Fetal liver stromal cells for hematopoietic stem cells

胎儿肝脏基质细胞用于造血干细胞

• 批准号: 8034855
• 负责人: Song Chou
• 金额: $ 5.68万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8034855
• 关键词: Adult; Angiopoietins; Apoptosis; B-Lymphocytes; Binding; Biological; Biological Assay; Blood; Blood Cells; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; CD3 Antigens; CD44 gene; Cell Count; Cell Death; Cell Surface Proteins; Cells; Clinical; Coculture Techniques; Confocal Microscopy; Culture Media; Data; Disease; Embryo; Fellowship; Fetal Liver; Fibroblast Growth Factor 1; Frozen Sections; Future; Goals; Growth Factor; Hematopoietic stem cells; Hepatocyte; Hormones; Human; IL2RA gene; Immunofluorescence Immunologic; Immunologic Deficiency Syndromes; Inborn Errors of Metabolism; Inherited; Insulin-Like Growth Factor II; Integral Membrane Protein; Lead; Literature; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Mus; PTPRC gene; Placenta; Population; Protein Binding; Proteins; Proto-Oncogene Protein c-kit; SLAM protein; Serum; Signal Transduction; Sorting - Cell Movement; Staining method; Stains; Stem Cell Factor; Stem cells; Stress; Stromal Cells; Supporting Cell; Surface; System; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Thrombopoietin; Umbilical Cord Blood; base; cell type; cytokine; gene therapy; improved; in vivo; leukemia; novel; research study; self-renewal; spatial relationship; success

DESCRIPTION (provided by applicant): Hematopoietic stem cells (HSCs) undergo dramatic expansion in the fetal liver. The long term goal of this proposal is to characterize the stromal cells for HSC expansion in fetal liver and identify the growth factors they secrete for expanding HSC numbers. This study will not only answer important basic biological questions, but it can lead to major improvements in the clinical use of HSCs in bone marrow transplantation for treatment of leukemia and other cancers. HSCs are also a promising cell target for developing gene therapies for treating a broad variety of inherited immunodeficiency syndromes and inborn errors of metabolism. Previous studies and our preliminary data indicate that certain subpopulations of fetal liver E15.5 CD3+ and B220+ cells express high levels of four growth factors: IGF2, AngptI3, SCF and TPO. When added together, these four growth factors are able to significantly expand both adult bone marrow and fetal liver HSCs ex vivo. The cells that express all of these four HSC expansion factors are potential HSC stromal cells since SCF is a transmembrane protein and is proposed to mediate one interaction between HSCs and their stromal cells by binding to its receptor c-Kit, which is expressed by all HSCs. In this proposal, FL B220+ and CD3+ cells will be further fractionated by FACS sorting and mRNAs for many growth factors quantitated by qPCR to determine their ability to produce these four key HSC expansion factors and to discover additional HSC expansion factors they secrete. Competitive repopulating experiment and limiting dilution assay will be used to determine that ability of characterized B220* and CDS* subpopulations to support ex vivo HSC expansion in medium without adding any growth factor. (Specific Aim 1) Parallel studies will also be conducted to determine whether similar cells exist in adult bone marrow and placenta and, if so, whether these cells also express the key HSC expansion factors and can support HSC self- renewal ex vivo. Bone marrow HSCs are normally quiescent; therefore they will also be studied under stressed conditions where they expand in numbers. (Specific Aim2) Finally immunofluorescence staining experiments will be carried out to determine whether characterized CDS* and B220+ subpopulations are located in close proximity to HSCs in fetal liver and thus are bona fide stromal cells for HSCs. (Specific Aim 3) Relevance: The goal of this study is to discover the cells that can cause blood stem cells to dramatically increase in numbers and find out what proteins they use to accomplish this. In the future we can produce the same proteins to generate much more blood stem cells to improve the success rate of bone marrow transplantation for leukemia and cancer as well as develop gene therapies for many inherited diseases.

描述（由申请人提供）：造血干细胞（HSC）在胎儿肝脏中经历了巨大的扩张。该提案的长期目标是表征胎儿肝脏中HSC扩展的基质细胞，并确定其分泌的HSC数量的生长因子。这项研究不仅会回答重要的基本生物学问题，而且可以导致HSC在骨髓移植中用于治疗白血病和其他癌症的临床使用。 HSC也是开发基因疗法的有希望的细胞靶标，用于治疗各种遗传性免疫缺陷综合症和代谢的先天误差。先前的研究以及我们的初步数据表明，胎儿肝E15.5 CD3+和B220+细胞的某些亚群表达了高水平的四个生长因子：IGF2，ANGPTI3，SCF和TPO。当加在一起时，这四个生长因子能够显着扩大成年骨髓和胎儿肝HSC。由于SCF是一种跨膜蛋白，因此表达所有这四个HSC扩展因子的所有HSC扩展因子中的所有细胞都是潜在的HSC基质细胞，并提议通过与所有HSC表示的受体C-KIT结合，通过与其受体C-KIT结合来介导HSC及其基质细胞之间的一种相互作用。在此提案中，FL B220+和CD3+细胞将通过FACS分类和mRNA进一步分级，用于QPCR定量的许多生长因子，以确定它们产生这四个关键HSC扩展因子的能力，并发现它们分泌的其他HSC扩展因子。竞争性重新注入实验和限制稀释测定将用于确定表征的B220*和CDS*亚群的能力，以支持培养基中的离体HSC扩展而无需添加任何生长因子。 （特定目的1）还将进行平行研究，以确定成人骨髓和胎盘中是否存在相似细胞，如果是的，这些细胞是否也表达了关键的HSC膨胀因子，并可以支持HSC自我更新的离体。骨髓HSC通常是静止的。因此，在压力条件下，它们也将在数量扩展的情况下进行研究。 （特定AIM2）最终将进行免疫荧光染色实验，以确定表征的CDS*和B220+亚群是否位于胎儿肝脏中HSC的附近，因此HSC的真正的基质细胞是HSC的真正的基质细胞。 （特定目的3）相关性：这项研究的目的是发现可能导致血干细胞大量增加数量并找出他们用来实现这一目标的蛋白质的细胞。将来，我们可以产生相同的蛋白质来产生更多的血干细胞，以提高白血病和癌症的骨髓移植的成功率，并为许多遗传疾病开发基因疗法。