Generation of Hematopoietic Stem and Progenitor Cells from Human iPSCs

从人类 iPSC 中生成造血干细胞和祖细胞

• 批准号: 10706178
• 负责人: Andre LaRochelle
• 金额: $ 92.74万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10706178
• 关键词: ATAC-seq; Activins; Address; Adult; Agonist; Algorithms; Animals; Aorta; Arteries; Autologous; Binding; Biological Assay; Blood; Blood Vessels; CD34 gene; Cell Count; Cell Line; Cell Therapy; Cells; ChIP-seq; Chemicals; Chromatin; Clinic; Clinical; Coculture Techniques; Control Groups; DNA; Data; Data Set; Development; Developmental Process; Dorsal; Doxycycline; Ectopic Expression; Elements; Embryo; Endothelial Cells; Endothelium; Engraftment; Enhancers; Erythroid; Frequencies; Gene Expression; Generations; Genes; Genetic Transcription; Genomic Segment; Globin; Gonadal structure; Growth; Harvest; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; IL3 Gene; Immune; Investigation; Lentivirus Vector; Libraries; Mesenchymal; Mesonephric structure; Methods; Mitogen-Activated Protein Kinases; Modeling; Molecular; Molecular Profiling; Morphology; Mus; Nodal; Oncogenic; Ontology; PTPRC gene; Pathway interactions; Patients; Phase Transition; Phenotype; Population; Production; Property; Protocols documentation; Publishing; Role; Sampling; Signal Pathway; Signal Transduction; Specific qualifier value; Stem Cell Development; Stem cell transplant; Stromal Cells; System; Testing; Transcription Coactivator; Translations; Transplantation; Vascular Endothelium; base; cell type; cellular engineering; clinically relevant; density; differentiation protocol; experimental study; genome-wide; healthy volunteer; hematopoietic differentiation; hematopoietic engraftment; hemogenic endothelium; human tissue; improved; in vitro activity; in vivo; induced pluripotent stem cell; inhibitor; lentivirally transduced; monolayer; mouse model; progenitor; programs; self-renewal; stem cell differentiation; synergism; tool development; transcription factor

Objective 1: Develop a scalable culture system for hematopoietic differentiation of hiPSCs To address the limitations of current iPSC differentiation approaches, we initially sought to develop a chemically-defined and scalable hematopoietic differentiation protocol requiring no replating, EB formation or co-culture on stromal elements. Through systematic optimization steps, we established a simple 21-day monolayer-based culture system that recapitulates ex vivo the emergence of human hematopoiesis. Under culture conditions that favored mesodermal specification of human iPSCs (Day 0 to 3), an adherent monolayer rapidly formed. With the subsequent addition of hematopoietic cytokines (Day 3 to 21), hematopoietic clusters emerged from the monolayer before their eventual release in the supernatant fraction. We systematically characterized cells arising from this system by harvesting supernatant and monolayer populations at regular intervals between day 5 and 21 of differentiation. Within the supernatant, hematopoietic cells (CD43+CD45+/-) underwent sequential development with features of primitive wave hematopoiesis (peak at day 7), definitive multilineage HSPCs with potent colony formation activity in vitro but limited engraftment potential in vivo (peak at day 12), and definitive erythroid-committed progenitors expressing adult-type globin chains (peak at day 17 to 21). To understand the possible causes underpinning the absence of engraftable HSPCs in this system, we examined the cellular constituents of the CD43-CD45- non-hematopoietic fraction that supports hematopoiesis during differentiation. We first identified a prevalent population of mesenchymal cells throughout differentiation. Perivascular mesenchymal cells are known to interact with HSPCs and maintain their activity in the adult BM niche, but their role in promoting definitive HSPC development during ontogeny has not been demonstrated. Importantly, arterial HE was largely absent within the supportive monolayer. Thus, in keeping with prevailing arterial-specification models, we deduced that the adherent monolayer was likely inadequate to support the generation of bona fide engrafting HSCs in culture. Objective 2. Optimize iPSC differentiation conditions to promote arterial HE formation To augment the production of arterial HE during iPSC differentiation, we postulated that simultaneous activation of key signaling pathways independently shown to control arterial fate during vascular development, including WNT/-catenin, activin/nodal/TGF, and mitogen-activated protein kinase (MAPK)/ERK pathways, might provide a synergy sufficient to further instruct definitive hematopoiesis with engraftment potential from human iPSCs. To test this possibility, we supplemented the culture medium with the WNT/-catenin agonist CHIR99021 (CHIR) and nodal/activin/TGF