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

人造血干细胞（HSC）移植可以治疗一系列血液恶性肿瘤， 遗传性血液疾病然而，由于缺乏最佳供体和干细胞数量少， 可从普通HSC来源获得。迄今为止，HSC的离体扩增用于增强体内植入， 患者在临床上无效。培养物中产生的细胞数量不足或细胞分化不良， 离体起始细胞群是这些细胞的不适当临床生物制造的促成因素。 目前的造血细胞生物制造技术是昂贵的，这进一步使其复杂化。 可扩展性和广泛的临床翻译。为了克服这些关键的障碍，帮助实现完整的生活- 为了节省HSC的潜力，体外维持和扩增患者来源的HSC的新方法是 needed.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，分泌和组织多方面的环境信号线索（干细胞龛）。去除干细胞 会破坏这种自我平衡HSC在骨骼中经历有限的自我更新 骨髓龛（BM），通常是静止的。相反，在胎儿肝脏中，HSC经历显著的扩增， 变得高度增殖，这表明胎肝龛为胎儿提供了一个独特的微环境。 HSC。然而，由于伦理约束，获得可行的人类胎肝是具有挑战性的。通过遗传 通过改造人类诱导多能干细胞（hiPSC），我们第一次可以产生胎儿肝脏， 具有造血生态位能力的组织。在我们的方法中，GATA 6的瞬时和非均匀脉冲 转录因子作用5天导致培养物中中胚层和内胚层的共同发育。的 培养物进一步自组织成功能性人胎肝组织（含有造血细胞）， 需要向培养物中添加外源性生长因子。我们的目标是发展一个普遍的， 用于扩增人HSC的通用平台是可扩展的、简单的和经济的。我们 假设我们的人类胎儿肝脏组织自主产生已知和未知的因子， 有助于造血，可以为我们提供一个“通用”和“可编程”的细胞 微环境，或利基为此目的。在目标1中，我们将采用GATA 6工程胎肝利基 （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，DESigner肝脏利基（DESLIN），在目标3中，我们将检查 FLIN-HSC培养物在基于微载体的搅拌悬浮生物反应器中的可扩展性。总体看 该平台的开发和优化有可能大大降低大规模生产的成本。 造血干细胞的生产，并将阐明生物学和影响造血的关键信号分子。 随后，所提供的知识和工具将适用于广泛的血液学 疾病，包括恶性肿瘤和遗传性疾病。

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.