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.

FIG. 1.

Baseline phenotype of human primary adipose-derived stromal cell (hASCs). (A) Bromodeoxyuridine (BrdU) incorporation assays at 2 and 4 days comparing n=3 patient samples. *P<0.01 in reference to sample 1. (B) Osteogenic differentiation assays comparing n=3 patient samples. After 5 days of supplementation with osteogenic differentiation medium (ODM), alkaline phosphatase (ALP) staining was performed. After 10 days of supplementation with ODM, Alizarin red (AR) staining was performed. Black scale bar=25 μm. (C) Flow cytometry analysis as presented in histograms. Dark gray line indicates stained hASC sample, whereas light gray line indicates unstained hASC sample (negative control). hASCs (passage 4) express typical mesenchymal stem cell markers, including CD73, CD44, CD90, and CD105. In contrast, hASCs were negative for CD45 or CD31 (hematopoietic and endothelial cell markers, respectively). Representative data shown from n=2 patient samples.

FIG. 2.

Cellular proliferation with NELL-1 or N-terminus of Sonic Hedgehog (SHH-N). BrdU incorporation assays at 4 days comparing baseline proliferation (control) with the effects of NELL-1 (100 and 300 ng/mL) or SHH-N (250 and 500 ng/mL). **P<0.01 in reference to identical sample under control conditions.

FIG. 3.

Endogenous Nell-1 and Hedgehog signaling. Human primary ASCs from 3 patient samples were assayed for Nell-1 and Hedgehog specific gene expression after 0 and 7 days of osteogenic differentiation by quantitative real time-polymerase chain reaction (PCR). (A) NELL-1 expression. (B) SHH expression. (C) Patched1 (PTC1) expression. **P<0.01 in reference to identical sample at 0 days of differentiation.

FIG. 4.

Osteogenic differentiation with NELL-1 and/or SHH-N. Recombinant human (rh) NELL-1 (100 and 300 ng/mL) or SHH-N (100 and 250 ng/mL) were supplemented to standard ODM. (A, B) ALP staining and quantification was performed after 5 days of differentiation. (C, D) AR staining and quantification was performed after 10 days of differentiation. All assays presented were performed on hASCs derived from patient 1.

FIG. 5.

Osteogenic differentiation with NELL-1 and/or cyclopamine. rhNELL-1 (300 ng/mL) or the smoothened antagonist cyclopamine (5 μM) was supplemented to standard ODM. (A, B) ALP staining and quantification was performed after 5 days of differentiation. (A, C) AR staining and quantification was performed after 10 days of differentiation. (D) Runt-related transcription factor 2 (RUNX2) gene expression as assessed by quantitative PCR at 5 days of differentiation. All assays presented were performed on hASCs derived from patient 5.

FIG. 6.

Adipogenic differentiation with NELL-1 and/or SHH-N. rhNELL-1 (100 and 300 ng/mL) or SHH-N (100 and 250 ng/mL) was supplemented to standard adipogenic differentiation medium. (A) Oil red O (ORO) staining after 7 days of differentiation. (B) ORO quantification via leaching and photometric quantification, normalized to total protein content.