A New Approach to Expand Cord Blood Derived Hematopoietic Stem Cells, Using Bioengineered Human Fetal Liver Tissue 3D-Constructs

Despite advances in ex-vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of both long- and short-term repopulating cells, capable of functional engraftment, to enable the treatment of an adolescent or adult patient. We have previously shown that CB-HSPC can be effectively expanded in a 2D serum-free culture system, using a feeder layer of adult human bone marrow-derived stromal cells; still, the percentage of the most primitive stem cells decreased with time. Because, during development, the fetal liver is the main site of HSC expansion and differentiation, we hypothesized that efficient expansion of functional HSPC could be achieved in vitro under more physiologic conditions provided by surrogate fetal liver microenvironments. Therefore, we compared bioengineered liver constructs made from a natural 3D liver extracellular matrix (3DExM) seeded with hepatoblasts (HB), fetal liver-derived stromal cells (FLSC), or bone marrow-derived stromal cells (BMSC), with a 2D culture system using FLSC or BMSC. Overall, 2D culture systems generated a higher yield of mature blood cells by day 14, mostly within the myelomonocytic lineages, with fold increases in total cell number in 2D-HB, 2D-FLSC, and 2D-BMSC cultures of 1145, 16151, and 229, respectively, while 3D cultures generated fold increases of 94.3, 492, and 110, respectively.   Nevertheless, the output and expansion of more primitive HSPC was significantly higher in 3D cultures, as determined by flow cytometry and colony-forming assays. Specifically, in 3D-HB cultures, the percentage of CD34+CD38- cells increased by 100% at day 2, while 3D-FLSC and 3D-BMSC cultures each supported a 90% increase in the percentage of CD34+CD38- cells during this period. By contrast, all of the 2D cultures experienced a 30% decrease in the percentage of CD34+CD38- cells during this same time period. In addition, only 3D conditions maintained CD34+CD38- cells until day 12 of culture. Colony-forming assays demonstrated that the CFU-GEMM output was higher in 3D cultures when compared with their 2D counterpart. Among the 3D cultures, the 3D-HB cultures had the highest number of CFU-GEMM at day 2 and day 4. In conclusion, we demonstrate that by integrating biological components in vitro to obtain structures that contain all the necessary elements to mimic the fetal liver microenvironmental niches, which are known to promote rapid expansion of HSC during development, we were able to achieve significant expansion and maintenance of CD34+CD38- cells. In addition, since little is known about fetal liver niches that support HSPC expansion, the 3D constructs will provide, for the first time, a model system in which to dissect the role of the individual cellular and matrix niche components in supporting CB HSPC maintenance, expansion, and differentiation.