Figure 5. PDPN+146-CD73+CD164+ expression identifies hSSC from other tissue sources.
A) Isolation of hSSCs from adult human femoral tissue.
(i) Experimental strategy (right) and representative FACS plots (left) showing gating scheme for isolation of hSSCs from adult human femurhead tissue (n=6).
(ii) Alcian blue stain showing cartilage tissue in a cross-section of micro-mass generated by adult hSSCs after differentiation in vitro (left). Scale: 500 μm. Alizarin Red stain showing bone tissue (osteoblasts) generated by adult hSSCs after differentiation in vitro (right). Scale: 100 μm.
(iii) Image of ossicle formed 4 weeks after transplant of 100,000 hSSCs isolated in A-(i) under the renal capsule of NSG mice. Scale: 2 mm.
(iv) MP stain of a cross-section of the ossicle shown in A-(iii). Scale: 500 μm.
(v) IHC staining of a cross-section of the ossicle shown in A-(iii) for nuclei with DAPI and cartilage, and bone with anti-Collagen II (COL2), and anti-Osteocalcin (OSC) antibodies, respectively. Scale: 100 μm; n=6.
B) Isolation of hSSCs after skeletal induction of human monocyte-derived iPSCs.
(i) Experimental strategy (right) and representative FACS plots (left) showing gating strategy for the isolation of hSSCs 2 weeks after skeletal induction of the iPSCs.
(ii) Alcian blue stain showing cartilage in a cross-section of micro-mass generated 2 weeks after differentiation of iPSC-derived hSSCs in vitro (left). Scale: 500 μm. Alizarin Red S stain showing bone (osteoblasts) generated 2 weeks after differentiation of iPSC-derived hSSCs in vitro (right). Scale: 100 μm.
(iii) Image of an ossicle formed 4 weeks after transplant of 100,000 hSSCs purified from skeletal induced iPSC cultures and then transplanted under the renal capsule of NSG mice. Scale: 2 mm.
(iv) MP stain of a cross-section of the ossicle shown in B-(iii). Scale: 200 μm.
(v) IHC stained image of a cross-section of the ossicle in B-(iii) for nuclei with DAPI and cartilage, and bone with anti-Collagen II (COL2), and anti-Osteocalcin (OSC) antibodies, respectively. Scale: 100 μm; n=6.
C) Isolation of hSSCs from BMP2-treated human adipose stroma (B-HAS).
(i) Experimental strategy for human adipose stroma (HAS) induction with either BMP2 alone or with co-delivery of BMP2 and sVEGFR.
(ii) FACS analysis of HAS (left), HAS-derived ossicle after induction with BMP2 (middle), and HAS-derived cartilage after induction with BMP2 and sVEGFR (right).
(iii) Image of a vascularized ossicle generated 4 weeks after subcutaneous transplantation of HAS treated with BMP2 in NSG mice (left). Scale: 2 mm. MP stain of a cross-section of the same ossicle (right). Scale: 200 μm.
(iv) Image of cartilage mass formation lacking vascularization generated 4 weeks after subcutaneous transplantation of HAS treated with BMP2 and sVEGFR in NSG mice (left). Scale: 2 mm. MP stain of a cross-section of the same subcutaneous cartilaginous mass (right). Scale: 200 μm; n=6.
D) Bar chart showing quantification of percent contribution by the different skeletal fates to total graft mass for grafts derived from fetal hSSCs, adult hSSCs, skeletal-induced-iPSC, B-HAS, or BMP2+sVEGFR-treated HAS after morphometric analysis.