Generation of BMEC Lines and in vitro BMEC-HSPC Co-culture Assays.

Endothelial cells (ECs) sustain the self-renewal and regeneration of adult hematopoietic stem and progenitor cells (HSPCs) via deployment of EC-derived paracrine factors, termed as angiocrine factors. Generation of durable ex vivo vascular niche that maintains EC identity and preserves the angiocrine profile of organ of origin offers platforms for in vitro dissection of the mechanism by which angiocrine factors execute their instructive function for stem cell maintenance and tissue regeneration. This protocol describes detailed methods to isolate primary bone marrow ECs (BMECs), to subsequently transduce lentiviral vector carrying myristoylated-Akt1 into primary BMECs, and to use the Akt1-BMECs to expand engraftable murine HSPCs. The BMEC-HSPC co-culture system serves as bioreactor prototype to generate scalable populations of the blood and immune systems.