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Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells.

ABSTRACT: A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

SUBMITTER: James AW

PROVIDER: S-EPMC3411358 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells.

James Aaron W AW Pang Shen S Askarinam Asal A Corselli Mirko M Zara Janette N JN Goyal Raghav R Chang Le L Pan Angel A Shen Jia J Yuan Wei W Stoker David D Zhang Xinli X Adams John S JS Ting Kang K Soo Chia C

Stem cells and development 20120222 12

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present ...[more]

PMID: 22264144

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Project description:While it has been postulated that tissue-specific bioscaffolds derived from the extracellular matrix (ECM) can direct stem cell differentiation, systematic comparisons of multiple ECM sources are needed to more fully assess the benefits of incorporating tissue-specific ECM in stem cell culture and delivery platforms. To probe the effects of ECM sourced from decellularized adipose tissue (DAT) or decellularized trabecular bone (DTB) on the adipogenic and osteogenic differentiation of human adipose-derived stem/stromal cells (ASCs), a novel detergent-free decellularization protocol was developed for bovine trabecular bone that complemented our established detergent-free decellularization protocol for human adipose tissue and did not require specialized equipment or prolonged incubation times. Immunohistochemical and biochemical characterization revealed enhanced sulphated glycosaminoglycan content in the DTB, while the DAT contained higher levels of collagen IV, collagen VI and laminin. To generate platforms with similar structural and biomechanical properties to enable assessment of the compositional effects of the ECM on ASC differentiation, micronized DAT and DTB were encapsulated with human ASCs within methacrylated chondroitin sulfate (MCS) hydrogels through UV-initiated crosslinking. High ASC viability (>90%) was observed over 14 days in culture. Adipogenic differentiation was enhanced in the MCS+DAT composites relative to the MCS+DTB composites and MCS controls after 14 days of culture in adipogenic medium. Osteogenic differentiation studies revealed a peak in alkaline phosphatase (ALP) enzyme activity at 7 days in the MCS+DTB group cultured in osteogenic medium, suggesting that the DTB had bioactive effects on osteogenic protein expression. Overall, the current study suggests that tissue-specific ECM sourced from DAT or DTB can act synergistically with soluble differentiation factors to enhance the lineage-specific differentiation of human ASCs within 3-D hydrogel systems.

Project description:Bone morphogenetic protein-2 (BMP-2) is a growth factor used to stimulate bone regeneration in clinical applications. However, there are contradicting reports on the functionality of BMP-2 in human adipose stem cells (hASCs), which are frequently used in tissue engineering. In this study, we analyzed the effects of BMP-2 on SMAD1/5 signaling, proliferation, and differentiation in hASCs. Our results indicated that BMP-2 induced dose-dependent (25-100 ng/ml) activation of SMAD signaling. Furthermore, the cell proliferation analysis revealed that BMP-2 (100 ng/ml) consistently decreased the proliferation in all the cell lines studied. However, the analysis of the differentiation potential revealed that BMP-2 (100 ng/ml) exhibited a donor-dependent dual role, inducing both osteogenic and adipogenic differentiation in hASCs. The quantitative alkaline phosphatase (qALP) activity and mineralization levels were clearly enhanced in particular donor cell lines by BMP-2 stimulus. On the contrary, in other cell lines, qALP and mineralization levels were diminished and the lipid formation was enhanced. The current study also suggests that hASCs have accelerated biochemical responsiveness to BMP-2 stimulus in human serum-supplemented culture medium compared with fetal bovine serum. The production origin of the BMP-2 growth factor is also important for its response: BMP-2 produced in mammalian cells enhanced signaling and differentiation responses compared with BMP-2 produced in Escherichia coli. These results explain the existing contradiction in the reported BMP-2 studies and indicate the variability in the functional end mechanism of BMP-2-stimulated hASCs.This study examined how bone morphogenetic protein-2 (BMP-2) modulates the SMAD signaling mechanism and the proliferation and differentiation outcome of human adipose stem cells (hASCs) derived from several donors. The results indicate that BMP-2 triggers molecular SMAD signaling mechanisms in hASCs and regulates differentiation processes in human serum-culture conditions. Importantly, BMP-2 has dual activity, inducing osteogenic and adipogenic differentiation, subject to hASC donor line studied. These findings explain contradictory previous results and highlight the importance of further studies to understand how signaling pathways guide mesenchymal stem cell functions at the molecular level.

Dataset Information

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells.

Publications

Additive effects of sonic hedgehog and Nell-1 signaling in osteogenic versus adipogenic differentiation of human adipose-derived stromal cells.

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