Utilization of cord blood-expanded stem cells for megakaryocyte and platelet production

 描述(申请人提供)：血小板输注是治疗血小板减少症最有效的方法。由于对志愿捐献者的依赖、保质期短、感染风险和同种异体免疫，对血小板输注日益增长的需求往往受到血小板(PTL)供应短缺的限制。因此，创建一种有效的、不依赖捐赠者的PTLS来源是至关重要的，它可以补充--甚至可能取代--当前依赖捐赠者的输血系统。造血干细胞扩增的最新进展加速了对用于输血的不同来源血小板的寻找，这成为一种有希望的体外生成血小板的来源。然而，在体外创造具有临床意义的血小板产品一直受到技术和数量限制的阻碍。体外培养的巨核细胞(MK)所能产生的PTLS数量较少，难以概括培养过程中人体生理性血小板生成的复杂性，是形成适合输血的PTLS的主要障碍。本申请中提出的研究旨在开发一种替代方法，通过创造一种MK细胞产品，在输注到患者体内后在体内产生血小板。创建这样的 该产品不仅将绕过与体外生产PTL相关的技术障碍和成本，而且还将为开发针对血小板减少症患者的基于细胞的疗法提供基础，并可能阻止同种异体免疫。此外，这项研究产生的单核细胞将进入制造阶段，以创造第一个用于研究的CD41+单核细胞的商业来源。MK的商业供应将极大地加快人类血栓形成研究的步伐。这一目标将通过实施新的培养系统方案来实现，其中包括MK祖细胞/前体、未成熟MK和成熟MK的MK细胞产品将从经过验证的脐带血CD34+造血干细胞来源中产生，该来源通过丙戊酸治疗表观遗传再编程的方式扩增。这些拟议的可行性研究旨在(1)表征所产生的MK的表型和功能，(2)在免疫缺陷小鼠模型的移植试验中确定MK的体外和体内效力，以及(3)测试MK在低温保存和储存后保持存活和功能的能力。这些研究将导致开发一个开发商业MK细胞产品的平台，用于临床治疗血小板减少症和实验室研究。

 DESCRIPTION (provided by applicant): Platelet transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for platelet transfusions is often limited by shortage in the platelets (PTL) supply due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. It is therefore critical to create an efficient, donor-independent source of PTLs that could complement-and possibly replace-the current donor- dependent transfusion system. The search for a different source of platelets for transfusion was accelerated by the recent advances in hematopoietic stem cell expansion which became a promising source for generating platelets ex vivo. However, creating a clinically relevant platelet product ex vivo has been hampered by technological and quantitative limitations. The major barrier in generating PTLs suitable for transfusion is the low number of PTLs that can be produced by cultured megakaryocytes (MK) ex vivo and the difficulties in recapitulating the complexity of physiological human thrombopoiesis in culture. The studies proposed in this application are designed to develop an alternative approach by creating a MK cell product that can generate platelets in vivo after its transfusion into patients. Creating such product will not only circumvent the technological hurdles and costs associated with ex vivo PTL production but also provide the foundation for developing a cell-based therapy for thrombocytopenic patients and possibly preclude alloimmunization. In addition, MKs generated from this study will be advanced to a manufacturing phase to create the first commercially available source of CD41+ MKs for use in research. A commercial supply of MKs would dramatically accelerate the pace of research in human thrombopoiesis. This goal will be accomplished by implementing a new culture system protocol in which a MK cell product comprising MK progenitors/precursors, immature MKs, and mature MKs, will be generated from a validated source of cord blood-derived CD34+ hematopoietic stem cells expanded by means of epigenetic reprograming by treatment with valproic acid. These proposed feasibility studies are designed to (1) characterize the resultant MKs phenotypically and functionally, (2) determine the MKs' potency ex vivo and in vivo in transplantation assays in an immunodeficient mouse model, and (3) test the MKs' ability to remain viable and functional following cryopreservation and storage. These studies will lead to development of a platform for developing a commercial MK cell product for clinical use to treat thrombocytopenia and for laboratory research.