The rhesus macaque as a preclinical model for induced pluripotent stem cells

恒河猴作为诱导多能干细胞的临床前模型

• 批准号: 8344862
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 35.04万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/8344862
• 关键词: Ablation; Achievement; Adult; Alkaline Phosphatase; Allogenic; Antibodies; Autologous; Binding; Biological Assay; CD34 gene; Cell Therapy; Cells; Characteristics; Clinical; Clinical Trials; Codon Nucleotides; Collaborations; Derivation procedure; Development; Ectoderm Cell; Ectopic Expression; Embryo; Embryonic Development; Endoderm Cell; Excision; FK506; Fibroblasts; Ganciclovir; Gene Expression; Generations; Genes; Genomics; Goals; Health; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Homing; Human; Immunodeficient Mouse; Implant; In Vitro; Inbreeding; Insertional Activations; Insertional Mutagenesis; Laboratory Procedures; Laboratory mice; Lentivirus Vector; Location; Longevity; Macaca; Macaca mulatta; Marrow; Mediating; Mesenchymal; Mesoderm Cell; Methods; Modeling; Morphology; Mouse Strains; Mus; National Institute of Dental and Craniofacial Research; Oral Administration; Organoids; Paper; Pharmaceutical Preparations; Physiological; Play; Pre-Clinical Model; Preparation; Prodrugs; Proliferating; Property; Proteins; Proto-Oncogenes; Protocols documentation; Publishing; Reagent; Regenerative Medicine; Relative (related person); Residual state; Resources; Retrieval; Retroviral Vector; Risk; Rodent Model; Role; Safety; Site; Skin; Somatic Cell; Specific qualifier value; Staging; Stem cells; Stromal Cells; Structure of beta Cell of islet; Suicide; System; T-Lymphocyte; TK Gene; Tacrolimus Binding Proteins; Teratoma; Testing; Thymidine Kinase; Tissues; Transcription factor genes; Transplantation; United States National Institutes of Health; Xenograft Model; Xenograft procedure; base; blastocyst; bone; caspase-9; cell killing; cell type; cross reactivity; cytokine; design; embryonic stem cell; gene therapy; graft vs host disease; human embryonic stem cell; immunogenic; immunogenicity; improved; in vivo; induced pluripotent stem cell; model development; neutrophil; nonhuman primate; novel; osteogenic; pluripotency; prevent; programs; scale up; small molecule; suicide gene; telomere; tissue regeneration; tool; transcription factor; tumor; vector

The re-programming of post-natal somatic cells to induced pluripotent stem cells (iPSCs) via ectopic expression of stem cell specifying transcription factors has many exciting potential applications for improving human health. iPSCs were initially developed in the murine model, and shown to have the potential to contribute to all tissues via blastocyst complementation assays. Just a few years later, human iPS cells were created using a similar panel of transcription factors, and demonstrated to form teratomas in immunodeficient mice and share functional and gene expression characteristics with human embryonic stem cells. However, there are numerous hurdles to moving iPSC forward into clinical regenerative medicine applications. First and most important are safety concerns. Both murine and human iPSCs were initially derived by introducing the required transcription factor genes into target cells using integrating vectors, associated with risks due to ongoing or reactivated ectopic expression of the transcription factors, or insertional activation of genomic proto-oncogenes. Novel non-integrating vectors or protein transfer systems have begun to surmount this problem. However, much more serious concerns relate to the consequences of administering primitive pluripotent cells that may have the potential to form tumors, if differentiation is incomplete or inefficient. Second, there are significant challenges to the efficient differentiation of iPSCs into functional adult tissues. Protocols for differentiation of iPSCs towards even well-characterized hematopoietic stem cells are inefficient, inconsistent and result in aberrant or embryonic hematopoiesis. Design of methods for direct delivery or facilitation of homing of iPSCs or their progeny to appropriate locations in the body will also be a major challenge. While murine models are invaluable tools, it will be critical to develop more relevant models for clinical development of iPSCs. Murine and human embryonic stem cells behave quite differently in culture, require different cytokines and handling, and may be derived from different stage of embryonic development. Generation of murine iPSCs appears to be at least an order of magnitude more efficient that generation of human iPSCs. Telomeres in inbred laboratory mice are significantly longer than human telomeres, and may impact on the relative ease of immortalization of murine versus human cells and thus oncogenicity. Human iPSCs can be implanted