A Genetically Engineered Human Fetal Liver Niche as a Novel Platform for Biomanufacturing of Hematopoietic Stem Cells

人类造血干细胞(HSCs)移植可以治疗一系列血液系统恶性肿瘤 遗传性血液疾病。然而，由于缺乏合适的捐献者和干细胞数量较少，成功受到限制。 可从常见的HSC来源获得。到目前为止，造血干细胞的体外扩增用于增强体内植入 患者在临床上一直无效。培养过程中产生的细胞数量不足或分化不良 在体外启动细胞群体一直是导致这些细胞临床生物制造不当的因素。 目前的造血细胞生物制造技术昂贵，这进一步复杂化了 可伸缩性和广泛的临床翻译。为了克服这些关键障碍，帮助实现完整的生活- 保存造血干细胞的潜力，在体外维持和扩增患者来源的造血干细胞的新方法是 需要的。干细胞的自我更新和分化是通过与邻近细胞的复杂串扰来调节的 类型，它们分泌和组织一个多方面的信号提示环境(干细胞利基)。清除干细胞 会破坏这种动态平衡。造血干细胞在骨骼中经历有限的自我更新 骨髓龛(BM)，通常是静止的。相比之下，在胎肝中，HSCs经历了显著的扩张和 变得高度增殖，这表明胎儿肝脏的生态位为 造血干细胞。然而，由于伦理限制，获得可存活的人类胎儿肝脏是具有挑战性的。由基因决定的 人类诱导多能干细胞(HiPSCs)的工程，我们第一次可以产生胎儿肝脏 具有造血龛能力的组织。在我们的方法中，GATA6的瞬变和非均匀脉冲 转录因子作用5天后，培养中胚层和内胚层共同发育。这个 进一步自组织培养成有功能的人胎肝组织(包含造血细胞)，不需要 需要在培养中加入外源生长因子。我们的目标是发展一种普遍的、 用于扩展人类HSC的通用平台，可扩展、简单且经济。我们 假设我们的人胎肝组织自主产生已知和未知的因子 有助于造血，并能为我们提供一个“通用的”和“可编程的”细胞 微环境，或为此目的的利基。在目标1中，我们将使用GATA6设计的胎儿肝脏利基 (FLIN)用于HSC的扩增。在目标2中，我们将通过以下方式询问造血生态位环境 设计一个可定制的胎儿肝脏，设计肝脏利基(Deslin)，在Aim 3中，我们将检查 微载体搅拌悬浮生物反应器中FLIN-HSC培养的可扩展性。总体而言， 该平台的开发和优化有可能极大地降低大规模的成本 并将阐明影响造血的生物学和关键信号分子。 随后，所提供的知识和工具将适用于广泛的血液学领域 包括恶性肿瘤和遗传病在内的疾病。

Human hematopoietic stem cells (HSCs) transplants can treat a range of hematological malignancies and genetic blood disorders. However, success is limited by a lack of optimal donors and low number of stem cells available from common HSC sources. To date, expansion of HSCs ex vivo for enhanced in vivo engraftment in patients has been clinically ineffective. Insufficient cell numbers generated in culture or poor differentiation of the starting cell population ex vivo has been contributing factors to improper clinical biomanufacturing of these cells. The current techniques of hematopoietic cell biomanufacturing are expensive which further complicates scalability and wide clinical translation. To overcome these critical barriers and help achieve the full life- saving potential of HSCs, novel approaches to maintain and expand patient-derived HSCs in vitro are needed. Stem cell self-renewal and differentiation are regulated through intricate crosstalk with neighboring cell types, which secrete and organize a multifaceted milieu of signaling cues (stem cell niche). Removing stem cells from their native environment can disrupt this homeostasis. HSCs experience limited self-renewal in the bone marrow niche (BM) and are typically quiescent. In contrast, in fetal liver, HSCs undergo marked expansion and become highly proliferative, which suggests that the fetal liver niche provides a unique microenvironment for HSCs. However, access to viable human fetal liver is challenging due to ethical constraints. By genetically engineering human induced pluripotent stem cells (hiPSCs), for the first time we could generate a fetal liver tissue with hematopoietic niche capacity. In our approach, a transient and heterogeneous pulse of GATA6 transcription factor for 5 days resulted in co-development of mesoderm and endoderm layers in culture. The culture further self-organized into a functional human fetal liver tissue (containing hematopoietic cells) without the need to add exogenous growth factors to the culture. Our objective is to develop a universal, and common platform for expansion of human HSCs that is scalable, simple, and economical. We hypothesize that our human fetal liver tissue autonomously produces known and unknown factors that contribute to hematopoiesis and can provide us with a “universal” and “programmable” cellular microenvironment, or niche for this purpose. In aim 1 we will employ a GATA6-engineered Fetal LIver Niche (FLIN) for the expansion of HSCs. In aim 2, we will interrogate hematopoietic niche environment through engineering a customizable fetal liver, DESigner Liver Niche (DESLIN) and in aim 3, we will examine scalability of FLIN-HSC cultures in microcarrier-based Stirred suspension bioreactors. Overall, the development and optimization of this platform has the potential to dramatically reduce the cost of large-scale production of HSCs and will shed light on the biology and key signaling molecules affecting hematopoiesis. Subsequently, the delivered knowledge and tools will be applicable in a broad spectrum of hematological diseases including malignancies and genetic disorders.