inhibitor SB431542 (SB) at day 2 of iPSC differentiation and activated MAPK/ERK signaling with LY294002 (LY) from day 3 to day 6 of culture. Control cultures contained no CHIR/SB/LY (non-treated), or were supplemented with CHIR/SB or LY only. Compared to control cultures, addition of CHIR/SB/LY led to a marked increase in percentages and numbers of CD144+CD34hiCD73-CD184+ arterial HE, peaking at day 5 of differentiation. We next investigated whether this early increase in arterial HE formation observed in the presence of CHIR/SB/LY influenced hematopoietic development. Addition of CHIR/SB/LY decreased overall CD43+/-CD45+ hematopoietic cell numbers but a notable rise in percentages of phenotypically defined definitive HSPCs (CD34+CD45RA-CD90+) was observed within the hematopoietic population at day 12 of differentiation compared to controls. In CFU assays, the frequency of progenitors with multilineage differentiation capacity was similar between control groups but significantly increased in the CHIR/SB/LY group. To further assess the self-renewal and differentiation capacity of iPSC-derived hematopoietic CD34+ cells, colonies derived from the first round of CFU plating were pooled and replated in secondary CFU assays. Notably, we observed a 3-fold increase in total CFU numbers from CD34+ cells derived from CHIR/SB/LY cultures compared to control groups. However, these cells did not sustain long-term hematopoietic engraftment after transplantation into NSG mouse recipients, indicating that additional revisions to this system are required. Objective 3. Identify HSC-specific superenhancers and associated master transcription factors While activation of an arterial program is required for HSC induction ex vivo, our data suggest that current culture conditions fail to modulate other critical molecular programs uncoupled from arterial development. We hypothesized that inadequate activation of core TF networks defining HSC identify could explain the lack of engraftment potential in HSPCs generated ex vivo. Because SEs represent dense binding platforms for cell-type specific master TFs and transcriptional co-activators, we first sought to delineate the SE landscape and the TFs they regulate in HSC-enriched populations. We conducted genome-wide integrative ChIP-seq and ATAC-seq analyses on phenotypically defined CD34+CD38- HSCs purified from mobilized PB samples obtained from 3 independent healthy volunteers. A total of 873 SEs were identified by elevated H3K27ac signal density; they represented 3.8% of total enhancers and their median size was 10-fold larger than genomic regions encompassed by typical-enhancers. By filtering ATAC-seq data for conserved domains, and applying binding motif enrichment and proximity algorithms, we identified 594 TFs encoded by genes regulated by SEs in human CD34+CD38- cells. The curated TF gene set was validated by interrogating published gene expression datasets in human CD34+CD38- cells and via unbiased gene ontology (GO) analysis. To identify putative master regulators of HSC identity, we narrowed our list of 594 SE-regulated TFs to include only transcription factors with binding motifs within their own SEs, denoting a distinctive auto-regulatory property of master TFs. A total of 34 master regulators were uncovered. Objective 4. Enhance cell engineering through manipulation of master transcriptional regulators To evaluate a potential role of the uncovered HSC-specific master TFs for the generation of functional HSPCs ex vivo, we independently cloned the assembled library of 34 TFs into doxycycline-inducible lentiviral vectors and transduced human iPSCs with the complete library or a subset of 13 TFs deemed more promising based on preliminary testing in CFU assays and absence of expression in iPSC-derived HSPCs. Transduced iPSCs were subjected to hematopoietic differentiation using our previously optimized protocol. Addition of doxycycline from day 5 of differentiation, when arterial HEs have formed and EHT begins, allowed for a stage-appropriate transient expression of the hematopoietic master regulators. To evaluate functionality of HSPCs obtained at day 12 of culture, cells were plated in CFU assays or transplanted intrafemorally into NBSGW immune-deficient murine recipients. Notably, we observed 2-fold increase in numbers of CFUs from cultures with enforced expression of master TFs compared to mock-transduced control experiments. Colony size was also enhanced and a preponderance of the most primitive CFU types (CFU-GM and CFU-GEMM) was noted with the introduction of master TFs during hematopoietic differentiation of human iPSCs. Efficient short-term engraftment of human iPSC-derived HSPCs (15% in PB) was observed two months after transplantation, but long-term engraftment was not sustained.

