Abstract

The early hematopoietic/endothelial progenitor (hemangioblast) has been recently described; however, HLA antigen expression of this progenitor was not characterized as yet. We have isolated blast-like cells (BLC) from ES/EB-based cultures and characterized their HLA-expression and ability to form hematopoietic and endothelial colonies. We used the two-step differentiation technology to obtain the bipotential blast cells from human embryonic stem cells (hES). This involved short differentiation in embryoid body (EB) system followed by differentiation in semisolid culture medium (Methocult H4436), supplemented with mixture of cytokines (BMP-4, VEGF, SCF, Flt3l, and TPO). First, we have shown that the occurrence of hemangioblast (BL-CFC) during EB differentiation (day 0–6) is transient and peaks on day 3. On this day about 125±35 out of 2400 EB cells form blast colonies. The emergence of this event was associated with the expression of T (early mesoderm gene) and was suppressed by the development of endoderm layer in subsequent days in culture. Similarly, the highest HB occurrence was associated with dramatic increase in expression of early hematopoietic/endothelial genes: CD34, CD31 and KDR. The similar pattern of expression of Patch1 and Gli1 genes suggests association with hedgehog (Hh) pathway activation during this process. The BL-CFC colonies were composed of 30–50 blast cells on day 6 of culture and had homogenous morphology in Wright-Giemsa stain with big nucleus containing disorganized chromatin and narrow rim of cytoplasm filled with large-size granules. However, as shown by FACS staining, they were quite heterogenous and expressed markers of both immature hematopoietic and endothelial progenitors (CD31, CD34, VE-cadherin, Flt-1) and mature differentiated cells (CD45, CD33, CD146). At least some of them expressed fetal and embryonic globin genes as shown by RT-PCR. Moreover, they could be characterized by high expression of HLA class I molecules by immunofluorescence, when compared to hES and EB cells. The ES cells, EB-derived cells and blast-like cells (BLC) were stained with FITC-labeled anti-HLA-A2 antibody BB7.2 and cells were observed under fluorescent microscope. While undifferentiated ES cells were negative for HLA-A2, the EB cells showed slight expression and the blast-like cells were strongly positive for HLA-A2 expression. These blast-like cells (BLC) could be successfully differentiated to hematopoietic cells in standard CFU assay. In these conditions, blast cells formed CFU-M colonies (63.4±0.8%) containing macrophages, BFU-E (19.5±3.5%) containing nucleated red blood cells and CFU-EM colonies (17.1±2.7%) composed of macrophages and nucleated erythrocytes. The cells of CFU-EM and BFU-E expressed both epsilon- and gamma-globin genes, but not adult-type beta-globin. When in endothelial cell culture conditions, the blast cells differentiated to endothelial cells which had the ability to uptake Dil-Ac-LDL and form complicated vascular networks in Matrigel. Concluding:

  1. Hemangioblasts transiently exist in early embryonic development and can form single cell-derived colonies,

  2. differentiation of hemangioblasts can be tracked by the use of chosen molecular markers

  3. blast-like colonies (BL-CFC) consist of cells having properties of endothelial and hematopoietic precursors, however, the issue if they can maintain dual properties over time needs to be further explored

  4. these blasts-like cells can be potentially used in regenerative medicine, however, strong expression of HLA molecules may limit their clinical applications in the future.

Disclosures: Carrier:Celgene: Speakers Bureau; Millenium: Speakers Bureau; Novartis: Speakers Bureau; America Stem Cell: Membership on an entity’s Board of Directors or advisory committees.

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