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In Vitro Cell Dev Biol Anim. 2016 Jan;52(1):49-61. doi: 10.1007/s11626-015-9945-4. Epub 2015 Sep 22.

Derivation of male germ cells from ram bone marrow mesenchymal stem cells by three different methods and evaluation of their fate after transplantation into the testis.

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Stem Cell Research Lab, Infertility and Reproductive Health Research Center, Health Research Institute, Babol University of Medical Sciences, Beginning of Babol-Amol Old Highway, P.O. Box: 47135-547, Babol, Iran.
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Bani Hashem Sq., Bani Hashem St., Resalat Highway, P.O. Box 19395-4644, Tehran, Iran.
Department of Andrology, Reproductive Biomedicine Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Urology, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran.


Mesenchymal stem cells (MSCs) have the capacity to differentiate into germ cells (GCs). This research, for the first time, has evaluated the fate of in vitro MSC-derived GCs generated by three different induction methods and compared them after transplantation into the testes of rams. Passage-3 ram bone marrow (BM)-MSCs were divided into three treatment groups: (1) 14-d treatment with 10 μM retinoic acid (RA; RA14), (2) 21-d treatment with 10 μM RA (RA21), and (3) 21-d treatment with 10 ng/ml transforming growth factor beta-1 (TGFb1). After confirmation of the existence of germ-like cells in the culture, the treated cells were labeled and transplanted into the testes of ram lambs. After 2 mo, we conducted histological evaluations of the rams' testes. Results showed that in vitro-derived GCs from all treatment groups survived in the testes. Some of these GCs homed at the basement membrane of seminiferous tubules and formed colonies. The homed cells and cell colonies were similar to testicular native spermatogonia and expressed PGP9.5. TGFb1 exhibited the highest efficiency for in vitro production of GCs as well as the highest capability for homing and colony formation in the testes. RA21 was less efficient than TGFb1, particularly in colony formation. RA14 was the weakest group. No further differentiation of the transplanted GCs was observed. From our results, it could be concluded that a 21-d treatment period of BM-MSCs with TGFb1 is the most efficient method for in vitro generation of spermatogonia-like cells that survive, home, and form colonies in the testes.


Fate; Germ cells; In vitro; Mesenchymal stem cells; Ram; Testis; Transplantation

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