Phase II Results of UC-MSCs in the Treatment of Type 2 Diabetes

Stem cells have always been promising for the treatment of diabetes. However, there has long been a lack of systematic and objective clinical evidence to support it. Just on May 3, researchers from the First Medical Center of the General Hospital of the Chinese People's Liberation Army published a paper titled "Efficacy and safety of umbilical cord-derived mesenchymal stem cells in Chinese adults with" in the journal "Stem Cell Research and Treatment". type 2 diabetes: a single-center, double-blinded, randomized, placebo-controlled phase II trial ”

The results of the Phase II clinical study well summarize the potential value of stem cell (umbilical cord mesenchymal stem cells used in the study) treatment for type 2 diabetes. The editor has excerpted some of the content for communication purposes only.

background

Type 2 diabetes mellitus (T2DM), a heterogeneous syndrome characterized by progressive deterioration of glycemic control due to reduced insulin sensitivity and decreased insulin secretion, has become a serious global human health concern due to its high prevalence and severe morbidity. threaten. Dietary control, physical exercise, and antidiabetic drugs have been shown to temporarily improve hyperglycemia, but cannot inhibit the pathogenesis of T2DM or reduce the incidence. Therefore, there is a need to develop more effective treatments for T2DM.

Mesenchymal stem cells (MSCs) are a type of adult stem cells that have strong anti-inflammatory and immunomodulatory capabilities by secreting a variety of cytokines and immunosuppressive molecules [1,2] . It has been successfully used to treat different types of autoimmune diseases, such as stroke [ 3] , myocardial infarction [4] , rheumatoid arthritis, systemic lupus erythematosus [5,6] and graft-versus-host disease [7] . Chronic inflammatory response and immune disorder in pancreatic islets can lead to insulin resistance, pancreatic β-cell destruction and the onset of T2DM. Therefore, MSCs transplantation may be a treatment option for T2DM. Previous animal studies have shown that MSCs treatment improves hyperglycemia by promoting islet recovery and alleviating insulin resistance [8, 9] . In addition, more and more clinical trials have reported the therapeutic effect and safety of MSCs transplantation in patients with T2DM [10,11,12,13,14,15,16,17,18,19,20] .

MSCs can be derived from various tissues (e.g., bone marrow, adipose tissue, and umbilical cord), and there is no ethical controversy in the acquisition or application of MSCs. MSCs were originally isolated from bone marrow [21] , which is considered the most accessible source of MSCs for the treatment of T2DM [22] . Several clinical trials have confirmed the therapeutic potential of bone marrow-derived MSCs (BM-MSCs) in T2DM [10, 12, 14, 15, 16, 17] . Umbilical cord-derived MSCs (UC-MSCs) have similar immunosuppressive properties to BM-MSCs and have clinical potential for the treatment of T2DM due to their low cost, painless, high yield, rapid collection, and non-immunogenic characteristics [19] . However, few clinical studies have focused on using UC-MSCs to treat T2DM, and few clinical trials have reported the impact of UC-MSCs on insulin resistance in T2DM patients. Therefore, a single-center, randomized, double-blind, placebo-controlled phase II trial was conducted to explore the efficacy and safety of intravenous infusion of UC-MSCs in patients with T2DM.

method

Study design and participants

This prospective, single-center, randomized, double-blind, placebo-controlled Phase II trial was conducted from October 2015 to December 2018 at the First Medical Center of the General Hospital of the People's Liberation Army of China. The research protocol was approved by the Ethics Committee of PLAGH First Medical Center ( approval number: 2013-107-01 ) and complied with the guidelines of the Declaration of Helsinki. All participants provided written informed consent prior to recruitment. The study is registered at ClinicalTrials.gov ( Registration Number: NCT02302599 ).

