Reversal of type 1 diabetes via islet β cell regeneration following immune modulation by cord blood-derived multipotent stem cells

Yong Zhao, Zhaoshun Jiang, Tingbao Zhao, Mingliang Ye, Chengjin Hu, Zhaohui Yin, Heng Li, Ye Zhang, Yalin Diao, Yunxiang Li, Yingjian Chen, Xiaoming Sun, Mary Beth Fisk, Randal Skidgel, Mark Holterman, Bellur Prabhakar, Theodore Mazzone, Yong Zhao, Zhaoshun Jiang, Tingbao Zhao, Mingliang Ye, Chengjin Hu, Zhaohui Yin, Heng Li, Ye Zhang, Yalin Diao, Yunxiang Li, Yingjian Chen, Xiaoming Sun, Mary Beth Fisk, Randal Skidgel, Mark Holterman, Bellur Prabhakar, Theodore Mazzone

Abstract

Background: Inability to control autoimmunity is the primary barrier to developing a cure for type 1 diabetes (T1D). Evidence that human cord blood-derived multipotent stem cells (CB-SCs) can control autoimmune responses by altering regulatory T cells (Tregs) and human islet β cell-specific T cell clones offers promise for a new approach to overcome the autoimmunity underlying T1D.

Methods: We developed a procedure for Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates lymphocytes from the whole blood and briefly co-cultures them with adherent CB-SCs before returning them to the patient's circulation. In an open-label, phase1/phase 2 study, patients (n=15) with T1D received one treatment with the Stem Cell Educator. Median age was 29 years (range: 15 to 41), and median diabetic history was 8 years (range: 1 to 21).

Results: Stem Cell Educator therapy was well tolerated in all participants with minimal pain from two venipunctures and no adverse events. Stem Cell Educator therapy can markedly improve C-peptide levels, reduce the median glycated hemoglobin A1C (HbA1C) values, and decrease the median daily dose of insulin in patients with some residual β cell function (n=6) and patients with no residual pancreatic islet β cell function (n=6). Treatment also produced an increase in basal and glucose-stimulated C-peptide levels through 40 weeks. However, participants in the Control Group (n=3) did not exhibit significant change at any follow-up. Individuals who received Stem Cell Educator therapy exhibited increased expression of co-stimulating molecules (specifically, CD28 and ICOS), increases in the number of CD4+CD25+Foxp3+ Tregs, and restoration of Th1/Th2/Th3 cytokine balance.

Conclusions: Stem Cell Educator therapy is safe, and in individuals with moderate or severe T1D, a single treatment produces lasting improvement in metabolic control. Initial results indicate Stem Cell Educator therapy reverses autoimmunity and promotes regeneration of islet β cells. Successful immune modulation by CB-SCs and the resulting clinical improvement in patient status may have important implications for other autoimmune and inflammation-related diseases without the safety and ethical concerns associated with conventional stem cell-based approaches.

Trial registration: ClinicalTrials.gov number, NCT01350219.

Figures

Figure 1
Figure 1
Overview of Stem Cell Educator therapy. A T1D participant (left) is connected to a Blood Cell Separator (right) and the Stem Cell Educator (bottom center) to form a closed system. Lymphocytes isolated from the T1D participant by the Blood Cell Separator travel through the Stem Cell Educator where they come in contact with CB-SCs attached to the interior surfaces of the device. Educated lymphocytes are returned to the patient's blood circulation. CB-SCs, cord blood stem cells; T1D, type 1 diabetes.
Figure 2
Figure 2
Improvement of β-cell function by Stem Cell Educator therapy. (A) Fasting C-peptide levels of T1D participants over 24 weeks. Group A and Group B participants (n = 6 per group) received one Stem Cell Educator treatment. Control group participants (n = 3) received sham therapy (no CB-SCs in the Stem Cell Educator). (B) 12-week follow-up C-peptide levels after OGTT at 2 hours in Group A T1D subjects with some residual β cells. (C) Comparison of C-peptide levels at glucose challenge after 40-week follow-up in Group B T1D subjects. The dashed red line indicates the lower limit for normal C-peptide levels in Chinese populations. The dashed purple line indicates the minimum detectable level (sensitivity) of C-peptide by radioimmunoassay (RIA). CB-SCs, cord blood stem cells; OGTT, oral glucose tolerance test; T1D, type 1 diabetes.
Figure 3
Figure 3
Markers of immune function in T1D patients after Stem Cell Educator therapy. Patient lymphocytes were isolated from peripheral blood by Ficoll-Hypaque (γ = 1.077) for flow cytometry analyses in T1D patients at baseline and 4 weeks after Stem Cell Educator therapy. Isotype-matched IgG served as control. (A) Flow Analysis of CD4+CD25+Foxp3+ Tregs demonstrating an increase in the percentage of Tregs at 4 weeks post-treatment. (B) Cytokine ELISAs demonstrating an increase in TGF-β1 but not IL-10 at 4 weeks post treatment. (C) Flow cytometry on co-stimulating molecules indicating increases in CD28 and ICOS at 4 weeks post-treatment with Stem Cell Educator therapy (top panels). Control group failed to show increases (bottom panels). (D) Flow analysis of intra-cellular cytokines demonstrating differential effects on key interleukins at 4 weeks post-treatment. Data are representative of preparations from all T1D participants (n = 12) that received Stem Cell Educator therapy. ELISA, enzyme-linked immunosorbent assay; ICOS, inducible costimulator; IgG, immunoglobulin G; IL10, interleukin 10; T1D, type 1 diabetes; Tregs, regulatory T cells.
Figure 4
Figure 4
Characterization of Aire in CB-SC. (A) Expression of Aire mRNA in CB-SCs. Real time PCR analysis for Aire mRNA expression followed by electrophoresis in 2% agarose gel. Data are representative of three CB-SC preparations. (B) Immunocytochemistry for Aire. Isotype-matched IgG served as control (left) for Aire staining (right) with magnification ×200. (C) Western blot shows the dose-dependent knockdown response of Aire following siRNA treatment. (D) Effects of Aire knockdown on PD-L1. Western blot demonstrates decreased expression of program death ligand-1 (PD-L1) in CB-SC following knockdown of Aire expression by siRNA. CB-SC cells transfected with negative control siRNA (NC siRNA) served as control for three pairs of human Aire-specific siRNA (P1, P2, and P3) at optimal concentration (50 nM). Representative data of those obtained from five experiments. (E) Effects of Aire knockdown on co-cultured lymphocytes. Flow analysis of Treg population following culture of lymphocytes alone, in the presence of phytohaemagglutinin (PHA, 10 μg/ml), in the presence of PHA and NC siRNA-treated CB-SCs, and in the presence of PHA and Aire siRNA (50 nM)-treated CB-SCs. Representative data obtained from three experiments. Aire, autoimmune regulator; CB-SCs, cord blood stem cells; IgG, immunoglobulin G: PCR, polymerase chain reaction; PHA, phytohaemagglutinin; siRNA, small interfering RNA; T1D, type 1 diabetes; Tregs, regulatory T cells.

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