Biological activity of stem cells We have noted that enumeration of the Colony Forming Unit-Fibroblast, which give rise to BMSC cultures, and a subset of which are stem cells, is highly dependent on the species from which they are derived, and the culture conditions. We have further optimized culture conditions by demonstrating that fetal bovine serum heat inactivation could significantly repress colony formation in mouse and human cultures, and that lots of serum that support mouse cultures do not necessarily support human cultures. Using standardized and improved culture conditions, the concentration of colony forming units - fibroblast, or colony forming efficiency (CFE), ranged in five inbred mouse strains from 3.5+1.0 to 11.5+4.0 per 1x105 nucleated cells. In normal human donors, CFE decreased slightly with age and averaged 52.2+4.1 for children and 32.3+3.0 for adults. Accurate determination of CFE was found to be highly dependent on appropriate culture conditions in both species. We continue to routinely grow HSF6 ES cells. These cells were 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. The role of BMSCs/SSCs in disease While BMSCs/SSCs are increasingly contemplated for stem cell therapy, their involvement in pathogenetic mechanisms of skeletal disorders is far less recognized. We compared BMSC colony forming efficiency (CFE) in cultures of mouse models of skeletal disease, as well as of patients with several skeletal, metabolic, and hematological disorders. In four transgenic lines with profound bone involvement, CFE was either significantly reduced or increased compared to their wild-type littermates. Human CFE was significantly reduced in patients with congenital generalized lipodystrophy, achondroplasia (SADDAN), pseudoachondroplasia, and Pagets disease of bone, and elevated in patients with alcaptonuria and sickle cell anemia. Our findings support the concept that CFE values may provide useful insights into bone/bone marrow pathophysiology. We have had a long-term interest in the somatic mosaic disease, fibrous dysplasia of bone (FD), caused by activating missense mutations of the GNAS gene that codes for the G protein, Gs-alpha. We investigated the role of somatic mosaicism in FD within the context of skeletal (mesenchymal) stem cells by assessing the frequency of mutated CFU-Fs, a subset of which are SSCs, from FD lesions, and in some cases, from unaffected sites, in a series of patients. There was a tight inverse correlation between the % mutant CFU-F vs. age, suggesting demise of mutant stem cells due to exuberant apoptosis noted in samples from young patients. In older patients, either partially or completely normal bone/marrow histology was observed. Upon in vivo transplantation, FD ossicles were generated only by cell strains in which mutant CFU-Fs were identified. Strains that lacked mutant CFU-F (but were mutation positive) failed to regenerate an FD ossicle. These data indicate that GNAS mutations are only pathogenic when in clonogenic skeletal stem cells. From these data, we have evolved the novel concept of normalization of FD. As a lesion ages, mutant stem cells fail to self-renew, and their progeny are consumed by apoptosis, whereas residual normal stem cells survive, self-renew and enable formation of a normal structure. This suggests that activating GNAS mutations disrupt a pathway that is required for skeletal stem cell self-renewal. The use of BMSCs/SSCs in tissue engineering and regenerative medicine Currently, bone defects are treated with either bone autograft or osteoconductive materials. As an alternative, engineered bone can be generated by BMSC transplantation. We demonstrated that BMSC transplants are capable of self-assembly into mature bone organs even when injected as suspensions of cells and HA/TCP particles through the skin. Human BMSCs were mixed with 40 mg aliquots of HA/TCP particles and with fibrinogen and thrombin to form discrete cohesive transplants. Transplants were placed either into the subcutaneous space of the back or onto the calvarium of immunocompromised mice, either via injection, or through a standard open procedure that maintained the shape of the transplant. Injected transplants were delivered using a 3 mm bone biopsy trocar set, fully disrupting their ex vivo organization. The extent of bone formation ranged from moderate to abundant and was equivalent between injected and open approaches. All transplants exhibited cortico-cancellous bone with abundant hematopoiesis, unaffected by the delivery method. Of injected and open approach transplants to the calvarium, all demonstrated bony union to the underlying mouse tissue. Mechanical properties of the transplants were similar in both groups, and in situ hybridization confirmed a human origin of the osteoblasts and osteocytes in the transplants. These results suggest that injection of BMSC transplants will not compromise the amount or quality of newly formed bone. These observations are central to our ability to close bone defects through minimal access approaches. We have shown previously that transplants of BMSCs combined with HA/TCP scaffolds successfully form cortico-cancellous bone to reconstruct the dog craniofacial skeleton. Yet, these transplants long-term stability in large animal models has not been evaluated. We performed a study to evaluate long-term BMSC transplant stability when used to augment the mandible in dogs. Autologous BMSC HA/TCP transplants were introduced on to the unilateral dog mandible as onlay grafts, while contralateral control mandibles received HA/TCP onlays alone. In all dogs, BMSC transplants formed significantly greater amounts of bone over their control counterparts. The new bone formed an extensive union with the underlying mandible. By quantitative CT, the extent of newly formed bone could be determined non-invasively. HA/TCP particles alone under went a high degree of resorption, while autologous cultured BMSCHA/TCP transplants provided long-term bony augmentation of the mandible. We have collaborated with a number of investigators to determine how BMSCs can be used clinically for non-orthopaedic injuries and diseases. With Dr. Joseph Frank, CC, we labeled BMSCs with iron oxide nano-particles, which allows for in situ imaging by MRI. We found that when labeled BMSCs are placed in a sites of neovascularization and inflammation in vivo that 10% of the label is passively taken up by bystander cells (primarily macrophages). This study suggests that care should be taken to validate donor origin of cells using an independent marker by histology prior to drawing conclusions about the in vivo behavior of transplanted cells. With Dr. Eva Mezey, NIDCR, we found that systemically infused murine BMSCs into mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without HLA matching, this suggests that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups. Lastly, with Dr. Joshua Zimmerberg, NICHD, we examined the mechanism by which BMSCs can be immunosuppressive and escape cytotoxic lympocytes. We found that BMSCs express high levels of Fas and FasL, but stimulation of Fas with anti-Fas antibody did not result in BMSC apoptosis, suggesting a disruption of the Fas activation pathway. However, BMSCs may utilize FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues.
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