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Clinical Trial
. 2012 Jan 10;10:3.
doi: 10.1186/1741-7015-10-3.

Reversal of Type 1 Diabetes via Islet β Cell Regeneration Following Immune Modulation by Cord Blood-Derived Multipotent Stem Cells

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Free PMC article
Clinical Trial

Reversal of Type 1 Diabetes via Islet β Cell Regeneration Following Immune Modulation by Cord Blood-Derived Multipotent Stem Cells

Yong Zhao et al. BMC Med. .
Free PMC article

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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