Umbilical Cord Mesenchymal Stromal Cell With Autologous ... · Camillo Ricordi,2,3,4,5,6 and...

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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 Diabetes Care 2016;39:149157 | 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 10 6 /kg UC-MSC, 106.8 3 10 6 /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 (AUC C-Pep ) during an oral glucose tolerance test at 1 year. Additional end points were safety and tolerability of the procedure, met- abolic control, and quality of life. RESULTS The treatment was well tolerated. At 1 year, metabolic measures improved in treated patients: AUC C-Pep increased 105.7% (6.6 6 6.1 to 13.6 6 8.1 pmol/mL/180 min, P = 0.00012) in 20 of 21 responders, whereas it decreased 7.7% in control subjects (8.4 6 6.8 to 7.7 6 4.5 pmol/mL/180 min, P = 0.013 vs. SCT); insulin area under the curve increased 49.3% (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 subjects (1,517.7 6 630.2 to 1,431.7 6 441.6 mmol/mL/180 min, P = 0.027 vs. SCT). HbA 1c decreased 12.6% (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) in the treated group, whereas it increased 1.2% in the control group (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). Fasting glycemia decreased 24.4% (200.0 6 51.1 to 151.2 6 22.1 mg/dL, P < 0.002) and 4.3% in control subjects (192.4 6 35.3 to 184.2 6 34.3 mg/dL, P < 0.042). Daily insulin requirements decreased 29.2% in only the treated group (0.9 6 0.2 to 0.6 6 0.2 IU/day/kg, P = 0.001), with no change found in control subjects (0.9 6 0.2 to 0.9 6 0.2 IU/day/kg, P < 0.01 vs. SCT). CONCLUSIONS Transplantation of UC-MSC and aBM-MNC was safe and associated with moderate improvement of metabolic measures in patients with established T1D. 1 Organ Transplant Institute, Fuzhou General Hospital, Xiamen University, Fuzhou, China 2 Diabetes Research Institute, Cell Transplant Center, University of Miami, Miami, FL 3 Diabetes Research Institute Federation, Holly- wood, FL 4 The Cure Alliance, Miami, FL 5 Department of Surgery, University of Miami Miller School of Medicine, Miami, FL 6 Department of Medicine, University of Miami Miller School of Medicine, Miami, FL Corresponding author: Jianming Tan, tanjm156@ xmu.edu.cn. Received 23 January 2015 and accepted 22 June 2015. Clinical trial reg. no. NCT01374854, clinicaltrials .gov. This article contains Supplementary Data online at http://care.diabetesjournals.org/lookup/ suppl/doi:10.2337/dc15-0171/-/DC1. A slide set summarizing this article is available online. J.Ca. and Z.W. contributed equally to this work. This article was prepared while A.Pi. was em- ployed at the University of Miami. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for prot, and the work is not altered. Jinquan Cai, 1 Zhixian Wu, 1 Xiumin Xu, 2,3,4,5 Lianming Liao, 1 Jin Chen, 1 Lianghu Huang, 1 Weizhen Wu, 1 Fang Luo, 1 Chenguang Wu, 1 Alberto Pugliese, 2,6 Antonello Pileggi, 2,3,4,5 Camillo Ricordi, 2,3,4,5,6 and Jianming Tan 1,3,4 Diabetes Care Volume 39, January 2016 149 IDF-ADA TRANSLATIONAL SYMPOSIUM

Transcript of Umbilical Cord Mesenchymal Stromal Cell With Autologous ... · Camillo Ricordi,2,3,4,5,6 and...

Page 1: Umbilical Cord Mesenchymal Stromal Cell With Autologous ... · Camillo Ricordi,2,3,4,5,6 and Jianming Tan1,3,4 Diabetes Care Volume 39, January 2016 149 IDF-ADA TRANSLATIONAL SYMPOSIUM.

Umbilical Cord MesenchymalStromal Cell With AutologousBoneMarrowCell Transplantationin Established Type 1 Diabetes:A Pilot Randomized ControlledOpen-Label Clinical Study toAssess Safety and Impact onInsulin SecretionDiabetes Care 2016;39:149–157 | 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 mononuclearcell (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 (1.13 106/kg UC-MSC, 106.83 106/kg aBM-MNCthrough supraselective pancreatic artery cannulation) or standard care (control).Patients were followed for 1 year at 3-month intervals. The primary end point wasC-peptide area under the curve (AUCC-Pep) during an oral glucose tolerance test at1 year. Additional end points were safety and tolerability of the procedure, met-abolic control, and quality of life.

RESULTS

The treatmentwaswell tolerated. At1 year,metabolicmeasures improved in treatedpatients: AUCC-Pep increased 105.7% (6.6 6 6.1 to 13.6 6 8.1 pmol/mL/180 min,P = 0.00012) in 20 of 21 responders, whereas it decreased 7.7% in control subjects(8.46 6.8 to 7.76 4.5 pmol/mL/180 min, P = 0.013 vs. SCT); insulin area under thecurve increased 49.3% (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 subjects (1,517.76 630.2 to 1,431.76441.6 mmol/mL/180 min, P = 0.027 vs. SCT). HbA1c decreased 12.6% (8.6 6 0.81%[70.06 7.1mmol/mol] to 7.56 1.0% [58.06 8.6mmol/mol], P< 0.01) in the treatedgroup, whereas it increased 1.2% in the control group (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). Fastingglycemia decreased 24.4% (200.06 51.1 to 151.26 22.1 mg/dL, P < 0.002) and4.3% in control subjects (192.4 6 35.3 to 184.26 34.3 mg/dL, P < 0.042). Dailyinsulin requirements decreased 29.2% in only the treated group (0.9 6 0.2 to0.66 0.2 IU/day/kg, P = 0.001), with no change found in control subjects (0.960.2 to 0.9 6 0.2 IU/day/kg, P < 0.01 vs. SCT).

