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Efficacy and safety of stem cells transplantation in patients with type 1 diabetes mellitus-a systematic review and meta-analysis.

Stem cells (SCs) therapy is a new promising therapeutic modality for type 1 diabetes (T1DM). We performed a systematic review and meta-analysis to evaluate the efficacy and safety of stem cells transplantation (SCT) in patients with T1DM. We searched five literature databases (MEDLINE, EMBASE, Web of Science, WanFang and CENTRAL) up to 31 October 2019. 29 studies (487 patients with T1DM) were included in our meta-analysis. There was no substantial publication bias. Meta-analysis showed the SCT had significant effect to decrease HbA1c (SMD, 1.40; 95% CI, 0.93 to 1.86; p < 0.00001; I2 = 89%) and to improve C-peptide levels (SMD, -0.62; 95% CI, -1.22 to -0.02; p = 0.04; I2 = 92%) at 1 year follow-up. Subgroup analyses showed the heterogeneity level of the results was high. Significant improvement of metabolic outcomes was observed in the subgroups of mesenchymal stem cells (MSCs) combined with hematopoietic stem cells (HSCs) and HSCs. The older age showed significant association with the efficacy in HSCs subgroup. The higher GADA positive rate before treatment also significantly associated with the decrease of daily insulin requirement. The transient insulin independence rate at last follow-up was 9.6 per 100 person-years (95% CI: 5.8-13.5%). The mean length of insulin independence was 15.6 months (95% CI: 12.3-18.9). The mortality of SCT was 3.4% (95% CI: 2.1-5.5%). Therefore, SCT is an efficacious and safe method for treating patients with T1DM especially in the subgroups of MSCs + HSCs and HSCs. Well designed, double blind and randomized controlled trails with large sample size and long-term follow-up are needed for further ‍evaluation.

transplantation has been developed in the past decades for the replacement of damaged β cells [2]. However, the development of autoantibodies, immunosuppression toxicity, limited donor supply, and high costs limit this therapy to patients with T1DM.
Due to the intrinsic regenerative potential and immunomodulatory abilities, the stem cells (SCs) therapy has opened up a breakthrough approach to cure autoimmune disease. Meta-analyses of studies on SCs therapy indicated a certain potential to treat liver cirrhosis [3][4][5], chronic liver disease [6], inflammatory bowel disease [7] and ischemic heart disease [8]. SCs therapy also showed a potential effect to halt or abolish the autoimmune destruction of β cells and to generate functional β cells which could achieve optimized glycemic control in T1DM [9][10][11][12][13]. Our systematic review and meta-analysis aims to explore the efficacy and safety of stem cells transplantation (SCT) in patients with T1DM. We also browsed the references of included studies and relevant systematic reviews.

Inclusion and exclusion criteria
Studies were identified carefully by two investigators working independently to determine whether an individual study was eligible for inclusion in the meta-analysis. When agreement could not be reached, a third researcher decided if the study was eligible, based on the same criteria of inclusion and exclusion. Inclusion criteria: individuals of any age, gender and population with T1DM according to the criteria of the American Diabetes Association before SCT. Exclusion criteria: Patients with malignancy, any acute or chronic infection, pregnancy, positive serology for human immunodeficiency virus, hepatitis B or C, underlying hematologic, nephrologic, cardiac, psychiatric, or hepatic disease, mental disorders, inborn or adaptive immunodeficiency and hypersensitivity. Case reported studies were excluded. Studies that did not report the detail data related to the efficacy or safety of SCT were also excluded.

Data extraction
Data were independently extracted by two investigators who reached a consensus on all of the items. Information extracted from each study was considered as follows: first author, year of publication, descriptions of patients, mode of injection and the outcomes we interested in, such as the daily insulin requirement, glycosylated hemoglobin (HbA1c) and C-peptide level in treatment group before and after treatment as well as the adverse and mortality effect. We defined severe complications contains bilateral pneumonia and pseudomonas aeruginosa sepsis, which may cause damage to vital organs. Dealing with missing data: we contacted original authors to clarify missing data or data which were not clearly reported.

Quality assessment of included studies
Two reviewers assessed methodological quality independently according to the Newcastle-Ottawa Scale (NOS). Any discrepancies were resolved by discussion, if disagreements cannot be resolved, a third reviewer were consulted.

