 ± 7.5 mmol/mol) to 8.8 ± 0.9% (73 ± 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 thereafter) (Fig. 2D and Supplementary Fig. 1D). At 12 months, FBG decreased 24.4% in SCT recipients (200.0 ± 51.1 to 151.2 ± 22.1 mg/dL) and 4.3% in control subjects (192.4 ± 35.3 to 184.2 ± 34.3 mg/dL) (Fig. 2D and Supplementary Fig. 1D).

Progressive and significant reductions in insulin dose requirements after transplantation 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 significantly different from SCT (P < 0.01 at 6, 9, and 12 months) (Fig. 2E and Supplementary Fig. 1E). The insulin requirement was reduced 29.2% in the SCT group (0.9 ± 0.2 to 0.6 ± 0.2 IU/day/kg, P = 0.001) and was unchanged (∼0%) in the control group (0.9 ± 0.2 to 0.9 ± 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 observed in the SCT group (0.03 ± 0.02 to 0.06 ± 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 ± 0.02 to 0.03 ± 0.02 pmol/mL, P not significant), with only 9 of 21 patients (42.9%) showing improvement (Fig. 2F and Supplementary Fig. 1F).

At baseline, patients in both groups demonstrated similar symptoms of anxiety 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).

Patient-reported severe hypoglycemic events were lower in the SCT group than in the control group (0.43 [0–2] vs. −0.048 [−1 to 1], P = 0.02). In the SCT group, transient abdominal pain was observed in one patient during cell transplantation, which resolved without sequel. Bleeding at the puncture site was observed in another patient (1 of 21 [4.7%]), which resolved after applying local pressure. Upper respiratory tract infections were comparable between groups, with seven cases in the SCT group (7 of 21 [33%]) and five in the control group (5 of 21 [23.8%]); all resolved with medical therapy (Table 2). No remarkable changes in C-reactive protein, white blood cell counts, hemoglobin, sCr, and alanine aminotransferase were observed (data not shown). No severe adverse events, such as malignant tumors, were observed during the follow-up period.

At baseline, the SCT and the control groups had similar GADA-positive rates (66.7% vs. 57.1%), IL-10 levels (4.7 ± 4.2 vs. 5.3 ± 4.4 pg/mL), IFN-γ levels (6.0 ± 3.0 vs. 7.2 ± 3.2 pg/mL), and ATP levels in CD4⁺ T cells (378.7 ± 52.8 vs. 376.0 ± 71.7 ng/mL) (P > 0.05 for all comparisons). At 1 year, patients in the SCT group showed a 75% increase in IL-10 levels (8.2 ± 7.7 vs. 4.7 ± 4.2 pg/mL; one-tailed t test P < 0.03), a 50.7% decrease in IFN-γ levels (3.0 ± 1.8 vs. 6.0 ± 3.0 pg/mL; two-tailed t test P < 0.001, 1 year vs. baseline in SCT group; P < 0.00004, SCT vs. control at 1 year), and a 9.7% decrease in ATP levels in CD4⁺ T cells (345.3 ± 43.6 vs. 378.7 ± 52.8 ng/mL; one-tailed t test P < 0.03, baseline vs. 1 year in SCT group; two-tailed t test P = 0.045, SCT vs. control at 1 year). These changes were significant compared with those in the control group (Supplementary Fig. 2). The overall GADA-positive rates at 1 year were not significantly different between the two groups (57% in SCT and 52% in control), with only two and one patients having converted from GADA positive to GADA negative in the SCT and control groups, respectively. None of the study subjects showed conversion from GADA negative to GADA positive.

Therapeutic strategies for T1D must address the autoreactive host immune system as well as pancreatic β-cell repair and regeneration. Most of the T1D clinical trials have been conducted in patients soon after disease onset (7,8) when it is more likely to expect a clinical benefit. However, there is increasing evidence that some level of insulin production is maintained in many patients years after diagnosis, and some recent trials have enrolled patients within 2 years from diagnosis. Herein, we describe the results of combined UC-MSC and aBM-MNC transplantation in patients with established T1D.

We show that cotransplantation of allogeneic Wharton’s jelly UC-MSC and aBM-MNC is followed by signs of improved insulin secretion and reduce insulin requirement, as indicated by significantly improved fasting C-peptide levels, AUC_C-Pep (primary end point), and AUC_Ins during OGTT performed at 1 year. As well, we observed reductions in HbA_1c, FBG, and insulin requirement compared with baseline and the control group. Although the absolute change in C-peptide is marginal, it is relatively significant in view of the long disease duration of the study patients, many of whom had no or barely detectable fasting C-peptide levels.