CONCLUSIONS

Transplantation of UC-MSC and aBM-MNCwas safe and associatedwithmoderateimprovement of metabolic measures in patients with established T1D.

1Organ Transplant Institute, Fuzhou GeneralHospital, Xiamen University, Fuzhou, China2Diabetes Research Institute, Cell TransplantCenter, University of Miami, Miami, FL3Diabetes Research Institute Federation, Holly-wood, FL4The Cure Alliance, Miami, FL5Department of Surgery, University of MiamiMiller School of Medicine, Miami, FL6Department of Medicine, University of MiamiMiller School of Medicine, Miami, FL

Corresponding author: Jianming Tan, [email protected].

Received 23 January 2015 and accepted 22 June2015.

Clinical trial reg. no. NCT01374854, clinicaltrials.gov.

This article contains Supplementary Data onlineat http://care.diabetesjournals.org/lookup/suppl/doi:10.2337/dc15-0171/-/DC1.

A slide set summarizing this article is availableonline.

J.Ca. and Z.W. contributed equally to this work.

This article was prepared while A.Pi. was em-ployed at the University of Miami.

© 2016 by the American Diabetes Association.Readers may use this article as long as the workis properly cited, the use is educational and notfor profit, and the work is not altered.

Jinquan Cai,1 ZhixianWu,1 Xiumin Xu,2,3,4,5

Lianming Liao,1 Jin Chen,1

Lianghu Huang,1 WeizhenWu,1 Fang Luo,1

Chenguang Wu,1 Alberto Pugliese,2,6

Antonello Pileggi,2,3,4,5

Camillo Ricordi,2,3,4,5,6 and

Jianming Tan1,3,4

Diabetes Care Volume 39, January 2016 149

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DATR

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Type 1 diabetes (T1D) remains a thera-peutic challenge because of its elusiveetiology (1,2). Intensive insulin treat-ment can lead to tight metabolic con-trol, and it reduces the incidence anddelays the progression of long-term di-abetes complications; however, main-taining normal glycemic levels is oftendifficult and associated with increasedfrequency of hypoglycemic episodes(3,4). Therapeutic interventions aimedat preserving b-cell mass at the time ofdiabetes onset thus far have showntransient and limited efficacy (2), mostlyconsisting of a less decline in insulin se-cretion but to improvement. Promisingresults in the experimental and clinicalsettings support the use of stem celltransplantation (SCT) or bone marrow(BM)–derived hematopoietic stem cells(HSCs) for the treatment of autoimmunediabetes (5–10).Mesenchymal stromal cells (MSCs)

are considered multipotent stem cellsthat can be isolated from BM, umbilicalcord (UC), adipose tissue, and placenta,among other tissues. The ability ofMSCsto modulate immune responses and tis-sue repair through paracrine mecha-nisms is well documented (11) andappealing for the treatment of T1D (9).Urban et al. (12) showed that transplan-tation of BM cells (BMCs) and MSCs insublethally irradiated diabetic mice im-proved glycemic and serum insulin lev-els along with tissue regeneration andrepair; in their study, combined BMCand MSC infusion appeared to be syn-ergistic. Recently, Thakkar et al. (13)reported that coinfusion of insulin-secreting adipose-derived MSCs andBM-HSCs is a clinically safe and viabletreatment option for T1D.Increasing evidence supports the per-

sistence of residual b-cell mass in pan-creatic specimens obtained frompatients with T1D and the persistenceof C-peptide production years after di-agnosis (14–16). These observationshave important repercussions on the ra-tionale for developing new interventionsaimed at the recovery of function in pa-tients with established diabetes. More-over, exploring the impact of immuneinterventions in this patient populationmay provide invaluable insight into theirsafety, mechanistic impact, and, to a cer-tain extent, efficacy, which could help tobetter tailor future T1D prevention andintervention trials. On the basis of these

premises, we conducted a pilot ran-domized controlled open-label trial toinvestigate the potential benefits onmetabolic control and safety of com-bined UC-MSC and autologous bonemarrow mononuclear cell (aBM-MNC)transplantation without immunother-apy in patients with established T1D.

RESEARCH DESIGN AND METHODS

PatientsThis single-center trial was conductedfrom January 2009 to December 2010.The study protocol was approved bythe Fuzhou General Hospital (FGH) in-stitutional review board affiliated withXiamen University. Written informedconsent was signed by all participants.Inclusion criteria were both sexes, age18–40 years, history of T1D $2 and#16 years (a time frame selected to al-low confirmation of T1D diagnosis andto 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 dailyinsulin requirements ,100 IU. Patientswith chronic renal dysfunction, prolifer-ative retinopathy, chronic liver dysfunc-tion, pancreatitis, abdominal aorticaneurysm, and chronic virus infectionswere excluded. The diagnosis of T1Dwas confirmed by measurement of se-rum levels of GAD antibodies (GADA) atthe time of onset andmeasured again attrial 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 healthydiet. At the end of this run-in phase, allpatients were individually interviewed,and 75 were entered into the screeningphase based on the inclusion criteria.Forty-two patients were finally enrolledand randomized into an SCT group (n =21 receiving UC-MSC + BM-MNC trans-plantation and standard clinical treat-ment) or a continued standard clinicaltreatment (control) group (n = 21) be-tween July and December 2009 andwere observed until December 2010 at3-month intervals (Fig. 1A). Because ofthe nature of the therapeutic procedures,the control group did not receive placebo

treatment; thus, patients were not blindedto group assignment.

