Abstract
Great progresses are promised for the development of stem cell-based regenerative medicine if we can manipulate stem cell self-renewal. Thus, one of the central tasks in stem cell biology is to understand how stem cell fate is determined. Hematopoietic stem cells (HSCs) are the best studied stem cells. Their in vivo self-renewal has been extensively studied, but its in vitro recapitulation remains so difficult. We previously reported that HSCs undergo asymmetrical self-renewal division in culture with stem cell factor (SCF) and thrombopoietin (TPO). Since then, we have sought any condition in which HSCs can symmetrically self-renew. We now report in vitro symmetrical self-renewal division of HSCs in the absence of Lnk. Lnk is an adaptor protein containing praline-rich domain, pleckstrin homology domain, and Src homology domain. Lnk-deficient mice have over 10-fold HSCs due to increased self-renewal capacity. CD34−Kit+Sca-1+Lin− cells were purified from bone marrow of wild-type or Lnk-deficient B6 mice, and were subjected to serum-free single cell cultures in the presence of a variety of cyokines. We found that Lnk-deficient CD34−Kit+Sca-1+Lin− cells are hypersensitive to TPO. Repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from Lnk-deficient mice increased after 3 day-culture with TPO or with SCF and TPO, but not after 3 day-culture with SCF. In contrast, repopulating activity in 40 CD34−Kit+Sca-1+Lin− cells from wild type mice did not significantly change after 3 day-culture with SCF, TPO, or SCF and TPO. Moreover, paired daughter cell-experiments clearly showed that Lnk-deficient, but not wild-type long-term repopulating cells are able to undergo symmetrical self-renewal division at least once in the presence of SCF and TPO. These data suggest that Lnk acts just like a tuner in the regulation of HSC self-renewal downstream of TPO/Mpl signaling. We further investigated TPO-mediated signal transduction pathways in CD34−Kit+Sca-1+Lin− cells. To this end, we developed a novel assay which allowed us to analyze signal transduction in a very limited number of cells. We detected enhanced up-regulation of STAT5 and Akt pathways, and inversely enhanced down-regulation of p38 MAPK pathway in Lnk-deficient CD34−Kit+Sca-1+Lin− cells, as compared with normal ones. These data suggest that these combinational changes in signal transduction lead to initiation of self-renewal in HSCs. We propose that stem cell self-renewal is determined by a balance of positive and negative signals in multiple signal transduction pathways rather than by any particular self-renewal signals.
Cytokine Signaling and Hematopoietic Homeostasis Are Disrupted in Lnk-deficient Mice