Hematopoietic Stem and Progenitor Cell Expansion

点击翻译按钮获取中文摘要

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 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.