Umbilical Cord Mesenchymal StemCellsTo ensure cellular homogeneity through-out the study, UC-MSCs used in the trialwere all obtained from a single humandonor UC. Written consent for the useof 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 FGHDepartment of Obstetrics and Gynecol-ogy. Themesenchymal tissue inWharton’sjelly was diced into cubes of;0.5 cm3 andcentrifuged at 250g for 5 min. The pellet(mesenchymal tissue) was washed withserum-free DMEM (HyClone) and then di-gested with collagenase type IV (1mg/mL;Life Technologies) at 378C for 16–18 h, di-lutedwith an equal volume of DMEM, andfurther digested with 0.005% trypsin(HyClone) at 378C for 60min. To neutralizethe excess trypsin, 25% human albumin(Alpha Therapeutic Corporation, Los An-geles, CA) was added to the mesenchymaltissue followed by two washes in DMEM.Cells were plated in DMEM supplementedwith 5% platelet lysate (Tagene Biotech,Xiamen, China), 100 units/mL penicillin,and 100 mg/mL streptomycin at a densityof 1 3 106 cells/mL in a 378C humidified5% CO2 incubator as previously described(19). The medium was renewed every 2–3days, and nonadherent cells were dis-carded. After reaching 80% confluence,UC-MSCswere harvestedwith 0.25% tryp-sin and 0.02% EDTA, replated at a densityof 0.5–13 106 cells in a 175-cm2

flask, andincubated for 5–7days.UC-MSCswere fro-zen at passage 2. Ten to 14 days beforetransplantation, cells were thawed andgrown again until passage 4 or 5. On theday of transplantation, UC-MSCs were in-cubatedwithM199medium (HyClone) for1 h at 378C humidified 5% CO2. Cells wereharvested with trypsin and washed twicewith PBS. UC-MSCs were resuspended inPBS for transplantation. As per standardpractice at our center (19) and in accor-dance with 2006 International Society forCellular Therapy criteria (20), we performcell surface marker analysis (Coulter EPICSXL Flow Cytometer acquisition report) andshowed that UC-MSCs were positive forCD29, CD73, CD90, and CD105 and nega-tive for CD34 and CD45. The differentia-tion potential was evaluated by culturingUC-MSCs in differentiation medium for

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21 days and staining them with alizarinred S and oil red O for osteocytes andadipocytes, respectively (data not shown).Batch testing for bacteria, mycoplasma,fungi, and endotoxin were performed be-fore release for transplantation.

Autologous Bone MarrowMononuclear CellsCell processing was performed at theFGH current good manufacturing prac-tices facility. Under local anesthesia with2% lidocaine, BMwas aspirated frombothiliac crests to obtain 300–375 mL; theaspirate was mixed with 20,000 unitsheparin, separated using a quadrupleblood collection bag (Terumo MedicalCorporation, Changchun, China), andcentrifuged (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 salinesolution were transported for immediatetransplantation along with UC-MSCs.

Transplantation ProceduresBefore transplantation, patients werefasted and received prophylactic octreo-tide. As reported by Wu et al. (21), the

catheterization procedure was carriedout under angiography guidance in allsubjects. The dorsal pancreatic arteryor its substitute was identified, and60–80 mL BM-MNCs (106.8 3 106/kg)plus 30–50 mL UC-MSCs (1 3 106/kg)were sequentially infused within 30min. Amylase levels were tested at day1 postinoculum to monitor for the oc-currence of pancreatitis.

End PointsThe primary endpointwas C-peptide areaunder the curve (AUCC-Pep) during a 3-horal glucose tolerance test (OGTT) per-formed after .12 h fasting since thelast insulin injection at 1 year after SCT.Blood samples for C-peptide and seruminsulin levels were collected at OGTT timepoints210,25, 30, 60, 90, 120, and 180min. The AUCC-Pep and insulin area underthe curve (AUCIns) calculations were per-formed using the trapezoidal methodwith subtraction of the baseline (22).

Secondary end points were safety,HbA1c, exogenous insulin requirement(daily dose), fasting blood glucose(FBG), fasting C-peptide, and serumAUCIns of OGTT. Blood samples were col-lected after overnight fasting beforeand every 3 months post-SCT for FBG

(hexokinase method, AU2700; Olym-pus), HbA1c (high performance liquidchromatography assay, Variant II; Bio-Rad), and C-peptide (chemiluminescentimmunoassay, ADVIA Centaur XP; Sie-mens) analysis. Safety parameters in-cluded close observation at 3-monthintervals for infectious diseases (e.g.,upper respiratory tract infection) andmonitoring of white blood cell countsas well as levels of C-reactive protein,hemoglobin, serum creatinine (sCr),and alanine aminotransferase. Immuneparameters analyzed were qualitativedetermination of GADA by ELISA (23),levels of T-cell activation and regulatoryT-cell (Treg)–related cytokines (inter-feron-g [IFN-g] and IL-10) measured byELISA (R&D Systems), and cellular im-mune status index based on CD4 T-cellATP released after mitogenic stimula-tion in vitro (ImmuKnow; Cylex) (24).Serum was collected at baseline and 1year after treatment.

Clinical ManagementDuring hospitalization and home care,fingertip glycemic monitoring was per-formed before meals, 2 h after meals orat bedtime by turns, one to two times aday. It was similar to a whole-day in-tense monitoring when those values in$1 week were pooled together. Insulindosing was based on FBG before mealsand 2 h postprandially, with target levelsof,110 mg/dL (6.1 mmol/L) and ,140mg/dL (7.8 mmol/L), respectively. If thepatient presented clinical symptoms ofhypoglycemia or blood glucose ,90mg/dL (5.0 mmol/L), the insulin dosewould be decreased, even when bloodglucose levels before meals or 2 h post-prandiallywere.110mg/dL (6.1mmol/L)or 140 mg/dL (7.8 mmol/L). Endocrinolo-gists periodically counseled patients onhealthy diet and exercise to avoid clinicalcare discrepancies or nonadherence. In-sulin doses were managed by the study’sendocrinologist (Z.W.).

