Cord Blood Expansion Inside a Bioengineered Liver

描述（由申请人提供）：脐带血（CB）是治疗癌症和遗传疾病的造血干/祖细胞（HSPC）的临床相关来源。使用CB的优点包括其容易获得，降低传播病毒感染的可能性，以及降低HLA不匹配受者诱导移植物抗宿主病的风险。 尽管如此，CB的移植速度延迟，以及每单位造血干细胞（HSC）数量相对较低，限制了其更广泛的应用。 尽管在CB-HSPC扩增方面取得了进展，但关于从单个单位获得足够数量的长期和短期再增殖细胞以治疗青少年或成年患者的能力仍然存在挑战。 我们之前已经证明，CB-HSPC可以使用成人骨髓源性基质细胞的饲养层向骨髓和淋巴谱系扩增和分化。使用该系统，我们优化了初始祖细胞含量和细胞因子浓度，并表明扩增的细胞具有植入免疫前胎羊的能力。 虽然长期移植的HSC的绝对数量在该培养系统中增加，但这些最原始的干细胞的相对百分比随着时间的推移而下降。 最近，我们已经开发了三维（3-D），肝细胞外基质（ECM）衍生的支架，并将其与胎儿成肝细胞和内皮细胞接种。这些细胞移植到肝脏ECM支架内的假定天然位置， 显示典型的内皮、肝和胆管上皮标记，从而在体外产生肝样组织。众所周知，在胚胎发育过程中，胎肝是HSC的主要部位 扩张与分化。 在胎肝内，HSC活跃地循环，并且这些细胞在移植时胜过成人HSC。因此，在肝组织内，存在促进不对称或对称自我更新分裂的细胞小生境，导致原始HSC的维持或扩增。此外，CB-HSPC的初始分裂行为高度依赖于环境。例如，在2-D培养物中，已经显示鼠源的基质细胞系AFT 024和胎儿成肝细胞分别有效地保持人和小鼠HSC的自我更新能力。 因此，我们假设CB-HPSC的功能性和有效的扩增可以在生物工程人肝构建体提供的生理条件下实现。我们的最终目标是开发一种新的平台，用于使用生物工程化的人肝组织有效扩增CB-HSPC。 为此，我们将：1）确定3-D生物工程化的人肝组织构建体支持CB衍生的HSPC的离体扩增的能力，和2）检查和定义由CB衍生的HSPC之间发生的相互作用引起的功能结果。HSPC和生物工程化肝组织小生境的单个细胞和基质组分。完成后，这些研究将增加对胎肝小生境如何支持HSC扩增的理解，更重要的是，将允许开发一种新的策略来功能性地扩增CB-HPSC。

DESCRIPTION (provided by applicant): Cord blood (CB) is a clinically relevant source of hematopoietic stem/progenitor cells (HSPC) to treat cancer and genetic diseases. The advantages of using CB include its ready availability, the reduced probability of transmitting vira infections, and the lower risk of inducing graft vs. host disease in HLA-mismatched recipients. Still, CB's delayed speed of engraftment, and the relatively low number of hematopoietic stem cells (HSC) per unit, limit its broader use. Despite the advancements made in CB-HSPC expansion, challenges remain regarding the ability to obtain, from a single unit, sufficient numbers of both long-and short-term repopulating cells, for treatment of an adolescent or adult patient. We have previously shown that CB-HSPC can be expanded and differentiated towards both the myeloid and lymphoid lineages, using a feeder layer of adult human bone marrow-derived stromal cells. Using this system, we optimized the initial progenitor content and cytokine concentrations, and showed that expanded cells had the ability to engraft pre-immune fetal sheep. While the absolute number of long-term engrafting HSC increased in this culture system, still, the relative percentage of these most primitive stem cells decreasd with time. Recently, we have developed three- dimensional (3-D), liver extracellular matrix (ECM)-derived scaffolds and seeded them with fetal hepatoblasts and endothelial cells. These cells engrafted in their putative native locations within the liver ECM scaffolds, and subsequently displayed typical endothelial, hepatic, and biliary epithelial markers, thus creating a hepatic-lik tissue in vitro. It is well known that, during development, the fetal liver is the main site of HSC expansion and differentiation. Within the fetal liver, HSC actively cycle and these cells outcompete adult HSC upon transplantation. Thus, within the hepatic tissue, cellular niches exist that promote asymmetric or symmetric self-renewal divisions, leading to maintenance or expansion of primitive HSC. In addition, the initial divisional behavior of CB-HSPC is highly dependent upon the environment. For example, the stromal cell line AFT024 and fetal hepatoblasts, both of murine origin, have been shown, in 2-D cultures, to effectively preserve the self- renewal capacity of human and mouse HSC, respectively. Therefore, we hypothesize that a functional and efficient expansion of CB-HPSC can be achieved under physiological conditions provided by the bioengineered human hepatic constructs. Our ultimate goal is to develop a novel platform for the efficient expansion of CB- HSPC using bioengineered human liver tissue. To this end, we will: 1) Determine the ability of 3-D bioengineered huma liver tissue constructs to support ex-vivo expansion of CB-derived HSPC~ and 2) Examine and define the functional outcome arising from interactions that occur between CB-HSPC and individual cellular and matrix components of the niches of the bioengineered liver tissue. Upon completion, these studies will add to the understanding of how fetal liver niches support HSC expansion, and, more importantly, will allow the development of a novel strategy to functionally expand CB-HPSC.