In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.
BACKGROUND
Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.


RESULTS
To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.


CONCLUSIONS
VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.
Yamauchi
Kaori
K
Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Hasegawa
Kouichi
K
Chuma
Shinichiro
S
Nakatsuji
Norio
N
Suemori
Hirofumi
H
D000818:Animals; D001483:Base Sequence; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D017077:Culture Media, Conditioned; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005718:Gametogenesis / Q000235:genetics; D015870:Gene Expression; D005819:Genetic Markers; D005854:Germ Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D016256:Lewis X Antigen / Q000235:genetics / Q000378:metabolism; D008252:Macaca fascicularis; D008297:Male; D008868:Microtubule Proteins / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010063:Ovum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013094:Spermatozoa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019089:Stem Cell Factor / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
C525809:BMP4 protein, human; D055415:Bone Morphogenetic Protein 4; D017077:Culture Media, Conditioned; D017931:DNA Primers; D005819:Genetic Markers; D016256:Lewis X Antigen; D008868:Microtubule Proteins; D050814:Octamer Transcription Factor-3; D011994:Recombinant Proteins; D019089:Stem Cell Factor; C044103:tektins; D014212:Tretinoin; D053487:DEAD-box RNA Helicases