Patients who met the following inclusion criteria were eligible: (1) Aged between 20 and 65 years old; (2) Diagnosed with T2DM < 20 years (HbA1c level between 7.0% and 12.0%); (3) Metformin stable insulin therapy (0.5-1.0 U/kg/day) Insufficient control ≥ 3 months; (4) Fasting C-peptide level ≥ 1 ng/mL; (5) Body mass index (BMI) 24-40 kg/m 2. If the patient has ketonuria, tumors, serum creatinine level < 175 μmol/L, previously diagnosed myocardial infarction, current angina or heart failure, > 1 major vascular event, retinopathy requiring laser treatment, malignant hypertension, unexplained Intervening endocrine disease, occupation precluding insulin therapy, serious co-morbidities that limit life expectancy, poor understanding of the study protocol, drug abuse, planned pregnancy, and allergies.

program

Human umbilical cords were obtained from healthy women who gave birth at the First Medical Center of the Chinese People's Liberation Army. All subjects provided informed consent. All procedures were performed in accordance with the guidelines of the Ethics Committee of the First Medical Center of the Chinese People's Liberation Army. UC-MSCs were isolated from gelatinous tissues surrounding veins and arteries and characterized by phenotypic analysis and cell differentiation assays as previously described [23, 24] . Patients who met the inclusion criteria were randomly assigned to receive intravenous infusion of UC-MSCs ( 100 mL ) or the same dose of placebo ( UC-MSCs suspension, consisting of normal saline containing 3% human albumin and 0.5 mL of multivitamins) into the elbow. The patient was infused into the joint intravenously 3 times with an interval of 4 weeks, and was discharged after 24 hours of observation without any adverse reactions. This study used 4 generations of UC-MSCs , and the total number of UC-MSCs per infusion was 1 × 10 6 /kg .

After discharge, patients were followed up every 12 weeks for 48 weeks and were asked to perform blood glucose self-monitoring at different times during the follow-up period ( ≥ 15 times per week, including the 5- point curve). Insulin titration is based on peripheral blood glucose levels, with a target fasting glucose level between 4.4 and 7.0 mmol/L . Patients are advised to maintain a regular diet and healthy lifestyle management during hospitalization and follow-up . If patients' total daily insulin dose was ≤0.2 U/kg at any time during the study , they were discontinued from exogenous insulin and given oral antidiabetic agents to prevent hypoglycemia. If patients experience uncontrolled blood sugar when the total daily insulin dose is > 1.5 U/kg , they will stop the study and adjust the blood sugar-lowering strategy according to the patient's condition to prevent diabetic complications.

Assess insulin needs and HbA1c levels every 12 weeks.

result

Characterization of human UC-MSCs

Cultured human UC-MSCs had bipolar spindle and fibroblast-like morphology, and the immunophenotypic characteristics and multilineage differentiation potential of adherent cells were subsequently examined. The cells expressed surface markers of UC-MSCs, including CD90, CD73, and CD105, while negative markers of UC-MSCs, including CD34, CD45, and HLA-DR, were not detected. In addition, UC-MSCs showed no effects on osteogenesis. Differentiation potential of cells and adipocytes.

study population

Between October 2015 and December 2018, 183 patients were screened for eligibility, and 91 patients were ultimately recruited. Enrolled subjects were randomly assigned to receive UC-MSCs transplantation (n = 45) or placebo (n = 46). A total of 73 (86.8%) patients completed the trial, including 37 (82.2%) patients in the UC-MSCs group and 36 (78.3%) patients in the placebo group. Two patients in the placebo group did not complete all treatment. Two patients withdrew due to adverse events, and 6 patients in both groups were lost to follow-up (Figure 1). Demographic and clinical characteristics at baseline were balanced between the two groups (Table 1).

Curative effect

Compared with the placebo group, more patients in the UC-MSCs group reached the primary endpoint . At 48 weeks after treatment, 20% of patients in the UC-MSCs group achieved the goals of HbA1c <7.0% and daily exogenous insulin reduction of ≥50%, compared with 4.55% in the placebo group (p<0.05, Table 2). The 95% CI for the difference in rates between the two groups was 2.25-28.66%.

