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

在上一财政年度，骨骼生物科报告了所有三个领域的活动。 1)干细胞的生物活性； &#8232； 人类多能干细胞有两个潜在的有吸引力的应用：基于细胞替代的治疗和药物发现。两者都需要高效地产生大量临床级别的干细胞，这些干细胞不会发生有害的基因组变化。目前使用的集落培养方法往往导致细胞产量低，不可避免地导致细胞群体的异质性，以及大量的染色体异常。我们比较了hPSC克隆/拟胚体和早期胚胎之间的结构关系，以期根据发育生物学的见解来优化现有的培养方法。我们确定了核心信号通路(包括那些由转化生长因子-β启动的通路)，这些通路是hPSCs多个上皮向间充质转化(EMT)、细胞异质性和染色体不稳定性的基础。我们还分析了非集落单层和悬浮培养等新兴方法，这些方法为hPSC的扩增和分化提供了可供选择的生长模型。基于细胞相互作用和信号通路的影响，我们提出了生产临床级hPSCs、干细胞前体和微型有机化合物的概念、策略和解决方案，这些都是未来临床应用所需的关键步骤。 2)疾病中的BMSCs/SSCs； 为了保持我们对骨骼疾病(特别是与NIDCR CSDB骨骼临床研究单位的Michael T.Collins博士合作的骨纤维异常增殖症)的兴趣，我们一直在继续研究这种疾病的可能治疗方法。纤维发育不良(FD)是一种罕见的骨骼疾病，导致畸形、骨折、功能障碍和疼痛。它是由SSCs/BMSCs功能失常引起的。双膦酸盐被认为是一种潜在的治疗方法，因为FD的病变往往以SSCs/BMSCs异常诱导的大量破骨细胞生成为特征。此外，在另一种脆性骨疾病中，成骨不完全，双膦酸盐被发现可以改善骨体积和密度。为了确定双膦酸阿伦磷酸酯的疗效，进行了为期两年的随机、双盲、安慰剂对照试验。多发性骨质疏松症的受试者被随机分组。阿仑磷酸钠用药周期为24个月，周期为6个月(开始6个月，关闭6个月)。主要终点是骨转换标记物，包括血清骨钙素和NTX-端肽。在骨转换标志物方面，阿伦磷酸酯组NTX-端肽减少，但骨钙素在两组之间没有显著差异。阿仑膦酸钠组腰椎正常骨和FD预定区域的面骨密度增加，但两组之间的疼痛评分、骨骼疾病负担评分和功能参数没有显著差异。这些结果表明，阿伦磷酸钠治疗确实导致骨吸收标记物NTX-端肽的减少和aBMD的改善，但对血清骨钙素、疼痛或功能参数没有显著影响。 3)组织工程和再生医学中的干细胞 将诱导多能干细胞(IPSCs)分化为固定的骨骼祖细胞的能力可以使此类细胞无限制地自体供应用于治疗用途。因此，我们尝试使用来自两个不同组织来源(皮肤成纤维细胞和骨髓间充质干细胞)的系和分化方法，以及我们之前为人类胚胎干细胞(HESCs)的成骨分化而设计的分化方法，以及其他出版物所建议的那样，尝试从人IPSCs中创建新的成骨细胞。使用体外方法对所得到的细胞进行检测，并将结果与体内移植检测结果进行比较。我们的结果表明，几个IPSC系的衍生物在体内形成了真正的骨，但体外实验的结果并不能预测成功的细胞系和分化方法。此外，来自皮肤或骨髓间充质干细胞的IPSCs也同样能很好地形成骨骼，这表明IPSCs没有保留对其前世生活的记忆。此外，IPSC来源的细胞系之一在体内形成了可验证的软骨，这同样没有通过体外试验进行预测。未来的研究将致力于采取更具发展性的方法来获得一致代的骨形成细胞。 虽然基于IPSC的细胞疗法在再生医学方面具有巨大的潜力，但它们也可能与肿瘤形成风险有关。目前的啮齿动物模型并不是临床应用效率和安全性的最佳预测因子。因此，我们开发了一个临床相关的非人类灵长类动物模型(恒河猴)，以评估未分化和分化的IPSCs在没有免疫抑制的自体环境中的致瘤潜力和体内疗效。未分化的自体IPSCs以剂量依赖的方式形成成熟畸胎瘤。然而，肿瘤的形成伴随着炎症反应。另一方面，IPSC来源的中胚层基质样细胞在体内形成新骨，没有任何畸胎瘤形成的证据。因此，我们首次在一个与人类生理非常相似的大型动物模型中表明，未分化的自体IPSC可以形成畸胎瘤，并且IPSC衍生的祖细胞可以在体内产生功能组织。

