Male germ-like cell differentiation potential of human umbilical cord Wharton’s jelly-derived mesenchymal stem cells in co-culture with human placenta cells in presence of BMP4 and retinoic acid

Document Type: Original Article


1 Department of Reproductive Biology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Anatomical Sciences, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran

3 IVF Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

4 Institute for Molecular Medicine and Cell Therapy, Düsseldorf, Germany and GENEOCELL, Advanced Molecular & Cellular Technologies, Tehran, Iran

5 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

6 Pediatric Urology Research Center, Children Hospital Medical Center, Tehran University of Medical Sciences, Tehran, Iran

7 Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran


Objective(s):Mesenchymal stem cells (MSCs) derived from Wharton’s jelly (WJ-MSCs) are now much more appealing for cell-based infertility therapy. Hence, WJ-MSCs differentiation toward germ layer cells for cell therapy purposes is currently under intensive study.
Materials and Methods: MSCs were isolated from human Wharton’s jelly and treated with BMP4, retinoic acid (RA) or co-cultured on human amniotic epithelial (HAE) and chorionic plate (HCP) placenta feeder cells. profile of POU5F1, Fragilis, Plzf, DDX4, Piwil2, Stra8, Dazl, β1- and α6-integrins (ITΒ1, ITA6) genes expression as germ cell markers were analyzed using RT-PCR and real-time PCR. Immunocytochemistry of surface markers were conducted.
Results: After 3 weeks treatment with different reagents and co-culture system, morphology of WJ-MSCs  changed to shiny clusters and germ cell specific markers in mRNA were up-regulated in both placental feeder + RA and BMP4 + RA. Induction of hWJ-MSCs with BMP4 in presence of RA resulted in significant up-regulation (P≤0.05) of all germ cell specific genes (c-Kit; 2.84±0.59, DDX4; 1.69±0.39, Piwil2; 1.14±0.21, Dazl; 0.65±0.25, α6 integrin; 1.26±0.53, β1 integrins; 1.18±0.65) compared to control and placental feeder cells + RA. Our results indicated that HAE and HCP followed by RA treatment were involved in human germ cell development.
Conclusion: We demonstrated that under the right conditions, hWJ-MSCs have the ability to differentiate to germ cells and this provides an excellent pattern to study infertility cause and treatment.


1. Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Nat Acad Sci 2003; 100:11457-11462.

2. Kee K, Gonsalves JM, Clark AT, Pera RAR. Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem cells Dev 2006; 15:831-837.

3. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427:148-154.

4. Easley IV CA, Phillips BT, McGuire MM, Barringer JM, Valli H, Hermann BP, et al. Direct differentiation of human pluripotent stem cells into haploid spermatogenic cells. Cell Reports 2012; 2:440-446.

5. Panula S, Medrano JV, Kee K, Bergström R, Nguyen HN, Byers B, et al. Human germ cell differentiation from fetal-and adult-derived induced pluripotent stem cells. Hum Mol Genet 2011; 20:752-762.

6. Bongso A, Fong C-Y. The therapeutic potential, challenges and future clinical directions of stem cells from the Wharton’s jelly of the human umbilical cord. Stem Cell Rev  2013; 9:226-240.

7. Troyer DL, Weiss ML. Concise review: wharton's jelly-derived cells are a primitive stromal cell population. Stem Cell  2008; 26:591-599.

8. Nekanti U, Dastidar S, Venugopal P, Totey S, Ta M. Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia. Int J Biol Sci 2010; 6:499.

9. Carlin R, Davis D, Weiss M, Schultz B, Troyer D. Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC) matrix cells. Reprod Biol Endocrinol 2006; 4:8.

10. Ewen‐Campen B, Schwager EE, Extavour CG. The molecular machinery of germ line specification. Mol Reprod Dev 2010; 77:3-18.

11. Shirazi R, Zarnani AH, Soleimani M, Abdolvahabi MA, Nayernia K, Kashani IR. BMP4 can generate primordial germ cells from bone‐marrow‐derived pluripotent stem cells. Cell Biol Int 2012; 36:1185-1193.

12. Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev 1999; 13:424-436.

13. Ohta K, Lin Y, Hogg N, Yamamoto M, Yamazaki Y. Direct effects of retinoic acid on entry of fetal male germ cells into meiosis in mice. Biol Reprod 2010; 83:1056-1063.

14. Iwahashi K, Yoshioka H, Low EW, McCarrey JR, Yanagimachi R, Yamazaki Y. Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells. Biol Reprod 2007; 77:697-706.

15. Morishita T, Yokoyama M, Nozaki M, Sano M, Nakano H. Improvement in blastocyst hatching of mouse embryos cocultured with human placental cells. J Assist Reprod Genet 1993; 10:463-467.

16. Suşman S, Rus-Ciucă D, Soriţău O, Tomuleasa C, Buigă R, Mihu D, et al. Pancreatic exocrine adult cells and placental stem cells co-culture. Working together is always the best way to go. Rom J Morphol Embryol 2011; 52:999-1004.

17. Miyamoto K, Hayashi K, Suzuki T, Ichihara S, Yamada T, Kano Y, et al. Human placenta feeder layers support undifferentiated growth of primate embryonic stem cells. Stem Cells 2004; 22:433-440.

18. Venugopal P, Balasubramanian S, Majumdar AS, Ta M. Isolation, characterization, and gene expression analysis of Wharton’s jelly-derived mesenchymal stem cells under xeno-free culture conditions. Stem Cells Cloning 2011; 4:39.

19. Soncini M, Vertua E, Gibelli L, Zorzi F, Denegri M, Albertini A, et al. Isolation and characterization of mesenchymal cells from human fetal membranes. J Tissue Eng Regen Med 2007; 1:296-305.

20. Lee J, Lako M, Armstrong L. Derivation of Human Sperm from Embryonic Stem Cells Dev 2009; 18:1111-1112.

21. Kee K, Angeles VT, Flores M, Nguyen HN, Pera RAR. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 2009; 462:222-225.

22. Mitchell KE, Weiss ML, Mitchell BM, Martin P, Davis D, Morales L, et al. Matrix cells from Wharton's jelly form neurons and glia. Stem Cells 2003;21:50-60.

23. Chao KC, Chao KF, Fu YS, Liu SH. Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes. PLoS One 2008; 3:e1451.

24. Huang P, Lin LM, Wu XY, Tang QL, Feng XY, Lin GY,  et al. Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like
cells in vitro. J Cell Biochem 2010; 109:747-754.

25. Fujiwara Y, Komiya T, Kawabata H, Sato M, Fujimoto H, Furusawa M, et al. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc Nat Acad Sci 1994; 91:12258-12262.

26. Nagano MC. In vitro gamete derivation from pluripotent stem cells: progress and perspective. Biol Reprod 2007; 76:546-551.

27. Sandlow JI, Feng H-L, Sandra A. Localization and expression of the< i> c-Kit</i> receptor protein in human and rodent testis and sperm. Urology 1997; 49:494-500.

28. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc Nat Acad Sci 2001; 98:6186-6191.

29. Shinohara T, Avarbock MR, Brinster RL. β1-and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc Nat Acad Sci 1999; 96:5504-5509.

30. Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, et al. Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Dev Biol 2003; 258:209-225.

31. Cox DN, Chao A, Lin H. Piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 2000; 127:503-514.

32. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev 1998; 12:3715-3727.

33. Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 1997; 124:2463-2476.

34. Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001; 19:971-974.

35. Kerr CL, Cheng L. The dazzle in germ cell differentiation. J Mol Cell Biol 2010; 2:26-29.

36. Ruggiu M, Saunders PT, Cooke HJ. Dynamic subcellular distribution of the DAZL protein is confined to primate male germ cells. J Androl 2000; 21:470-477.

37. Lin YM, Chen CW, Sun HS, Tsai SJ, Lin JSN, Kuo PL. Presence of DAZL transcript and protein in mature human spermatozoa. Fertil Steril 2002; 77:626-629.