Hematopoietic Stem and Progenitor Cell Expansion

造血干细胞和祖细胞扩增

基本信息

批准号：
10706179
负责人：
Andre LaRochelle
金额：
$ 30.91万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10706179
关键词：
Adult Autologous Biological Assay CD34 gene Cell Nucleus Cells Cellular Membrane Clinical Development Dose Elements Endoplasmic Reticulum Engineering Engraftment Extracellular Domain Fibronectins Flow Cytometry Frequencies Gene Expression Profile Genes Genetic Transcription Goals Heat-Shock Proteins 90 Heat-Shock Response Hematological Disease Hematology Hematopoiesis Hematopoietic Stem Cell Transplantation Hematopoietic stem cells Homologous Transplantation Human Hypoxia Hypoxia Pathway IgG1 Immunoglobulin G Impairment Investigation Ligands Marrow Measures Mediating Mediator of activation protein Mus Oxygen Patients Phenotype Proliferating Proteins Protocols documentation Publishing Recovery Reporting Role Series Signal Pathway Signal Transduction Stimulus Supplementation Time Transplantation Umbilical Cord Blood Undifferentiated Work base clinically significant cytokine endoplasmic reticulum stress fitness gene correction heat-shock factor 1 hematopoietic stem cell expansion hypoxia inducible factor 1 in vivo neutrophil normoxia notch protein nuclease peripheral blood preservation progenitor protein complex proteostasis receptor response small molecule inhibitor stem cell function stem cell genes stem cells therapeutic gene

项目摘要

Objective 1: Notch-mediated ex vivo expansion of human HSPCs by culture under hypoxia To investigate whether hypoxia can facilitate superior ex vivo expansion of human HSPCs than normoxia in the presence of Delta1ext-IgG, a total of 1 x 105 human MPB CD34+ cells were cultured under normoxic or hypoxic conditions in vessels coated with fibronectin alone or combined with increasing concentrations of Delta1ext-IgG (2.5, 5, 10 and 20 g/mL). After 21 days in culture, cells were counted and characterized by flow cytometry and functional assays. We demonstrate that ex vivo culture of human adult HSPCs with Delta1ext-IgG under low oxygen tension (2% O2) limits ER stress in LTR-HSCs and, to a lesser extent, in lineage committed progenitors compared to normoxic (21% O2) cultures. A distinct HSC gene expression signature was upregulated in cells cultured with Delta1ext-IgG in hypoxia and, after 21 days of culture, the frequency of long-term repopulating (LTR) HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared to the normoxia group, as measured by limiting dilution analysis in NSG mice. Notch and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured CD34+ cells, and both hypoxia inducible factor-1 and the intracellular domain of Notch1 receptor were central in the convergence point between the two signaling pathways. Thus, our work underscores the importance of mitigating ER stress perturbations to preserve functional HSCs in extended cultures, and offers a clinically feasible platform for the expansion of human HSPCs. This work was published Stem Cell reports 2021. Objective 2: Notch-mediated ex vivo expansion of human HSPCs by culture with 17-AAG In a recent study, murine and human cord blood-derived HSPCs cultured over a 10-day period in standard medium/cytokines supplemented with 17-AAG were functionally similar to fresh HSCs. However, 17-AAG alone did not support significant HSC expansion. In FY22, we have initiated investigations to determine whether highly proliferative conditions, such as those promoted in culture with Delta1ext-IgG, could act synergistically with Hsf1 inhibition and promote HSC expansion ex vivo. We have optimized doses of 17-AAG for culture of human adult HSPCs and long-term xenogeneic transplants are ongoing to assess efficacy of this approach.

目标 1：通过缺氧培养，Notch 介导的人 HSPC 离体扩增为了研究在 Delta1ext-IgG 存在的情况下，缺氧是否可以比常氧更好地促进人 HSPC 的离体扩增，在常氧或缺氧条件下，在单独涂有纤连蛋白或与增加纤连蛋白结合的血管中培养总共 1 x 105 个人 MPB CD34+ 细胞。 Delta1ext-IgG 浓度（2.5、5、10 和 20 μg/mL）。培养 21 天后，对细胞进行计数并通过流式细胞术和功能测定进行表征。我们证明，与常氧 (21% O2) 培养物相比，在低氧张力 (2% O2) 下用 Delta1ext-IgG 体外培养人类 HSPC 可以限制 LTR-HSC 中的 ER 应激，并且在较小程度上限制谱系定型祖细胞中的 ER 应激。。在缺氧条件下用 Delta1ext-IgG 培养的细胞中，独特的 HSC 基因表达特征上调，培养 21 天后，长期再增殖 (LTR) HSC 的频率相对于未培养的细胞增加了 4.9 倍，与未培养的细胞相比增加了 4.2 倍。常氧组，通过 NSG 小鼠的有限稀释分析进行测量。 Notch 和缺氧途径交叉以维持培养的 CD34+ 细胞中未分化的表型，缺氧诱导因子 1 和 Notch1 受体的细胞内结构域位于两条信号传导途径之间的汇合点的中心。因此，我们的工作强调了减轻 ER 应激扰动以在扩展培养物中保留功能性 HSC 的重要性，并为人类 HSPC 的扩展提供了临床上可行的平台。这项工作发表在《2021 年干细胞报告》上。目标 2：通过 17-AAG 培养，Notch 介导的人 HSPC 离体扩增在最近的一项研究中，鼠和人脐带血来源的 HSPC 在补充有 17-AAG 的标准培养基/细胞因子中培养 10 天，其功能与新鲜 HSC 相似。然而，单独的 17-AAG 并不支持显着的 HSC 扩增。在 2022 财年，我们开始研究以确定高度增殖条件（例如在 Delta1ext-IgG 培养中促进的条件）是否可以与 Hsf1 抑制协同作用并促进 HSC 离体扩增。我们已经优化了用于人类成人 HSPC 培养的 17-AAG 剂量，并且正在进行长期异种移植以评估这种方法的功效。