BIOCHARACTERISTICS OF c-kit+ HUMAN AMNIOTIC FLUID-DERIVED MESENCHYMAL STEM CELLS ANDTHEIR DIFFERENTIATION INTO CARDIOMYOCYTES IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

BAI Jing ¹ , WANG Yiru ² , LIU Lifeng ² , CHEN Jie ² , WANG Yu ²

1Department of Cardiology, the First Affiliated Hospital of General Hospital of Chinese PLA, Beijing, 100048, P.R.China;;
2Instituteof Geriatric Cardiology, General Hospital of Chinese PLA.;

Corresponding author：

Keywords：

c-kit+ human amniotic fluid-derived mesenchymal stem cells, Cardiomyocyte differentiation, Myocardium regeneration treatment ,Biocharacteristic

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【Abstract】 Objective To investigate the biocharacteristics of c-kit+ human amniotic fluid-derived mesenchymal stem
cells (HAFMSCs) and its capacity to differentiate into cardiomyocytes in vitro. Methods Fifty samples of human amniotic
fluid were obtained from amniocenteses or voluntary termination of pregnancy and were expanded in vitro. c-kit+ HAFMSCs were sorted by flow cytometry and were recultured in the same media. c-kit+ HAFMSCs were amplified and identified, then exposed to osteogenic , adi pogenic, and myogenic media. The flow cytometry, and immunocytochemistry were used for identifying the cell phenotype, von Kossa staining for osteogenic differentiation, oil red O staining for adi pogenic differentiation, and real-time fluorescent quantitative PCR for the expressions of NKx2.5, Tbx5, GATA-4, and α-MHC genes. Results After the selection procedure, the percentage of c-kit+ HAFMSCs was 3.07% ± 1.03% of the total adherent cells. The cells expressed MSCs markers (CD29, CD44, CD73, CD90, CD105), and did not express hematopoietic stem cells markers (CD34, CD45). The cells were positive for human leukocyte antigen (HLA)-ABC, and negative for HLA-DR. They also expressed Oct-4, which characterized the undifferentiated stem cell state. The growth curves of c-kit+ HAMFSCs at passages 5, 10, and 15 were similar. Li pid droplet was observed by oil red O staining and calcium deposition by von Kossa staining in the cells at 21 days after adi pogenic and osteogenic induction. The myocardium special gene expressions of Tbx5, Nkx2.5, GATA-4, and α-MHC were significantly ber after myogenic induction than those before myogenic induction (P lt; 0.05). Conclusion Selected c-kit+ HAFMSCs by flow cytometry is a group of pure MSCs, which has potential to differentiate into cardiomyocytes and can be used as seeding cells for myocardium regeneration treatment.

Citation： BAI Jing,WANG Yiru,LIU Lifeng,CHEN Jie,WANG Yu. BIOCHARACTERISTICS OF c-kit+ HUMAN AMNIOTIC FLUID-DERIVED MESENCHYMAL STEM CELLS ANDTHEIR DIFFERENTIATION INTO CARDIOMYOCYTES IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(2): 152-157. doi: Copy

1.	Jeremy L, Herrmann, Aaron M, et al. Cell-based therapy for ischemic heart disease: a clinical update. Ann Thorac Surg, 2009, 88(5): 1714-1722.
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6.	Prusa AR, Marton E, Rosner M, et al. OCT-4 expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003, 18(7): 1489-1493.
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8.	De Coppi P, Bartsch G Ir, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
9.	Cabral AL, Anqelo PC, Leite HV, et al. Isolation, differentiation and biochemical aspects of amniotic fluid stem cell. Rev Assoc Med Bras, 2008, 54(6): 489-493.
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12.	Kim J, Lee Y, Kim H, et al. Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif, 2007, 40(1): 75-90.
13.	Delo DM, De Coppi P, Bartsch G Jr, et al. Amniotic fluid and placental stem cells. Methods Enzymol, 2006, 419: 426-438.
14.	Fleischman RA. From white spots to stem cells: the role of the Kit receptor in mammalian development. Trends Genet, 1993, 9(8): 285-290.
15.	Perin L, Sedrakyan S, Giuliani S, et al. Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis. PLoS One, 2010, 5(2): e9357.
16.	Arnhold S, Glüer S, Hartmann K, et al. Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure. Stem Cells Int, 2011, 2011: 715341.
17.	Phermthai T, Odglun Y, Julavijitphong S, et al. A novel method to derive amniotic fluid stem cells for therapeutic purposes. BMC Cell Biol, 2010, 11: 79.
18.	Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2011, 177(4): 2011-2021.
19.	Sieqel N, Rosner M, Unbekandt M, et al. Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet, 2010, 19(17): 3320-3331.
20.	Yeh YH, Wei HJ, Lee WY, et al. Cellular cardiomyoplasty with human amniotic fluid stem cells: in vitro and in vivo studies. Tissue Eng Part A, 2010, 16(6): 1925-1936.

1. Jeremy L, Herrmann, Aaron M, et al. Cell-based therapy for ischemic heart disease: a clinical update. Ann Thorac Surg, 2009, 88(5): 1714-1722.
2. Beltrami AP, Urbanek K, Kajstura J, et al. Evidence that human cardiac myocytes divide after myocardial infarction. N Eng J Med, 2001, 344(23): 1750-1757.
3. Kajstura J, Ubannek K, Perl S, et al. Cardiomyogenesis in the adult human heart. Circ Res, 2010, 107(2): 305-315.
4. Lloyd-Jones D, Adams RJ, Brown TM, et al. Executive summary: heart disease and stroke statistics——2010 update: a report from the American Heart Association. Circulation, 2010, 121(7): 948-954.
5. 顾东风, 黄广勇, 河江, 等. 中国心力衰竭流行病学调查及其患病率. 中华心血管病杂志, 2003, 31(1): 3-6.
6. Prusa AR, Marton E, Rosner M, et al. OCT-4 expressing cells in human amniotic fluid: a new source for stem cell research? Hum Reprod, 2003, 18(7): 1489-1493.
7. Wolbank S, Stadler G, Peterbauer A, et al. Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics. Tissue Eng Part A, 2009, 15(7): 1843-1854.
8. De Coppi P, Bartsch G Ir, Siddiqui MM, et al. Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 2007, 25(1): 100-106.
9. Cabral AL, Anqelo PC, Leite HV, et al. Isolation, differentiation and biochemical aspects of amniotic fluid stem cell. Rev Assoc Med Bras, 2008, 54(6): 489-493.
10. Prusa AR, Hengstschlager M. Amniotic fluid cells and human stem cell research: a new connection. Med Sci Monit, 2003, 8(11): RA253-257.
11. Da Sacco S, Sedrakyan S, Boldrin F, et al. Human amniotic fluid as a potential new source of organ specific precursor cells for future regenerative medicine applications. J Urol, 2010, 183(3): 1193-1200.
12. Kim J, Lee Y, Kim H, et al. Human amniotic fluid-derived stem cells have characteristics of multipotent stem cells. Cell Prolif, 2007, 40(1): 75-90.
13. Delo DM, De Coppi P, Bartsch G Jr, et al. Amniotic fluid and placental stem cells. Methods Enzymol, 2006, 419: 426-438.
14. Fleischman RA. From white spots to stem cells: the role of the Kit receptor in mammalian development. Trends Genet, 1993, 9(8): 285-290.
15. Perin L, Sedrakyan S, Giuliani S, et al. Protective effect of human amniotic fluid stem cells in an immunodeficient mouse model of acute tubular necrosis. PLoS One, 2010, 5(2): e9357.
16. Arnhold S, Glüer S, Hartmann K, et al. Amniotic-fluid stem cells: growth dynamics and differentiation potential after a CD-117-based selection procedure. Stem Cells Int, 2011, 2011: 715341.
17. Phermthai T, Odglun Y, Julavijitphong S, et al. A novel method to derive amniotic fluid stem cells for therapeutic purposes. BMC Cell Biol, 2010, 11: 79.
18. Hauser PV, De Fazio R, Bruno S, et al. Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol, 2011, 177(4): 2011-2021.
19. Sieqel N, Rosner M, Unbekandt M, et al. Contribution of human amniotic fluid stem cells to renal tissue formation depends on mTOR. Hum Mol Genet, 2010, 19(17): 3320-3331.
20. Yeh YH, Wei HJ, Lee WY, et al. Cellular cardiomyoplasty with human amniotic fluid stem cells: in vitro and in vivo studies. Tissue Eng Part A, 2010, 16(6): 1925-1936.

Chinese Journal of Reparative and Reconstructive Surgery

BIOCHARACTERISTICS OF c-kit+ HUMAN AMNIOTIC FLUID-DERIVED MESENCHYMAL STEM CELLS ANDTHEIR DIFFERENTIATION INTO CARDIOMYOCYTES IN VITRO

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