in immunodeficient mouse strains and form teratomas, but the next steps in development, requiring functional differentiation and appropriate delivery or homing, may be impossible to model in xenografts. Scale-up of laboratory procedures developed in mice to human therapies would also be very difficult to develop solely using murine-murine or human-murine xenograft models. The rhesus macaque non-human primate (NHP) model will be a valuable resource to clear hurdles preventing clinical development. The close physiologic and genomic relationship between humans and NHPs results in cross-reactivity for most cytokines, antibodies and other reagents. Teratoma formation and other safety issues can be directly assessed utilizing autologous rhesus iPSCs. Differentiation, homing and other parameters critical for efficacy can be modeled. Tissue damage models such as pancreatic beta cell or hematopoietic stem cell ablation are well established in macaques. Rhesus embryonic stem cells were isolated prior to human ESCs, and their properties are well-characterized. Development of rhesus iPSCs at the NIH takes advantage of our unique expertise in NHP transplantation and in the development of novel cell and gene therapies in this valuable model. Our plans also focuses on the development of a suicide gene strategy to increase safety of utilization of iPSCs for tissue regeneration. There is a significant risk that residual pluripotent cells remaining following direct differentiation of iPSCs could form tumors in vivo. If integrating vectors are used to generate iPSCs, tumors could result from vector-related insertional mutagenesis or re-activation of reprogramming factors. Even if differentiation is complete and successful, iPSC-derived progeny might localize or proliferate inappropriately. In all these scenarios, the ability to ablate iPSCs in vivo would be desirable. Several promising suicide gene strategies have been developed over the past decade, allowing efficient killing of cells carrying the suicide gene vector via administration of a non-toxic drug. Several clinical trials utilizing allogeneic T cells carrying the herpes thymidine kinase (tk) suicide gene have been performed. Ganciclovir, a pro-drug only toxic to cells expressing herpes tk, was shown to ablate alloreactive T cells and successfully treat graft-versus-host disease. To avoid the immunogenicity of herpes tk, another promising suicide system utilizes human caspase 9 fused to the FK506 binding domain, allowing inducible dimerizer and caspase 9 activation following administration of an oral small molecule dimerizer. These suicide gene strategies hold great promise for iPSC safety, but need further clinical development in a relevant model such as the rhesus macaque. Thsi project began this year, and we have already published a paper taking advantage of our expertise in vector integration site retrieval and analysis to demonstrate that in human iPSCs, vector integration sites do not appear to play a role in promoting successful reprogramming of iPSCs. This is reassuring for at least preclinical and model development, allowing continued use of lentiviral vectors for reprogramming, given their much greater efficiency compared to non-integrating vectors. We have now optimized conditions to derive rhesus iPSCs, and have successfully shown that rhesus iPSCs can be created from rhesus marrow mesenchymal cells or from skin fibroblasts, utilizing either retroviral or lentiviral vectors. These clones are pluripotent as assayed in a murine teratoma assay, express all pluripotency markers, and can be differentiated to endodermal, mesodermal and ectodermal cell types. The conditions utilized for murine and human ESCs and iPSCs were not successful using rhesus cells, and we have developed new conditons, based on the optimal conditions for growing rhesus ESCs. Our rhesus iPSCs express alkaline phosphatase, shut off the reprogramming vectors and morphologically resemble rhesus ESCs. For in vivo studies in autologous rhesus, excision of the potentially-immunogenic reprogramming factors is likely required, so we have now created rhesus iPSCs with an exicisable reprogramming cassette, and shown all retained functions following cre-mediated excision. These cells are about to be tested in an autologous rhesus teratoma model. We have developed an in vivo bone organoid model in the rhesus, in collaboration with Pam Robey's group in NIDCR. We are currently testing the model with rhesus MSCs, and plan to move into rhesus iPSCs in the next several months. We have also begun to differentiate rhesus iPSCs to mature neutrophils, and will test their lifespan and function in vivo. Finally, we have introduced suicide genes into rhesus iPSCs in preparation for beginning in vivo suicide ablation studies.

