Establishment of bone marrow and hematopoietic niches in vivo by reversion of chondrocyte differentiation of human bone marrow stromal cells

Abstract

Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived "mesenchymal stem cells") can establish the hematopoietic microenvironment within heterotopic ossicles generated by transplantation at non-skeletal sites. Here we show that non-mineralized cartilage pellets formed by hBMSCs ex vivo generate complete ossicles upon heterotopic transplantation in the absence of exogenous scaffolds. These ossicles display a remarkable degree of architectural fidelity, showing that an exogenous conductive scaffold is not an absolute requirement for bone formation by transplanted BMSCs. Marrow cavities within the ossicles include erythroid, myeloid and granulopoietic lineages, clonogenic hematopoietic progenitors and phenotypic HSCs, indicating that complete stem cell niches and hematopoiesis are established. hBMSCs (CD146(+) adventitial reticular cells) are established in the heterotopic chimeric bone marrow through a unique process of endochondral bone marrow formation, distinct from physiological endochondral bone formation. In this process, chondrocytes remain viable and proliferate within the pellet, are released from cartilage, and convert into bone marrow stromal cells. Once explanted in secondary culture, these cells retain phenotype and properties of skeletal stem cells ("MSCs"), including the ability to form secondary cartilage pellets and secondary ossicles upon serial transplantation. Ex vivo, hBMSCs initially induced to form cartilage pellets can be reestablished in adherent culture and can modulate gene expression between cartilage and stromal cell phenotypes. These data show that so-called "cartilage differentiation" of BMSCs in vitro is a reversible phenomenon, which is actually reverted, in vivo, to the effect of generating stromal cells supporting the homing of hematopoietic stem cells and progenitors.

Human umbilical cord blood-borne fibroblasts contain marrow niche precursors that form a bone/marrow organoid in vivo

“…2, top panels); hematopoietic and bone marrow sinusoidal cells (endothelial and peri-vascular cells) did not stain, confirming their murine origin. Furthermore, in CB ossicles, as previously demonstrated for BM ossicles (Serafini et al, 2014), we detected the presence of human CD146-positive stromal cells associated with the vessel wall (Fig. 2, bottom panels).…”

“…The results presented here provide further elucidation of the biology of CB-BFs, demonstrating that CBBFs were able to generate complete pellet-derived ossicles in vivo in which a functional HSC niche was created. The progressive substitution of cartilage by marrow in the ossicles likely occurred through an 'endochondral myelogenesis' process previously observed in cartilage pellets obtained from BMSCs (Serafini et al, 2014). Interestingly, CB ossicles presented a different ratio of red and yellow marrow compared with the BMSC-derived counterpart, and showed a higher prevalence of red marrow, as demonstrated by histomorphological analyses and by the number of the hematopoietic cells harvested from the transplants.…”

“…In this system, cartilage pellets, produced ex vivo and subsequently transplanted in vivo, remodelled into miniature ossicles with perfect architecture (Serafini et al, 2014). These pellet-derived ossicles include donor-derived bone and marrow stroma, as a result of phenotypical reversion of differentiated chondrocytes, that become subsequently filled by a functional hematopoietic tissue provided by the host.…”

Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes

“…The ability of chondrocytes to dedifferentiate was previously suggested by Song and Tuan following in vitro transdifferentiation assays with human mesenchymal stem cells (hMSCs) (Song and Tuan, 2004). More recently, serial transplantation assays in vivo demonstrated that chondrogenically differentiated hMSCs regained self-renew properties and multilineage potential (Serafini et al, 2014). While our data does not demonstrate that chondrocytes regain multipotency, we do show that activation of pluripotency programs has a functional role during fracture healing and propose that this allows for epigenetic remodeling to switch off chondrogenic programs and induce osteogenic programs.…”

Skeletal stem cells

“…However, like the osteogenic and adipogenic assays, loose interpretation of pellet culture results plagues its use (Robey et al, 2014). Cartilage pellets can in turn be transplanted, to the effect of generating ossicles through a unique developmental sequence (Serafini et al, 2014).…”