Proliferation in culture of primordial germ cells derived from embryonic stem cell: induction by retinoic acid.
An in vitro system that supports primordial germ cells (PGCs) survival and proliferation is useful for enhancement of these cells and efficient transplantation in infertility disorders. One approach is cultivation of PGCs under proper conditions that allow self-renewal and proliferation of PGCs. For this purpose, we compared the effects of different concentrations of retinoic acid (RA), and the effect of PGCs co-culture (Co-C) with SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) cells on the proliferation of embryonic stem cells (ESCs)-derived PGCs. One-day-old embryoid body (EB) was cultured for 4 days in simple culture system in the presence of 5 ng/ml bone morphogenetic protein-4 (BMP4) (SCB group) for PGC induction. For PGC enrichment, ESCs-derived germ cells were cultured for 7 days in the presence of different doses (0-5  μM) of RA, both in the simple and STO Co-C systems. At the end of the culture period, viability and proliferation rates were assessed and expression of mouse vasa homologue (Mvh),  α6 integrin,  β1 integrin, stimulated by retinoic acid 8 (Stra8) and piwi (Drosophila)-like 2 (Piwil2) was evaluated using quantitative PCR. Also, the inductive effects were investigated immunocytochemically with Mvh and cadherin1 (CDH1) on the selected groups. Immunocytochemistry/PCR results showed higher expression of Mvh, the PGC-specific marker, in 3  μM RA concentrations on the top of the STO feeder layer. Meanwhile, assessment of the Stra8 mRNA and CDH1 protein, the specific makers for spermatogonia, showed no significant differences between groups. Based on the results, it seems that in the presence of 3 μM RA on top of the STO feeder layer cells, the majority of the cells transdifferentiated into germ cells were PGCs.
Department of Anatomical Sciences, Faculty of Medicine, Fasa University of Medical Sciences, Fasa 74616-86688, Iran.
Zohreh
Z
Makoolati
Department of Anatomical Sciences, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-175, Iran movahed.m@modares.ac.ir.
Mansoureh
M
Movahedin
Department of Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115-175, Iran.
Mehdi
M
Forouzandeh-Moghadam
D048868:Adaptor Proteins, Signal Transducing / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D000818:Animals; D060565:Argonaute Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism; D064200:Cdh1 Proteins / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D054338:Cell Transdifferentiation / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques; D053487:DEAD-box RNA Helicases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D007150:Immunohistochemistry; D039121:Integrin alpha6beta1 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D013093:Spermatogonia / Q000166:cytology* / Q000378:metabolism; D059467:Transcriptome; D014212:Tretinoin / Q000494:pharmacology*
D048868:Adaptor Proteins, Signal Transducing; D060565:Argonaute Proteins; D015415:Biomarkers; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D064200:Cdh1 Proteins; C577585:Fzr1 protein, mouse; D039121:Integrin alpha6beta1; C486868:PIWIL2 protein, human; C102019:Stra8 protein, mouse; D014212:Tretinoin; C090864:Ddx4 protein, mouse; D053487:DEAD-box RNA Helicases
embryonic stem cell; primordial germ cell; proliferation; retinoic acid