Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells

Abstract

Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division. (Blood. 2008;112:560-567) IntroductionThe hematopoietic system of the adult mouse is responsible for the daily production of billions of differentiated blood cells of various types. Because most of these cells have a limited lifespan and proliferative ability, they must be continuously generated from a population of more primitive cells that collectively have life-long self-sustaining ability. This function is restricted to a tiny subset of multipotent cells generally referred to as hematopoietic stem cells (HSCs). Historically, HSCs have been both defined and quantified retrospectively by their ability to generate clones containing both lymphoid and myeloid blood cells for at least 4 months when transplanted into irradiated recipients at limiting dilutions. 1 Analyses of such mice have also allowed the long-term differentiation activity of individual HSCs to be characterized. 2,3 Methods have now been developed for isolating purified populations in which 20% to 60% of the cells display this durability of reconstituting activity, indicating that intravenously injected HSCs can have very high seeding efficiencies in irradiated mice. [4][5][6][7] The ability to isolate such highly purified HSC populations has also permitted a more direct and complete analysis of their in vivo differentiation activity in single-cell transplant experiments. 6,[8][9][10][11] More recently, serial transplants of such clonally repopulated mice have been performed. Together, these experiments have revealed that HSCs possess one of 4 distinct differentiation programs that are propagated over many generations in vivo. 10,12 In addition, these studies have shown that extensive self-renewal ability in vivo is strongly associated with the display of either a...

Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential

“…Hence the purity of this type of HSC in the E-SLAM fraction of adult BM was 43% ( Figure 2B). These values are much higher than we previously reported for other powerful HSC isolation strategies (eg, CD45 mid Rho Ϫ SP cells 12,17 and E-Rho Ϫ cells 34 ). Secondary mice transplanted with BM cells harvested individually from 4 primary mice that had each been repopulated with a single adult BM E-SLAM cell more than 26 weeks before confirmed that, in each case, progeny HSCs had been generated from the original input E-SLAM cell (data not shown).…”

“…However, to obtain high HSC purities, additional elements are required, and these typically differ to accommodate the differing phenotypic characteristics of fetal and adult HSCs 14,30,31 caused by their different cycling status. 14,31 In preliminary studies, we found that a high expression of EPCR, a powerful discriminator of adult BM HSCs, 11 was stable on HSCs that had been proliferating in vitro for 5 to 7 days (data not shown). This finding suggested that selection of EPCR ϩ cells within the CD48 Ϫ CD150 ϩ fraction (E-SLAM cells) might allow fetal and adult HSCs to be purified using the same simple protocol.…”

“…10 Variations in external cues may be one contributing parameter, at least in vivo, because it is known that self-renewal responses can be directly and rapidly modulated in this way in vitro. 11,12 In addition, there is some evidence of predetermined heterogeneity in HSC self-renewal potential. This is exemplified by the differences in regenerative activity of HSCs from fetal and adult sources 13,14 and the finding of a consistent association of short-term and long-term multilineage WBC outputs with distinct phenotypes of adult bone marrow (BM) cells (eg, according to their expression of CD49b and CD34).…”

Identification of DNA methylation changes atcis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

“…[5][6][7] Gene expression studies have revealed important insights into how biological processes are regulated at the transcriptional level in haematopoietic stem and progenitor cells (HSPC). [8][9][10] However, the global molecular mechanisms acting during physiological HSC commitment remain elusive. In a recent resource study, we investigated the global molecular mechanisms acting during physiological HSC commitment by performing an integrative analysis of proteome, transcriptome and DNA methylome, which enabled a detailed characterization of the regulatory networks active during early HSC commitment.…”

Negative Regulation of Haematopoiesis: Role of Inhibitory Adaptors

“…In contrast to SH2-B and APS, Lnk is mainly expressed in haematopoietic cells, notably in haematopoietic stem cells (HSC), and haematopoietic (lymphoid and myeloid) progenitors. Moreover, Lnk expression is upregulated by certain cytokines important for the development and function of these haematopoietic cells, such as SCF, thrombopoietin [TPO], and erythropoietin [EPO] (Kent et al, 2008;Buza-Vidas et al, 2006;Gerry et al, 2009aGerry et al, , 2009bBaran-Marszak et al, 2010). Interestingly, Lnk is also highly expressed in endothelial cells and its expression is also induced by Tumor Necrosis Factor (TNF)- (Fitau et al, 2006;Kwon et al, 2009).…”