WISP-1 drives bone formation at the expense of fat formation in human perivascular stem cells

Abstract

The vascular wall within adipose tissue is a source of mesenchymal progenitors, referred to as perivascular stem/stromal cells (PSC). PSC are isolated via fluorescence activated cell sorting (FACS), and defined as a bipartite population of pericytes and adventitial progenitor cells (APCs). Those factors that promote the differentiation of PSC into bone or fat cell types are not well understood. Here, we observed high expression of WISP-1 among human PSC in vivo, after purification, and upon transplantation in a bone defect. Next, modulation of WISP-1 expression was performed, using WISP-1 overexpression, WISP-1 protein, or WISP-1 siRNA. Results demonstrated that WISP-1 is expressed in the perivascular niche, and high expression is maintained after purification of PSC, and upon transplantation in a bone microenvironment. In vitro studies demonstrate that WISP-1 has pro-osteogenic/anti-adipocytic effects in human PSC, and that regulation of BMP signaling activity may underlie these effects. In summary, our results demonstrate the importance of the matricellular protein WISP-1 in regulation of the differentiation of human stem cell types within the perivascular niche. WISP-1 signaling upregulation may be of future benefit in cell therapy mediated bone tissue engineering, for the healing of bone defects or other orthopaedic applications.

Figure 1

Immunohistochemical detection of WISP-1 protein in human adipose tissue. (A) WISP-1 immunoreactivity within the vascular wall of small caliber vessels in cross section. (B) WISP-1 immunoreactivity within a venule (center) and branching capillaries in longitudinal cross section. (C,D) WISP-1 immunoreactivity in larger caliber veins. Scale bars = 25 μm.

Figure 2

WISP-1 expression in human PSC after FACS isolation and upon orthopaedic transplantation. (A) Fluorescence activated cell sorting (FACS) of human lipoaspirate was performed per established protocols to isolate perivascular stem/stromal cells (PSC). After isolation of CD31−CD45− non-endothelial/non-hematopoietic cells (not shown), PSC were further defined as adventitial progenitor cells (APC, CD34+CD146−) or microvascular pericytes (CD146+CD34−). (B) Quantitative RT-PCR for WISP1 transcripts, examined in passage one PSC or unpurified ASC (adipose-derived stromal cells) from the same patient sample. Performed in technical triplicate. (C–F) H&E appearance of rat spine fusion segments. (C,E) H&E appearance of PSC-treated spinal fusion segments with new bone formation, incorporation of demineralized bone matrix (DBM), and conspicuous bone-lining osteoblasts (arrowheads). (D,F) H&E appearance of control-treated spinal fusion segments, with little new bone formation, DBM particles without connections to each other and embedded in fibrous stroma, and inactive bone lining cells (arrowheads). (G–J) WISP-1 indirect immunohistochemical staining of PSC treated spine fusion segments. WISP-1 immunoreactivity appears brown, while nuclear Hematoxylin counterstain appears blue. (G,I) Strong WISP-1 immunoreactivity in bone lining cells among PSC treated samples. (H,J) Weak to intermediate WISP-1 immunoreactivity in inflammatory cells and stromal fibroblasts among control treated samples. *P < 0.05. Scale bars (parts C,D,G,H) 25 µm; Scale bar (parts E,F,I,J) 100 µm.

Figure 3

Osteogenic and adipogenic differentiation of human PSC with WISP-1 knockdown. WISP-1 siRNA or scramble siRNA treated PSC were evaluated for osteogenic and adipogenic differentiation. (A) Knockdown efficiency of WISP-1 in human PSC, assessed by qRT-PCR. (B–D) Osteogenic differentiation of human PSC with or without WISP-1 knockdown. (B) Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2 (Runt-related transcription factor 2), ALP (Alkaline Phosphatase), COL1A1 (Type I Collagen), and OCN (Osteocalcin). (C) Alkaline phosphatase (ALP) staining and photometric quantification after 12 days of osteogenic differentiation with or without WISP-1 knockdown. (D) Bone nodule formation examined by Alizarin red (AR) staining after 12 days of osteogenic differentiation with or without WISP-1 knockdown. (E) Expression levels of markers of BMP and Wnt signaling activity by qRT-PCR at 3 days of osteogenic differentiation with or without WISP-1 knockdown, including ID1 (Inhibitor of DNA binding 1) and AXIN2 (Axis Inhibition Protein 2). (F) PSC were treated with rBMP2 (50 ng/mL), with or without WISP-1 knockdown. Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2, ALP, and OCN. (G) Adipogenic differentiation of human PSC with or without WISP-1 knockdown. Adipocytic gene markers assessed by quantitative RT-PCR at 3 days of differentiation, including PPARγ (Peroxisome proliferator-activated receptor gamma), CEBPα (CCAAT/enhancer-binding protein alpha), FABP4 (Fatty acid binding protein 4), and LPL (Lipoprotein lipase). *P < 0.05; **P < 0.01. Scale bars (part C) 10 mm; scale bars (part D) 200 µm.

Figure 4

Osteogenic and adipogenic differentiation of human PSC with WISP-1 overexpression or rWISP-1 protein. (A–D) WISP-1 overexpression or control plasmid treated PSC were evaluated for osteogenic and adipogenic differentiation. (A) Efficacy of WISP-1 plasmid in human PSC, assessed by qRT-PCR at 6, 9, and 15 days. (B) Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2 (Runt-related transcription factor 2), ALP (Alkaline Phosphatase), COL1A1 (Type I Collagen), and OCN (Osteocalcin). (C) Adipocytic gene markers assessed by quantitative RT-PCR at 3 days of differentiation, including PPARγ (Peroxisome proliferator-activated receptor gamma), CEBPα (CCAAT/enhancer-binding protein alpha). (D) Oil red O staining of human PSC with or without WISP-1 overexpression, 15 days of differentiation. (E,F) Osteogenic differentiation of human PSC with or without recombinant (r)WISP-1 protein (200 ng/mL). (E) Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2, ALP, and OCN. (F) Effects of rWISP-1 (200 ng/mL) and rBMP2 (50 ng/mL) combined application in human PSC. Expression levels of ALP assessed by qRT-PCR at 3 days of osteogenic differentiation. *P < 0.05; **P < 0.01. Scale bars: 100 µm.