Cord Blood Expansion Inside a Bioengineered Liver

描述（由申请人提供）：脐带血（CB）是治疗癌症和遗传病的造血干细胞/祖细胞（HSPC）的临床相关来源。使用CB的优点包括其易于获得，传播病毒感染的可能性降低，以及在hla不匹配的受体中诱导移植物抗宿主病的风险较低。然而，CB的移植速度较慢，每单位造血干细胞（HSC）的数量相对较低，限制了其广泛应用。尽管在CB-HSPC扩展方面取得了进展，但对于青少年或成人患者的治疗，从单个单位获得足够数量的长期和短期再生细胞的能力仍然存在挑战。我们之前已经证明，使用成人骨髓来源的基质细胞喂养层，CB-HSPC可以向髓系和淋巴系扩展和分化。利用该系统，我们优化了初始祖细胞含量和细胞因子浓度，结果表明扩增后的细胞具有移植免疫前胎羊的能力。在该培养体系中，长期移植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减少。最近，我们开发了三维（3-D）肝细胞外基质（ECM）衍生的支架，并将其植入胎儿肝母细胞和内皮细胞。这些细胞在肝脏ECM支架内被植入其假定的天然位置，随后

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.