Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지
DOI QR코드

DOI QR Code

Transdifferentiation of α-1,3-Galactosyltransferase Knock Out (GalT KO) Pig Derived Bone Marrow Mesenchymal Stromal Cells (BM-MSCs) into Pancreatic Cells by Transfection of hPDX1

hPDX1 유전자의 삽입에 의한 직접 췌도세포 분화

  • Ock, Sun A (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Oh, Keon Bong (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Hwang, Seongsoo (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Kim, Youngim (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Kwon, Dae-Jin (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration) ;
  • Im, Gi-Sun (Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration)
  • 옥선아 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 오건봉 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 황성수 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 김영임 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 권대진 (농촌진흥청 국립축산과학원 동물바이오공학과) ;
  • 임기순 (농촌진흥청 국립축산과학원 동물바이오공학과)
  • Received : 2015.09.01
  • Accepted : 2015.09.23
  • Published : 2015.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of ${\beta}-cells$, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from ${\alpha}$-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3~4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in ${\beta}-cell$ replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

Keywords

References

  1. Ahn KS, Kim YJ, Kim M, Lee BH, Heo SY, Kang MJ, Kang YK, Lee JW, Lee KK, Kim JH, Nho WG, Hwang SS, Woo JS, Park JK, Park SB and Shim H. 2011. Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts. Theriogenology. 75:933-999. https://doi.org/10.1016/j.theriogenology.2010.11.001
  2. Allahverdi A, Abroun S, Jafarian A, Soleimani M, Taghikhani M and Eskandari F. 2015. Differentiation of human mesenchymal stem cells into insulin producing cells by using a lentiviral vector carrying PDX1. Cell J. 17:231-242.
  3. Association AD. 2013. Standards of medical care in diabetes- 2013. Diabetes Care. 36(Suppl 1):S11-66. https://doi.org/10.2337/dc13-S011
  4. Choi JB, Uchino H, Azuma K, Iwashita N, Tanaka Y, Mochizuki H, Migita M, Shimada T, Kawamori R and Watada H. 2003. Little evidence of transdifferentiation of bone marrowderived cells into pancreatic beta cells. Diabetologia. 46: 1366-1374. https://doi.org/10.1007/s00125-003-1182-9
  5. Ianus A1, Holz GG, Theise ND and Hussain MA. 2003. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J. Clin. Invest. 111:843-850. https://doi.org/10.1172/JCI200316502
  6. Karnieli O, Izhar-Prato Y, Bulvik S and Efrat S. 2007. Generation of insulin-producing cells from human bone marrow mesenchymal stem cells by genetic manipulation. Stem Cells. 25:2837-2844. https://doi.org/10.1634/stemcells.2007-0164
  7. Kim SK and MacDonald RJ. 2002. Signaling and transcriptional control of pancreatic organogenesis. Curr. Opin. GenetDev. 12:540-547. https://doi.org/10.1016/S0959-437X(02)00338-6
  8. Kumar SS, Alarfaj AA, Munusamy MA, Singh AJ, Peng IC, Priya SP, Hamat RA and Higuchi A. 2014. Recent developments in $\beta$-cell differentiation of pluripotent stem cells induced by small and large molecules. Int. J. Mol. Sci. 15: 23418-23447. https://doi.org/10.3390/ijms151223418
  9. Kunisada Y, Tsubooka-Yamazoe N, Shoji M and Hosoya M. 2012. Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells. Stem. Cell. Res. 8:274-284. https://doi.org/10.1016/j.scr.2011.10.002
  10. Miyazaki S, Yamato E and Miyazaki J. 2004. Regulated expression of pdx-1 promotes in vitro differentiation of insulinproducing cells from embryonic stem cells. Diabetes. 53: 1030-1037. https://doi.org/10.2337/diabetes.53.4.1030
  11. Pellegrini S, Ungaro F, Mercalli A, Melzi R, Sebastiani G, Dotta F, Broccoli V, Piemonti L and Sordi V. 2015. Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo. Acta. Diabetol. [Epub ahead of print]
  12. Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F and Brevini TA. 2013. Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc. Natl. Acad. Sci. USA. 110:8948-8953. https://doi.org/10.1073/pnas.1220637110
  13. Pokrywczynska M1, Lewandowska MA, Krzyzanowska S, Jundzill A, Rasmus M, Warda K, Gagat M, Deptula A, Helmin- Basa A, Holysz M, Nowacki M, Buchholz L, Bodnar M, Marszalek A, Grzanka A, Jozwicki W, Michalkiewicz J and Drewa 2015. Transdifferentiation of bone marrow mesen chymal stem cells into the islet-like cells: The role of extracellular matrix proteins. T. Arch. Immunol. Ther. Exp. (Warsz). [Epub ahead of print]
  14. Thompson P, Badell IR, Lowe M, Cano J, Song M, Leopardi F, Avila J, Ruhil R, Strobert E, Korbutt G, Rayat G, Rajotte R, Iwakoshi N, Larsen CP and Kirk AD. 2011. Islet xenotransplantation using gal-deficient neonatal donors improves engraftment and function. Am. J. Transplant. 11: 2593-602. https://doi.org/10.1111/j.1600-6143.2011.03720.x
  15. Xu ZF, Pan AZ, Yong F, Shen CY, Chen YW and Wu RH. 2012. Human umbilical mesenchymal stem cell and its adipogenic differentiation: Profiling by nuclear magnetic resonance spectroscopy. World J. Stem. Cells. 4: 21-27. https://doi.org/10.4252/wjsc.v4.i4.21
  16. Yuan H, Liu H, Tian R, Li J and Zhao Z. 2012. Regulation of mesenchymal stem cell differentiation and insulin secretion by differential expression of Pdx-1. Mol. Biol. Rep. 39: 7777-7783. https://doi.org/10.1007/s11033-012-1619-7
  17. Zalzman M1, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O, Zern MA, Fleischer N and Efrat S. 2003. Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc. Natl. Acad. Sci. USA. 100(12):7253-7258. https://doi.org/10.1073/pnas.1136854100
  18. Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y and Deng H. 2009. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res. 19:429-438. https://doi.org/10.1038/cr.2009.28
  19. 옥선아, 오건봉, 황성수, 임석기, 김영임, 박진기. 2013. $\alpha$-1,3-Galactosyltransferase Knock Out(GalT KO) 돼지유래 골수 중간엽 줄기세포의 특성 규명. 한국수정란이식학회지. 28: 281-287.