目标 1：开发可扩展的 hiPSC 造血分化培养系统 为了解决当前 iPSC 分化方法的局限性，我们最初寻求开发一种化学定义的、可扩展的造血分化方案，不需要重新铺板​​、EB 形成或基质元素共培养。通过系统的优化步骤，我们建立了一个简单的 21 天单层培养系统，该系统在体外重现了人类造血的出现。在有利于人类 iPSC 中胚层特化的培养条件下（第 0 天至第 3 天），贴壁单层迅速形成。随着随后添加造血细胞因子（第3天至第21天），造血簇从单层中出现，然后最终释放到上清液部分中。 我们通过在分化第 5 天到第 21 天之间定期收集上清液和单层细胞群，系统地表征了该系统产生的细胞。在上清液中，造血细胞 (CD43+CD45+/-) 经历了连续发育，具有原始波造血功能（第 7 天达到峰值），最终的多系 HSPC 在体外具有有效的集落形成活性，但体内植入潜力有限（第 12 天达到峰值） ），以及表达成人型珠蛋白链的确定的红系定型祖细胞（在第​​ 17 天至第 21 天达到峰值）。为了了解该系统中缺乏可移植 HSPC 的可能原因，我们检查了在分化过程中支持造血作用的 CD43-CD45-非造血部分的细胞成分。我们首先鉴定了分化过程中普遍存在的间充质细胞群。已知血管周围间充质细胞与 HSPC 相互作用并在成体 BM 生态位中维持其活性，但它们在个体发育过程中促进最终 HSPC 发育的作用尚未得到证实。重要的是，支持性单层内基本上不存在动脉 HE。因此，根据流行的动脉特异性模型，我们推断贴壁单层可能不足以支持培养中真正移植的 HSC 的生成。 目标2.优化iPSC分化条件以促进动脉HE形成 为了在 iPSC 分化过程中增加动脉 HE 的产生，我们假设同时激活关键信号通路独立地显示出控制血管发育过程中的动脉命运，包括 WNT/-catenin、activin/nodal/TGF 和丝裂原激活蛋白激酶 (MAPK) )/ERK 途径，可能提供足以进一步指导具有人类 iPSC 植入潜力的确定性造血作用。为了测试这种可能性，我们在 iPSC 分化的第 2 天向培养基中添加了 WNT/-catenin 激动剂 CHIR99021 (CHIR) 和 nodal/activin/TGF 抑制剂 SB431542 (SB)，并使用 LY294002 (LY) 激活了 MAPK/ERK 信号传导培养第 3 天至第 6 天。对照培养物不含CHIR/SB/LY（未处理），或仅补充CHIR/SB或LY。与对照培养物相比，添加 CHIR/SB/LY 导致 CD144+CD34hiCD73-CD184+ 动脉 HE 的百分比和数量显着增加，在分化第 5 天达到峰值。接下来我们研究了在 CHIR/SB/LY 存在下观察到的动脉 HE 形成的早期增加是否影响造血发育。添加 CHIR/SB/LY 降低了总体 CD43+/-CD45+ 造血细胞数量，但与对照相比，在分化第 12 天的造血细胞群中观察到表型明确的最终 HSPC (CD34+CD45RA-CD90+) 的百分比显着增加。在 CFU 测定中，对照组之间具有多谱系分化能力的祖细胞频率相似，但 CHIR/SB/LY 组显着增加。为了进一步评估 iPSC 衍生的造血 CD34+ 细胞的自我更新和分化能力，将来自第一轮 CFU 铺板的集落合并并在第二次 CFU 测定中重新铺板。值得注意的是，与对照组相比，我们观察到源自 CHIR/SB/LY 培养物的 CD34+ 细胞的总 CFU 数量增加了 3 倍。然而，这些细胞在移植到 NSG 小鼠受体后并没有维持长期的造血移植，这表明需要对该系统进行额外的修改。 目标 3. 鉴定 HSC 特异性超级增强子和相关的主转录因子 虽然体外 HSC 诱导需要激活动脉程序，但我们的数据表明，当前的培养条件无法调节与动脉发育无关的其他关键分子程序。我们假设定义 HSC 识别的核心 TF 网络激活不充分可以解释离体产生的 HSPC 缺乏植入潜力。由于 SE 代表细胞类型特异性主 TF 和转录共激活因子的密集结合平台，因此我们首先试图描绘 SE 景观及其在富含 HSC 的群体中调节的 TF。我们对从 3 名独立健康志愿者获得的动员 PB 样本中纯化出的表型定义的 CD34+CD38- HSC 进行了全基因组综合 ChIP-seq 和 ATAC-seq 分析。通过升高的 H3K27ac 信号密度总共鉴定了 873 个 SE；它们占增强子总数的 3.8%，其中值大小比典型增强子所包含的基因组区域大 10 倍。通过过滤 ATAC-seq 数据中的保守域，并应用结合基序富集和邻近算法，我们在人类 CD34+CD38- 细胞中鉴定了由 SE 调控的基因编码的 594 个 TF。通过询问人类 CD34+CD38- 细胞中已发表的基因表达数据集并通过无偏见的基因本体 (GO) 分析来验证精心策划的 TF 基因集。为了确定 HSC 身份的假定主调节因子，我们缩小了 594 个 SE 调节 TF 的列表，仅包括在其自身 SE 内具有结合基序的转录因子，这表明主 TF 具有独特的自动调节特性。共查获34个主要监管机构。 目标 4. 通过操纵主转录调节因子增强细胞工程 为了评估未发现的 HSC 特异性主 TF 对于离体生成功能性 HSPC 的潜在作用，我们独立地将 34 个 TF 的组装文库克隆到强力霉素诱导的慢病毒载体中，并用完整文库或 13 个文库的子集转导人类 iPSC根据 CFU 检测的初步测试以及 iPSC 衍生的 HSPC 中不存在表达，TF 被认为更有前途。使用我们之前优化的方案对转导的 iPSC 进行造血分化。从分化第 5 天开始，即动脉 HE 形成且 EHT 开始时，添加多西环素，允许造血主调节因子出现适合阶段的瞬时表达。为了评估培养第 12 天获得的 HSPC 的功能，将细胞进行 CFU 测定或股内移植到 NBSGW 免疫缺陷小鼠受体中。值得注意的是，与模拟转导的对照实验相比，我们观察到强制表达主转录因子的培养物中的 CFU 数量增加了 2 倍。在人类 iPSC 的造血分化过程中，随着主 TF 的引入，集落大小也有所增加，并且最原始的 CFU 类型（CFU-GM 和 CFU-GEMM）占优势。移植后两个月观察到人类 iPSC 衍生的 HSPC（PB 中 15%）的有效短期植入，但长期植入并未持续。