Hematopoietic Stem and Progenitor Cell Expansion

目的1：缺氧条件下Notch介导的人HSPC体外扩增 为了探讨在Delta1ext-Ig G存在下，低氧是否比常氧更有利于人HSPC的体外扩增，在常氧或低氧条件下，将1×105个人MPB CD34+细胞培养在单独包被纤维连接蛋白或与增加Delta1ext-Ig G浓度(2.5、5、10和20g/m L)联合培养的血管中。培养21d后，用流式细胞仪和功能分析对细胞进行计数和鉴定。我们证明，与常氧(21%O2)培养相比，在低氧压(2%O2)下用Delta1ext-Ig G体外培养人成年HSPC可以限制LTR-HSCs的内质网应激，并且在较小程度上限制了谱系承诺的祖细胞的内质网应激。在低氧条件下用Delta1ext-Ig G培养的细胞中有明显的HSC基因表达上调，培养21天后，NSG小鼠的极限稀释分析表明，长期再填充(LTR)HSC的频率比未培养的细胞增加了4.9倍，与正常氧组相比增加了4.2倍。在培养的CD34+细胞中，Notch通路和低氧通路交叉以维持未分化的表型，而低氧诱导因子-1和Notch1受体的胞内区都位于这两个信号通路之间的汇聚点。因此，我们的工作强调了减轻内质网应激干扰以在扩展培养中保留功能性HSC的重要性，并为人类HSPC的扩增提供了一个临床上可行的平台。这项工作正在接受同行审查，《临床调查杂志》正在审查中(2020)。 目的：利用CRISPRa和CRISPRi瞬时调控HSPC自我更新关键调控因子的表达。 异位表达HOXB4是一种含有高度保守的DNA结合基序的同源结构域的转录因子，在体外和体内都能促进HSPC的自我更新，并被认为是一种扩增HSPC的方法。然而，使用逆转录病毒载体过表达HOXB4会在大型动物模型中导致白血病，并且被认为风险太高，不能在任何临床环境中考虑。在HSPC中瞬时诱导HOXB4的表达可能是利用HOXB4在HSPC上的力量的另一种方法。例如，可溶的重组HOXB4蛋白诱导转导的HSPC快速体外扩增，从而避免使用整合的逆转录病毒载体，同时受益于HOXB4的自我更新能力。然而，事实证明，重组蛋白的半衰期很短，这是一个实际障碍，需要替代方法。在最近的研究中，包括DNMT3A、TET2和ASXL1在内的关键表观遗传调控因子的功能丧失的体细胞突变也被证明赋予HSPC增殖优势，导致与年龄相关的克隆性造血(CH)。对小鼠模型中这些基因的遗传突变的分析表明，它们与增强的HSPC自我更新有关。与HOXB4类似，DNMT3A、TET2或ASXL1的永久失活也可能与驱动疾病表型的继发性突变协同作用而易于发生恶性肿瘤。因此，我们假设DNMT3A、TET2或ASXL1活性的瞬时失活可能允许HSPC在体外扩增，而不会增加对恶性肿瘤的易感性。 为了研究这种可能性，基于CRISPR/Cas9的转录激活(CRISPRa)和抑制(CRISPRi)分别被开发来瞬时诱导或抑制所选择的靶基因的内源表达。CRISPRa和CRISPRi利用人工合成的单引导RNA(SgRNAs)将核酸酶失活的死亡Cas9(DCas9)与转录激活因子(例如VP64、p65)或抑制因子(例如KRAB)结构域融合在一起。目前的CRISPRa和CRISPRi方法依赖于电穿孔庞大的外源质粒DNA或通过慢病毒载体转导HSPC来传递所需的Cas9/sgRNA激活剂和抑制物成分。然而，使用质粒会对HSPC产生显著的细胞毒性，慢病毒转导会带来插入突变的风险，并且由于载体整合，不容易诱导成功扩增HSPC所需的细胞发育程序的瞬时变化。相比之下，先前的CRISPR工作使用核酸酶活性的Cas9对HSPC进行基因组编辑，证明了通过电穿孔作为核糖核蛋白(RNP)复合体的Cas9/sgRNA系统比基于质粒的试剂更有效，并且对HSPC只产生最小的毒性。重要的是，RNPs还受制于细胞固有的蛋白酶体活性，因此显示出有限的细胞内半衰期，这是瞬时基因激活或抑制的一个重要特征。 在20财年，我们已经初步开发了重组生产和纯化dCas9-激活物或dCas9-阻遏物结构域融合蛋白的方法。然而，由于大多数蛋白质超过160 kDa，产量通常较低。为了克服这一限制，我们使用了一种改进的方法，将激活因子和抑制因子结构域从dCas9中分离出来(Liao等人，Cell 2017)。尽管MPH和MS2-KRAb结构域比Cas9融合蛋白小得多(分别为56 kDa和26 kDa)，而且更容易产生，但我们在原代CD34+细胞中观察到高毒性，阻碍了进一步的发展。我们建立了基于修饰的mRNA瞬时表达dCas9融合蛋白的CRISPRi和CRISPRa方法。这种方法促进了靶基因的有效上调和下调，并正在进行利用该系统瞬时激活或抑制HSPC基因表达的工作，作为促进HSPC体外扩增的一种新方法。

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 is under peer-review Journal of Clinical Investigation, under review (2020). Objective 2: Transiently modulate the expression of key regulators of HSPC self-renewal using CRISPRa and CRISPRi. Ectopic expression of HOXB4, a transcription factor containing a highly conserved DNA-binding motif known as the homeodomain, has been found to enhance HSPC self-renewal in vitro and in vivo and has been suggested as an approach to expand HSPCs. However, overexpression of HOXB4 using retroviral vectors resulted in leukemia in large animal models and is considered too risky to be considered in any clinical settings. Transient induction of HOXB4 expression in HSPCs could represent an alternative approach to harnessing the power of HOXB4 on HSPCs. For instance, a soluble recombinant HOXB4 protein induced rapid ex vivo expansion of transduced HSPCs, thereby avoiding the use of integrating retroviral vectors while benefiting from the self-renewal capacity of HOXB4. However, the short half-life of the recombinant protein has proven a practical hurdle and alternative approaches are needed. In