In both groups, daily insulin requirements progressively decreased at weeks 9, 20, 32 and 48 compared with their respective baseline values ​​(p<0.01 compared with baseline in both groups). However, at 9, 20, 32 and 48 weeks, patients in the UC-MSCs group still had lower insulin requirements than the placebo group (0.50±0.18 vs 0.63±0.19 U/kg/day, p<0.01; 0.50±0.24vs0 .58±0.24U/kg/day, p > 0.05; 0.49±0.24vs0.59±0.27 U/kg/day, p > 0.05; 0.45±0.25vs0.57±0.26U/kg/day, p< 0.05) (Figure 2A). Similar patterns were observed for total insulin dose in both groups at the same time points (38.80±15.89vs49.75±14.92U/day, p<0.01; 37.81±19.87vs45.56±19.14U/day, p>0.05; 37.32 ± 19.63 vs. 46.19±21.85 U/day, p > 0.05; 34.51±20.19 vs. 45.19±21.21 U/day, p<0.05) (Figure 2B). At 9, 20, 32 and 48 weeks, the percentage of insulin reduction in the UC-MSCs group was significantly higher than that in the placebo group (30.00% vs 12.77%, p<0.01; 27.83% vs 15.25%, p<0.01; 24.44 % vs 10.24%, p < 0.05, 27.78% vs 15.62%, p < 0.05) (2C). Overall, 13.5% (5/37) of patients were insulin-free within 8–24 weeks (12±7.6 weeks) after UC-MSCs transplantation, and no insulin was repeated within 37.2±15.2 weeks . and all patients except those in the placebo group.

HbA1c levels decreased after UC-MSCs transplantation, with the greatest decrease at 9 weeks after treatment and a slight increase at 20, 32, and 48 weeks of follow-up; however, they remained lower than baseline values. In addition, HbA1c levels in the placebo group decreased slightly at 9 weeks and then gradually increased to higher levels. During the follow-up period, the HbA1c level in the UC-MSCs group was lower than that in the placebo group (p < 0.01 at 9 and 48 weeks, p < 0.05 at 20 weeks, and p < 0.05 at 32 weeks in both groups) (Figure 2D). The HbA1c level of the UC-MSCs group decreased by 1.31% at 48 weeks, while the control group only decreased by 0.63% (p=0.0081). Compared with the placebo group, more participants with UC-MSCs achieved target HbA1c levels (<7.0%) at 9, 20, 32, and 48 weeks. These results demonstrate the therapeutic potential of UC-MSCs transplantation in T2DM.

Fasting C-peptide levels in the UC-MSCs group did not change significantly after treatment, but decreased in the placebo group (Figure 3A). The fold change of glucagon-stimulated C-peptide levels in the UC-MSCs group increased at 9 weeks and gradually decreased at 20 and 48 weeks. Fold changes in C-peptide levels gradually decreased in the placebo group. AUCC-pep in the UC-MSCs group increased at 9 and 20 weeks, decreased slightly at 48 weeks, and gradually decreased in the placebo group. These results indicate that UC-MSCs did not significantly improve islet β-cell function in T2DM patients.

HEC results showed that UC-MSCs transplantation effectively reduced insulin resistance in T2DM patients. The GIRs of the placebo group at baseline and 9 and 48 weeks were 3.26, 3.26, and 3.60 mg/min/kg, respectively (p > 0.05 compared with baseline) , and the GIRs of the UC-MSCs group were 3.12, 3.97, and 4.76, respectively. mg/min/kg (p<0.01 at weeks 9 and 48 compared to baseline) (p<0.05 at week 9 and p<0.01 at week 48 between the two groups) (Figure 3D) .

safety

All adverse events are summarized in Table 3. Two patients in the UC-MSCs group withdrew from the trial. A female patient with a 10-year history of hypertension and hyperlipidemia developed cerebral infarction 1 month after the third infusion of UC-MSCs. He recovered after two months of treatment. A male patient suffered a femoral neck fracture due to an accident 3 months after the third infusion of UC-MSCs. After discussion with the independent adjudication committee, it was deemed that these events were not related to UC-MSCs treatment.

