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1.
FIG. 4.

FIG. 4. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Osteogenic differentiation of ASCs with cyclopamine. (A) Runx2 and Col1 expression with cyclopamine and/or Shh-N (500 ng/mL) at 4 days of differentiation, by qRT-PCR. (B) ALP staining and quantification with cyclopamine and/or Shh-N after 7 days. (C) AR staining and quantification with cyclopamine and/or Shh-N after 14 days. Photographs are of 20× magnification; values are normalized to control expression levels, a,b,cp < 0.01. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.
2.
FIG. 3.

FIG. 3. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Osteogenic differentiation with Shh-N. Osteogenic differentiation was examined through a 14 day time course. (A) Runx2 and collagen type I (Col1) expression by qRT-PCR after 4 days. (B) ALP staining after 7 days, appearing blue. ALP quantification after 7 days was normalized to total protein content. (C) AR staining and quantification after 14 days. (D) Osteopontin (Opn) and Osteocalcin (Oc) expression by qRT-PCR after 14 days. Photographs are of 20× magnification; values are normalized to control expression levels, ap < 0.01. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.
3.
FIG. 1.

FIG. 1. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Hedgehog signaling during osteogenic and adipogenic differentiation. (A) Progression of mouse adipose-derived stromal cell (ASC) osteogenic differentiation through 2 weeks, shown by alkaline phosphatase (ALP) and alizarin red (AR) staining. (B) Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), Gli1, and patched (Ptc1) expression at 0, 1, and 2 weeks of osteogenic differentiation, normalized to GAPDH. (C) Mouse ASC adipogenic differentiation, shown by oil red O staining. (D) Shh, Ihh, Gli1, and Ptc1 expression at 0 and 1 week of adipogenic differentiation, normalized to GAPDH. Photographs are of 20× magnification; means and significance levels are calculated relative to 0 week expression, a,b,c,dp < 0.01. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.
4.
FIG. 5.

FIG. 5. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Cooperation of Hedgehog and bone morphogenetic protein (BMP) signaling. (A) Bmp2 and Bmp4 expression after 48 h of Shh-N treatment, by qRT-PCR. (B) Hedgehog pathway activity after 48 h of recombinant (r)BMP-2 treatment, by qRT-PCR. (C) ALP staining (left) and quantification (right) after 7 days with Shh-N and/or rBMP-2. Bars represent mean ALP enzymatic activity, normalized to total protein content. (D) AR staining (left) and quantification (right) after 14 days with Shh-N and/or rBMP-2. Bars represent mean AR staining, as measured by absorbance after cetylpyridium chloride leaching. Photographs are of 20× magnification; values are normalized to control expression levels, a,bp < 0.01. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.
5.
FIG. 2.

FIG. 2. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Hedgehog pathway activation and adipogenic differentiation in ASCs. (A) Hedgehog signaling elements following culture with N-terminal Sonic Hedgehog (Shh-N) for 48 h by quantitative real-time polymerase chain reaction (qRT-PCR), including Ptc1, Gli1, Gli2, and Gli3. (B) Hedgehog ligand expression after Shh-N exposure for 48 h, including Shh, Ihh, and Dhh. (C–F) Adipogenic differentiation with Shh-N or cyclopamine. (C) Oil red O staining with Shh-N at day 7 adipogenic differentiation (250–750 ng/mL). (D) Oil red O staining with cyclopamine at day 7 differentiation (5–20 μM). (E) PPAR-γ expression with Shh-N at 7 days, by qRT-PCR. (F) PPAR-γ expression with cyclopamine at 7 days of differentiation. Means and significance levels are calculated relative to control expression levels, a,b,c,dp < 0.01. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.
6.
FIG. 6.

FIG. 6. From: Sonic Hedgehog Influences the Balance of Osteogenesis and Adipogenesis in Mouse Adipose-Derived Stromal Cells.

Grafting of ASCs in tibial defects. (A, B) A 1-mm monocortical defect was surgically created in the proximal mouse tibia; spatiotemporal gene expression after injury was then examined by qRT-PCR and in situ hybridization. (A) Hedgehog signaling expression from 1 to 6 days postinjury (Gli1, Smo, and Ptc1). (B) Ptc1 in situ in which positive staining cells appear purple; dotted lines demarcate the cortical bone. (C) Schematic of the grafting of green fluorescent protein (GFP)–positive ASCs into a monocortical tibial defect. (D, E) Histology of defects with Shh-N- or control-treated ASCs. From top to bottom, green fluorescent protein immunohistochemistry appearing gray, ALP staining appearing purple, pentachrome staining in which bone appears yellow, and phospho-Smad 1/5/8 immunohistochemstry appearing brown. Black arrows D indicate examples of positively stained cells for phospho-Smad 1/5/8. Color images available online at www.liebertonline.com/ten.

Aaron W. James, et al. Tissue Eng Part A. 2010 August;16(8):2605-2616.

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