1) Biological activity of stem cells 

 During the past year, we have been analyzing the data generated by molecular profiling of different clones of BMSCs (originating from a single colony forming unit-fibroblast, CFU-F) in order to determine what may distinguish multipotent clones, able to form bone, hematopoietic stroma and marrow adipocytes in vivo, from clones that are only able to make bone, or only fibrous tissue. We have identified a number of genes that are upregulated (e.g., Secreted Frizzled Related Protein-2, SFRP2) and downregulated (e.g., Calponin 1, CNN1) in multipotent clones. We have obtained two lines of mice, one deficient in Sfrp2 and the other deficient in Cnn1, and are studying the skeletal phenotype and the activity of BMSCs/SSCs in these two strains. Based on our extensive knowledge of how to induce BMSCs to form bone by in vitro expansion and in vivo transplantation, we developed techniques for generation of bone by human embryonic stem cells (hESCs), and are currently applying these techniques to induced pluripotent stem cells (iPSCs) that were derived from skin fibroblasts and BMSCs using polycistronic lentiviral vectors containing the four reprograming factors, Oct4, Sox2, Klf4 and cMyc. Preliminary studies indicate that iPSCs from both skin and BMSCs-derived iPSCs are capable of forming small foci of bone upon in vivo transplantation. Current studies are aimed at improving the quality and quantity of osteogenic differentiation. 


 2) BMSCs/SSCs in disease

 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, G-alpha(s), that leads to overproduction of cAMP. We are continuing to study the downstream effects of mutant G-alpha(s) activity by analyzing changes in a number of signaling pathways, in addition to that of cAMP-induced activation of PKA, due to the potential cross-talk between pathways. In particular, we are focusing on the Wnt pathway, which is mediated by binding of Wnt to frizzled co-receptors, which are G protein coupled receptors. Although the Wnt/beta-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. We investigated interactions of the four major G-alpha protein families G-alpha(s), G-alpha(i/o), G-alpha(q/11), and G-alpha(12/13) with the Wnt/beta-catenin signaling pathway and identified a causative role of Wnt/beta-catenin signaling in FD. The activating G-alpha(s) mutations that cause FD potentiated Wnt/beta-catenin signaling, and removal of G-alpha(s) led to reduced Wnt/beta-catenin signaling and decreased bone formation. We also showed that activation of Wnt/beta-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of beta-catenin levels rescued differentiation defects of FD patient-derived BMSCs. G-alpha proteins may act at the level of beta-catenin destruction complex assembly by binding to Axin. Our results indicate that activated G-alpha proteins differentially regulate Wnt/beta-catenin signaling but, importantly, are not required core components of Wnt/beta-catenin signaling. Our data also suggest that activated G-alpha proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.

 In another study, we investigated the role that BMSCs play in modulating changes in hematopoiesis. It is well established that one of the major rolls that BMSCs play is in the support of hematopoiesis. It is also known that inflammation alters hematopoiesis, often by decreasing erythropoiesis and enhancing myeloid output. However, the mechanisms behind these changes and how bone marrow stromal cells contribute to this process are not well understood. For these reasons, we examined changes in BMSCs in the setting of murine Toxoplasma gondii infection, which is known to cause a rapid crash in erythropoiesis. Our data revealed that infection alters early myeloerythroid differentiation, blocking erythroid development beyond the Pre MegErythroblast stage, while expanding the Granulocyte-Macrophage Precursor (GMP) population. IL-6 was found to be a critical mediator of these differences, independent of hepcidin-induced iron restriction, which also causes a decrease in erythropoiesis. Comparing bone marrow with the spleen showed that the hematopoietic response was driven by the local marrow microenvironment, and bone marrow chimeras demonstrated that radioresistant cells (which are for the most part BMSCs) were the relevant source of IL-6 in vivo. Finally, direct ex vivo sorting revealed that VCAM(+)CD146(lo) BMSCs significantly increase IL-6 secretion after infection. These data suggest that BMSCs regulate the hematopoietic changes during inflammation via IL-6. In addition to these studies, we have established BMSC cultures and created in vivo transplants from patients with a variety of diseases, including a patient with idiopathic juvenile osteoporosis (has three possible genetic defects: compound heterozygosity C20orf26;new dominant PIK3CD;synergistic heterozygosity LAMA2 + LAMC2), Hajdu-Cheney syndrome (NOTCH2 mutation), and from a patient with gnatho-diaphyseal dysplasia (ANO5 mutation). The cells from these patients are available for studying the downstream effects of mutation. 3) BMSCs/SSCs in tissue engineering and regenerative medicine

 Autologous transplantation of human BMSCs has been successfully used for bone reconstruction. However, in order to advance this approach into the mainstream of bone tissue engineering, the conditions for BMSC cultivation and transplantation must be optimized. In a recent report, cultivation with dexamethasone (Dex) was reported to significantly increase bone formation by human BMSCs in vivo. Based on this important conclusion, we analyzed the data accumulated by our laboratory, where human BMSCs have been routinely generated using media both with and without a combination of two osteogenic supplements: Dex at 10(-8) M and ascorbic acid phosphate (AscP) at 10(-4)  M. Our data demonstrated that for virtually all donors, BMSC strains propagated with and without Dex/AscP formed similar amounts of bone in vivo. Thus, human BMSCs do not appear to need induction to osteogenic differentiation ex vivo prior to transplantation. Similarly, for virtually all donors, BMSC strains cultured with and without Dex/AscP formed hematopoietic territories to a comparable extent. While Dex/AscP did not increase bone formation, they significantly stimulated BMSC in vitro proliferation without affecting the number of BMSC colonies formed by the colony-forming units-fibroblasts. We conclude that for the substantial majority of donors, Dex/AscP have no effect on the ability of BMSCs to form bone and myelosupportive stroma in vivo. However, due to increased BMSC proliferation, the total osteogenic population obtained from a single marrow sample is larger after cultivation with Dex/AscP than without them. In addition to use in bone regeneration, BMSCs are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. Working with our colleagues in the Cell Processing Section, DTM, CC, which is partner in the NIH Bone Marrow Stromal Cell Transplantation Center, we have established a bank of BMSC products from healthy third party donors. These cells are currently being used to treat patients with acute GVHD, and patients with cardiovascular disease.

Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2012
Total Cost
$1,363,253
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
Zip Code
Featherall, Joseph; Robey, Pamela G; Rowe, David W (2018) Continuing Challenges in Advancing Preclinical Science in Skeletal Cell-Based Therapies and Tissue Regeneration. J Bone Miner Res 33:1721-1728
Chen, Kevin G; Mallon, Barbara S; Park, Kyeyoon et al. (2018) Pluripotent Stem Cell Platforms for Drug Discovery. Trends Mol Med 24:805-820
Chen, Kevin G; Johnson, Kory R; McKay, Ronald D G et al. (2018) Concise Review: Conceptualizing Paralogous Stem-Cell Niches and Unfolding Bone Marrow Progenitor Cell Identities. Stem Cells 36:11-21
Ren, Jiaqiang; Ward, Dawn; Chen, Steven et al. (2018) Comparison of human bone marrow stromal cells cultured in human platelet growth factors and fetal bovine serum. J Transl Med 16:65
Chen, Kevin G; Johnson, Kory R; Robey, Pamela G (2017) Mouse Genetic Analysis of Bone Marrow Stem Cell Niches: Technological Pitfalls, Challenges, and Translational Considerations. Stem Cell Reports 9:1343-1358
Pievani, Alice; Sacchetti, Benedetto; Corsi, Alessandro et al. (2017) Human umbilical cord blood-borne fibroblasts contain marrow niche precursors that form a bone/marrow organoid in vivo. Development 144:1035-1044
Robey, Pamela (2017) ""Mesenchymal stem cells"": fact or fiction, and implications in their therapeutic use. F1000Res 6:
Liu, Shutong; de Castro, Luis F; Jin, Ping et al. (2017) Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers. Sci Rep 7:46731
Corsi, Alessandro; Ippolito, Ernesto; Robey, Pamela G et al. (2017) Bisphosphonate-induced zebra lines in fibrous dysplasia of bone: histo-radiographic correlation in a case of McCune-Albright syndrome. Skeletal Radiol 46:1435-1439
Sacchetti, Benedetto; Funari, Alessia; Remoli, Cristina et al. (2016) No Identical ""Mesenchymal Stem Cells"" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels. Stem Cell Reports 6:897-913

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