Cord Blood Expansion Inside a Bioengineered Liver

描述(申请人提供)：脐带血(CB)是临床上相关的造血干/祖细胞(HSPC)来源，用于治疗癌症和遗传性疾病。使用CB的优点包括容易获得，减少传播病毒感染的可能性，以及在人类白细胞抗原不相合的受者中诱发移植物对宿主疾病的风险较低。尽管如此，CB植入速度的延迟，以及单位造血干细胞(HSC)数量的相对较低，限制了其更广泛的使用。尽管在CB-HSPC扩展方面取得了进展，但在从单个单位获得足够数量的长期和短期再生细胞用于青少年或成年患者治疗的能力方面仍然存在挑战。我们之前已经证明，使用成人骨髓来源的基质细胞饲养层，CB-HSPC可以向髓系和淋巴系扩增和分化。利用该体系，我们对扩增细胞的初始祖细胞含量和细胞因子浓度进行了优化，表明扩增的细胞具有植入免疫前胚胎绵羊的能力。在该培养体系中，虽然长期移植的HSC的绝对数量增加，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，我们开发了三维(3D)、肝细胞外基质(ECM)来源的支架，并将其与胎儿肝母细胞和血管内皮细胞种植。这些细胞被植入其在肝脏ECM支架内假定的天然位置，随后 显示典型的内皮、肝和胆管上皮标志物，从而在体外形成类似肝脏的组织。众所周知，在发育过程中，胎肝是HSC的主要部位。 拓展和差异化。在胎肝内，HSC活跃地循环，这些细胞在移植后胜过成年HSC。因此，在肝组织内，存在促进不对称或对称的自我更新分裂的细胞生态位，导致原始HSC的维持或扩张。此外，CB-HSPC的初始划分行为高度依赖于环境。例如，基质细胞系AFT024和胎肝母细胞都来自小鼠，已被证明在二维培养中分别有效地保存了人和小鼠HSC的自我更新能力。因此，我们假设CB-HPSC可以在生物工程构建的人肝脏提供的生理条件下实现功能和有效的扩增。我们的最终目标是开发一种利用生物工程人肝组织高效扩增CB-HSPC的新平台。为此，我们将：1)确定3D生物工程人肝组织构建物支持CB来源的HSPC体外扩增的能力；2)检查并确定CB-HSPC与生物工程肝组织壁龛的单个细胞和基质成分之间发生的相互作用所产生的功能结果。完成后，这些研究将增加对胎肝壁龛如何支持HSC扩增的理解，更重要的是，将允许开发一种新的策略来从功能上扩大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.