Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34+ hematopoietic cells. Co-transplantation of CD34+ HSCs and CD34− MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34+ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34+ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34+ cells. Based on additional in vitro results of endothelial differentiation of CD34+ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34+ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34+ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs

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

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

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

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

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