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


 
 Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-based therapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are free from harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields, unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. We have compared the structural relationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methods based on the insights from developmental biology. We identified core signaling pathways (including those initiated by TGF-beta) that underlie multiple epithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyzed emerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth models for hPSC expansion and differentiation. Based on the influence of cellcell interactions and signaling pathways, we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and mini-organoids, which are pivotal steps needed for future clinical applications. 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 have continued to study possible treatments for this disease. Fibrous dysplasia (FD) is a rare skeletal disorder, resulting in deformity, fracture, functional impairment, and pain. It arises from mis-functioning SSCs/BMSCs. Bisphosphonates have been advocated as a potential treatment, due to the fact that FD lesions are often characterized by exuberant osteoclastogenesis, induced by the abnormal SSCs/BMSCs. Furthermore, in another brittle bone diseases, Osteogenesis imperfect, bisphosphonates were found to improve bone volume and density. To determine the efficacy of the bisphosphonate, alendronate, a two-year randomized, double-blind, placebo-controlled trial was carried out. Subjects with polyostotic FD were randomized and stratified by age. Alendronate was administered over a 24 month period in 6 month cycles (6 months on, 6 months off). Primary endpoints were bone turnover markers, including serum osteocalcin and NTX-telopeptides. With respect to bone turnover makers, there was a decline in NTX-telopeptides in the alendronate group, but no significant difference in osteocalcin between groups. The alendronate group had an increase in areal bone mineral density in normal bone at the lumbar spine, and in pre-determined regions of FD, but there were no significant differences in pain scores, skeletal disease burden scores, or functional parameters between the groups. These results showed that alendronate treatment did lead to a reduction in the bone resorption marker, NTX-telopeptides, and improvement in aBMD, but no significant effect on serum osteocalcin, pain, or functional parameters was observed. 3) Stem cells in tissue engineering and regenerative medicine The ability to differentiate induced pluripotent cells (iPSCs) into committed skeletal progenitors could allow for an unlimited autologous supply of such cells for therapeutic uses. Therefore we attempted to create novel bone-forming cells from human iPSCs using lines from two distinct tissue sources (skin fibroblasts and BMSCs), and methods of differentiation that we previously devised for osteogenic differentiation of human embryonic stem cells (hESCs), and as suggested by other publications. The resulting cells were assayed using in vitro methods, and the results compared to those obtained from in vivo transplantation assays. Our results showed that true bone was formed in vivo by derivatives of several iPSC lines, but that the successful cell lines and differentiation methodologies were not predicted by the results of the in vitro assays. In addition, bone was formed equally well from iPSCs originating from skin or BMSCs, suggesting that the iPSCs did not retain a memory of their previous life. Furthermore, one of the iPSC-derived cell lines formed verifiable cartilage in vivo, which likewise was not predicted by in vitro assays. Future studies will aim to take a more developmental approach to obtain consistent generation of bone forming cells. While iPSC-based cell therapies have a great potential for regenerative medicine, they are also potentially associated with tumorigenic risks. Current rodent models are not the optimal predictors of efficiency and safety for clinical application. Therefore, we developed a clinically relevant non-human primate model (Rhesus monkey) to assess the tumorigenic potential and in vivo efficacy of both undifferentiated and differentiated iPSCs in an autologous setting without immunosuppression. Undifferentiated autologous iPSCs formed mature teratomas in a dose-dependent manner. However, tumor formation was accompanied by an inflammatory reaction. On the other hand, iPSC-derived mesodermal stromal-like cells formed new bone in vivo without any evidence of teratoma formation. We therefore showed for the first time in a large animal model that closely resembles human physiology that undifferentiated autologous iPSCs form teratomas, and that iPSC-derived progenitor cells can give rise to a functional tissue in vivo.