Vascularization of <i>in vivo</i> implanted tissue grafts.

<p>H&E staining. A. Microporous calcium phosphate (CP) scaffolds seeded with mesenchymal stem/progenitor cells (MSCs) alone showed minimal vascularization of the micropores of the implant. B. Co-transplantation of hematopoietic stem/progenitor cells (HSCs) and MSCs resulted in substantial numbers of blood vessels (black arrows) in the micropores of the CP scaffolds.</p

Collagen apposition in tissue grafts <i>in vivo</i>.

<p>A–D. Masson's trichrome staining (blue) shows increased pre-mineralizing collagen deposition and osteoid formation in co-transplantation of MSCs and HSCs without VEGF (C1) and VEGF-delivered MSC and HSC co-transplantation sample (D1), in contrast to MSC transplantation alone (A1) and VEGF-delivered MSC transplantation sample (B1). A2–D-2. Magnification of red boxes in A1–D1, respectively.</p

Expression of human osteocalcin and mineral deposition in tissue grafts <i>in vivo</i>.

<p>Human osteocalcin immunostaining displays increased levels of expression in MSC and HSC co-transplantation sample (C) and VEGF-delivered MSC and HSC co-transplantation sample (D), in contrast with MSC transplantation alone sample (A) and VEGF-delivered MSC transplantation sample (B). Osteoblast-like cells are observed lining the calcium phosphate scaffold (CP) (black arrow in D). E. Quantified human osteocalcin content confirms significantly increased human osteocalcin expression of the co-transplantation group of MSCs and HSCs (n = 5, p<0.05). Interestingly, VEGF delivery decreased human osteocalcin expression, despite the higher level of vascularization (see detailed discussion in text). F–I. Undecalcified H&E stained sections of in vivo implanted scaffolds show increased dark mineralized tissue throughout the co-transplanted MSC and HSCs cell-seeded scaffold (yellow arrows in H) and VEGF-delivered, co-transplanted MSC and HSC-seeded scaffold (yellow arrows in I) groups, in contrast to MSC transplantation alone (F) and VEGF-only (G).</p

Isolation of hematopoietic stem/progenitor cells and mesenchymal stem/progenitor cells from a single bone marrow aspiration.

<p>A. Human bone marrow is aspirated from the iliac crest of donor patients. B. Mesenchymal stem/progenitor cells (MSC) isolated from human bone marrow attach to tissue culture plates and assume typical spindle, fibroblast-like shape. C. Von Kossa stained MSC-derived osteoblasts in osteogenic differentiation medium. Black stained mineralized nodules are observed as well as pericellular staining throughout the plate. D. Hematopoietic stem/progenitor cells (HSCs) are isolated from the same human bone marrow sample. E. HSCs are expanded in suspension culture, smaller than MSCs, and non-adherent, in addition to maintaining spherical shape. F. MSCs are seeded on the surfaces of the micropores of the 3D cylindrical calcium phosphate (CP) scaffold. Culture expanded HSCs with or without VEGF are then seeded in Matrigel and infused into the micropores of the 3D CP scaffolds to complete implant fabrication (controls included Matrigel with no HSCs, or with VEGF alone). G. Carboxyfluoroscein diacetate (CFDA) labeled MSC and HSCs labeled with red CM-DiI are visualized in the micropores of the 3D graft. Green MSC are on the surface of the micropores of the CP scaffold, whereas red HSCs are suspended in Matrigel that is infused into MSC-occupied pore surface. H. Scaffolds are implanted subcutaneously in the dorsum of immunocompromized mice.</p

Endothelial differentiation of HSCs <i>in vitro</i>.

<p>A. Hematopoietic stem/progenitor cells (HSCs) propagated in suspension culture, assuming spherical shape. B. HSCs form endothelial-like colonies in fibronectin-coated plates. C. Formation of tubular intercellular structures in 3D Matrigel culture. Uptake of acetylated-LDLs (red) (blue: DAPI) (D) and vWF (von Willebrand Factor) immunofluorescent stain (green) (E). F. Quantification of vWF measured by ELISA showing substantial expression of vWF in HSC-derived endothelial-like cells, in comparison with dermal fibroblasts as controls.</p

Transplanted human HSCs and MSCs engraft <i>in vivo</i> and into vascular endothelium of host vasculature.

<p>Immunostaining (brown) of human specific nuclei of tissue grafts by MSC transplantation alone (without HSCs) (A), MSC transplantation with exogenous VEGF (B), co-transplantation of MSCs and HSCs without VEGF delivery (C), or co-transplantation of MSCs and HSCs with VEGF delivery (D). Red arrows point to human nuclei that engraft to forming blood vessel wall surrounding functional lumen (L) filled with red blood cells.</p