Hematopoietic Stem and Progenitor Cell Expansion

目的1：Notch介导的低氧培养人HSPCs的体外扩增 为了研究在Delta 1 ext-IgG存在下低氧是否可以促进人HSPC的比常氧上级的离体扩增，在常氧或低氧条件下，在用单独的纤连蛋白或与增加浓度的Delta 1 ext-IgG（2.5、5、10和20 μ g/mL）组合的纤连蛋白包被的血管中培养总共1 × 105个人MPB CD 34+细胞。培养21天后，通过流式细胞术和功能测定对细胞进行计数和表征。我们证明，离体培养的人成年HSPCs与Delta 1 ext-IgG在低氧张力（2%O2）限制ER应力LTR-HSC，并在较小程度上，在谱系定向祖细胞相比，常氧（21%O2）的文化。一个独特的HSC基因表达的签名是上调与Delta 1 ext-IgG在缺氧培养的细胞，培养21天后，长期再增殖（LTR）HSC的频率增加4.9倍，相对于未培养的细胞和4.2倍相比，常氧组，在NSG小鼠有限稀释分析测量。Notch和缺氧途径共同维持培养的CD 34+细胞的未分化表型，缺氧诱导因子-1和Notch 1受体的胞内结构域是这两条信号通路的交汇点。因此，我们的工作强调了减轻ER应激扰动以在扩展培养中保留功能性HSC的重要性，并为人类HSPC的扩增提供了临床可行的平台。这项工作发表在Stem Cell reports 2021上。 目的2：通过与17-AAG一起培养Notch介导的人HSPC的离体扩增 在最近的一项研究中，在补充有17-AAG的标准培养基/细胞因子中培养10天的鼠和人脐带血来源的HSPC在功能上与新鲜HSC相似。然而，单独的17-AAG不支持显著的HSC扩增。在2022财年，我们已经启动了研究，以确定是否高度增殖的条件，如在培养中与Delta 1 ext-IgG促进，可以协同作用与Hsf 1抑制和促进HSC体外扩增。我们已经优化了17-AAG的剂量用于培养人成年HSPC，并且正在进行长期异种移植以评估该方法的功效。

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.