Quality-of-Life MeasuresGlobal anxiety and depression status wasassessed separately at baseline and 1 yearafter SCT by the participants and thestudy physician, who was unaware ofthe group assignment, using the Self-Rating Anxiety Scale (range 20–80, withhigher scores indicating greater anxiety),the Self-Rating Depression Scale (range20–80,with higher scores indicatingmoresevere depression), and the summary

Figure 1—Study chart and HLA proportions. A: Screening, randomization, and completion of1-year evaluations. B: Representation of the distribution of patients in the two study groups onthe 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).

care.diabetesjournals.org Cai and Associates 151

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scores for the physical andmental quality-of-life (QOL) components of the MedicalOutcomes Study 36-Item Short-Form Sur-vey (range 0–100, with higher scores in-dicating better health status).

Statistical AnalysisA computer-generated block randomiza-tion was used to assign each subject toone of theexperimental groups. Statisticalanalysis was performed using SPSS ver-sion 10.1, GraphPad Prism 6, and Micro-soft Excel software. Data are presentedas mean 6 SD. The x2 test, independentt test, two-factor repeated-measuresANOVA, and mixed-effects linear modelwere used for two-group numerationdata comparison, two-group measure-ment data comparison, repeated-measurescomparison (normal distribution), andrepeated-measures comparison (nonnor-mal distribution), respectively. Tests yield-ing P , 0.05 were considered statisticallysignificant. Power and sample size consid-erations assumea50% increaseofAUCC-Pepat 1 year after treatment from an average6.67 pmol/mL/180min of Chinese patientswith T1D. Student t test of independenceconsidered two independent groups of21 patients each as having adequate powerto detect this assumed difference (type Ierror = 0.05, 90% power).

RESULTS

Patient CharacteristicsForty-twopatients (22 female and20male)with established T1D were enrolledand randomized to receive SCT or stan-dard treatment (Fig. 1A). Both groupswere well matched in terms of baselinecharacteristics (Table 1 and Fig. 1B),with no statistically significant dif-ferences between the SCT and controlconditions in terms of mean age at thetime 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 (59.506 8.42and 60.336 10.76 kg), BMI (21.996 1.78and 22.06 6 2.46 kg/m2), HbA1c (8.56 60.81% [70.06 6.5 mmol/mol] and 8.6860.87% [71.0 6 7.1 mmol/mol]), FBG(200.06 6 51.09 and 192.43 6 35.318mg/dL), insulin dose (0.91 6 0.23 and0.90 6 0.20 IU/day/kg), and sCr(68.95 6 14.79 and 73.90 6 13.26mmol/L). At the time of enrollment,66.67% of patients (14 of 21) in the SCTgroup and 55.14% (12 of 21) in the controlgroup were GADA positive (Table 1 andSupplementary Table 2), which did not

appear to correlate with disease duration(data not shown). The frequency of HLAalleles associatedwith T1D riskwere com-parable among the study subjects in bothgroups (Fig. 1B and Supplementary Tables1 and 2); 95% of patients (n = 20) in theSCT group and 86% (n = 18) in the controlgroup had at least one predisposing al-lele, 62% and 52% of SCT and controlgroup patients had at least the HLA-DRallele associated with T1D (DR9, DR4,and/or DR3) (25). Of these, 43% (n = 9)in the SCT group and 38% (n = 8) in thecontrol group had at least one HLA-DRplus 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 thecontrol group had none of the risk alleles.

Therapeutic Efficacy of SCTAt 1 year, metabolic measures improvedin SCT recipients. The AUCC-Pep increased105.7% from basal (6.6 6 6.1 to 13.6 68.1 pmol/mL/180 min, P = 0.00012),with 15 of 21 patients (71.4%) showingincreased levels at 1 year. In contrast,AUCC-Pep decreased 7.7% in con-trol subjects (8.4 6 6.8 to 7.7 64.5 pmol/mL/180 min, P = 0.013 vs. SCT),with 8 of 21 patients (38.0%) showingimproved values at 1 year (Fig. 2A andSupplementary Fig. 1A). The AUCIns in-creased in SCT recipients 49.3% frombasal (1,477.8 6 1,012.8 to 2,205.5 61,194.0 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 6441.6 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 12months (repeated-measures ANOVA P, 0.01), whereas theyremained stable in the control groupduringthe follow-up period (SCT vs. control P ,0.01 for all time points) (Fig. 2C and Sup-plementary Fig. 1C). HbA1c decreased12.6% in the SCT group from 8.66 0.81%(70.0 6 7.1 mmol/mol) to 7.5 6 1.0%(58.068.6mmol/mol) (P,0.01),whereasit increased 1.2% in control subjects from8.760.9% (72.067.5mmol/mol) to8.860.9% (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 itdecreased significantly in the SCT groupat 3, 6, 9, and 12 months (P , 0.002 vs.baseline for all time points in SCT) andwas significantly lower than in the controlgroup (P , 0.042 at 3 months, P , 0.01

thereafter) (Fig. 2D and SupplementaryFig. 1D). At 12 months, FBG decreased24.4% in SCT recipients (200.0 6 51.1 to151.2 6 22.1 mg/dL) and 4.3% in con-trol subjects (192.4 6 35.3 to 184.2 634.3mg/dL) (Fig. 2D and SupplementaryFig. 1D).

Progressive and significant reductionsin insulin dose requirements after trans-plantation were observed in the SCTgroup at 3, 6, 9, and 12 months (P ,0.002), whereas they were unchangedin 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 SCTgroup (0.9 6 0.2 to 0.6 6 0.2 IU/day/kg,P = 0.001) and was unchanged (;0%) inthe control group (0.9 6 0.2 to 0.9 60.2 IU/day/kg, P , 0.01 vs. SCT) (Fig. 2Eand Supplementary Fig. 1E).

Fasting C-peptide levels were mainlyunchanged in the control group (Fig. 2Fand Supplementary Fig. 1F), whereasthey markedly increased in the SCTgroup at 9 and 12 months (comparedwith baseline, P , 0.01) (Fig. 2F andSupplementary Fig. 1F). Fasting C-peptidein the SCT group was significantly higherthan in the control group at 9 and12 months (P , 0.01 and P = 0.00001,respectively). Comparing baseline with12-month data, an increase was ob-served in the SCT group (0.03 6 0.02to 0.06 6 0.03 pmol/mL, P , 0.01),with 20 of 21 patients (95.2%) showingimprovement, whereas no change wasfound in the control group (0.02 6 0.02to 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 MeasuresAt baseline, patients in both groupsdemonstrated similar symptoms of anx-iety and depression and QOL scores(Table 2). At 12 months, patients in theSCT group showed decreased anxietyand depression symptoms and improvedQOL score, whereas these measures didnot changemarkedly in the control group(Table 2).

SafetyPatient-reported severe hypoglycemicevents were lower in the SCT groupthan in the control group (0.43 [0–2] vs.20.048 [21 to 1], P = 0.02). In the SCTgroup, transient abdominal pain was

152 UC-MSC and Autologous BM-MNC Transplant for T1D Diabetes Care Volume 39, January 2016

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Table

1—Base

linech

aracteristicsofpatients

Infusion

HLA

allele

Studygroup

andpt.no.*

Sex

Age

atonset

(years)

T1D

duration

(years)

Body

wt(kg)

BMI(kg/m

2)

GADApos.

aten

roll.

AUCC-Pep

(pmol/mL/

180min)

HbA1c(%

)FBG(m

g/dL)

Insulin

dose

(IU/

day/kg)

sCr

(mmol/L)

MSC

(310

6/kg)

BM-M

NC

(310

6/kg)

