KDR--A NOVEL FUNCTIONAL HEMATOPOIETIC STEM CELL MARKER

KDR--一种新型功能性造血干细胞标记物

• 批准号: 6629030
• 负责人: CESARE PESCHLE
• 金额: $ 30.01万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6629030
• 关键词: CD34 molecule; NOD mouse; SCID mouse; angiogenesis; biomarker; bone marrow; cell differentiation; cell population study; cell transplantation; embryogenesis; flow cytometry; gene expression; genetic transduction; growth factor receptors; hematopoiesis; hematopoietic stem cells; hepatocyte growth factor; human tissue; immunomagnetic separation; protein structure function; sheep; stainings; tissue /cell culture; vascular endothelial growth factors; xenotransplantation

DESCRIPTION: (Investigator's abstract) The major hurdle in studies on hematolymphopoietic stem cells (HSCs) has been the lack of a HSC specific marker, comparable to CD34 for hematopoietic progenitor cells (HPCs). Vascular endothelial growth factor receptor 2 (VEGFR2, Flk1 in mouse and KDR in men) is a key functional marker in embyronic hemoangiogenesis. We have investigated the expression of KDR on repopulating HSCs from human post-natal hematopoietic tissues. CD34+ cells comprise 0.1-0.5 percent cells expressing KDR. The KDR marker distinguishes HSCs from HPCs. The KDR+ fraction comprises virtually all HSCs, i.e., repopulating HSCs assayed in NOD-SCID mice and fetal sheep xenografts, and putative HSCs assayed in 12-wk extended Dexter type long term culture (ELTC) as ELTC-initiating cells (ELTC-ICs) or cobblestone area forming cells (CAFSs). Conversely, the KDR- fraction comprises virtually all oligo-unipotent HPCs with no self-renewal capacity. The HSC frequency in bone marrow (BM) KDR+ cells, evaluated by limiting dilution, is 20 percent by NOD-SCID mice assay and 25 percent by 12-wk LTC assay. The latter frequency value rises to 53 percent in LTC supplemented with VEGF, thus suggesting a functional role for KDR in HSCs; it further rises to virtually 100 percent in the KDR+ cell subfraction resistant to prolonged GF starvation in culture. Similar results were obtained in cord blood (CB), normal or mobilized peripheral blood (PB, MPB). On this basis, we hypothesize that in post-natal life KDR is a key functional marker for long-term repopulating CD34+ HSCs. To test this hypothesis, we propose (I) to investigate HSCs activity in CD34+ KDR+ cells. Namely: (Ia) To follow through previous studies on the assay of KDR+ repopulating HSCs, e.g. to test if repopulating HSCs are "pure" in the KDR+ subset resistant to GF starvation. (Ib) To investigate the self-renewal, differentiation and survival capacity of KDR+ HSCs, including in vivo HSC long-term engaftment in fetal sheep and ex vivo HSC expansion. (II) To transduce the KDR/FLK1 gene into CD34+KDR- cells, to investigate the functional role of Flk1/KDR in HSCs (preliminary studies suggest that Flk1 gene transfer largely suppresses HPC clonogenic activity, but partially rescues HSC activity in ELTC). Since preliminary studies suggest that a discrete number of CD34-lin-KDR+ cells engraft NOD-SCID mice and fetal sheep, while containing ELTC-ICs, we hypothesize that KDR is a key marker for CD34- HSCs. Therefore, we propose (III) to assay HSC activity in CD34-lin-KDR+. We will also attempt to convert in vitro CD34-lin-KDR+ [sic] cells into CD34+lin-KDR+ cells. Other informative studies on CD34+KDR+ and KDR- cells will be comparatively extended to CD34-lin-KDR+ and KDR- cells. We propose that these studies may provide significant contribution to the identification, characterization and utilization of CD34+ and CD34- HSCs, which will reflect at not only biological but also at clinical level, specifically for HSC transplantation, blood cell production in vitro and HSC gene therapy.

描述：（研究者的摘要）研究的主要障碍 造血干细胞（HSC）一直缺乏HSC特异性 标记物，与造血祖细胞 (HPC) 的 CD34 相当。血管 内皮生长因子受体 2（小鼠中的 VEGFR2、Flk1 和男性中的 KDR）是 胚胎血管生成的关键功能标志物。我们已经调查了 KDR 在人产后造血再生 HSC 中的表达 组织。 CD34+细胞包含0.1-0.5%表达KDR的细胞。科德罗 标记将 HSC 与 HPC 区分开来。 KDR+ 部分几乎包括所有 HSC，即在 NOD-SCID 小鼠和胎羊中检测的再生 HSC 异种移植物和推定的 HSC 在 12 周延长 Dexter 型长期中进行测定 培养（ELTC）作为 ELTC 起始细胞（ELTC-IC）或鹅卵石区域形成 细胞（CAFS）。相反，KDR-部分几乎包括所有 寡聚单能 HPC，没有自我更新能力。骨中 HSC 频率 通过有限稀释评估，骨髓 (BM) KDR+ 细胞为 20% NOD-SCID 小鼠测定和 1​​2 周 LTC 测定的 25%。后者的频率 在补充 VEGF 的 LTC 中，该值上升至 53%，因此表明 KDR 在 HSC 中的功能作用；进一步上升至几乎 100% KDR+ 细胞亚组分对培养中的长期 GF 饥饿具有抵抗力。 在正常或动员的脐带血 (CB) 中也获得了类似的结果 外周血（PB、MPB）。 在此基础上，我们假设 KDR 在产后生活中是一个关键的功能 长期再生 CD34+ HSC 的标记。为了检验这个假设，我们 建议 (I) 研究 CD34+ KDR+ 细胞中的 HSC 活性。即：(Ia)至 遵循之前关于 KDR+ 再生 HSC 测定的研究，例如到 测试重新填充的 HSC 是否在对 GF 具有抵抗力的 KDR+ 子集中是“纯的” 饥饿。 (Ib) 研究自我更新、分化和生存 KDR+ HSC 的能力，包括体内 HSC 在胎儿体内的长期植入 绵羊和离体 HSC 扩增。 (II) 将KDR/FLK1基因转导至 CD34+KDR- 细胞，研究 Flk1/KDR 在 HSC 中的功能作用 （初步研究表明Flk1基因转移很大程度上抑制了HPC 克隆形成活性，但部分挽救了 ELTC 中的 HSC 活性）。 由于初步研究表明离散数量的 CD34-lin-KDR+ 细胞 移植 NOD-SCID 小鼠和胎羊，同时含有 ELTC-IC，我们 假设 KDR 是 CD34- HSC 的关键标记。因此，我们建议 (III)测定CD34-lin-KDR+中HSC的活性。我们还将尝试转换 将体外 CD34-lin-KDR+ [原文如此] 细胞转化为 CD34+lin-KDR+ 细胞。其他信息 CD34+KDR+和KDR-细胞的研究将相对扩展至 CD34-lin-KDR+ 和 KDR- 细胞。 我们认为这些研究可能会为以下方面做出重大贡献： CD34+ 和 CD34- HSC 的鉴定、表征和利用， 不仅会反映在生物学层面，而且会反映在临床层面，特别是 用于 HSC 移植、体外血细胞生产和 HSC 基因治疗。