Clinical trials of MSC therapy for the treatment of acute graft-versus-host disease following allogeneic hematopoietic SCT (26) and to improve outcome in allogeneic renal transplantation (19,27), among other applications (28), have shown encouraging results. Carlsson et al. (9) recently reported on the beneficial effect of BM-MSC in newly diagnosed individuals with T1D. Hu et al. (29) reported metabolic improvements (fasting and postprandial glycemia, HbA_1c, fasting C-peptide) paralleled by reductions of exogenous insulin requirements following administration of UC-MSCs in newly diagnosed patients with T1D. Moreover, Thakkar et al. (13) recently reported the safety and efficacy of coinfusion of insulin-secreting adipose-derived MSCs and BM-HSCs in patients with T1D in which the use of autologous inoculum appeared to confer better long-term control of hyperglycemia compared with allogenic SCT.

Compared with new-onset T1D, MSCs may be less effective in long-standing T1D because of the fewer inflammatory signals in the pancreatic microenvironment, which are essential for homing MSCs toward the pancreas. Moreover, MSCs infused intravenously undergo a pulmonary first-pass effect and are likely to be sequestered in the lungs (30). Therefore, cells were injected through the pancreatic artery in the present study to promote homing of stem cells directly to the pancreas (31). Of note, we did not observe any patient with abnormal amylase levels, which indicated the safety of pancreatic arterial infusion. Furthermore, the patients’ QOL significantly improved, possibly reflecting the positive impact of improved metabolic control following SCT. Protocol-associated side effects were mild and self-limiting.

Although the use of autologous SCT may prevent sensitization to allogeneic antigens, it may introduce the issue of diverse yields of stem cell numbers or failure to expand cells during culture (data not shown) in established T1D due to impaired function of stem cells obtained from individuals with diabetes (32–34). Conversely, UC stem cells obtained from healthy donor tissues have the advantage of abundant yield, which could guarantee homogeneity and similar quantities of infused cells. Therefore, in designing the present trial, we reasoned that combination of aBM-MNCs with allogeneic adjuvant cells, such as UC-MSCs with, reportedly, hypoimmunogenicity, may represent a viable strategy toward retaining and recovering stem cell properties and increasing efficacy of SCT for the treatment of T1D. Urbán et al. (12) suggested that MSCs alone might be inadequate for tissue regeneration and repair in experimental models of T1D, therefore requiring a complementary treatment. Moreover, BM-MNCs comprising multiple cell fractions of undifferentiated stem cells and differentiated cells are appealing owing to their tissue regeneration and repair potential (35); thus, they could be used in combination 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 present study, we could investigate T-cell activation 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-γ (T-helper 1 cytokine) and ATP production by CD4⁺ T cells (ImmuKnow), indicating reduced T-cell activation (24). Residual β-cell mass has been described in pancreatic specimens obtained from cadaveric donors with T1D, suggesting that interventions able to dampen inflammation may be beneficial toward achieving recovery of function (14). Whether SCT influence on T-cell and Treg function is one of the mechanisms involved in the current study remains controversial. Zhao et al. (38) showed that treatment of established T1D with UC-MSCs provided lasting reversal of autoimmunity. In the present study, we could also assess GADA levels, which remained largely unchanged; however, this is only a partial assessment of autoimmune responses, and more studies are needed to address the effects of anti-islet responses.

Similarly, BMCs were shown to contribute to β-cell expansion and to develop into functionally competent pancreatic β-cells when homed to pancreatic islets after transplantation in experimental animals (35). It could be speculated that the positive effects of SCT on insulin secretion could also result from the regeneration of residual β-cells or from the generation of new β-cells from BMC precursors; however, this hypothesis cannot be tested in the absence 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 contribution of each cell product (namely, UC-MSCs and aBM-MNCs) was not assessed separately. Although metabolic control improved in patients receiving SCT, insulin independence was not achieved. Additionally, the lack of a placebo group may generate bias in the QOL measurements, which should be verified in a future large-scale study. Assessment of long-term safety is paramount, considering the potential risk of tumors generated from unwanted MSC differentiation or from other unknown factors related to MSCs. Of note, we did not find abnormalities in chromosome numbers in the UC-MSCs used in the current trial. Others have reported the stability of cultured MSCs regarding the development of abnormal chromosomes after several passages well beyond that used in the present trial (39,40). Patients are counseled to have regular health checks to determine early any malignancy that may develop during follow-up.

In conclusion, we established the safety of the approach and proof of concept that SCT may lead to measurable improvements of metabolic function in patients with established T1D. The encouraging results point to a number of issues that should be addressed in the 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 Foundation 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 manuscript. 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 performing 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 Federation's 2015 World Diabetes Congress, Vancouver, Canada, 30 November–4 December 2015.