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（HSC）在临床移植中被常规使用，但缺乏合适的供体细胞来源仍然是剥夺许多患者这种潜在治愈性治疗的主要限制因素。主要研究者的最终目标是从胚胎干细胞（ESC）中产生HSC，作为适合临床移植的新细胞来源。我们提出了一个深入的研究计划，研究恒河猴胚胎干细胞造血的发育生物学，并建立恒河猴模型作为相关的临床前大动物模型，用于评估胚胎干细胞衍生的造血干细胞的有效性和安全性。使用诸如与鼠骨髓来源的间充质基质细胞（MSC）共培养的方法，非人灵长类动物和人ESC已经分化成原始造血细胞，如通过替代体外测定所定义的。然而，这些衍生的细胞已被证明在移植到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重现这些过程，从而促进恒河猴ESCs有效分化为能够在体内植入和长期造血重建的HSC。在目标-1中，我们将从恒河猴胚胎干细胞中产生MSC细胞，并将在共培养实验中测试其作为造血支持微环境的潜力，以从恒河猴胚胎干细胞中产生HSC。在目的-2中，我们将验证我们的假设，即通过基因转移或外源性递送HOXB 4蛋白，在恒河猴ESCs中过表达HOXB 4（一种在胚胎造血中起关键作用的转录因子）可以提高其分化为HSC的效率。在目的-3中，我们将评估源自恒河猴ESC的造血细胞单独或当与恒河猴ESC衍生的MSC共移植到NOD/SCID小鼠中时的体内植入潜力，因为我们假设恒河猴ESC衍生的MSC的共移植将潜在地提供适当的生态位，从而增强恒河猴ESC衍生的HSC的体内植入潜力。这些研究的结果将有助于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。胚胎干细胞具有产生无限数量的用于人类治疗应用的不同分化细胞（包括造血干细胞）的潜力。我们建议开发一种恒河猴移植模型，以研究这些细胞的有效性和安全性，因为在将其转移到人类临床应用之前，应在适当的临床前动物模型中进行仔细测试。 (End摘要）

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)