Identification of a subpopulation of marrow MSC-derived medullary adipocytes that express osteoclast-regulating molecules: marrow adipocytes express osteoclast mediators.

Increased marrow medullary adipogenesis and an associated decrease in bone mineral density, usually observed in elderly individuals, is a common characteristic in senile osteoporosis. In this study we investigated whether cells of the medullary adipocyte lineage have the potential to directly support the formation of osteoclasts, whose activity in bone leads to bone degradation. An in vitro mesenchymal stem cell (MSC)-derived medullary adipocyte lineage culture model was used to study the expression of the important osteoclast mediators RANKL, M-CSF, SDF-1, and OPG. We further assessed whether adipocytes at a specific developmental stage were capable of supporting osteoclast-like cell formation in culture. In vitro MSC-derived medullary adipocytes showed an mRNA and protein expression profile of M-CSF, RANKL, and OPG that was dependent on its developmental/metabolic stage. Furthermore, RANKL expression was observed in MSC-derived adipocytes that were at a distinct lineage stage and these cells were also capable of supporting osteoclast-like cell formation in co-cultures with peripheral blood mononuclear cells. These results suggest a connection between medullary adipocytes and osteoclast formation in vivo and may have major significance in regards to the mechanisms of decreased bone density in senile osteoporosis

Human bone marrow-derived cells and their differentiation to the adipocyte lineage.

<p>A) Phase contrast image of bone marrow-derived mesenchymal stem cells grown for 12 days in growth medium. B) Phase contrast images of parallel cultures grown in adipogenic medium for 12 days. MSC-derived adipocyte cultures show a heterogenous mixture of cell morphologies including lipid-laden cells (<i>black arrows</i>) and non-lipid laden cells (<i>white arrows</i>).</p

RANKL is present in a sub-population of adipocytes.

<p>(<b>A</b>) Immunostaining was performed in MSC-derived adipocyes at day 12 of differentiation (green). Two sub-populations were identified based on the presence (white arrows) or absence of lipid vacuoles (gray arrows). Adipocytes that show positive RANKL staining primarily were non-lipid containing cells. Cells were further sub-fractionated into (<b>B</b>) non-lipid-laden and (<b>C</b>) lipid-laden MSC-derived adipocyte populations. Scale bar in C = 200 µm for panels B-C. (<b>D</b>) RANKL protein was quantified over time in both sub-populations of cells, and confirmed that lipid-laden adipocytes represented a very low percentage of the total RANKL-positive cells. (<b>E</b>) This observation was confirmed through quantitative RT-PCR analysis to detect RANKL transcript. Lipid-laden populations (lipid fraction) showed an approximately 10-fold lower level of RANKL mRNA compared to the non-lipid containing fraction (adipo-fibroblast). Scale bars in panels A-C = 200 µm. <i>n</i> = <i>3–4 donors</i>.</p

Osteoclast-like formation of adipofibroblast/PBMNC co-cultures.

<p>(<b>A-D</b>) the osteoclast regulating potential of the adipofibroblast derived from MSC-derived adipocyte cultures was assessed by TRAP staining. After 15 days of co-culture with peripheral blood mononuclear cells (PBMNC) and was compared to (<b>C</b>) positive controls where PBMNCs cultures were given 50 ng/ml of M-CSF and 60 ng/ml RANKL in DMEM:RPMI (1/1) medium and (<b>D</b>) untreated controls where PBMNC were cultured in the same mixed basal medium only. (<b>C</b>) Multinucleated cells formed after treatment with M-CSF and RANKL for 15 days showing a distinct osteoclast like cell phenotype including multi-nucleation, TRAP positivity and a large diameter. Positive osteoclasts-like cells were numerous in (<b>A</b>) co-cultures with adipofibroblasts and (<b>C</b>) positive controls (<i>black arrows</i>) while scarce in (<b>D</b>) co-cultures with dermal fibroblasts. (<b>A</b>) Multi-nucleated TRAP positive osteoclast-like cells formed in ‘adipofibroblast’ PBMNC co-cultures after 15 days in culture. Osteoclast-like cell formation was comparable to that of the (<b>B</b>) positive controls while no significant formation of multi-nucleated TRAP-positive osteoclast-like cell formation was seen in (<b>B</b>) dermal fibroblast and PBMNC co-cultures. Scale bar in panel A = 200 µm for panels A-B and 300 µm for panels C-D. <i>n</i> = <i>3–4 donors for both MSC and PBMNC donors respectively</i>.</p

RANKL protein is regulated during adipocyte differentiation.

