Human embryonic stem cells (HSF-6) show greater proliferation and apoptoses when grown on glioblastoma cells than mouse embryonic fibroblasts at day 19 in culture: comparison of proliferation, survival, and neural differentiation on two different feeder cell types.
Phenotypic guidance of embryonic stem (ES) cell fate is paramount if these cells are to be used for tissue repair and regeneration. Our objective was to compare two different cell culture feeders and their effect on proliferation, apoptosis, and differentiation of human (h) ES cells. HSF-6 hES cells were grown in Knockout Dulbecco's modified Eagle medium (DMEM) on mouse embryonic fibro-blasts (MEFs) or U87 glioblastoma cells at densities of 50,000, 100,000, and 150,000 cells/well of a six-well plate for 7, 12, and 19 days. Immunocytochemistry was performed for bromodeoxyuridine (BrdU), TUNEL, and neural differentiation markers including class III beta-tubulin, NeuN, nestin, and doublecortin. Slides were examined by laser confocal microscopy with semiquantitative analyses of marker expression. BrdUand TUNEL-positive cells were primarily, but not exclusively, at edges and between established colonies. BrdU expression was higher on U87 feeders at low and intermediate densities at day 19. Both feeders demonstrated higher BrdU expression at day 7 compared to days 12 and 19. U87 produced more TUNEL-positive cells than MEFs with increasing numbers with increasing density and time in culture. Nuclear Oct-4 staining was seen only at day 7. MEFs appeared to promote greater neural differentiation of hES cells than U87. We conclude hES cells grown on U87 feeders demonstrate greater numbers of apoptotic cells and BrdU-positive cells at day 19. Independent of the feeders, proliferation and apoptosis may be positively correlated. We speculate differences in proliferation, apoptosis, and neural differentiation may be due to differential elaboration of specific cytokines by MEFs and U87.
Ozolek
John A
JA
Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA. ozolekja@upmc.edu
Jane
Esther P
EP
Krowsoski
Leandra
L
Sammak
Paul J
PJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005909:Glioblastoma; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology*
D015415:Biomarkers