DR

AB

SCT 1

F14

754

20.58

Yes

21.24

7.5

306.0

0.74

761.05

151.85

1\14

2\3

7\35

2F

2011

5519

.26

No

12.70

7.8

201.6

1.18

841.12

136.36

12\–

3\24

7\51

3M

2316

8023

.89

Yes

1.90

8.0

61.2

0.83

691.04

77.50

3\9

2\24

46\75

4M

196

6419

.97

Yes

12.14

7.9

190.8

0.80

100

1.36

57.81

3\9

11\33

51\58

5M

282

4420

.09

No

2.53

8.4

203.4

0.70

640.95

61.36

3\9

11\–

51\60

6M

1411

6524

.46

Yes

19.90

8.4

203.4

0.80

920.96

29.23

3\7

33\74

44\58

7M

514

5719

.72

Yes

1.90

7.9

230.4

1.19

731.24

133.33

4\15

11\24

61\62

8F

2215

6025

.30

Yes

1.90

8.9

221.4

0.83

551.07

43.33

3\–

11\33

58\–

9F

139

5521

.48

No

14.85

10.5

282.6

0.80

431.01

103.64

3\9

2\–

46\58

10F

2215

5622

.43

Yes

2.02

8.3

142.2

1.00

671.58

67.86

4\9

2\24

48\60

11F

185

58.2

22.45

No

2.10

9.2

176.4

1.00

480.88

116.84

4\9

2\11

56\70

12F

195

58.2

22.45

No

7.02

7.9

199.8

1.17

521.57

147.77

9\14

2\–

46\51

13F

252

5722

.27

Yes

3.17

8.5

203.4

1.12

560.91

138.60

8\11

2\11

60\75

14F

166

5722

.27

No

2.18

10.4

244.8

1.37

720.89

161.40

4\12

24\32

39\51

15M

2115

6521

.97

Yes

4.84

9.2

235.8

0.58

741.39

132.31

8\12

2\11

60\75

16M

1910

7024

.80

No

5.01

7.9

185.4

0.44

821.23

52.86

1\11

24\32

39\51

17F

1411

5221

.10

Yes

4.60

8.9

226.8

1.00

631.04

182.69

11\12

11\–

51\55

18F

1115

5021

.36

Yes

2.27

7.8

169.2

1.16

520.87

164.00

15\11

11\24

13\60

19F

2012

5119

.43

Yes

8.98

9.2

140.4

0.84

690.96

178.43

8\12

11\24

60\–

20M

234

7123

.72

Yes

5.33

8.8

172.8

0.72

720.99

30.99

9\13

2\33

46\58

21M

183

7022

.86

Yes

1.93

8.4

203.4

0.79

850.96

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

dd

d

Ctrl. 22

M20

1162

22.77

Yes

2.62

9.3

210.6

1.00

73d

d14

\15

11\24

35\60

23F

206

5221

.64

Yes

4.35

8.1

176.4

1.08

63d

d9\14

2\11

61\62

24M

217

6319

.88

Yes

8.31

7.7

237.6

0.94

86d

d12

\15

2\24

46\56

25M

153

6824

.38

No

9.43

8.2

165.6

1.06

76d

d4\8

2\24

61\–

26F

168

4919

.63

Yes

1.92

8.7

187.2

0.98

54d

d7\12

11\30

13\60

27M

2411

6419

.97

No

9.02

10.3

237.6

0.97

93d

d4\13

26\30

38\70

28F

2113

4720

.08

No

7.81

9.4

199.8

1.23

67d

d8\10

1\2

37\60

29F

225

5220

.83

Yes

4.52

7.5

142.2

1.08

79d

d3\13

11\33

58\60

30F

204

5521

.22

Yes

3.65

8.4

136.8

0.95

81d

d4\14

11\24

13\38

31F

166

4922

.37

No

16.23

9.1

190.8

0.67

47d

d4\9

11\24

60\61

32F

2710

5722

.27

Yes

30.88

8.3

241.2

1.19

54d

d1\3

24\26

35\60

33M

245

6822

.46

Yes

4.25

9.9

273.6

0.76

78d

d12

\–2\24

61\75

34M

245

7626

.30

Yes

1.90

10.2

207

0.61

86d

d4\14

2\31

54\75

35M

1913

7226

.45

No

12.95

8.2

147.6

0.61

99d

d8\13

3\–

7\46

36M

2210

7926

.70

Yes

3.79

8.8

176.4

0.72

68d

d12

\311

\24

51\60

37F

253

4818

.99

No

5.95

7.7

185.4

1.02

59d

d12

\–2\24

51\58

38M

222

7522

.89

Yes

5.21

9.6

207

0.71

75d

d12

\411

\74

51\60

Con

tinuedon

p.15

4

care.diabetesjournals.org Cai and Associates 153

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observed in one patient during cell trans-plantation, which resolved without se-quel. Bleeding at the puncture site wasobserved in another patient (1 of 21[4.7%]), which resolved after applyinglocal pressure. Upper respiratory tractinfections were comparable betweengroups, with seven cases in the SCT group(7 of 21 [33%]) and five in the controlgroup (5 of 21 [23.8%]); all resolvedwith medical therapy (Table 2). No re-markable changes in C-reactive protein,white blood cell counts, hemoglobin,sCr, and alanine aminotransferase wereobserved (data not shown). No severeadverse events, such as malignant tu-mors, were observed during the follow-up period.

Immunological ParametersAt baseline, the SCT and the controlgroups had similar GADA-positive rates(66.7% vs. 57.1%), IL-10 levels (4.76 4.2vs. 5.3 6 4.4 pg/mL), IFN-g levels (6.0 63.0 vs. 7.26 3.2 pg/mL), and ATP levels inCD4+ T cells (378.7 6 52.8 vs. 376.0 671.7 ng/mL) (P . 0.05 for all compari-sons). At 1 year, patients in the SCT groupshowed a 75% increase in IL-10 levels(8.26 7.7 vs. 4.76 4.2 pg/mL; one-tailedt test P, 0.03), a 50.7%decrease in IFN-glevels (3.06 1.8 vs. 6.06 3.0 pg/mL; two-tailed t test P, 0.001, 1 year vs. baselinein SCT group; P, 0.00004, SCT vs. controlat 1 year), and a 9.7% decrease in ATPlevels in CD4+ T cells (345.3 6 43.6 vs.378.7 6 52.8 ng/mL; one-tailed t testP, 0.03, baseline vs. 1 year in SCT group;two-tailed t test P = 0.045, SCT vs. controlat 1 year). These changes were significantcomparedwith those in the control group(Supplementary Fig. 2). The overallGADA-positive rates at 1 year were notsignificantly different between the twogroups (57% in SCT and 52% in control),with only two and one patients havingconverted from GADA positive to GADAnegative in the SCT and control groups,respectively. None of the study subjectsshowed conversion from GADA negativeto GADA positive.

CONCLUSIONS

Therapeutic strategies for T1D must ad-dress the autoreactive host immune sys-tem as well as pancreatic b-cell repairand regeneration. Most of the T1D clin-ical trials have been conducted in pa-tients soon after disease onset (7,8)when it is more likely to expect a clinicalbenefit. However, there is increasing

evidence that some level of insulin pro-duction is maintained in many patientsyears after diagnosis, and some recenttrials have enrolled patients within2 years from diagnosis. Herein, we de-scribe the results of combined UC-MSCand aBM-MNC transplantation in pa-tients with established T1D.

We show that cotransplantation ofallogeneic Wharton’s jelly UC-MSC andaBM-MNC is followed by signs of im-proved insulin secretion and reduce in-sulin requirement, as indicated bysignificantly improved fasting C-peptidelevels, AUCC-Pep (primary end point), andAUCIns during OGTT performed at 1year. As well, we observed reductionsin HbA1c, FBG, and insulin requirementcompared with baseline and the controlgroup. Although the absolute change inC-peptide is marginal, it is relatively sig-nificant in view of the long disease du-ration of the study patients, many ofwhom had no or barely detectable fast-ing C-peptide levels.

Clinical trials of MSC therapy for thetreatment of acute graft-versus-hostdisease following allogeneic hematopoi-etic SCT (26) and to improve outcome inallogeneic renal transplantation (19,27),among other applications (28), haveshown encouraging results. Carlssonet al. (9) recently reported on the ben-eficial effect of BM-MSC in newly diag-nosed individuals with T1D. Hu et al. (29)reportedmetabolic improvements (fast-ing and postprandial glycemia, HbA1c,fasting C-peptide) paralleled by reduc-tions of exogenous insulin requirementsfollowing administration of UC-MSCs innewly diagnosed patients with T1D.Moreover, Thakkar et al. (13) recentlyreported the safety and efficacy of coin-fusion of insulin-secreting adipose-derived MSCs and BM-HSCs in patientswith T1D in which the use of autologousinoculum appeared to confer betterlong-term control of hyperglycemiacompared with allogenic SCT.

Compared with new-onset T1D, MSCsmay be less effective in long-standingT1D because of the fewer inflammatorysignals in the pancreatic microenviron-ment, which are essential for homingMSCs toward the pancreas. Moreover,MSCs infused intravenously undergo apulmonary first-pass effect and arelikely to be sequestered in the lungs(30). Therefore, cells were injectedthrough the pancreatic artery in the

Table

1—Continued

Infusion

HLA

allele

Studygroup

andpt.no.*

Sex

Age

atonset

(years)

T1D

duration

(years)

Body

wt(kg)

BMI(kg/m

2)

GADApos.

aten

roll.