通过异位表达干细胞指定转录因子，将产后体细胞重新编程以诱导多能干细胞（IPSC），这在改善人类健康方面具有许多令人兴奋的潜在应用。 IPSC最初是在鼠模型中开发的，并有可能通过胚泡互补测定法对所有组织做出贡献。几年后，使用类似的转录因子创建了人IPS细胞，并证明可以在免疫缺陷的小鼠中形成畸胎瘤，并与人类胚胎干细胞共享功能和基因表达特征。 但是，将IPSC转向临床再生医学应用有许多障碍。 首先也是最重要的是安全问题。鼠和人IPSC最初是通过使用整合载体引入靶细胞中所需的转录因子基因的，这与由于转录因子的持续或重新激活的异位表达或基因组原始原始癌基因的插入激活而引起的风险相关。新型的非整合载体或蛋白质转移系统已经开始解决这个问题。但是，如果分化不完全或效率低下，则更严重的关注与给予可能形成肿瘤的原始多能细胞的后果有关。其次，IPSC有效分化为功能性成年组织面临重大挑战。 IPSC将IPSC分化为即使是特征良好的造血干细胞的方案效率低下，不一致且导致异常或胚胎造血。设计直接交付或促进IPSC或其后代到体内适当位置的方法的设计也将是一个重大挑战。 尽管鼠模型是宝贵的工具，但为IPSC的临床开发开发更相关的模型至关重要。鼠和人类胚胎干细胞在培养中的表现截然不同，需要不同的细胞因子和处理，并且可以源自胚胎发育的不同阶段。鼠的IPSC产生至少是人类IPSC的产生的数量级。 近交实验室小鼠中的端粒明显比人端粒长大，并且可能会影响鼠与人类细胞的永生化相对易于不变，从而影响了致癌性。人IPSC可以植入免疫缺陷的小鼠菌株并形成畸胎瘤，但是在异种移植物中可能不可能建模开发的下一步，需要功能分化和适当的递送或归因。仅使用鼠类摩rine或人类异种移植模型，在小鼠中开发的针对人类疗法的实验室程序的扩展也将非常困难。 恒河猕猴非人类灵长类动物（NHP）模型将是清除防止临床发展的障碍的宝贵资源。 人与NHP之间的密切生理和基因组关系导致大多数细胞因子，抗体和其他试剂的交叉反应性。畸胎瘤和其他安全问题可以通过自体恒河体IPSC直接评估。 可以对功效至关重要的分化，归巢和其他参数进行建模。猕猴中良好地确定了组织损伤模型，例如胰腺β细胞或造血干细胞消融。 在人类ESC之前，将恒河猴的胚胎干细胞分离出来，它们的特性得到了充分的特征。 在NIH上开发恒河所IPSC，利用了我们在NHP移植方面的独特专业知识，以及在这个有价值的模型中开发新细胞和基因疗法。 我们的计划还着重于开发自杀基因策略，以提高IPSC用于组织再生的安全性。 在直接分化IPSC后，残留的多能细胞可能在体内形成肿瘤。 如果使用载体用于生成IPSC，则肿瘤可能是由载体相关的插入诱变或重新编程因子的重新激活而引起的。 即使差异化是完整且成功的，IPSC衍生的后代也可能不适当地定位或扩散。在所有这些情况下，都需要在体内消融IPSC的能力。在过去的十年中，已经开发了几种有希望的自杀基因策略，从而有效地杀死了携带自杀基因载体的细胞，该细胞通过施用无毒药物。已经进行了携带疱疹胸苷激酶（TK）自杀基因的同种异体T细胞的几项临床试验。 Ganciclovir是一种仅对表达疱疹TK细胞的毒性毒性的毒物，显示用于消融同种异体T细胞，并成功地治疗了移植物抗宿主病。为了避免疱疹TK的免疫原性，另一种有希望的自杀系统利用了与FK506结合结构域融合的人caspase 9，从而使诱导型二聚体和caspase 9激活后，在口服小分子二聚体后进行了激活。这些自杀基因策略对IPSC安全具有很大的希望，但是在恒河猴等相关模型中需要进一步的临床发展。 THSI项目始于今年，我们已经发表了一篇论文，利用了我们在向量集成站点的检索和分析方面的专业知识，以证明在人IPSC中，向量集成站点似乎在促进IPSC的成功重新编程中似乎并不发挥作用。对于至少临床前和模型开发而言，这令人放心，鉴于与非整合向量相比，它们的效率更高，因此可以继续使用慢病毒载体进行重编程。现在，我们有优化的条件来推导恒河猴IPSC，并成功地表明，可以利用逆转录病毒或慢病毒载体来从恒河骨髓间充质细胞或皮肤成纤维细胞中创建恒河猴IPSC。 这些克隆是在鼠畸胎瘤测定中进行的，表达所有多能标记物，并且可以分化为内胚层，中胚层和外皮细胞类型。使用恒河猴的鼠类ESC和IPSC所使用的条件并不成功，并且我们基于增长的恒河猴ESC的最佳条件开发了新的孔子。我们的恒河猴表达碱性磷酸酶，关闭重编程载体，形态学上类似于恒河猴。对于体内研究，可能需要切除潜在的免疫原性重编程因子，因此我们现在创建了带有可加入的重编程盒的恒河体IPSC，并且在CRE介导的切除后显示了所有保留的功能。这些细胞将在自体恒河类畸胎瘤模型中进行测试。我们已经与NIDCR的Pam Robey小组合作，在恒河猴中开发了一个体内骨骼器官模型。我们目前正在使用恒河所MSC测试该模型，并计划在接下来的几个月中搬入恒河部IPSC。我们还开始将恒河猴分化为成熟的中性粒细胞，并将在体内测试其寿命和功能。 最后，我们将自杀基因引入恒河猴IPSC，以准备开始体内自杀研究。