A bio-engineered hepatic niche for ex vivo expansion of HSCs

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Abstract / Project Summary New Directions in Hematology Research (SHINE-II) PAS-18-730 NIDDK Title: Expansion of bone marrow hematopoietic stem cells in modular human liver culture platforms The hematopoietic stem cell (HSC) resides at the top of the hierarchy of blood cells and is responsible for the generation of all the progenitor and mature cells of the hematopoietic system. How HSCs are capable of generating the vast diversity of hematopoietic cell types while still retaining their self-renewal and proliferative capacity is a topic of active research in the field. Many studies have pointed to the niche as the critical source of extrinsic factors that instruct and support HSC cell fate decisions. The primary niche for homeostatic hematopoiesis in adult mammals is the bone marrow. Much debate still exists regarding the specific cell types of the bone marrow niche, but it is generally accepted that in the bone marrow HSCs are primarily quiescent and infrequently activate for expansion and differentiation of progenitor cell types. Likewise, experiments that have attempted to expand HSCs ex vivo using bone marrow stromal cells or cytokine cocktails in tissue culture plastic have not been successful in generating robust and renewable stem cells capable of long-term, multi-lineage reconstitution of irradiated recipients. We hypothesize that an approach based on the ontogeny of hematopoietic system will allow for a more successful ex vivo expansion of HSCs. Previous findings have shown that HSCs expand very rapidly during embryonic development in the fetal liver. Furthermore, under stress or due to certain pathological conditions, HSCs migrate to the adult liver where they undergo extramedullary hematopoiesis (EmH). Following these physiological cues, we propose to bioengineer ex vivo co-culture systems of increasing complexities using primary human hepatocytes (PHH), supportive fibroblasts, and primary human liver non- parenchymal cells (NPC). We will first utilize our long-term micropatterned co-culture (MPCC) system of PHH colonies surrounded by supportive fibroblasts (3T3-J2 murine embryonic fibroblasts and human liver portal fibroblasts or LPF) to which we will introduce adult bone marrow HSCs cultured in an optimized serum-free culture medium supplemented with hematopoietic cytokines. We will monitor HSC expansion in MPCCs and characterize them phenotypically via flow cytometry prior to assessing their functional potential in vitro and in vivo in mouse transplantation assays. Next, we will incorporate HSCs into novel 3-dimensional (3D) human liver microtissues that we have shown to enable optimal interactions of PHHs and primary human liver sinusoidal endothelial cells (LSEC); the PHH-LPF-LSEC microtissues will be further augmented with quiescent hepatic stellate cells and Kupffer cells to elucidate the role of these cell types in modulating HSC expansion ex vivo. Lastly, we will induce EmH in mice to prime HSCs for the liver microenvironment and then harvest and expand them in human liver platforms of increasing cellular complexities as above. In conclusion, the overall goal of this focused SHINE II proposal is to create physiologically-relevant, long-term, and modular human liver platforms for the expansion and study of adult HSCs and their interactions with key cell types within the liver.