Publication Date:
2005-11-16
Description:
Factors responsible for long-term survival and proliferation of human hematopoietic stem cells (hHSC), and their mechanisms of action, remain to be defined. We previously showed that specific O-sulfated heparan sulfate (OS-HS) improves human long-term culture initiating cell (LTC-IC) maintenance for up to 5 wks in vitro. This is related to the ability of OS-HS to bind to and modulate the activity of heparin-binding cytokines on primitive hematopoietic progenitors (PHP). Recent studies indicate that bone morphogenetic proteins (BMPs) influence the development of embryonic hematopoiesis and also augment short-term survival and proliferation of hHSC. Since HS may modulate BMP activity, we examined how combinations of OS-HS, BMPs and specific BMP antagonists influence PHP in umbilical cord blood (UCB). First, we confirmed by real-time quantitative RT-PCR (qRT-PCR) that UCB CD34+ and/or CD34+/CD38− cells (using linear mRNA amplification) constitutively express transcripts for BMP-4 and its inhibitor Chordin, BMP receptors BMPR-IA, BMPR-IB, BMPR-II, AcvR-II and AcvR-IIB, downstream signaling proteins SMAD-1 and -5, and target genes upregulated by BMPs including Inhibitors of DNA binding (Id) proteins 1–4, and determined their relative levels of expression. BMP-4 upregulated Id2 expression 17-fold, confirming that the BMP signaling pathway is functionally active in CD34+ cells. Next, we demonstrated that OS-HS may protect BMP-4 from its inhibitors, using Western immunoblotting of immunoprecipitated proteins to show that direct binding of the antagonist Chordin to BMP-4 is inhibited by OS-HS in a dose-dependent manner. Finally, we examined the effect of exogenous supplementation with 6 BMPs, or inhibition of endogenous BMPs by 7 antagonists, on short-term (2 wk) and long-term (5 wk) LTC-IC maintenance in UCB CD34+/CD38− cells cultured in presence of OS-HS, Flt3-ligand and thrombopoietin. Long-term LTC-IC maintenance was enhanced by BMP-4 (LTC-IC maintenance: 164 +/− 11% compared to culture without BMP-4; P=0.0001) but reduced by BMP-2 or BMP-7 (P
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
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