The role of post-natal skeletal stem cells in health and disease

产后骨骼干细胞在健康和疾病中的作用

• 批准号: 8344110
• 负责人: PAMELA G ROBEY
• 金额: $ 146.3万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344110
• 关键词: AKT1 gene; Actins; Acute Graft Versus Host Disease; Address; Adipocytes; Adipose tissue; Adolescent; Affect; Alleles; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Area; Aspirate substance; Binding; Biological; Biological Assay; Biological Markers; Biology; Blood; Bone Marrow; Bone Marrow Stem Cell; Bone Regeneration; Bone Tissue; Brain; CD34 gene; Cardiovascular Diseases; Cartilage; Cell Communication; Cell Count; Cell Culture Techniques; Cell Line; Cell Transplantation; Cell physiology; Cells; Characteristics; Clathrin; Clinical; Clinical Protocols; Clinical Trials; Clone Cells; Code; Collaborations; Collagen; Colony-forming units; Complex; Connective Tissue; Connective Tissue Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; DNA Sequence; Data; Defect; Dental Cementum; Dental Pulp; Dentin; Development; Disease; Disintegrins; Endocytosis; Exhibits; Extracellular Matrix Proteins; FDA approved; Fibroblasts; Fibronectins; G-Protein-Coupled Receptors; GNAS gene; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Gene Expression Profile; Generations; Genes; Goals; Gold; Growth Factor; Hajdu-Cheney Syndrome; Health; Hematopoiesis; Hematopoietic stem cells; Hereditary Disease; Homeostasis; Human; Immune; In Vitro; Injury; Integrin Signaling Pathway; Integrins; Intrinsic factor; Knowledge; Lentivirus Vector; Marrow; McCune-Albright Syndrome; Mediating; Mediator of activation protein; Mesenchymal Stem Cells; Mesoderm; Metabolism; Metalloproteases; Missense Mutation; Molecular Profiling; Mosaicism; Mouse Mammary Tumor Virus; Mus; Mutation; National Human Genome Research Institute; Natural regeneration; Nature; Neuroectoderm; Oligonucleotides; Oncogenes; Organ; Osteitis Fibrosa Disseminata; Osteoclasts; Osteogenesis; Osteoporosis; Oxidative Stress; Pathway interactions; Patients; Pattern; Periodontal Ligament; Periosteal Cell; Pharmaceutical Preparations; Phenotype; Phosphorylation; Play; Population; Predisposition; Preparation; Property; Proteins; Proteus; Proteus Syndrome; Proto-Oncogene Proteins c-akt; Protocols documentation; Regenerative Medicine; Regulation; Role; Sampling; Signal Pathway; Signal Transduction; Site; Skeleton; Skin; Source; Stem cells; Stromal Cells; Structure; Study Section; Surrogate Markers; Techniques; Testing; Tissue Engineering; Tissues; Transforming Growth Factor-Beta Induced Protein IGH3; Translations; Transplant Recipients; Transplantation; United States National Institutes of Health; base; beta catenin; biological adaptation to stress; bone; bone cell; bone loss; bone resorbing activity; c-Myc Staining Method; cell motility; cell type; clinical effect; connective tissue growth factor; craniofacial; cytokine; human embryonic stem cell; improved; in vivo; induced pluripotent stem cell; integrin beta5; integrin-linked kinase; intercellular communication; interest; meetings; mutant; osteogenic; peripheral blood; preclinical study; programs; receptor; reconstruction; rho; skeletal; skeletal abnormality; substantia spongiosa; tumor

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.

