Using divisional history to measure hematopoietic stem cell self-renewal and differentiation.
OBJECTIVE
The purpose of this study was to investigate cell fates and long-term repopulating potential of a primitive hematopoietic stem cell (HSC) population (i.e., FR25Lin(-) cells) in vitro.


METHODS
FR25Lin(-) cells were isolated by elutriation and cell sorting and cultured with a combination of cytokines for 7 days. Utilizing the membrane dye PKH-26, cultured cells were separated into two subsets based on their proliferation rates and assayed for progenitors and HSC.


RESULTS
Fresh FR25Lin(-) cells were mostly quiescent; however, some of this population entered cell cycle after cytokine exposure reaching a peak 4 to 5 days after culture. Two subsets of cultured cells were isolated: 1) cells that had divided several times (PKH(dull) cells) and 2) cells that remained undivided or divided only once or twice (PKH(bright) cells). The PKH(dull) cells accounted for 94% of total viable cells in culture after 5 days. The PKH(dull) subset contained all the multi-potential in vivo progenitors (CFU-S) and 10 times more committed progenitors (CFU-C). Quantitative analysis of HSC engraftment from the PKH(bright) subset demonstrated stem cell maintenance. For the PKH(dull) subset, on day 5, HSC numbers increased. By day 7, increased differentiation in the PKH(dull) population supports expanding differentiation divisions.


CONCLUSIONS
Our primitive HSC population underwent different types of cell divisions stimulated by cytokines, resulting in subsets with different self-renewal and differentiation potentials. This in vitro/in vivo model provides a useful tool for studies of early events during HSC self-renewal and differentiation.
Yan
Feng
F
Oncology Center, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Collector
Michael I
MI
Tyszko
Sara
S
Sharkis
Saul J
SJ
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured / Q000166:cytology / Q000187:drug effects; D003433:Crosses, Genetic; D004357:Drug Synergism; D005260:Female; D006085:Graft Survival; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D011828:Radiation Chimera; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology
D015850:Interleukin-6; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D013926:Thrombopoietin