Human Haemato-Endothelial Precursors: Cord Blood CD34+ Cells Produce Haemogenic Endothelium
Figure 3
Functional characterization of adherent cells in MH-CM culture.
A- left panel -Clonogenetic assay of adherent cells generated by CD34+ cultured in MH-CM for 30 days, carried out in semi-solid culture supplemented with the indicated medium. Right panel - Light microscopy image of a single cell-derived colony grown in semi-solid EGM2 endothelial medium. A representative experiment out of 5 is shown. B-Phase-contrast morphology of adherent cells cultured on matrigel supplemented with EGM-2 for 12 hours (original magnification 10x). A representative experiment out of 5 is shown. C- Immunohistochemistry evaluation of adherent cells, generated by CD34+ cultured in MH-CM for 30 days, engrafted in breast cancer-bearing NSG mice (n = 5). Human CD34 (i,ii,iii) and human CD31 (iv) detect functional vessels containing red blood cells. These antibodies are human-specific and do not cross react with mouse vessels (vi). A representative experiment out of 5 is shown. (v) Tumor volume quantification showing that the volumes of tumors where breast cancer cells were co-injected with the adherent cells generated by CD34+ cells are significantly higher (* p<0.04) than the volume of tumors where breast cancer cells alone were injected in NSG mice (n = 5/group). D-Confocal laser-scanning of human CD34 antigen distribution in a tumor section of a MB-MDA436 plus adherent cells, generated by CD34+ cultured in MH-CM for 30 days. Images were acquired using a Leica TCS SP5 confocal microscope and sequential Z-stacks were performed using a 63×1.4NA oil immersion objective, zoom 3X, 0.3 µm z step. Snapshot images are orthogonal sections of the z-stacks taken at points along the vessel’s cavity. A representative picture of one tumor out of 5 is shown. E- Expression levels of haemopoietic and endothelial marker genes obtained from microarray analysis of fresh CB CD34+ cells (CD34), the adherent cellular progeny obtained after 53 days of culture in MH-CM (Adherent Cells in MH-CM) and this adherent cellular progeny grown in endothelial medium (EGM2) for additional 7 days (Adherent Cells in Endot. Medium). Expression values were normalized using CD34+ cells as control. A global up-regulation of endothelial marker genes was observed in adherent cells grown in MH-CM, both prior to and after instruction by EGM2, whereas haemopoietic markers were either unvaried or down-regulated. A representative analysis out of 3 is shown. F- Growth curve of the adherent cellular progeny obtained from CB CD34+ cells grown in MH-CM when transferred into EGM2 endothelial medium. A representative experiment out of 10 is shown.