Bone marrow HSPCs and BMMSCs share a common microenvironmental niche wherein intercellular and intracellular network signaling communications direct stem cell fate activation, proliferation, development, and tissue differentiation [34, 35]. Limited comparative information is available on the molecular signaling behavior of undifferentiated BMMSCs and HSPCs. Defining the signaling mechanisms expressed in adult undifferentiated stem cells is an essential step toward understanding the developmental and regenerative capabilities. Here we report a comprehensive evaluation, of mRNA gene transcripts for 94 signaling molecules, in which 11 transcripts were equally expressed in both HSPCs and BMMSCs, 19 overexpressed in HSPCs compared with BMMSCs, 27 overexpressed BMMSCs compared with HSPCs, 16 expressed only in HSPCs, 14 expressed only in BMMSCs and seven expressed in neither cell population. To our knowledge, this is the first study to report simultaneous determination of multiple cell signaling molecules in highly purified undifferentiated stem cell populations under standardized conditions. Flow cytometric analysis showed that the transcriptional levels of CD45, c-kit, Sca-1, KDR (Flk-1/Vegfr2), Pdgfra, and Ddr2 were consistent with the cell surface translational levels of protein expression.
Of the 90 PTKs, 58 are categorized as RTKs and 32 as cytoplasmic non-RTKs . Of the 37 RTK gene transcripts we evaluated, 23 gene transcripts were either exclusively confined to or more highly expressed in BMMSCs. Transcripts for Aatk, and Csfr1were more highly expressed in HSPCs than in BMMSCs, while transcripts Flt3, Kit, Tek, and Tie1were found to be exclusively expressed in HSPCs, all known receptors for ligands that have been shown to be important in primitive HSPC survival, quiescence, activation, proliferation, mobilization and/or differentiation [36–41]. In contrast, we found in BMMSCs a different set of transcripts for genes encoding signaling receptors linked to stem cell survival and growth (Axl, Pdgfr and Egfr), self-renewal (Egfr and Ephr), maintenance of stem cells in the dedifferentiate state (Egfr, Fgfr), and recruitment of cells to injured tissue (Met, Mstl1R, and Pdgfr) [42–47]. Furthermore signaling molecules that modulate osteogenesis/chondrogenesis (Ror1, Ror2, Ddr1, and Ddr2) and neuronal cell development (Ret) were either exclusively or differentially expressed in BMMSCs. We show transcription of Gas6, a secreted vitamin-K-dependent protein ligand for Axl, Mertk, and Tyro3 known to play a role in reversible cell growth arrest, survival, proliferation, cell adhesion, long-term hematopoiesis, and erythropoiesis [48–50], is ubiquitously expressed in HSPCs and BMMSCs, although transcript expression was 381-fold greater in BMMSCs.
Non-RTKs are integral components of the signaling cascades triggered by RTKs and by other cell surface receptors such as G protein-coupled receptors and growth factor/cytokine receptors of the immune system . Not surprisingly, transcripts for Csf1r, Csf2ra, Csf2rb2, Csf3r, IL6ra, IL7r, Epor, Mpl, flt3, Kit, Tie-1 and Tek receptors for the corresponding cytokine ligands M-CSF, GM-CSF, G-CSF, IL-6, IL-7, EPO, TPO, FLT3L, SCF, and angiopoietin-1 were found to be HSPC specific, whereas receptors of leukemia inhibitor factor (Lifr), nerve growth factor (Ngfr) and oncostatin-M (Osmr, stimulates BMMSCs to produce stromal-derived growth factor) were mainly expressed in BMMSCs. As hematopoietic supportive cells, BMMSCs constitutively expressed transcripts for M-CSF, IL-6, IL-11, LIF, SCF and Flt3 ligand, and inflammatory cytokine stimulation of BMMSCs with IL-1α induces G-CSF, and GM-CSF expression . These findings highlight the importance of BMMSCs in the context of the HSPC niche where they support HSPC survival (anti-apoptotic action) and quiescence . Furthermore, we found that the expression of G-protein chemokine receptors for the cell trafficking molecules MIP-1, RANTES, TARC, and MCP-1 (Cccr4), MIP-3β (Ccr7) and IP-10, I-TAC and Mig (Cxcr3) was exclusive in HSPCs. These data suggest and are consistent with the notion that quiescent HSPCs are poised for mobilization.
Consistent with the RTK and non-RTK findings, HSPCs were notably enriched in Tec kinases (Tec, Btk, Bmx, and Txk), SRC kinases (Fgr and Lck), SFK kinases (Hck), Syk Kinases (Syk and Zap-70), Janus kinase/STAT kinases (Jak2 and Jak3) and c-fes kinases (Fes). These intracellular regulated transcripts are known to be important in early HSPC decisions, and may play a key role in HSPC self-renewal, quiescence and lineage-specific differentiation. In contrast, BMMSCs expressed higher transcript levels of Abl1, Fert2, Fyn Ptk2, Tnk2, and Frk, which have been shown in other cell types to have cytoplasmic and/or nuclear regulatory functions in during cell differentiation, cell remodeling, cell division, cell adhesion and cell migration [54–59]; however, their roles in BMMSCs are unknown and further evaluation is needed.
Our findings in this report are subject to several limitations. First, we compared cell signaling receptors of cultured early passaged BMMSCs to freshly isolated HSPCs. It is possible that some of the differential expression in these genes is solely due to the fact that BMMSCs were cultured whereas the HSPCs were not. This may account for an over-representation of RTKs in BMMSCs compared with HSPCs. Second, it is accepted that in vivo conditions are different from the in vitro experimental culture conditions wherein most of the niche microenvironmental conditions are absent. Third, gene expression is under regulatory control at many different stages, and therefore it is difficult to equate mRNA levels with gene expression levels. Fourth, future studies are needed to determine the signaling profiles during times of stress, injury, inflammation or repair. Lastly, the gene expression data generated and the conclusions need to be verified in situ in localized cells at specific anatomic sites using immunochemistry and laser capture microdissection or other techniques .