Isolation and characterization of CD146+ multipotent mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) represent a bone marrow (BM) population, classically defined by five functional properties: extensive proliferation, ability to differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells−supporting hematopoiesis. However, research progress in this area has been hampered by lack of suitable markers and standardized procedures for MSC isolation. We have isolated a CD146+ multipotent MSC population from 20 human BM donors displaying the phenotype of self-renewing osteoprogenitors; an extensive 12-week proliferation; and the ability to differentiate in osteoblasts, chondrocytes, adipocytes, and stromal cells supporting hematopoiesis. Furthermore, the CD146+ MSCs secrete a complex combination of growth factors (GFs) controlling hematopoietic stem cells (HSCs) function, while providing a >2-log increase in the long-term culture (LTC) colony output in 8-week LTC over conventional assays. The hematopoietic stromal function exhibited by the MSCs was further characterized by manipulating LTCs with the chemical inhibitors Imatinib or SU-5416, targeting two GF receptors (GFRs), KIT or VEGFR2/1, respectively. Both treatments similarly impaired LTC colony output, indicating key roles for these two GF/GFR interactions to support LTC-initiating cell activity. CD146+ MSCs may thus represent a tool to explore the MSC-HSC cross-talk in an in vitro surrogate model for HSC “niches,” and for regenerative therapy studies. In addition, the MSC microRNA (miRNA) expression profile was analyzed by microarrays in both basic conditions and chondrogenic differentiation. Our analysis revealed that several miRNAs are modulated during chondrogenesis, and many of their putative targets are genes involved in chondrogenic differentiation

KDR receptor: A key marker defining hematopoietic stem cells

Studies on pluripotent hematopoietic stem cells (HSCs) have been hindered by lack of a positive marker, comparable to the CD34 marker of hematopoietic progenitor cells (HPCs). In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34+cells expressed vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR). Pluripotent HSCs were restricted to the CD34+KDR+cell fraction. Conversely, lineage-committed HPCs were in the CD34+KDR-subset. On the basis of limiting dilution analysis, the HSC frequency in the CD34+KDR+fraction was 20 percent in bone marrow (BM) by mouse xenograft assay and 25 to 42 percent in BM, peripheral blood, and cord blood by 12-week long-term culture (LTC) assay. The latter values rose to 53 to 63 percent in LTC supplemented with VEGF and to greater than 95 percent for the cell subfraction resistant to growth factor starvation. Thus, KDR is a positive functional marker defining stem cells and distinguishing them from progenitors

Distinct Roles of Bcl-2 and Bcl-Xl in the Apoptosis of Human Bone Marrow Mesenchymal Stem Cells during Differentiation

Background: Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells. Principal Findings: Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs. Significance: In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions

IL-3 and oncogenic Abl regulate the myeloblast transcriptome by altering mRNA stability

The growth factor interleukin-3 (IL-3) promotes the survival and growth of multipotent hematopoietic progenitors and stimulates myelopoiesis. It has also been reported to oppose terminal granulopoiesis and to support leukemic cell growth through autocrine or paracrine mechanisms. The degree to which IL-3 acts at the posttranscriptional level is largely unknown. We have conducted global mRNA decay profiling and bioinformatic analyses in 32Dcl3 myeloblasts indicating that IL-3 caused immediate early stabilization of hundreds of transcripts in pathways relevant to myeloblast function. Stabilized transcripts were enriched for AU-Response elements (AREs), and an ARE-containing domain from the interleukin-6 (IL-6) 3′-UTR rendered a heterologous gene responsive to IL-3-mediated transcript stabilization. Many IL-3-stabilized transcripts had been associated with leukemic transformation. Deregulated Abl kinase shared with IL-3 the ability to delay turnover of transcripts involved in proliferation or differentiation blockade, relying, in part, on signaling through the Mek/ Erk pathway. These findings support a model of IL-3 action through mRNA stability control and suggest that aberrant stabilization of an mRNA network linked to IL-3 contributes to leukemic cell growth. © 2009 Ernst et al

Effect of Abelson murine leukemia virus on granulocytic differentiation and interleukin-3 dependence of a murine progenitor cell line

The murine diploid hematopoietic cell line 32D C13 strictly requires interleukin-3 (IL-3) for proliferation. When 32D C13 cells are transferred to IL-3-free medium which contains recombinant human granulocyte colony stimulating factor (rhG-CSF), the cell number increases four- to five-fold, and after 14 days the whole cell population is differentiated into morphologically normal and myeloperoxidase- and lactoferrin-positive metamyelocytes and granulocytes. Infection with Abelson murine leukemia virus (A-MuLV) of 32D C13 cells growing in the presence of IL-3 induces, within 2 weeks, the appearance of cells that are IL-3-independent for growth. The latter cells lack myeloid, T and B cell markers, and are unable to differentiate, even in the presence of very high doses of rhG-CSF. However, once the 32D C13 cells have been exposed to G-CSF, they become resistant to the transforming effects of A-MuLV as judged by the appearance of the IL-3-independent clones. These findings suggest that the ability of Abelson virus to transform immature progenitor cells is due to interference of the v-abl gene product with the mechanism that control the commitment of the cells to differentiate

Stage-related proliferative activity determines c-myb functional requirements during normal human hematopoiesis.

To determine if MYB protein is preferentially required during specific stages of normal human hematopoiesis we incubated normal marrow mononuclear cells (MNC) with c-myb antisense oligodeoxynucleotides. Treated cells were cultured in semisolid medium under conditions designed to favor the growth of specific progenitor cell types. Compared with untreated controls, granulocyte-macrophage (GM) CFU-derived colonies decreased 77% when driven by recombinant human (rH) IL-3, and 85% when stimulated by rH GM colony-stimulating factor (CSF); erythroid burst-forming unit (BFU-E)- and CFU-E-derived colonies decreased 48 and 78%, respectively. In contrast, numbers of G-CSF-stimulated granulocyte colonies derived from antisense treated MNC were unchanged from controls, though the numbers of cells composing these colonies decreased approximately 90%. Similar results were obtained when MY10+ cells were exposed to c-myb antisense oligomers. When compared with untreated controls, numbers of CFU-GM and BFU-E colonies derived from MY10+ cells were unchanged, but the numbers of cells composing these colonies were reduced approximately 75 and greater than 90%, respectively, in comparison with controls. c-myc sense and antisense oligomers were without significant effect in these assays. Using the reverse transcription-polymerase chain reaction, c-myb mRNA was detected in developing hematopoietic cells on days 0-8. At day 14 c-myb expression was no longer detectable using this technique. These results suggest that c-myb is required for proliferation of intermediate-late myeloid and erythroid progenitors, but is less important for lineage commitment and early progenitor cell amplification