title: RA induces differentiation of multipotent P19 cells towards male germ cell.
abstract: Generating male germ cells in vitro from multipotent stem cells is still a challenge for stem cell biologists. The difficulty is caused by the lack of knowledge about spermatogenesis molecular-controlling mechanisms. In vivo, PGCs differentiate into male germ cells in a very complicated environment through many middle steps. In this study, we use the pluripotent p19 cells to test their responses to different retinoic acid (RA) concentrations by evaluating markers for stem cells (bmp4, egr3), primordial germ cells (ddx4), spermatogonia (c-kit), premeiotic cells (stra8), and male germ cells (dazl and plzf). We have found that cyp26b1, which will catalyze RA, increases dramatically in p19 cells 1 d after RA treatment. Bmp3, egr3, and stra8 are stimulated after 1 d of RA treatment and then recover to normal after 3 d of RA treatment. C-kit keeps being expressed when treated with 10 nM-4 μM RA. Dazl and plzf are gained after 3 d of stimulation. The morphology of RA (100 nM-4 μM)-treated cells changes distinctively, and cell colonies are formed. Typical neural cell-like and germ cell-like morphologies appear in the 100 nM and 4 μM RA groups, respectively. We conclude that 100-500 nM RA can cause responses in p19 cells, but a high concentration of RA (1-4 μM) can drive these pluripotent cells' differentiation towards male germ cells. However, high concentrations of RA are also toxic. Some colonies that survived from 4 μM RA begin to express ddx4 and c-kit. Selection of the c-kit(+), dazl(+), and ddx4(+) cells after RA stimulation and creating a special culture medium for their propagation might benefit successful spermatogenesis induction in vitro.
authors.affiliation: Shanghai First Maternity and Infant Hospital, Tongji University, Shanghai, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Jiangjing
authors.initials: J
authors.lastname: Tang
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Haines
authors.forename: Huai
authors.initials: H
authors.lastname: Feng
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Teng
authors.forename: Yibing
authors.initials: Y
authors.lastname: Han
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D048430:Cell Shape / Q000187:drug effects; D005110:Extracellular Space / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D005854:Germ Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D008297:Male; D051379:Mice; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*
chemical_list: C508515:DAZL protein, mouse; D051741:Kruppel-Like Transcription Factors; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C513267:Zbtb16 protein, mouse; D014212:Tretinoin; D019009:Proto-Oncogene Proteins c-kit