Platelet-Derived Mitochondria Display Embryonic Stem Cell Markers and Improve Pancreatic Islet β-cell Function in Humans

Yong Zhao, Zhaoshun Jiang, Elias Delgado, Heng Li, Huimin Zhou, Wei Hu, Marcos Perez-Basterrechea, Anna Janostakova, Qidong Tan, Jing Wang, Mao Mao, Zhaohui Yin, Ye Zhang, Ying Li, Quanhai Li, Jing Zhou, Yunxiang Li, Eva Martinez Revuelta, Jose Maria García-Gala, Honglan Wang, Silvia Perez-Lopez, Maria Alvarez-Viejo, Edelmiro Menendez, Thomas Moss, Edward Guindi, Jesus Otero, Yong Zhao, Zhaoshun Jiang, Elias Delgado, Heng Li, Huimin Zhou, Wei Hu, Marcos Perez-Basterrechea, Anna Janostakova, Qidong Tan, Jing Wang, Mao Mao, Zhaohui Yin, Ye Zhang, Ying Li, Quanhai Li, Jing Zhou, Yunxiang Li, Eva Martinez Revuelta, Jose Maria García-Gala, Honglan Wang, Silvia Perez-Lopez, Maria Alvarez-Viejo, Edelmiro Menendez, Thomas Moss, Edward Guindi, Jesus Otero

Abstract

Diabetes is a major global health issue and the number of individuals with type 1 diabetes (T1D) and type 2 diabetes (T2D) increases annually across multiple populations. Research to develop a cure must overcome multiple immune dysfunctions and the shortage of pancreatic islet β cells, but these challenges have proven intractable despite intensive research effort more than the past decades. Stem Cell Educator (SCE) therapy-which uses only autologous blood immune cells that are externally exposed to cord blood stem cells adhering to the SCE device, has previously been proven safe and effective in Chinese and Spanish subjects for the improvement of T1D, T2D, and other autoimmune diseases. Here, 4-year follow-up studies demonstrated the long-term safety and clinical efficacy of SCE therapy for the treatment of T1D and T2D. Mechanistic studies found that the nature of platelets was modulated in diabetic subjects after receiving SCE therapy. Platelets and their released mitochondria display immune tolerance-associated markers that can modulate the proliferation and function of immune cells. Notably, platelets also expressed embryonic stem cell- and pancreatic islet β-cell-associated markers that are encoded by mitochondrial DNA. Using freshly-isolated human pancreatic islets, ex vivo studies established that platelet-releasing mitochondria can migrate to pancreatic islets and be taken up by islet β cells, leading to the proliferation and enhancement of islet β-cell functions. These findings reveal new mechanisms underlying SCE therapy and open up new avenues to improve the treatment of diabetes in clinics. Stem Cells Translational Medicine 2017;6:1684-1697.

Keywords: Diabetes; Immune; Islet β cell; Mitochondria; Platelet; Stem cell; Stem cell educator; Type 1 diabetes.

© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
Long‐term follow‐up studies of SCE therapy in Chinese T1D and T2D subjects. All Chinese subjects received one treatment with the SCE therapy. (A): Kinetic examination of fasting C‐peptide levels during the 4‐year follow‐up period. Before receiving a SCE therapy, subject #1 diagnosed with T1D for 8 months with glycated hemoglobin (HbA1C) at 11.3%, islet autoantibodies IA‐2A+ and GAD+; subject 2 diagnosed with T1D for 5 months with HbA1C at 9.4%, islet autoantibodies IA‐2A+ and GAD+ and IAA+. (B): Comparison of C‐peptide levels at a 75‐g oral glucose challenge after 4‐year follow‐up in T1D subjects 1 and 2. (C): Recovered fasting and high glucose‐stimulated C‐peptide levels were retained in subject 3 through the follow‐up at 4 years post‐treatment with a SCE therapy. (D): Comparison of fasting and high glucose‐stimulated C‐peptide levels after 4‐year follow‐up in long‐standing severe T2D subjects (N = 4). Data are presented as mean ± SD. Abbreviation: OGTT, oral glucose tolerance test.
Figure 2
Figure 2
Modulation of platelets by SCE therapy in diabetic patients. (A–C): Clinical data were summarized from the clinical trials in Chinese diabetic subjects. Both T1D (N = 10) and T2D (N = 16) received one treatment with SCE therapy. A complete blood count (CBC) test was performed at baseline (before the treatment) and 1 month after the treatment with SCE therapy. Age‐ and gender matched T1D (N = 7) and T2D (N = 10) subjects receiving conventional therapy served as controls. (A): Increase in the platelet count. (B): Increase in the platelet distribution width (PDW). (C): Decrease in the mean platelet volume (MPV). (D): Increase in the platelet count of Caucasian T1D subjects after receiving one treatment with SCE therapy, N = 8. Data are presented as mean ± SD.
Figure 3
Figure 3
Expression of immune modulation‐related markers in platelets and mitochondria. (A): Flow cytometry showed expression of co‐inhibitory surface markers PD‐L1, CD270, ICOS, and Galectin 9 on CB‐platelets (n = 9). (B): Flow cytometry showed the expression of co‐inhibitory surface markers PD‐L1, CD270, ICOS, and Galectin 9 on PB‐platelets (n = 8). (C): Intra‐cellular staining showed the expression of TGF‐β1 in CB‐platelets (n = 8). (D): Intra‐cellular staining showed the expression of TGF‐β1 in PB‐platelets (n = 4). (E): Intra‐cellular staining showed the expression of FoxP3 in CB‐ and PB‐platelets (n = 9 and 4, respectively). (F): Western blotting showed the expression of AIRE in seven cord blood preparations. (G): Double staining showed the expression of AIRE in CD41+ CB‐platelets (n = 5). (H): Western blotting showed the expression of AIRE in nine adult blood preparations. (I): Double staining showed the expression of AIRE in CD41+ PB‐platelets (n = 3). (J): Flow cytometry showed the expression of CD270 and CD274 (PD‐L1) on CB platelet‐releasing mitochondria labeled with MitoTracker Red. N = 7. (K): Phase contrast microscopy. Human PBMCs were activated with Dynabeads coupled with anti‐CD3 and anti‐CD28 antibodies, 30 U/ml rIL‐2 for 4 days, in absence (middle panels) and presence (right panels) of 70 μg/ml mitochondria. Untreated PBMCs (left right panel) served as control. Original magnification, ×40. (L): Quantification of cell proliferation after the treatment for 2 days. (M): Flow cytometry showed the increase in the percentage of CD4+PD1+ T cells. (N): Flow cytometry showed the increase in the percentage of CD8+PD1+ T cells. Data are presented as mean ± SEM from four experiments. Isotype IgGs serve as negative control for flow cytometry. Abbreviations: PBMC, peripheral blood‐derived mononuclear cells; ICOS, inducible costimulatory.
Figure 4
Figure 4
Expression of pancreatic islet β cell‐related markers in platelets. (A): Real time PCR analysis of pancreatic islet‐related hormone products and β‐cell‐related functional markers in CB‐platelets (n = 6). Freshly isolated human islets served as positive controls. (B): Real time PCR analysis of pancreatic islet β‐cell‐related transcription factors in CB‐platelets (n = 6). (C): Western blotting shows the protein expression of an islet β cell‐specific transcription factor MAFA in CB‐platelets. (D): Flow cytometry for human pancreatic islet‐related hormone products in freshly‐isolated human pancreatic islet cells. (E): Flow cytometry for the pancreatic islet‐related hormone products by double staining with platelet markers CD41 or CD42 in CB‐platelets. (F): Flow cytometry for pancreatic islet β‐cell‐related transcription factors by double staining with platelet markers CD41 or CD42 in CB‐platelets (n = 7). (G): Real time PCR analysis of pancreatic islet‐related hormone products and β‐cell‐related functional markers in PB‐platelets (n = 6). (H): Real time PCR analysis of pancreatic islet β‐cell‐related transcription factors in PB‐platelets (n = 6). (I): Flow cytometry for pancreatic islet‐related hormone products by double staining with platelet markers CD41 or CD42 in PB‐platelets (n = 15). (J): Flow cytometry of pancreatic islet β‐cell‐related transcription factors in PB‐platelets (n = 8). (K): Confocal microscopy of human CB‐ and PB‐platelets after triple immunostainings with insulin (blue), dense granule marker ADP (red), and α granule marker vWF (green). Isotype‐matched IgGs served as controls (inserted yellow dashed rectangle). Scale bars, 5 μm. Representative data were from six experiments.
Figure 5
Figure 5
Platelets express human ES cell markers. (A): Analysis of purified CB‐platelets by flow cytometry. The gated platelets in dot plot (top left panel, blue) were analyzed by using platelet markers CD41 and CD42, together with ES marker OCT4. Representative data of those obtained from eight experiments. (B): Flow cytometry after double staining with CD41 and ES markers in CB‐platelets (n = 8). (C): Induced pluripotent stem cells (iPSCs) as positive control express the ES cell markers, with isotype‐matched IgGs as negative controls (grey). (D): Flow cytometry after double staining with CD41 and ES markers in PB‐platelets (n = 4). (E): Gene expressions of ES markers in CB‐platelets are demonstrated by electrophoresis of real time PCR products. Their expressions in iPSCs served as positive controls. (F): Western blot showed the protein expression of ES markers in CB‐platelets. (G): Real time PCR showed gene expressions of ES markers in PB‐platelets. (H): Western blot showed the protein expression of ES markers in PB‐platelets.
Figure 6
Figure 6
Improve pancreatic islet β‐cell function by the platelet‐derived mitochondria. (A): Flow cytometry showed the basal release of mitochondria from platelets in dot plot (left) and histogram (right). (B): Effects of platelet aggregators on the releasing of mitochondria. Representative data of those obtained from four experiments. (C): Co‐culture of freshly‐isolated human pancreatic single islet cells (top chamber) with platelets labeled with MitoTracker Deep Red (bottom chamber) in Transwells. (D): Kinetic measurements by flow cytometry demonstrated that human pancreatic islet cells take up MitoTracker Deep Red‐labeled mitochondria released from platelets (bottom) in Transwell co‐culture. (E): Expression of chemokine and chemokine receptors on CB‐platelet‐derived mitochondria (n = 11). (F): Expression of adhesion molecules and chemokine receptors on human islet β cells. Representative data of those obtained from three experiments. (G): Migration and the taking up of mitochondria by human islet β cells were markedly declined in the presence of blocking Abs with anti‐CD29 and/or anti‐TLR4. (H): Cell viability was increased after single islet β cells co‐cultured with platelets in Transwells. (I): The number of islet β cells was increased after co‐culture with platelets in Transwells. (J): Co‐culture of freshly‐isolated whole human pancreatic islets (top chamber) with platelets labeled with mitochondria (bottom chamber) in Transwells. The pore size of transmembrane is 0.4 μm. (K): Islet cell viability was increased after co‐cultured with mitochondria. (L): The average islet size was increased after co‐cultured with mitochondria. (M): Flow cytometry indicates that the percentage of Insulin+Ki67+ islet β cells was increased after co‐cultured with mitochondria in Transwells. Mitochondria were purified from CB‐platelets (n = 4). (N): Functional analysis demonstrated that the C‐peptide release from islet β cells was enhanced in the presence of different insulin secretagogues. Data are presented as mean ± SEM (n = 3 experiments). (O): Immunohistochemistry of human pancreatic tissues from diabetic patients (N = 6) showed the migration of platelets into islets, with a formation of platelet clusters (green color, a platelet marker CD42a) at different sizes and close to islet β cells (red color, a β‐cell marker insulin). Scale bar, 10 μm. Abbreviations: ADP, adenosine diphosphate; ARA, arachidonic acid; Pct, percentage.

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