AUCC-Pep

(pmol/mL/

180min)

HbA1c(%

)FBG(m

g/dL)

Insulin

dose

(IU/

day/kg)

sCr

(mmol/L)

MSC

(310

6/kg)

BM-M

NC

(310

6/kg)

DR

AB

39F

138

5320

.20

No

13.36

8.1

169.2

1.04

73d

d12

\15

2\33

13\39

40M

227

5619

.61

Yes

8.25

9.2

212.4

0.86

82d

d10

\41\–

60\–

41M

205

7525

.06

No

4.34

7.5

172.8

0.53

85d

d7\–

24\–

39\60

42F

155

4719

.56

No

17.50

8.10

163.8

0.81

74d

d14

\–24

\26

37\38

Mean

10/11

20.38

7.00

60.33

22.06

12/21

8.39

8.68

192.43

0.90

73.90

dd

dd

d

Ctrl.,control;en

roll.,enrollm

ent;pos.,p

ositive;pt.no.,patientnumber.

154 UC-MSC and Autologous BM-MNC Transplant for T1D Diabetes Care Volume 39, January 2016

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present study to promote homing ofstem cells directly to the pancreas (31).Of note, we did not observe any patientwith abnormal amylase levels, which in-dicated the safety of pancreatic arterialinfusion. Furthermore, the patients’QOL significantly improved, possibly re-flecting the positive impact of improvedmetabolic control following SCT. Protocol-associated side effects weremild and self-limiting.Although the use of autologous SCT

may prevent sensitization to allogeneic

antigens, it may introduce the issue ofdiverse yields of stem cell numbers orfailure to expand cells during culture(data not shown) in established T1Ddue to impaired function of stem cellsobtained from individuals with diabetes(32–34). Conversely, UC stem cells ob-tained from healthy donor tissues havethe advantage of abundant yield, whichcould guarantee homogeneity and simi-lar quantities of infused cells. Therefore,in designing the present trial, we rea-soned that combination of aBM-MNCs

with allogeneic adjuvant cells, such asUC-MSCs with, reportedly, hypoimmu-nogenicity, may represent a viable strat-egy toward retaining and recoveringstem cell properties and increasing effi-cacy of SCT for the treatment of T1D.Urban et al. (12) suggested that MSCsalone might be inadequate for tissue re-generation and repair in experimentalmodels of T1D, therefore requiring a com-plementary treatment. Moreover, BM-MNCs comprising multiple cell fractionsof undifferentiated stem cells and differ-entiated cells are appealing owing to theirtissue 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 effectof MSC and SCT in patients with T1D arenot yet fully understood. First, the impactof immunomodulation by stem cellsshould be noted (13,36,37). In the pres-ent study, we could investigate T-cell ac-tivation and Treg-related cytokinesbefore and after SCT. We found thatSCT was associated with increased serumlevels of IL-10, a regulatory cytokine, withdecreased serum levels of IFN-g (T-helper1 cytokine) and ATP production by CD4+ Tcells (ImmuKnow), indicating reducedT-cell activation (24). Residual b-cellmass has been described in pancreaticspecimens obtained from cadaveric do-nors with T1D, suggesting that interven-tions able to dampen inflammation maybe beneficial toward achieving recoveryof function (14). Whether SCT influenceon T-cell and Treg function is one of themechanisms involved in the current studyremains controversial. Zhao et al. (38)showed that treatment of established T1Dwith UC-MSCs provided lasting reversal ofautoimmunity. In the present study, wecould also assess GADA levels, which re-mained largely unchanged; however, thisis only a partial assessment of autoimmuneresponses, andmore studies are needed toaddress the effects of anti-islet responses.

Similarly, BMCs were shown to con-tribute to b-cell expansion and to de-velop into functionally competentpancreatic b-cells when homed to pan-creatic islets after transplantation in ex-perimental animals (35). It could bespeculated that the positive effects ofSCT on insulin secretion could also resultfrom the regeneration of residualb-cellsor from the generation of new b-cellsfrom BMC precursors; however, this

Figure 2—Metabolic function. A: The AUCC-Pep increased markedly from baseline in the SCTgroup at 1 year after transplantation (two-tailed t test P = 0.0012). The change was significantcompared with the control group at 1 year (two-tailed t test P = 0.013). B: AUCIns significantlyincreased in the SCT group at 1 year comparedwith baseline levels (two-tailed t test P = 0.01) andthe control group at 1 year (one-tailed t test P = 0.027, SCT vs. control). C: HbA1c levels signif-icantly decreased from baseline in the SCT group (repeated-measures ANOVA P , 0.01 for all)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 controlgroup, whereas they improved over time in the SCT group compared with baseline (repeated-measures ANOVA P, 0.002) and respective values of the control group during follow-up (P ,0.042 at 3 months, P , 0.01 thereafter). E: Exogenous insulin requirements were significantlylower 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(P , 0.01, SCT vs. control from 6 to 12 months). F: The SCT group showed improved fastingC-peptide levels from baseline to 9 and 12 months (repeated-measures ANOVA P , 0.01) andcompared with the same time points in the control group (two-tailed t test P , 0.001 at9 months, P = 0.00001 at 12 months). *P, 0.05 comparison of changes from baseline betweenthe two groups.

care.diabetesjournals.org Cai and Associates 155

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hypothesis cannot be tested in the ab-sence of biopsy data.The limitations of this pilot study

include a relatively small sample sizeand the short duration of follow-up.Moreover, the independent contribu-tion of each cell product (namely, UC-MSCs and aBM-MNCs) was not assessedseparately. Although metabolic controlimproved in patients receiving SCT, in-sulin independence was not achieved.Additionally, the lack of a placebo groupmay generate bias in the QOL measure-ments, which should be verified in a fu-ture large-scale study. Assessment oflong-term safety is paramount, consider-ing the potential risk of tumors generatedfrom unwanted MSC differentiation orfrom other unknown factors related toMSCs. Of note, we did not find abnormal-ities in chromosome numbers in the UC-MSCs used in the current trial. Othershave reported the stability of culturedMSCs regarding the development of ab-normal chromosomes after several pas-sages well beyond that used in thepresent trial (39,40). Patients are coun-seled to have regular health checks to de-termine early any malignancy that maydevelop during follow-up.In conclusion, we established the

safety of the approach and proof of con-cept that SCT may lead to measurableimprovements of metabolic function inpatients with established T1D. The en-couraging results point to a number ofissues that should be addressed in the

design of future large-scale trials to helpto improve clinical outcomes.

