Ex vivo hematopoietic stem cell growth mediated by the heat shock response

摘要 造血干细胞（HSC）移植用于治疗几种恶性和非恶性肿瘤。 血液病不幸的是，即使经过几十年的研究，HSC也不能很好地维持或维持。 体外扩增，这使得许多患者没有足够的潜在挽救生命的HSC来源。 即使在优化条件下的短培养时间对HSC也是有害的，限制了HSC的机会。 扩增或基因编辑。在初步研究中，我们建立了一个支持持续表达的培养系统， 体内HSC生长。在该系统中，10个纯化的HSC可扩增成~104个造血干细胞， 在10天的时间内，祖细胞，而没有任何连续的长期多谱系重建活性的损失。 这种培养系统的发展是基于我们发现HSC具有较低的蛋白质表达率。 造血干细胞合成比其他血细胞少，而蛋白质合成的适度增加损害了HSC的功能。我们 确定增加的蛋白质合成通过降低蛋白质组质量来损害HSC。引人注目的是，HSC 在体外表现出蛋白质合成的大量增加，这降低了蛋白质组的质量并破坏了蛋白质 体内平衡（蛋白质平衡）。因此，我们寻求一种方法来规避这种崩溃时，HSC的蛋白质稳定， 从它们的体内环境中移除，以便允许它们的维持和扩张， 应用.热休克反应是机体对蛋白毒性应激反应的主要途径。 细胞质热休克反应的主要调节因子是热休克因子1（Hsf 1）。条件下 蛋白毒性应激，Hsf 1诱导热休克蛋白的转录，该热休克蛋白协调蛋白质折叠、运输 和降解以促进蛋白质稳定和细胞存活。我们已经确定，Hsf 1促进离体 HSC维持，因为条件性Hsf 1缺失显著加剧了体外HSC损失。而且我们 鉴定了在培养的HSC内增强Hsf 1活化的小分子，并且这些小分子 以Hsf 1依赖方式显著增强离体HSC生长和维持。基于这些 数据，我们假设蛋白毒性应激损害HSC自我更新，并导致HSC耗竭， 增加Hsf 1活性的干预措施可以通过增强蛋白抑制作用促进离体HSC生长 容量在目标1中，我们将使用一套新技术来测试Hsf 1活性的获得和丧失如何影响 HSC内的蛋白稳态。在目标2中，我们将在存在Hsf 1的情况下培养成年小鼠和人HSC。 活化剂，并通过进行有限稀释移植来测试这些处理是否能够使HSC扩增。在Aim中 3我们将使用细胞表面分析和单克隆抗体来确定离体生长如何影响HSC的身份。 细胞RNA测序。我们的研究代表了一种新的方法，促进离体HSC的生长，通过激活 热休克反应和增强蛋白质稳定能力。确定HSC维护模式， 扩展为患有各种造血系统疾病的患者提供了巨大的治疗潜力。

ABSTRACT Hematopoietic stem cell (HSC) transplants are used to treat several malignant and non-malignant hematological diseases. Unfortunately, even after decades of research, HSCs cannot be well maintained or expanded ex vivo, which has left many patients without a sufficient source of potentially life-saving HSCs. Even short culture times in optimized conditions are deleterious to HSCs, limiting opportunities for HSC expansion or gene editing. In preliminary studies, we established a culture system that supports sustained ex vivo HSC growth. In this system, ten purified HSCs can be expanded into ~104 hematopoietic stem and progenitor cells over a 10-day period, without any loss of serial long-term multilineage reconstituting activity. The development of this culture system was based on our discovery that HSCs have lower rates of protein synthesis than other blood cells, and that modest increases in protein synthesis impair HSC function. We determined that increased protein synthesis impairs HSCs by reducing proteome quality. Strikingly, HSCs exhibit a massive increase in protein synthesis in vitro that reduces proteome quality and disrupts protein homeostasis (proteostasis). We thus sought a way to circumvent this collapse in proteostasis when HSCs are removed from their in vivo environment so as to allow their maintenance and expansion for therapeutic applications. The heat shock response is the principal pathway that responds to proteotoxic stress in the cytoplasm. The master regulator of the heat shock response is Heat shock factor 1 (Hsf1). Under conditions of proteotoxic stress, Hsf1 induces transcription of heat shock proteins that coordinate protein folding, trafficking and degradation to promote proteostasis and cell survival. We have established that Hsf1 promotes ex vivo HSC maintenance, as conditional deletion of Hsf1 significantly exacerbates HSC loss in vitro. Furthermore, we identified small molecules that enhance Hsf1 activation within cultured HSCs and these small molecules significantly enhance ex vivo HSC growth and maintenance in a Hsf1-dependent manner. Based on these data, we hypothesize that proteotoxic stress impairs HSC self-renewal and contributes to HSC depletion in vitro, and interventions that increase Hsf1 activity can promote ex vivo HSC growth by enhancing proteostasis capacity. In Aim 1 we will use a suite of new technologies to test how gain and loss of Hsf1 activity influence proteostasis within HSCs. In Aim 2 we will culture adult mouse and human HSCs in the presence of Hsf1 activators and test if these treatments enable HSC expansion by performing limiting dilution transplants. In Aim 3 we will determine how ex vivo growth influences the identity of HSCs using cell surface profiling and single cell RNA-sequencing. Our studies represent a new approach for promoting ex vivo HSC growth by activating the heat shock response and enhancing proteostasis capacity. Identifying a modality for HSC maintenance and expansion holds enormous therapeutic potential for patients with diverse hematopoietic disorders.