Hematopoietic potential of histocompatible embryonic stem cell lines

描述(由申请人提供)： 我的职业目标是成为干细胞生物学领域的一名独立资助的研究人员，拥有ES细胞重新编程和分化的基本机制方面的专业知识，特别是在血液方面。独立之路奖(K99/R00)将通过为指导阶段和独立阶段提供资金，极大地帮助我过渡到完全独立。在不久的将来，这个奖项将使我在乔治·Q·戴利博士的指导下，在干细胞和发育生物学以及血液学方面获得深入的专业知识。儿童医院和哈佛医学院血液科/肿瘤科的培训计划汇集了这两个机构以及哈佛干细胞研究所的资源，并为在指导阶段完成培训提供了一个出色的环境。这将极大地促进我向独立的平稳过渡。 这项提案中描述的研究旨在为造血移植疗法创造替代组织来源。所有因缺乏合适的供体来源(例如匹配的脐带血、骨髓或血液干细胞)而无法接受造血干细胞移植的患者(例如患有儿童白血病或其他遗传病的患者)都将受益。小鼠血液病的原理证明(如下所述的研究)可能允许我们扩展到其他组织和器官的移植模型，以及患者。 这项建议的具体目标是：特定目标1：利用微细胞介导的人和鼠染色体转移产生组织相容的ctES细胞(无卵母细胞；独立时相)。特定目的2：用体外和体内方法检测ctES细胞的造血潜能。 核移植(NT)ES和孤雌生殖(P)ES细胞技术(卵母细胞介导法；指导阶段)所需的人类卵母细胞的可获得性有限，这对产生定制的胚胎干细胞构成了重大障碍。因此，需要其他方法来生成类似类型的单元格。体细胞(Sc)和胚胎干细胞(ES)融合以重新编程体细胞，已经证明了用无卵母细胞的方法产生ES细胞。然而，从得到的四倍体SC-ES杂交ES细胞中去除遗传物质以形成二倍体ES细胞还没有实现。作为另一种方法(具体目标1)，我建议将含有主要组织相容性复合体(MHC)的小鼠sc-17号染色体(人sc-6号染色体)的微细胞介导的转移到ES细胞。由此产生的复制的17号ES染色体(人类6号ES染色体)将被移除，以在小鼠和人类中产生二倍体的ctES细胞。 这项研究的目标是创造组织相容的患者特异性造血干细胞，这种干细胞除了具有潜在的治疗价值外，还将为深入研究重新编程和分化提供新的工具。

DESCRIPTION (provided by applicant): My career goal is to become an independently funded researcher in the field of stem cell biology with expertise in basic mechanisms of ES cell reprogramming and differentiation, especially with respect to blood. The pathway to Independence Award (K99/R00) will greatly assist my transition to full independence by providing funding for both mentored and independent phases. In the immediate future, this award will allow me to gain in depth expertise in stem cell and developmental biology, and hematology, under the mentorship of Dr. George Q. Daley. The training program in the hematology/oncology division at Children's Hospital and Harvard Medical School brings together the resources of both institutions as well as those of the Harvard Stem Cell Institute, and provides an outstanding environment for the completion of training during the mentored phase. This will greatly facilitate my smooth transition to independence. The research described in this proposal is designed to create alternative sources of tissue for hematopoietic transplantation therapies. All patients (e.g. those with childhood leukemia or other genetic disease) unable to receive hematopoietic stem cell transplantation due to lack of suitable donor sources (e.g. matched cord blood, bone marrow, or blood stem cells) stand to benefit. The proof of principle for hematologic disease in the mouse (studies described below) may allow us to expand to transplantation models in other tissues and organs, as well as to patients. The Specific Aims of this proposal are: Specific Aims 1: Generation of histocompatible ctES cells (oocyte free; independent phase) using micro cell mediated chromosome transfer in mouse and human. Specific Aim 2: Testing of hematopoietic potential in ctES cells by in vitro and in vivo methods. The limited availability of human oocytes required for both nuclear transferred (nt) ES and parthenogenetic (p) ES cell techniques (oocyte-mediated methods; mentored phase) poses a significant obstacle to generating customized embryonic stem cells. Therefore, alternative methods to generate similar types of cells are needed. Generation of ES cells by oocyte free methods already has been demonstrated by fusion of somatic cells (sc) and embryonic stem (es) cells to reprogram the somatic cells. However, removal of the genetic material from the resultant tetraploid sc-es hybrid ES cells to make diploid ES cells has not been achieved. As an alternative approach (Specific Aim 1), I propose micro cell-mediated transfer to ES cells of mouse sc-chromosome 17 (human sc-chromosome 6), which contains the major histocompatibility complex (MHC). The resultant duplicated es-chromosome 17 (human es-chromosome 6) will then be removed to make diploid ctES cells in mouse and human. The goal of this research is to create histocompatible patient specific hematopoietic stem cells, which in addition to their potential therapeutic value will provide novel tools for in-depth study of reprogramming and differentiation.