Acknowledgments. The authors thank JinhuaChen (Statistics Office, Fuzhou General Hospital,Xiamen University, Fuzhou, China) for criticalcontributions to the statistical work of this study.Funding. This study was supported by theFujian Province (Major Research Project Fund2009Y4001, Technology Innovation PlatformProject Fund 2008J1006 and 2010Y2006, andSpecial Program for Key Science Research2012YZ0001), the People’s Liberation ArmyClinical Innovation Major Project Fund(2010gxjs026), and the Natural Science Founda-tion of Fujian Province (2012J01408). Generoussupport by the Diabetes Research InstituteFoundation, Hollywood, FL, is acknowledged.Duality of Interest. No potential conflicts ofinterest relevant to this article were reported.Author Contributions. J.Ca. contributed toperforming the study and writing the manu-script. Z.W. contributed to performing thestudy, collecting and analyzing the data, andwriting the manuscript. X.X., L.L., A.Pu., A.Pi.,and C.R. contributed to analyzing the data andwriting the manuscript. J.Ch. and L.H. preparedthe cells. W.W. and C.W. contributed to per-forming the study. F.L. contributed to collectingthe data. J.T. designed the study. J.T. is theguarantor of this work and, as such, had fullaccess to all the data in the study and takesresponsibility for the integrity of the data andthe accuracy of the data analysis.Prior Presentation. Parts of this study werepresented at the International Diabetes Federa-tion’s 2015 World Diabetes Congress, Vancouver,Canada, 30 November–4 December 2015.

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2. Skyler JS, Ricordi C. Stopping type 1 diabetes:attempts to prevent or cure type 1 diabetes inman. Diabetes 2011;60:1–83. The Diabetes Control and Complications TrialResearch Group. The effect of intensive treat-ment of diabetes on the development andprogression of long-term complications in insulin-dependent diabetesmellitus. N Engl JMed 1993;329:977–9864. Zgibor JC, Songer TJ, Kelsey SF, et al. Theassociation of diabetes specialist care withhealth care practices and glycemic control inpatients with type 1 diabetes: a cross-sectionalanalysis from the Pittsburgh Epidemiology ofDiabetes Complications Study. Diabetes Care2000;23:472–4765. Beilhack GF, Scheffold YC, Weissman IL, et al.Purified allogeneic hematopoietic stem celltransplantation blocks diabetes pathogenesisin NOD mice. Diabetes 2003;52:59–686. Couri CE, deOliveiraMC, Simões BP. Risks, ben-efits, and therapeutic potential of hematopoieticstem cell transplantation for autoimmune diabe-tes. Curr Diab Rep 2012;12:604–6117. Voltarelli JC, Couri CE, Stracieri AB, et al. Autol-ogous nonmyeloablative hematopoietic stem celltransplantation innewly diagnosed type1diabetesmellitus. JAMA 2007;297:1568–15768. Li L, Shen S, Ouyang J, et al. Autologous hema-topoietic stem cell transplantation modulates im-munocompetent cells and improves b-cellfunction in Chinese patients with new onset oftype 1 diabetes. J Clin Endocrinol Metab 2012;97:1729–17369. Carlsson PO, Schwarcz E, Korsgren O, LeBlanc K. Preserved b-cell function in type 1 di-abetes by mesenchymal stromal cells. Diabetes2015;64:587–59210. D’Addio F, ValderramaVasquezA, BenNasrM,et al. Autologous nonmyeloablative hematopoieticstem cell transplantation in new-onset type 1 di-abetes: a multicenter analysis. Diabetes 2014;63:3041–304611. Wang J, Liao L, Tan J. Mesenchymal-stem-cell-based experimental and clinical trials:

Table 2—QOL and adverse events

SCT group Control group P value*

Anxiety (SAS) scoreBefore treatment 38.5 6 7.1 38.3 6 9.3 NSAfter treatment 34.6 6 5.4 39.7 6 8.9 0.0053

Depression (SDS) scoreBefore treatment 38.0 6 6.6 40.4 6 6.6 NSAfter treatment 33.3 6 4.7 39.7 6 6.3 0.0091

QOL (SF-36) scoreBefore treatment 78.6 6 8.4 79.4 6 8.1 NSAfter treatment 82.4 6 5.9 78.6 6 7.9 0.0368

Adverse event type URTI Bleeding Abdominal pain URTINumber of occurrences 7* 1* 1* 5Time after treatment (months) 3 (1–6) 0† 0‡ 4 (2–6)

Adverse event grade Mild Mild Mild MildAttribution Unrelated Definite Definite UnrelatedIntervention Medical Local pressure None MedicalOutcome Resolved Resolved Resolved Resolved

Data are mean 6 SD or mean (range). SAS, Self-Rating Anxiety Scale; SDS, Self-Rating Depression Scale; SF-36, Medical Outcomes Study 36-ItemShort-Form Survey; URTI, upper respiratory tract infection. *P, 0.05 comparedwith the occurrence rate in the control group. †Occurred in theward3 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.

156 UC-MSC and Autologous BM-MNC Transplant for T1D Diabetes Care Volume 39, January 2016

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