During this year, studies were completed in several areas. Biological activity of stem cells Bone marrow stromal cells (BMSC) are being used not only for bone regeneration, but also for immunomodulatory and anti-inflammatory applications. However, the properties responsible for these effects are not completely understood. In collaboration with Drs. Ren, Sabatino and Stroncek in the NIH Clinical Center, human BMSC were characterized to identify factors that might be responsible for their clinical effects and biomarkers for assessing their quality. Early passage BMSCs prepared from marrow aspirates of seven healthy subjects were compared with three human embryonic stem cell (hESC) samples, CD34+ cells, which are highly enriched in hematopoietic stem cells (HSCs) from three healthy subjects, and three fibroblast cell lines. The cells were analyzed with oligonucleotide expression microarrays with more than 35,000 probes. BMSC gene expression signatures differed from those of HSCs, hESC and fibroblasts. Genes upregulated in BMSCs were involved with cell movement, cell-to-cell signaling and interaction, and proliferation. The upregulated genes belonged to pathways for integrin signaling, integrin-linked kinase (ILK) signaling, NF-E2-related factor (NFR2)-mediated oxidative stress response, regulation of actin-based motility by Rho, actin cytoskeletal signaling, caveolar-mediated endocytosis, clathrin-mediated endocytosis and Wingless-type MMTV integration site (Wnt/ beta catenin signaling. Among the most highly upregulated genes were structural extracellular matrix (ECM) proteins (alpha5 and beta5 integrin chains, fibronectin and collagens type IIIA1 and type VA1), and functional EMC proteins e.g.;connective tissue growth factor (CTGF), transforming growth factor beta-induced protein (TGFBI) and a disintegrin and metalloproteinase (ADAM12). Global analysis of human BMSC suggests that they are mobile, metabolically active, proliferative and interactive cells that make use of integrins and integrin signaling. They produce abundant ECM proteins that may contribute to their clinical immune modulatory and anti-inflammatory effects. Current studies are focused on performing 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 from clones that are only able to make bone, or only fibrous tissue. 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. 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, Gs-alpha, that leads to overproduction of cAMP. We are continuing to study the downstream effects of mutant Gs-alpha 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. These studies may help to explain some of the phenotypes found in FD tissue that are not explained solely on the basis of overproduction of cAMP. In addition, we established BMSC cultures and created in vivo transplants from patients with a variety of diseases, including several patients with idiopathic juvenile osteoporosis (as part of the Undiagnosed Disease Program), Hajdu-Cheney syndrome (also in the Undiagnosed Disease Program), and Proteus syndrome (in collaboration with Lindhurst and Biesecker, NHGRI). Proteus syndrome is characterized by overgrowth in a mosaic pattern including skin, bone, brain, and other tissues. The in vivo transplants of Proteus-derived cells recapitulated the nature of the diseased bone, and cells were provided to our collaborators for their quest to find gene defects. They performed massively parallel sequencing of DNA from tissues and cells, comparing affected to unaffected tissues and cells. Twenty six of 29 patients with Proteus syndrome had a somatic activating mutation in the oncogene, AKT1. Tissues and cell lines from patients with Proteus syndrome contained various levels of mutant alleles that varied from 1% to 50%. Most importantly, a pair of single cell clones established from the same starting culture and differing by their mutation status had differential phosphorylation of AKT1. These data validate the hypothesis of somatic mosaicism in Proteus syndrome, and demonstrate that AKT/PI3K pathway activation causes the characteristic clinical findings of tissue overgrowth and tumor susceptibility. BMSCs/SSCs in tissue engineering and regenerative medicine The field of tissue engineering, specifically for bone regeneration, has made great strides in the past several decades. A number of cell sources would appear to be available, including cells derived from periosteal tissue, trabecular bone, dental pulp and periodontal ligament, and MSCs (mesenchymal stem cells) from non-hard tissues such as adipose-derived cells, as an example. We have tested many of the bone and non-bone marrow sources of MSCs by in vivo transplantation, which is the gold standard by which to evaluate differentiation capacity. By rigorous criteria, including in vivo transplantation, only periosteal cells, trabecular bone cells and BMSCs were capable of forming bone, and only BMSCs were capable of forming not only bone, but of supporting hematopoiesis. While dental pulp cells and periodontal ligament cells were able to make dentin and a pulp-like complex, and cementum and a periodontal ligament-like structure, respectively, MSCs from other tissues rarely formed bone or any other physiologically mineralized tissue, and never supported hematopoiesis. The importance of formation of stroma that supports hematopoiesis is that skeletal stem cells (SSCs) are a part of this specialized stroma. Thus, support of hematopoiesis serves as a surrogate marker for the presence of SSCs. Consequently, to date, BMSCs represent the primary source for use in bone regeneration. However, the development of ex vivo expansion facilities that not only meet FDA requirements, but also maintain the biological properties of the cells as assessed by several critical assays are essential for clinical translation. Activities with the Cell Processing Section (Ren, Sabatino, Stroncek, DTM, NIH CC) as a part of the NIH Bone Marrow Stromal Cell Transplantation Center, have led to the development of an FDA-approved Drug Master File, and to submission of several clinical protocols and INDs for the use of BMSCs in the treatment of acute graft versus host disease, and cardiovascular disease. Other protocols and INDs are currently under development.

Project Start
Project End
Budget Start
Budget End
Support Year
28
Fiscal Year
2011
Total Cost
$1,462,980
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
Zip Code
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
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
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