Generation of mesenchymal stromal cells from HOXB4-expressing human embryonic stem cells

HOXB4 transcription factor plays an important role in embryonic and adult hematopoiesis. Overexpression of HOXB4 in murine and human embryonic stem cells (ESC) has been used to generate hematopoietic stem cells (HSC) via the embryoid body formation method.

We used FuGENE 6-based transfection of YPL2-HOXB4 vector to generate HOXB4-expressing colonies from human ESC line H9 and investigated the potential of these cells for differentiation into primitive CD34(+) hematopoietic cells, via co-culture methodology with OP9 murine bone marrow stromal cells. Expression of HOXB4 in transfected human ESC colonies and their derivatives was verified using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR).

Utilizing OP9 stromal cell co-culture methodology, we generated CD34(+) cells from HOXB4-expressing H9 human ESC at a frequency similar to, and not higher than, non-transfected human ESC. However, we observed that some colonies of HOXB4-expressing human ESC not co-cultured on OP9 cells, differentiated into mesenchymal stromal cells (MSC) while preserving their HOXB4 expression. These HOXB4-expressing MSC expressed CD29, CD73, CD44, CD90, CD105 and HLA-class I, were negative for the expression of CD34, CD45, CD54, CD71, CD106 and HLA-DR, and could be differentiated into adipocytes and osteocytes.

In our specific experimental system we observed that overexpression of HOXB4 in human ESC did not improve the generation of CD34(+) hematopoietic cells via OP9 co-culture methodology. Furthermore, we could generate MSC from human ESC over-expressing HOXB4.