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

热休克反应介导的离体造血干细胞生长

• 批准号: 10319623
• 负责人: Robert A.J. Signer
• 金额: $ 51.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319623
• 关键词: Adult; Anemia; Autoimmune Diseases; Biological Assay; Blood; Blood Cells; Buffers; Cell Maintenance; Cell Nucleus; Cell Survival; Cell surface; Cells; Clinical; Cytoplasm; Data; Defect; Deficiency Diseases; Development; Disease; Engraftment; Environment; Exhibits; Genes; Genetic Transcription; Growth; Heat Losses; Heat shock proteins; Heat-Shock Response; Hematological Disease; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Human; Immune; Immunodeficient Mouse; Impairment; In Vitro; Intervention; Left; Life; Longevity; Maintenance; Malignant - descriptor; Mediating; Modality; Molecular Chaperones; Mus; Non-Malignant; Pathway interactions; Patients; Phenotype; Population; Protein Biosynthesis; Proteins; Proteome; Quality Control; Research; Savings; Source; Stress; Support System; Surface; System; Testing; Therapeutic; Time; Transplantation; base; bone marrow failure syndrome; cancer genetics; curative treatments; heat-shock factor 1; hematopoietic stem cell expansion; hematopoietic stem cell self-renewal; in vitro testing; in vivo; misfolded protein; multicatalytic endopeptidase complex; new technology; novel strategies; overexpression; prevent; protein folding; proteostasis; proteotoxicity; reconstitution; single-cell RNA sequencing; small molecule; stem cell function; stem cell growth; stem cells; trafficking; transcriptome; transcriptome sequencing

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.

抽象的 造血干细胞（HSC）移植用于治疗几种恶性和非恶性肿瘤 血液学疾病。不幸的是，即使经过数十年的研究，HSC也无法得到很好的维护或 扩大的Ex Vivo，它使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期文化时代也对HSC有害，这限制了HSC的机会 扩展或基因编辑。在初步研究中，我们建立了一个支持持续Ex的文化系统 体内HSC生长。在该系统中，可以将十个纯化的HSC扩展到〜104个造血茎和 祖细胞在10天的时间内，而不会损失连续的长期多核重建活性。 这种培养系统的发展是基于我们发现HSC的蛋白质速率较低的 合成比其他血细胞，而蛋白质合成的适度增加会损害HSC功能。我们 确定增加蛋白质合成通过降低蛋白质组质量会损害HSC。令人惊讶的是，HSC 体外蛋白质合成大量增加，可降低蛋白质组质量并破坏蛋白质 体内平衡（蛋白抑制）。因此，我们寻求一种方法来规避HSC时蛋白质的这种崩溃 从其体内环境中删除，以允许其维护和扩展以进行治疗 申请。热休克响应是对蛋白质毒性应激反应的主要途径 细胞质。热休克响应的主调节器是热休克因子1（HSF1）。在条件下 蛋白毒性应激，HSF1诱导热休克蛋白的转录，该热休克蛋白是协调蛋白质折叠，运输的转录 和降解以促进蛋白质的和细胞的存活。我们已经确定HSF1促进了离体 HSC维护，作为HSF1的条件缺失，在体外显着加剧了HSC损失。此外，我们 鉴定出可以增强培养的HSC和这些小分子内HSF1激活的小分子 以HSF1依赖性方式显着增强了体内HSC的生长和维护。基于这些 数据，我们假设蛋白毒性应力会损害HSC的自我更新，并有助于HSC耗竭 体外以及增加HSF1活性的干预措施可以通过增强蛋白质症来促进离体HSC的生长 容量。在AIM 1中，我们将使用一套新技术来测试HSF1活动的收益和损失如何影响 HSC中的蛋白质静脉曲张。在AIM 2中，我们将在HSF1存在下培养成年小鼠和人类HSC 激活剂并测试这些治疗方法是否通过进行限制稀释移植来实现HSC扩展。目标 3我们将通过细胞表面分析和单个单个的生长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通过激活促进离体HSC增长的新方法 热休克响应并增强蛋白质的能力。确定HSC维护的方式和 扩张具有多种造血疾病的患者的巨大治疗潜力。