Statistical analysis
Review Manager 5 and Comprehensive Meta Analysis software were used to perform the analyses. For studies reported quantitative data, a meta-analysis was undertaken when studies were sufficiently homogenous. If only two or three studies were to be identified, we performed a fixed-effects model meta-analysis. If more than three studies were to be identified, we performed randomeffects analysis. Publication bias was assessed by the funnel plots. Statistical heterogeneity was assessed by the forest plots. Subgroups were classified based on various sources of SCs, including mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), MSCs combined with HSCs (MSCs + HSCs), umbilical cord blood (UCB) and bone marrow mononuclear cells (BM-MNCs). We investigated the heterogeneity using the I 2 statistic which means the proportion of the unexplained heterogeneity by the estimates variability among studies. I 2 values of <25%, 25-50% and >50% represent minimal, moderate and substantial heterogeneity, respectively [14]. If the heterogeneity was high, meta-regression analyses were performed to test the association between the factors and the outcomes. All summary effects were presented with 95% CI [15].

Characteristics of studies
We retrieved 885 references from the electronic database, as showed on the flow diagram (Fig. 1). The 29 eligible studies met the inclusion and exclusion criteria . Main study characteristics were described in Table 1. In general, the total number of patients who underwent SCT was 487. Among them, 5 studies (including 45 participants) underwent MSCs transplantation, 13 studies (including 275 participants) underwent HSCs transplantation, 3 studies (including 52 participants) underwent MSCs + HSCs transplantation, 6 studies (including 83 participants) underwent UCB transplantation, and 2 studies (including 32 participants) underwent BM-MNCs transplantation. The ages of T1DM patients receiving SCT ranged from 3.02 to 40.3 years. These patients had less than 2 months to 11 years of disease duration before SCT. The follow-up periods ranged from 3 to 60 months. The overall quality of the included studies was optimal ( Table 2). There was no substantial publication bias be observed (Fig. 2). Fig. 3 and Table 3 showed the meta-analysis of daily insulin requirement (U/kg per day), HbA1c (%) and Cpeptide (ng/mL) in patients accepting SCT after 12 months follow-up. The overall analysis demonstrated that SCT had significant effect to decrease HbA1c (SMD, 1.40; 95% CI, 0.93 to 1.86; p < 0.00001; I 2 = 89%) and to improve C-peptide levels (SMD, -0.62; 95% CI, -1.22 to -0.02; p = 0.04; I 2 = 92%). SCT showed no significant effect to reduce daily insulin requirement (SMD, 0.55; 95% CI, -0.01 to 1.10; p = 0.05; I 2 = 90%), but the p was on the critical value.
Because of the high heterogeneity still observed in HSCs group, we further used meta-regression analysis to investigate the factors which might affect the efficacy of HSCs transplantation. The results showed that the older age and the higher GADA positive rate before treatment were significantly associated with the decrease of daily insulin requirement (p = 0.00017 and p = 0.00041 respectively) ( Fig. 4A and Fig. 4B). The older age was associated with the decrease of HbA1c and the improvement of C-peptide levels (p = 0.00077 and p = 0.00065  respectively) (Fig. 4C and Fig. 4D). However, the number of transplanted cells and the duration of T1DM showed no significant association with the outcomes in HSCs group (Table 4).