在今年，研究在多个领域完成。 干细胞生物学活性 骨髓基质细胞（BMSC）不仅用于骨再生，还用于免疫调节和抗炎应用。 但是，尚未完全理解负责这些影响的属性。 与Drs合作。 Ren，Sabatino和Stroncek在NIH临床中心，人类BMSC的特征是确定可能导致其临床效果和生物标志物评估其质量的因素。 将来自七个健康受试者的骨髓抽吸物制备的早期通过BMSC与三个人类胚胎干细胞（HESC）样品CD34+细胞进行了比较，CD34+细胞高度富集了来自三个健康受试者的造血干细胞（HSC）和三个成纤维细胞系。 用超过35,000个探针用寡核苷酸表达微阵列分析细胞。 BMSC基因表达特征与HSC，HESC和成纤维细胞的特征不同。 BMSC中上调的基因与细胞运动，细胞对细胞信号传导和相互作用以及增殖有关。 上调的基因属于整联蛋白信号传导，整联蛋白连接激酶（ILK）信号传导，NF-E2相关因子（NFR2）介导的氧化应激反应，通过Rho调节基于肌动蛋白的运动性，肌动蛋白细胞骨架骨骼信号，洞穴 - cave层 - 核介导的内细胞和粘液内细胞的氧化应激反应，基于肌动蛋白的运动性， (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分解蛋白和金属蛋白酶酶（ADAM12）。 目前的研究集中于进行BMSC的不同克隆（起源于单个集菌形成单位成纤维细胞，CFU-F）的分子分析，以确定哪些可以区分多功能克隆与仅能够制造骨骼或仅纤维组织的克隆的克隆。 基于我们对如何通过体外扩张和体内移植诱导BMSC形成骨骼的广泛知识，我们开发了通过人类胚胎干细胞（HESC）生成骨骼的技术，目前正在将这些技术应用于诱导的多能干细胞（IPSC）中，这些技术使用皮肤纤维纤维效应多个bmmsscss，并将其应用于多发性bmmsscss，并将其应用于多发性bmmsscsss ront。 Oct4，Sox2，KLF4和CMYC的因素。 初步研究表明，来自皮肤和BMSC衍生的IPSC的IPSC能够在体内移植中形成骨头的小焦点。 当前的研究旨在提高成骨分化的质量和数量。 疾病中的BMSC/SSC 我们对体细胞镶嵌性疾病，骨骼（FD）的纤维发育不良（FD）的长期兴趣是由GNA基因的错义突变引起的，该突变代码为G蛋白GS-Alpha代码，导致CAMP过度生产。 由于途径之间的潜在跨态，我们仍在继续研究突变体GS-α活性的下游效应，除了cAMP诱导的PKA激活之外，除了cAMP诱导的PKA激活外，我们还将继续研究突变体GS-Alpha活性的下游效应。 特别是，我们专注于Wnt途径，Wnt途径是由Wnt与毛躁的共受体的结合介导的，后者是G蛋白偶联受体。 这些研究可能有助于解释FD组织中发现的某些表型，而这些表型并非仅根据cAMP过度生产而被解释。 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综合征的特征是在马赛克模式中过度生长，包括皮肤，骨骼，脑和其他组织。 Proteus衍生细胞的体内移植概括了患病骨的性质，并向我们的合作者提供了细胞，以寻求寻找基因缺陷。 他们对组织和细胞的DNA进行了大规模平行的测序，并将影响与未受影响的组织和细胞进行了比较。 29例Proteus综合征患者中有26例在癌基因AKT1中具有体细胞激活突变。 来自Proteus综合征患者的组织和细胞系包含各种水平的突变等位基因，从1％到50％不等。 最重要的是，一对从相同的起始培养物中建立并因其突变状态而不同的单细胞克隆具有Akt1的差异化磷酸化。 这些数据验证了Proteus综合征中体细胞镶嵌的假设，并证明AKT/PI3K途径激活导致组织过度生长和肿瘤敏感性的特征性临床发现。 BMSC/SSC在组织工程和再生医学中 在过去的几十年中，组织工程领域，专门用于骨骼再生。 作为示例，许多细胞源似乎可用，包括源自骨膜组织，小梁骨，牙髓和牙周韧带的细胞，以及来自非脂肪衍生细胞等非硬化组织的MSC（间充质干细胞）。 我们已经通过体内移植测试了MSC的许多骨头和非骨髓来源，这是评估分化能力的黄金标准。 通过严格的标准，包括体内移植，仅骨膜细胞，小梁骨细胞和BMSC能够形成骨骼，并且仅BMSC不仅能够形成骨骼，而且能够支撑造血。 尽管牙髓细胞和牙周韧带细胞能够使牙本质和牙髓样复合物和牙骨质和牙周韧带样结构分别产生，但来自其他组织的MSC很少形成骨骼或任何其他生理矿化的组织，并且从未支持造血。 支持造血的基质形成的重要性是骨骼干细胞（SSC）是该专业基质的一部分。 因此，造血的支持是SSC存在的替代标记。 因此，迄今为止，BMSC代表了用于骨再生的主要来源。 但是，不仅符合FDA要求的离体扩展设施的发展，而且通过几种关键测定评估的细胞的生物学特性也是临床翻译至关重要的。 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. 目前正在开发其他协议和IND。