An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation.

While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC.