In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells

(Pages: 701-710)
Morteza Abouzaripour, Ph.D, 1Parichehr Pasbakhsh, Ph.D, 1Nader Atlasi, B.Sc, 1Abdol Hossein Shahverdi, Ph.D, 2Reza Mahmoudi, Ph.D, 3Iraj Ragerdi Kashani, Ph.D, 1,*
Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Medicine, ACECR, Tehran, Iran
Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Department of Embryology, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Medicine, ACECR, Tehran, Iran
Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
*Corresponding Address: P.O.Box: 1417613151 Department of Anatomical Sciences School of Medicine Tehran University of Medical Sciences Tehran Iran Email:ragerdi@sina.tums.ac.ir
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Abouzaripour Morteza, Pasbakhsh Parichehr, Atlasi Nader, Shahverdi Abdol Hossein, Mahmoudi Reza, Kashani Iraj Ragerdi. In Vitro Differentiation of Insulin Secreting Cells from Mouse Bone Marrow Derived Stage-Specific Embryonic Antigen 1 Positive Stem Cells. Cell J. 2016; 17(4): 701-710.

Abstract

Objective

Bone marrow has recently been recognized as a novel source of stem cells for the treatment of wide range of diseases. A number of studies on murine bone mar- row have shown a homogenous population of rare stage-specific embryonic antigen 1 (SSEA-1) positive cells that express markers of pluripotent stem cells. This study focuses on SSEA-1 positive cells isolated from murine bone marrow in an attempt to differentiate them into insulin-secreting cells (ISCs) in order to investigate their differentiation potential for future use in cell therapy.

Materials and Methods

This study is an experimental research. Mouse SSEA-1 positive cells were isolated by Magnetic-activated cell sorting (MACS) followed by characteriza- tion with flow cytometry. Induced SSEA-1 positive cells were differentiated into ISCs with specific differentiation media. In order to evaluate differentiation quality and analysis, dithizone (DTZ) staining was use, followed by reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and insulin secretion assay. Statistical results were analyzed by one-way ANOVA.

Results

The results achieved in this study reveal that mouse bone marrow contains a population of SSEA-1 positive cells that expresses pluripotent stem cells markers such as SSEA-1, octamer-binding transcription factor 4 (OCT-4) detected by immunocytochem- istry and C-X-C chemokine receptor type 4 (CXCR4) and stem cell antigen-1 (SCA-1) detected by flow cytometric analysis. SSEA-1 positive cells can differentiate into ISCs cell clusters as evidenced by their DTZ positive staining and expression of genes such as Pdx1 (pancreatic transcription factors), Ngn3 (endocrine progenitor marker), Insulin1 and Insulin2 (pancreaticβ-cell markers). Additionally, our results demonstrate expression of Pdx1 and Glut2 protein and insulin secretion in response to a glucose challenge in the differentiated cells.

Conclusion

Our study clearly demonstrates the potential of SSEA-1 positive cells to differentiate into insulin secreting cells in defined culture conditions for clinical ap- plications.