recent studies, loss-of-function somatic mutations in key epigenetic regulators, including DNMT3A, TET2 and ASXL1, have also been shown to confer a proliferative advantage on HSPCs, resulting in age-related clonal hematopoiesis (CH). Analysis of genetic mutations in these genes in mouse models have suggested their association with enhanced HSPC self-renewal. Similar to HOXB4, permanent inactivation of DNMT3A, TET2 or ASXL1 may also predispose to the development of malignancies in cooperation with secondary mutations that drive disease phenotype. Therefore, we hypothesized that transient inactivation of DNMT3A, TET2 or ASXL1 activity might allow HSPC expansion in vitro without increasing susceptibility to malignancies. To investigate this possibility, CRISPR/Cas9-based transcriptional activation (CRISPRa) and inhibition (CRISPRi) are developed to transiently induce or repress, respectively, the endogenous expression of select target genes. CRISPRa and CRISPRi utilize synthetic single guide RNAs (sgRNAs) to direct to a gene-of-interest a nuclease-inactive dead Cas9 (dCas9) fused to a transcriptional activator (e.g. VP64, P65) or repressor (e.g. KRAB) domain. Current approaches to CRISPRa and CRISPRi rely on electroporation of bulky exogenous plasmid DNA or on transduction of HSPCs with lentiviral vectors to deliver the required Cas9/sgRNA activator and repressor components. However, use of plasmids results in pronounced cytotoxicity to HSPCs and lentiviral transduction introduces the risk of insertional mutagenesis and, due to vector integration, is not readily amenable to the induction of transient changes to the cellular developmental program required for successful HSPC expansion. In contrast, prior CRISPR work using a nuclease-active Cas9 for genome editing of HSPCs demonstrated that the Cas9/sgRNA system delivered by electroporation as a ribonucleoprotein (RNP) complex is more effective than plasmid-based reagents and results in only minimal toxicity in HSPCs. Importantly, RNPs are also subject to the intrinsic proteasomal activity of the cell and thus display a limited intracellular half-life, a feature of interest for transient gene activation or repression. In FY20, we have primarily developed approaches to recombinantly produce and purify dCas9-activator or dCas9-repressor domain fusion proteins. However, yields are typically low as most proteins exceed 160 kDa. To overcome this limitation, we used a modified approach whereby the activator and repressor domains were separated from dCas9 (Liao et al, Cell 2017). Although the MPH and MS2-KRAB domains are much smaller (56 kDa and 26 kDa, respectively) than Cas9 fusion proteins and could be more readily produced, we observed high toxicity in primary CD34+ cells that prevented further development. We have developed CRISPRi and CRISPRa approach based on transfection of modified mRNA for transient expression of dCas9 fusion proteins. This approach has facilitated efficient up- and down-regulation of target genes and work is underway to transiently activate or repress gene expression in HSPCs with this system, as a novel approach to promote HSPC expansion ex vivo.