wnt3a but not wnt11 supports self-renewal of embryonic stem cells.
wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.
Singla
Dinender K
DK
Cardiovascular Research Institute, Department of Medicine, University of Vermont, Colchester, VT 05446, USA. Dinender.Kumar@uvm.edu
Schneider
David J
DJ
LeWinter
Martin M
MM
Sobel
Burton E
BE
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D015850:Interleukin-6 / Q000494:pharmacology; D053605:Leukemia Inhibitory Factor; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D013234:Stem Cells / Q000378:metabolism*; D013997:Time Factors; D051153:Wnt Proteins / Q000378:metabolism / Q000502:physiology*; D060508:Wnt3 Protein; D060509:Wnt3A Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D003470:Culture Media; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D051153:Wnt Proteins; C508957:Wnt11 protein, mouse; D060508:Wnt3 Protein; D060509:Wnt3A Protein; C555511:Wnt3a protein, mouse; D048051:p38 Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases