Oncostatin M-Mediated Regulation of KIT-Ligand-Induced Extracellular Signal-Regulated Kinase Signaling Maintains Hematopoietic Repopulating Activity of Lin−CD34+CD133+ Cord Blood Cells

Abstract

We investigated whether KIT signaling was sufficient to maintain human hematopoietic stem cells in culture or whether, as with murine stem cells, signaling through glycoprotein 130 (gp130) is additionally required. Sorted CD34 ؉ CD133؉ (CD33/CD38/CD71) ؊ cells from human umbilical cord blood (UCB) were cultured in the presence of combinations of KIT-ligand (KL) and the gp130 stimulating molecule oncostatin M (OSM). We found that OSM increased KL-induced proliferation, which was accompanied by an expansion in numbers of mature progenitors colonyforming cells (CFC, CAFCw2). More primitive progenitors, CAFCw6 and long-term culture-CFC, were not maintained by KL as a single factor. Although addition of OSM did not improve survival, the KL/OSM combination showed improved maintenance of immature progenitors as well as higher CD34 expression. Similarly, both KL and OSM were required to maintain NOD/SCID-repopulating activity. In experiments to investigate the underlying mechanism, we found that extracellular signal-regulated kinase (ERK) and its downstream target p90 ribosomal S6 kinase were activated by KL and downregulated by the inclusion of OSM during stimulation. The p38 mitogen-activated protein kinase (p38 MAPK) was not modulated by either KL or OSM. Indeed, many of the effects of OSM (increased cell division, maintenance of CFC, and maintenance of high CD34 expression) could be mimicked by using the mitogen-activated protein kinase kinase inhibitor U0126. More importantly, NOD/SCID-repopulating activity was preserved in the KL/ U0126-stimulated cells, but not in cells stimulated with a combination of KL and the p38 MAPK inhibitor SB203580. Our results show that the loss of repopulating activity during KL stimulation is counteracted by OSM through the downregulation of ERK pathway signaling. STEM CELLS

Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

“…Further lineage depletion of CD34 þ cells was not done because of primary sample size constraints. In some experiments, CD34-enriched cells were labeled for 10 minutes at 37 C with 2 mmol/L of the fluorescent dye 5-(and 6-)carboxy-fluorescein succinimidyl ester (CFSE; Molecular Probes) in Iscove's modified Dulbecco's medium (GIBCO; Invitrogen), 1% fetal calf serum, and 10 mmol/L HEPES (Gibco) as described (25). To determine the location of undivided cells for cell division tracking, a control culture was set up in the same manner but was additionally supplemented with colcemid (Karyomax; Gibco) as described (26).…”

“…Immature hematopoietic stem/progenitors were determined after long-term culture on the FBMD-1 stromal cell line in the presence of TPO and FL as described (25). After 6 weeks of culture, the entire culture was harvested by trypsin detachment and assayed for the presence of long-term, culturederived colony-forming cells (LTC-CFC) in methylcellulose.…”

“…Cells were placed in pre-chilled tubes containing ice-cold PBS with Na 2 VO 4 (1 mmol/L; Sigma) and washed once in cold PBS with Na 2 VO 4 . Cell lysis, SDS-PAGE, and immunoblotting were done as described (25). Antibodies to FLT3 (S-18, sc-480; Santa Cruz Biotechnology) and pFLT3 (#3461; Cell Signaling Technologies), AKT1/pS473 and AKT1/pT308 AKT (all from Cell Signaling Technologies), ERK1 (K-23; Santa Cruz Biotechnology), pY204-ERK1 (E4; Santa Cruz Biotechnology), b-actin (Sigma), pY694STAT5 and STAT5 (Cell Signaling Technologies), and phosphotyrosine (4G10 and PY20; Transduction Laboratories) were used as described in manufacturers' instructions.…”

Umbilical Cord Blood Hematopoietic Stem and Progenitor Cell Expansion for Therapeutic Use

“…Additional factors or small molecules that enhance UCB HSPC proliferation or function include the Notch Delta-like ligand 1 (DLL1), StemRegenin1 (SR 1), the copper chelator tetraethylenepentamide (TEPA), GSK3 inhibitors of WNT signaling, p18 a speciic inhibitor of cyclin-dependent kinase (CDK), pyrimidoindole derivatives such as UM1 1, speciic miRNAs, and epigenetic modiiers such as histone deacetylase (HDAC) inhibitors and nicotinamide, as well as additional growth factors such as the designer cytokine hyper-IL-6, oncostatin M, IL-11, angiopoietin-like 5, and other angiopoietin-like molecules and IGFBP2 [120][121][122][123][124][125][126][127][128][129][130][131][132][133][134][135][136][137][138][139]. Coculture of UCB cells with mesenchymal stromal cells has also been examined [140].…”

Comparative Gene Expression Profiling of Stromal Cell Matrices that Support Expansion of Hematopoietic Stem/Progenitor Cells

“…Non-canonical Wnt4 enhances murine HSPC expansion through a planar cell polarity-like pathway resulting in diverse consequences like cell differentiation, proliferation/survival, migration and adhesion [95,96] and Angpt2, which is an angiocrine factor that controls vascular morphogenesis and homeostasis in the niche through the angiopoietin-tie system [39,97] were up-regulated in MX2 cells, indicating their role in maintaining more committed progenitors. However, HSC related pathways like ECM remodeling, cell matrix glycoconjugates and oncostatin M via Jak/Stat and MAPK signaling were down-regulated in step 2 [54,98] ( Figure 6). …”