In the placebo group, one patient with positive insulin autoantibodies (IAA) and one patient with proliferative anemia withdrew from the trial. One patient was diagnosed with papillary thyroid cancer and one patient was diagnosed with prostate cancer at the last visit; therefore, they discontinued the study. No treatment-related deaths were reported during the study period. Other patients had no positive tumor markers and no tumors were found in imaging examinations. Physical examination and laboratory tests revealed no other UC-MSCs-related side effects during the 48 weeks of follow-up. All patients (n=91) remain under follow-up for potential late side effects.

discuss

This prospective, single-center, randomized, double-blind, placebo-controlled Phase II trial demonstrated that intravenous infusion of UC-MSC into the elbow joint resulted in a higher proportion of patients achieving target HbA1c levels compared with placebo. 7.0% and reduce daily insulin dosage to ≥50% at 48 weeks. UC-MSCs treatment reduced daily insulin requirements, lowered HbA1c levels, and improved insulin resistance in a time-dependent manner . No major adverse events related to UC-MSCs transplantation occurred. These results indicate that UC-MSCs transplantation is safe and effective in the treatment of T2DM.

In 2009, Bhansali et al. first showed that BM-MSCs transplantation significantly reduced insulin requirements and increased stimulated C-peptide levels in 10 T2DM patients [10]. Subsequently, multiple studies confirmed the effectiveness and safety of BM-MSCs and placenta-derived MSCs in the treatment of T2DM [11,12,13,14,15,16,17] , including three prospective, randomized, single Blind placebo-controlled studies [14,15,17] . UC-MSCs have low immunogenicity, are easy to obtain and do not involve ethical issues [25] . Therefore, they may be a new source of cell therapies for diabetes . Three studies used UC-MSCs transplantation to treat T2DM in humans. Patients in one study had insulin requirements reduced by ≥50% of subjects). However, these indices worsen over the next 3-6 months [18] . In another study, 18 T2DM patients received three intravenous infusions of UC-MSCs, 2 weeks apart, for a total of 6 months. The results showed that 8 out of 18 patients responded to treatment, manifested by reduced fasting and postprandial blood glucose levels; however, there was no significant difference in insulin dosage before and after treatment [ 19 ] .

In another trial, 6 T2DM patients were treated with two intravenous infusions of UC-MSCs, 2 weeks apart, with a follow-up period of ≥24 months. One-half of the patients stopped taking insulin between 25 and 43 months. The remaining patients continued to require insulin injections, but at significantly reduced doses [20] . These studies have initially confirmed the efficacy of UC-MSCs in the treatment of T2DM. However, results on post-treatment insulin requirements are inconsistent. In this double-blind, randomized, placebo-controlled study, more patients in the UC-MSCs group achieved the primary endpoint 48 weeks after treatment compared with the placebo group and had higher insulin requirements after UC-MSCs treatment. It showed a time-dependent decrease, which indicates the efficacy of UC-MSCs in the treatment of T2DM. However, the percentage of insulin-reduced and insulin-free patients in our UC-MSCs group was lower than reported in other studies, which may be due to the heterogeneity of enrolled subjects.

Previous studies have shown that HbA1c levels in T2DM patients gradually decreased by 1.2% after UC-MSCs transplantation, with the largest decreases observed at 3 and 6 months after treatment, and a slight increase at 12 months [18 ] . In this study, the HbA1c level in the UC-MSCs group decreased by 1.31%, and the percentage of patients reaching the target HbA1c level (<7%) was highest (55.56%) at week 9 and maintained at approximately 40%. The trend of HbA1c changes in our UC-MSCs group is similar to that published in other studies using UC-MSCs transplantation to treat T2DM patients. In addition, compared with other common antidiabetic drugs for the treatment of T2DM, our UC-MSCs infusion achieved a similar reduction in HbA1c levels, which confirmed the high efficiency of UC-MSCs in the treatment of T2DM .

Animal studies have shown that mesenchymal stem cell transplantation promotes the replication of residual β cells, improves islet autophagy function, promotes phenotypic dedifferentiation of islet β cells and polarized repair of macrophages in the local microenvironment of islets [26, 27 ] . In addition, some clinical studies have explored the effect of UC-MSCs on pancreatic β-cell function in T2DM patients. They found that fasting serum C-peptide levels gradually increased to a peak 6 months after UC-MSCs transplantation and then decreased slightly at 12 months. Similarly, another study found no significant changes in plasma C-peptide levels at four time points (OGTT 0, 1, 2, and 3 hours) after UC-MSCs infusion [ 19] . No significant improvement in fasting plasma C-peptide levels was observed in the UC-MSCs group . Glucagon-stimulated C-peptide levels and AUCC-pep transiently increased after UC-MSCs infusion and then gradually decreased to basal levels. Animal studies indicate that β-cell dedifferentiation rather than apoptosis is the major factor in T2DM. UC-MSCs treatment is a promising strategy to reverse β-cell dedifferentiation in T2DM. Since the reversal effect of UC-MSCs is stronger in the early stages of β-cell differentiation [28] , the improvement of pancreatic β-cell function in patients with long-term disease is not obvious.

MSCs infusion has been shown to improve insulin resistance in animal T2DM models. BM-MSCs infusion improves insulin resistance in T2DM rats by activating the insulin receptor substrate 1/protein kinase B signaling pathway [9] . UC-MSCs reduce insulin resistance in type 2 diabetic rats by inducing adipose tissue macrophages into an anti-inflammatory phenotype [29] . In addition, adipose-derived mesenchymal stem cells improve hyperglycemia in type 2 diabetic rats by regulating hepatic glucose metabolism [30] . It has been reported that UC-MSCs improve insulin resistance in the liver and adipose tissue of T2DM rats by inhibiting NLRP3 inflammasome-mediated inflammation [31] . Few clinical studies have focused on the effect of UC-MSCs on insulin resistance in T2DM. In this study, we demonstrated that UC-MSCs infusion significantly alleviated insulin resistance in a time-dependent manner, indicating that UC-MSCs treatment can effectively improve insulin resistance in T2DM.

The therapeutic effect of UC-MSCs on various diseases has been reported, and no obvious adverse reactions have been reported [32, 33] . In our study, one patient suffered cerebral infarction due to long-term uncontrolled hypertension and hyperlipidemia, which was not related to the infusion of UC-MSCs, and no cell-related immune response or tumor formation was found. Safety evaluation showed that UC-MSCs administered via intravenous infusion were well tolerated in T2DM patients . However, further observation of possible transplant complications is needed.

This study was a single-center trial enrolling a small number of Chinese patients. The enrolled subjects were highly heterogeneous in terms of age, T2DM disease duration, pancreatic β-cell function status, and insulin resistance. Therefore, the results cannot be extended to all patients with T2DM. Since our patients underwent short-term follow-up, long-term follow-up is needed to validate the current findings. In the future, well-controlled studies with more cases are needed to clarify the effectiveness and safety of intravenous infusion of UC-MSCs in the treatment of T2DM.

in conclusion

This prospective, single-center, randomized, double-blind, placebo-controlled trial demonstrates that administration of UC-MSCs via intravenous infusion is a safe and effective method to reduce exogenous insulin requirements and alleviate insulin resistance in patients with T2DM. UC-MSCs transplantation may be a potential treatment option for T2DM.