Embryonic Stem Cell Derived HSC Transplantation in Rhesus Macaque Model

描述(由申请人提供)： 尽管成人造血干细胞(HSCs)正被常规用于临床移植，但缺乏合适的供体细胞来源仍然是许多患者无法进行这种潜在治愈治疗的主要限制因素。首席研究员的最终目标是从胚胎干细胞(ESCs)中产生HSCs，作为一种适合临床移植的新细胞来源。我们提出了一项深入的研究计划，以研究恒河猴胚胎干细胞的造血发育生物学，并建立恒河猴模型，作为相关的临床前大型动物模型，用于评估胚胎干细胞来源的造血干细胞的有效性和安全性。采用与小鼠骨髓来源的间充质基质细胞(MSC)共培养等方法，非人类灵长类和人类ESCs已在体外分化为原始造血细胞，如替代试验所定义的那样。然而，这些来源的细胞在移植到NOD/SCID免疫缺陷小鼠后，仅具有有限的体内植入潜力。我们的基本假设是，通过提供在胚胎造血中起关键作用的适当的外在和内在诱导机制，我们可以在体外有效地概括这些过程，从而促进恒河猴胚胎干细胞高效分化为能够在体内植入和长期造血重建的干细胞。在AIM-1中，我们将从恒河猴ESCs中培养出MSC细胞，并将在共培养实验中测试它们作为造血支持微环境的潜力，以便从恒河猴ESCs中产生HSCs。在AIM-2中，我们将验证我们的假设，即通过基因转移或外源传递HOXB4蛋白在恒河猴ESCs中过表达HOXB4，可以提高其向HSC分化的效率。HOXB4是一种在胚胎造血中起关键作用的转录因子。在AIM-3中，我们将评估单独或与恒河猴ESC来源的MSCs联合移植到NOD/SCID小鼠体内的造血细胞植入潜力，因为我们假设联合移植恒河猴ESC来源的MSCs可能会提供合适的利基环境，从而增强恒河猴ESC来源的HSCs在体内的植入潜力。这些拟议研究的结果将帮助我们设计一种合理的方法，将这些细胞转移到实际的恒河猴移植实验中。胚胎干细胞有可能产生无限数量的不同分化细胞，用于人类的治疗应用，包括造血干细胞。我们建议开发一种恒河猴移植模型来研究这种细胞的有效性和安全性，因为在进入人类临床应用之前，它们应该在适当的临床前动物模型中进行仔细的测试。 (摘要结束)

DESCRIPTION (provided by applicant): Although adult hematopoietic stem cells (HSCs) are being used routinely in clinical transplantation, the lack of a suitable source of donor cells remains a major limiting factor depriving many patients of this potentially curative treatment. The ultimate goal of the Principal Investigator is to generate HSCs from embryonic stem cells (ESCs) as a novel source of cells suitable for clinical transplantation. We are proposing an intensive research program to study the developmental biology of hematopoiesis from rhesus ESCs, and to establish the rhesus macaque model as a relevant pre-clinical large animal model for the evaluation of the efficacy and safety of ESCderived HSCs. Using methodologies such as co-culturing with mesenchymal stromal cells (MSC) of murine bone marrow origin, non-human primate and human ESCs have been differentiated into primitive hematopoietic cells as defined by surrogate in vitro assays. However, those derived cells have been shown to possess only limited in vivo engraftment potential upon transplantation into NOD/SCID immunodeficient mice. Our underlying hypothesis is that by providing the appropriate extrinsic and intrinsic inductive mechanisms that play critical roles in embryonic hematopoiesis, we can effectively recapitulate those processes in vitro and thus promote the efficient differentiation of rhesus ESCs into HSCs that will be capable of engraftment and long-term hematopoietic reconstitution in vivo. In aim-1 we will generate MSC cells from rhesus ESCs and will test their potential as a hematopoietic supportive microenvironment in coculture experiments for the generation of HSCs from rhesus ESCs. In aim-2 we will test our hypothesis that the over-expression of HOXB4, a transcription factor which plays a key role in embryonic hematopoiesis, in rhesus ESCs via gene transfer, or the exogenous delivery of the HOXB4 protein, can improve the efficiency of their differentiation into HSCs. In aim-3 we will assess the in vivo engraftment potential of hematopoietic cells derived from rhesus ESCs alone or when co-transplanted with rhesus ESC-derived MSCs into NOD/SCID mice as we hypothesize that the co-transplantation of rhesus ESC-derived MSCs would potentially provide an appropriate niche and thus enhance the in vivo engraftment potential of rhesus ESCderived HSCs. The results of these proposed studies would help us to design a rational approach toward moving such cells into actual rhesus transplantation experiments. Embryonic stem cells have the potential to generate an unlimited number of diverse differentiated cells for human therapeutic applications including hematopoietic stem cells. We are proposing to develop a rhesus monkey transplantation model to study the efficacy and safety of such cells as they should be carefully tested in appropriate pre-clinical animal models before being moved into human clinical applications. (End of Abstract)