<p>MSC cultures were induced to form MSC-derived adipocytes, and RANKL protein was detected using immunofluorescence. Cells were counterstained with Hoechst33342, and RANKL protein levels were quantified per cell number by manual counting. Analysis was done on (<b>A</b>) day 0, (<b>B</b>) day 3, (<b>C</b>) day 9, (<b>D</b>) day 12 and (<b>E</b>) day 18. (<b>F</b>) Dermal fibroblasts in culture between 3–12 days were used as a control. There is an increase in both the number and intensity (<i>white arrows</i>) of RANKL positive cells between days 3 and 12 after differentiation. Scale bar in panel A = 200 µm for all panels. (<b>G</b>) To quantify changes in RANKL protein levels cells, the percentage of stained cells was counted at different times in culture. <i>n</i> = <i>3–4 donors</i>.</p

Production of osteoclast-regulating molecules during adipogenesis 3-day conditioned medium from high density untreated MSC in control medium and MSC-derived adipocyte cultures were collected at days 3, 6, 12, and 24.

<p>An ELISA was performed to quantify (A) SDF-1, (B) M-CSF, and (C) OPG. Representative graphs from each experiment show that protein levels of SDF-1 and M-CSF within conditioned medium from MSC-derived adipocytes is higher when compared to MSCs during the early to mid stages of culture while OPG levels are increased only during the late/mature phase of MSC-derived adipocyte cultures. * indicates a p-value <0.05.</p

Adipogenic lineage markers and osteoclast mediator expression profile in MSC-derived adipocytes.

<p>Total RNA from MSC-derived medullary adipocytes was isolated during the 25-day differentiation process and analyzed for (<b>A</b>) known adipogenic lineage markers PPARγ2 C/EBPα, adiponectin, and leptin. (<b>B</b>) The expression profiles of regulators of osteoclast development were mapped using the same analysis to quantify M-CSF, SDF-1, RANKL, and OPG. Measurements of transcript levels were performed using quantitative RT-PCR and graphed relative to levels of transcripts in MSCs at day 0. Error bars indicate variation between samples and is representative of all donors tested. <i>n</i> = <i>3–4 donors</i>.</p

RANKL shows variant co-localization between MSC and adipocyte markers.

<p>RANKL Immunofluorescent co-staining was done to show RANKL (<i>green</i>) co-localization tendencies between the common MSC markers (<b>A</b>) CD90 (<i>red</i>) and (<b>B</b>) CD105 (<i>red</i>) and the adipocyte transcription factors (<b>C</b>) PPARγ2 (<i>red</i>) and (<b>D</b>) C/EBPα (<i>red</i>). The majority of RANKL-positive cells did not co-localize with (<b>A</b>) CD90-positive cells (<b>B</b>) CD105, or (<b>C</b>) PPARγ2 but showed some co-localization with (<b>D</b>). C/EBPα (<i>gray arrows</i>). (<b>Ai-Ci</b>) Insets shows representative instances in which there was some co-localization. (<b>Di</b>) Inset shows representative instances where co-localization was minimal.</p

Direct Comparison of Umbilical Cord Blood versus Bone Marrow Derived Endothelial Precursor Cells in Mediating Neovascularization in Response to Vascular Ischemia

Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culturederived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of eovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29% ± 7.41%) compared with BM (19.69% ± 5.49%; P = .021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P < .05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P = .119; P = .177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses