During the last fiscal year, the Skeletal Biology Section reports activities in all three areas. 1) Biological activity of stem cells 

 We have continued to study the biological nature of BMSCs in comparison to stem/progenitor populations present in other connective tissues. Our data shows very clearly that when BMSCs are expanded ex vivo in an appropriate fashion, they maintain the ability to form bone, hematopoiesis-supportive stroma and marrow adipocytes by in vivo transplantation with an appropriate scaffold, and cartilage in cell pellet cultures. Mesenchymal stem cells derived from skeletal tissues such as bone proper, periosteum, etc., have similar, but not identical properties;they make bone, but do not support hematopoiesis. It is the ability of BMSCs to support hematopoiesis that makes them unique compared to stem/progenitor cells of other skeletal tissues. However, MSCs from non-skeletal tissues do not have the capacity to make a bone/marrow organ. Confusion in the field has arisen from the fact that cell surface markers used to characterize MSCs are characteristic of any adherent fibroblastic cell population, whether it has been determined to contain a subset of stem cells or not, by rigorous criteria (the ability of the progeny of a single cell to produce a functional tissue, and the ability to self-renew). In addition to the use of non-specific cell surface markers, artifactual in vitro assays that do not faithfully predict the in vivo differentiation capacity of the cells from many tissues are also widely used. Based on these unreliable assays, it has come to be thought that MSCs with identical differentiation properties exist in virtually all connective tissues, and some have suggested that MSCs from adipose tissue are useful for bone regeneration. However, by in vivo transplantation, we have shown that adipose-derived cells derived by a number of methods do not make bone, unless heavily treated with BMPs. BMPs will induce any fibroblastic cell to temporarily become osteogenic, but osteogenesis is not sustained as BMP signaling wanes. We continue to aim to develop assays that will faithfully predict differentiation capacity of stem/progenitor cells from different hard and soft connective tissues. Based on our extensive knowledge of how to induce osteogenic differentiation of BMSCs and human embryonic stem cells (hESCs) by in vitro expansion and in vivo transplantation, we are also currently developing techniques for generation of bone by human induced pluripotent stem cells (hiPSCs) that were derived from skin fibroblasts and BMSCs using lentiviral vectors containing the four reprograming factors, Oct4, Sox2, Klf4 and cMyc. Studies indicate that iPSCs derived from both skin and BMSCs are capable of forming small foci of bone upon in vivo transplantation after treatment with osteogenic conditions and growth on hydroxyapatite. However, the amount of bone formed is far less than that made by BMSCs, and not even equivalent to that made by hESCs. Surprisingly, we found that in one type of culture condition, one line made cartilage in the in vivo transplants. Further studies are in progress using cell lines with stage-specific reporters to take a more developmentally oriented approach to generate bone and cartilage. In addition, in keeping with our long term goal of using large numbers of iPSCs for bone tissue engineering, we have started to develop and utilize approaches that will circumvent the need for the use of irradiated mouse feeder cell layers and colony-picking, both of which represent hurdles to be overcome when thinking about the generation of clinical grade iPSCs and their derivatives. For this reason, our colleagues in the NIH Stem Cell Unit have developed a non-clonal monolayer style of culture for hESCs that we have tested and utilized for expansion of iPSCs. The cells retained their pluripotency using this technique as determined by their expression of pluripotency markers, and the ability to differentiate into cell types of all three embryonic layers, in vitro and in vivo. 2) BMSCs/SSCs in disease

 In keeping with our interests in skeletal diseases (in particular, fibrous dysplasia of bone in collaboration with Dr. Michael T. Collins in the Skeletal Clinical Studies Unit, CSDB, NIDCR), we participate in the Undiagnosed Diseases Program (UDP) (Dr. William Gahl, NHGRI). One patient with idiopathic juvenile osteoporosis has been assayed for colony forming efficiency (the closest approximation to the number of skeletal stem cells in bone marrow), and the patients BMSCs have been analyzed by the in vivo transplantation assay. Less bone was formed and less hematopoiesis was supported by these BMSCs than those of a normal age-matched donor. The patient has been treated with a bone anabolic agent for several years with some improvement in bone mineral density. Bone marrow will again be assayed to determine the effect of treatment on skeletal stem cell number and the ability to form bone and support hematopoiesis. Whole exome sequencing has revealed four possible genetic causes: compound heterozygosity of C20orf26;a new dominant PIK3CD mutation;synergistic heterozygosity of LAMA2 and LAMC2, and interestingly, a mutation in NOTCH2. Another patient, but with Hajdu-Cheney syndrome, was also found to have a NOTCH2 mutation (although in a different location). While there is some phenotypic overlap between the two patients, they are not identical, suggesting one of the other potential candidate genes in the UDP patient contributes to the phenotype noted, or that there is a phenotypic distinction based on the location of the NOTCH mutation in the molecule. 3) BMSCs/SSCs in tissue engineering and regenerative medicine In addition to use in bone regeneration, BMSCs are being used to treat a variety of conditions based on their extensive secretion of growth factors and cytokines. 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 a 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, patients with cardiovascular disease, and with inflammatory bowel disease. However, BMSC expansion is limited by senescence. Transcriptome analysis of 10 early and 15 late passage BMSC samples from 5 subjects revealed 2193 differentially expressed genes;those highly expressed in early passage cells were overrepresented in skeletal system development, embryonic morphogenesis and tube morphogenesis, while those highly expressed in the late passage BMSCs were overrepresented in nucleosome assembly, chromatin assembly and DNA packaging. 57 BMSC samples from 7 donors were analyzed for the transition from an early to late passage molecular signature;155 genes were highly correlated with BMSC senescence and a set of 24 genes was predictive of BMSC age. The change from an early to a late passage signature varied among donors;occurring between passage 3 and 5. In contrast, functional and phenotype measures including senescence-associated beta-galactosidase staining and colony formation efficiency changed at passage 6 or 7. The age predictive gene set identified 3 of 12 clinical BMSC lots not meeting release criteria. In conclusion, the onset of senescence-associated molecular changes was variable and preceded changes in other indicators of BMSC quality and senescence. It is hoped that the 24 BMSC lifespan predictive genes will be useful in assessing the quality of BMSC products.

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National Institute of Dental & Craniofacial Research
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Hong, So Gun; Winkler, Thomas; Wu, Chuanfeng et al. (2014) Path to the clinic: assessment of iPSC-based cell therapies in vivo in a nonhuman primate model. Cell Rep 7:1298-309
Chen, Kevin G; Mallon, Barbara S; Johnson, Kory R et al. (2014) Developmental insights from early mammalian embryos and core signaling pathways that influence human pluripotent cell growth and differentiation. Stem Cell Res 12:610-21
Mallon, Barbara S; Hamilton, Rebecca S; Kozhich, Olga A et al. (2014) Comparison of the molecular profiles of human embryonic and induced pluripotent stem cells of isogenic origin. Stem Cell Res 12:376-86
Boyce, Alison M; Kelly, Marilyn H; Brillante, Beth A et al. (2014) A randomized, double blind, placebo-controlled trial of alendronate treatment for fibrous dysplasia of bone. J Clin Endocrinol Metab :jc20141371
Phillips, Matthew D; Kuznetsov, Sergei A; Cherman, Natasha et al. (2014) Directed differentiation of human induced pluripotent stem cells toward bone and cartilage: in vitro versus in vivo assays. Stem Cells Transl Med 3:867-78
Robey, Pamela Gehron; Kuznetsov, Sergei A; Riminucci, Mara et al. (2014) Bone marrow stromal cell assays: in vitro and in vivo. Methods Mol Biol 1130:279-93
Chen, Kevin G; Hamilton, Rebecca S; Robey, Pamela G et al. (2014) Alternative cultures for human pluripotent stem cell production, maintenance, and genetic analysis. J Vis Exp :
Chen, Kevin G; Mallon, Barbara S; McKay, Ronald D G et al. (2014) Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell 14:13-26
Zhao, W; Petit, E; Gafni, R I et al. (2013) Mutations in NOTCH2 in patients with Hajdu-Cheney syndrome. Osteoporos Int 24:2275-81
Bianco, Paolo; Barker, Roger; Brustle, Oliver et al. (2013) Regulation of stem cell therapies under attack in Europe: for whom the bell tolls. EMBO J 32:1489-95

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