Formation of hematopoietic territories and bone by transplanted human bone marrow stromal cells requires a critical cell density.? ? Bone marrow stromal cells (BMSCs) include multipotent cells with the ability to form mature bone organs upon in vivo transplantation. Hematopoiesis in these bone organs has been ascribed to the action of skeletal stem cells, which are capable of differentiating towards bone and hematopoiesis-supporting stroma. Yet, the creation of hematopoietic territories may be in part a natural consequence of the formation of a sufficiently mature and large bone microenvironment. We have studied the relationship between BMSC numbers and the extent of bone/hematopoiesis formation in vivo. Human BMSCs were transplanted along with hydroxyapatite/tricalcium phosphate, utilizing a spectrum of dosages, into immunocompromised mice and the transplants were followed for up to 29 months. The extent of bone and hematopoiesis formation increased with increasing BMSC numbers; however, the relationship was sigmoid in character, and a threshold number of BMSCs was necessary for extensive bone formation or any hematopoiesis. Hematopoiesis only occurred in conjunction with extensive bone formation, and no hematopoiesis occurred where bone formation was poor. Consistent with our earlier studies of long-term BMSC transplantation, the transplants underwent a change in bone morphology (bone remodeling lead to generation of thick and even trabeculae) but not bone content (meaning, there was no bone overgrowth) after 8 weeks. Our results have provided evidence that the formation of both hematopoiesis and a mature bone organ is as much a consequence of a sufficiently high local density of bone marrow stromal cells as it is the product of skeletal stem cell action.? ? Skeletal stem cells (mesenchymal stem cells) in bone marrow are pericytes.? ? The anatomic localization of the clonogenic skeletal progenitors found in bone marrow stroma has long remained elusive. Finding the identity of these cells is of interest not only to develop better strategies for their isolation, but also in order to determine how these cells establish the hematopoietic microenvironment in human bone marrow, and how they support the maintenance of the hematopoietic stem cell. We found that MCAM/CD146-expressing, subendothelial cells (pericytes) in human bone marrow stroma are in fact the clonogenic subset of cells found in bone marrow stromal cell (BMSC) populations. CD146+ cells are capable of transferring the hematopoietic microenvironment to within a miniature bone/marrow organ created under the skin in immunocompromised mice, coincident with the establishment of identical subendothelial cells (pericytes). Establishment of pericytes by stromal cells in developing heterotopic bone marrow in vivo occurs via specific, dynamic interactions with developing in-growing sinusoids. Subendothelial stromal cells residing on the sinusoidal wall are major producers of Angiopoietin-1 (a pivotal molecule of the hematopoietic stem cell niche involved in vascular remodeling). This study has revealed the functional relationships between establishment of the hematopoietic microenvironment in vivo, the establishment of skeletal progenitors in bone marrow sinusoids, and angiogenesis. ? ? Characterization of craniofacial defects in mice with abnormal PTH/PTHrP receptor signaling.? ? Parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) regulate calcium homeostasis, and PTHrP further regulates growth and development. Ligand-induced signaling of PTH and PTHrP is mediated via a common G-protein coupled receptor, the PTH/PTHrP receptor. A transgenic mouse carrying the constitutively active PTH/PTH-rP receptor (HKrk-H223R) under the control of the mouse bone and odontoblast specific alpha1(I) collagen promoter (Col1-caPPR) has been developed to demonstrate the complex actions of this mutant receptor in hard tissue formation. We previously determined that there is exuberant abnormal bone formation in the long bones of these mice that is normalized with time due to the loss of skeletal stem cells. We have now further characterized Col1-caPPR mice abnormalities in the craniofacial region as a function of development. Col1-caPPR mice exhibited a delay in embryonic bone formation in the cranium. After birth, expansion of a number of craniofacial bones including the maxilla and mandible was noted, with abnormal trabecular bone structure, reminiscent of the abnormality in the long bones. Transgenic mice also exhibited a delay in tooth eruption. Transgenic teeth were noted for an expansion of the pulpal tissue and a highly disorganized odontoblastic layer that was unable to form tubular dentin. Finally, the temporomandibular joint (TMJ) exhibited a completely disorganized structure, with hypertrophic cartilage that is normal present, being almost completely absent, and replaced by a dense fibroblastic tissue. These abnormalities lead to a compete disfiguration of the TMJ by 4 months of age. These findings suggest that the Col1-caPPR mouse is a useful model for characterization of the downstream effects of the constitutively active receptor during development and growth, and as a model for development of treatments of human diseases with similar characteristics.? ? Pre-clinical studies on iron oxide nanoparticle-labeled bone marrow stromal cells.? ? There is increasing interest in using exogenous labels such as bromodeoxyuridine (BrdU) or dextran coated superparamagnetic iron oxide nanoparticles (SPION) to label cell populations to identify transplanted cells and monitor their migration by fluorescent microscopy or in vivo magnetic resonance imaging (MRI). However, direct transplantation of cells into target tissue can result in >80% cell death due to trauma or apoptosis. Bystander uptake of labeled cells by activated macrophages can confound the interpretation of the results. We investigated the frequency of BrdU or SPION uptake by activated macrophages using a Boyden chamber model of inflammation. SPION labeled bone marrow stromal cells (BMSCs) or HeLa cells, and mouse fibroblasts with BrdU were mixed in various ratios in Matrigel, in the upper chamber and incubated for up to 96 hours. FACS analysis of activated macrophages from the lower chamber using anti-CD68, anti-dextran antibodies revealed from 10 to 20% dextran or up to 10% BrdU positive activated macrophages after 96 hours of incubation. Transfer of iron to activated macrophages was < 10% of the total iron load in labeled cells. The uptake of BrdU and SPION was dependent on the density of labeled cells to inflammatory cells and microenvironmental conditions. Direct implantation of BrdU labeled cells into target tissue can result in uptake of label by activated macrophages, however, care should be taken to validate BrdU labeled cells by staining for bystander cell markers and microscopy. The minimal uptake of SPION by activated macrophages would suggest that the observed signal changes on cellular MRI originate primarily from the implanted cells. ? ? Human embryonic stem cells? ? We continue to gain expertise in routinely growing HSF6 cells. These cells have been cultured in a number of different ways to induce their differentiation into mesoderm and into osteogenic tissues. Such cells have been transplanted in vivo using a number of different scaffolds to determine the efficacy of the differentiation conditions that have been utilized.
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