Discussion
The prevalence of T1DM has been increasing steadily over the past decades. Although the rapid developments of modern diabetes treatment have improved the life quality of T1DM patients, the long-term complications of T1DM cannot be completely avoided. More than a decade ago, a new research field named SCs therapy emerged in health science. Many medical research institutes began nearly a decade research on SCT in patients with T1DM.
To draw a more definitive conclusion of the efficacy and safety of SCT in T1DM, we performed a metaanalysis of 29 published articles. As indicated by the pooled effect in our meta-analysis, MSCs + HSCs and HSCs groups showed significant effects to reduce daily insulin requirement and HbA1c levels, as well as to improve C-peptide levels at 1 year follow-up compared to the baseline. Whereas the MSCs group only showed significant effect to decrease HbA1c levels but no significant effect to reduce daily insulin requirement or to improve C-peptide levels, which was different from the meta-analysis conducted by Gan J et al. [45]. As we know that, β cells function will decrease rapidly within years during the natural course of T1DM. The analysis suggest that MSCs, MSCs + HSCs and HSCs groups could be effective in maintaining β cells function at 1 year follow-up. However, further studies are needed to confirm the long-term effectiveness. Our data also showed that the older age was significantly associated with the decrease of daily insulin requirement, the decrease of HbA1c and the improvement of C-peptide levels in the HSCs subgroup. We further explored the detail data of these studies and found that the mean disease durations before treatment in these patients were less than 1 year, especiailly most of them were less than 6 months. Thus, if newly diagnosed older patients with T1DM could undergo HSCs transplantation within 1 year, the better outcomes would be achieved. In addition, we also found that the higher GADA positive rate before treatment showed significant association with the decrease of daily insulin requirement. The positive of GADA represented the definitely autoimmunity dierected towards the islet β cells. The immunomodulatory of SCT might play a role on the glycemic control and the decreasd insulin dosage. Initially, the SCs therapeutic potential was considered as the ability of differentiation into insulin-producing cells. However, this dogma has changed when new results had been published regarding to the immunomodulatory properties of SCs [46]. Vanikar et al. reported that the potential therapeutic effects of SCs was attributed to their intrinsic regenerative capacity and immunomodulatory properties, which could arrest autoimmune β cells destruction, protect residual β cells, promote endogenous regeneration, improve innate/alloimmune graft rejection, and restore β-cell-specific unresponsiveness without chronic immunosuppression and to reverse hyperglycemia [12].
While the UCB group showed no significant effect to decrease HbA1c levels or to improve C-peptide levels, but even significantly increased daily insulin requirement after treatment. In addition to the small sample of the studies, some other factors might actually limit the capacity of UCB to preserve β cells function. First, an insufficient number of cells that carried regenerative or immunoregulatory capacity may have been transferred to patients with T1DM. Second, the ongoing autoimmune response in newly-onset T1DM may contain memory T cells, which are refractive to regulate by UCB [47], that facilitate the ongoing autoimmune destruction of endoge-nous or de novo β cells. Parker MJ et al. suggested that a therapeutic approach involving transient immune depletion and subsequent induction of immune regulation is optimal [48]. Because of containing an abundant number of immature unprimed highly functional regulatory T cells (Tregs) which may facilitate bystander suppression of effector T cells, UCB still owns therapeutic potential on T1DM. Now the clinical trials are currently underway in patients with T1DM using expanded Tregs isolated from UCB (NCT02932826). We are looking forward to see the results to explore the therapeutic potential of UCB.
In addition, we analyzed the insulin independence length after SCT. The mean length of patients who achieved transient insulin independence after SCT was 15.6 months. As we all know, newly diagnosed T1DM patients may enter the honeymoon phase after initiation of insulin therapy. During this period, patients can maintain a good glycemic control with a little or no insulin treatment. This period usually lasts no longer than 1.5 years. After that, they will need more insulin to control their blood glucose. To eliminate the honeymoon period, we analyzed RR from the 4 RCT studies, the result suggested the SCT showed significant effect to improve insulin independence rate compared to control group. Among the 4 RCT studies, only one patient achieved 7months insulin independence in the insulin group [42]. Thus, we preliminary draw the conclusion that SCT might prolong the insulin independence in T1DM. And more RCT studies with long times follow-up and large sample size analyses are needed to further investigation.
Although SCT appears to be effective, the safety is an important issue. The common adverse events contained alopecia, fever, vomiting, nausea, gastric tract symptoms, leucopenia, rash and diarrhea. Gu et al. reported that most of the early adverse events were caused by the mobilization and conditioning drugs used during HSCs transplantation, whereas later adverse events were unremarkable compared with patients using insulin therapy [42]. D'Addio et al. found that, most of the adverse events due to the administration of a high-dose immunosuppressive regimen. This observation indicated the need for reducing the dosage of immunosuppressive or for eventually changing the immunosuppressive agents and for a stronger prophylaxis for infections to obtain a safer approach [31]. Our meta-analysis showed that the incidence rate of severe adverse events and the mortality rate were 3.9% and 3.4% respectively. Two patients died due to pseudomonas sepsis after autologous HSCs transplantation reported by two studies respectively. Thus, further studies and more clinical evidence are needed to ensure the safety of autologous HSCs transplantation before it can be more widely used in clinical.
In conclusion, our systematic review and meta-analysis suggested that MSCs + HSCs and HSCs transplantation have potential to significantly improve the β cells function of patients with T1DM. However, a complete cure of T1DM is yet to be achieved. Since the number of patients with T1DM is increasing, there needs more controlled and randomized trials with larger number of patients and long-term observations to draw more credible results of SCs therapy. In order to find the best approach, it is worthwhile exploring the precise mechanisms, dosage, timing and frequency to consummate the therapy.   No., number; Δdaily insulin requirement, decrease of daily insulin requirement after treatment; GADA+ (%), GADA positive rate before treatment; ΔHbA1c, decrease of HbA1c after treatment; ΔC-peptide, increase of C-peptide after treatment.