(B) Bar graphs reflect human CD45+ cell engraftment in the BM and PB as well as frequency of CD34+ cells in the BM of transplanted mice. LMPPs were observed. cultures conditions supporting the growth of multipotent HSPCs has been reported within the last years8C12. One encouraging strategy employs a feeder-based co-culture system to mimic the bone marrow (BM) stem cell niche for the growth of multipotent HSPCs for experimental, pre-clinical as well as clinical methods13C16, examined in17,18. The quantification of multipotent HSPCs is commonly performed according to the lineage-relationships proposed by the classical model KRCA-0008 of human hematopoiesis. According to this classical model, HSCs and multipotent progenitors (MPPs) are the only cells made up of both myeloid as well as lymphoid differentiation potentials. However, the classical model of hematopoiesis has in the mean time been challenged by several groups proposing option lineage-relationships and read-outs for multipotent HSCs/MPPs19C22. In this context, we have shown that human CD133+CD45RA?CD34+ HSPCs are enriched for multipotent HSPCs19. growth, we recently re-evaluated the reported potential of murine stromal cell lines (AFT024, OP9, MS5) as well as human mesenchymal stromal cell (MSCs) from numerous tissues to support the growth of UCB-derived HSCs/MPPs15. In these experiments, none of the KRCA-0008 tested culture conditions supported the growth or maintenance of primitive CD133+ HSPCs with erythroid differentiation potentials. However, all tested conditions exhibited strong growth of phenotypical and functional LMPPs. While these experiments were exclusively performed with a mono-layer of murine stromal cells or human MSCs, the cellular composition of the BM stem cell niche is known Rabbit polyclonal to ZKSCAN4 to be much more complex and involves a variety of different cell types, signaling molecules as well as other soluble/cell-bound factors27C31. Another crucial cellular component of the stem cell niche and being a major KRCA-0008 contributor to HSC maintenance has recently KRCA-0008 been attributed to endothelial cells (ECs)32,33. Synergistically with MSCs, both cell types were shown to be essential components for HSC maintenance, and knockout of either cell type led to specific depletion of phenotypically and functionally unique HSC/MPP subsets32,33. Based on these findings, we decided to investigate whether main ECs either alone or in combination with MSCs support the growth and/or maintenance of CD133+ HSPCs with erythroid differentiation potential. Furthermore, we tested the growth capabilities of HSCs/MPPs in an environment, i.e. in a xenograft repopulation model in immunodeficient NSG (Non-obese diabetic scid gamma) mice. Results Main ECFCs and HUVECs are phenotypically and functionally homogeneous Human ECs can be very easily generated from numerous tissues. KRCA-0008 Here, we raised ECs from five impartial UCB models termed endothelial colony forming cells (ECFCs) and from umbilical veins of five different umbilical cords, classically termed human umbilical vein endothelial cells (HUVECs). Within our analyses, we did not detect any striking phenotypic differences between ECFCs and HUVECs. All ECs homogenously expressed the cell surface markers CD31, CD73, CD105, CD144, VEGFR2 and bound the lectin Ulex (Figs?1B, S1). Expression of hematopoietic (CD15 and CD45) and mesenchymal (CD90) cell surface markers was not detected (Figs?1B, S1)34. ECs were able to take up acetylated low-density lipoprotein (AcLDL), to store Von Willebrand Factor (vWF) in Weibel-Palade body and to form tube-like structures in Matrigel assays (Figs?1C, S2)34. In summary, all obtained main ECFCs and HUVECs fulfilled the widely-accepted criteria of bona fide ECs. ECFCs and HUVECs promote growth of CD133+CD34+ HSPCs To test.