MESOP9cell coculture system for haematopoietic differentiation

Our mES/OP9-cell coculture regime (Protocol 2) is summarized schematically in Figure 1. It represents a modification of the original system (13,15), in order to allow the sequential expansion of primitive and definitive haematopoietic cells.

The key steps are as follows:

• Undifferentiated mES cells, growing exponentially on MEF feeder layers, are reseeded sparsely onto confluent monolayers of OP9 stromal cells, in medium lacking LIF but supplemented with a higher level of FCS, and with vascular endothelial cell growth factor (VEGF) and bone morphogenetic protein 4 (BMP-4), to promote mesoderm induction.

• Following the preliminary, mesodermal differentiation of mES cells, coculture is continued in the presence of the growth factors, erythropoietin (Epo) and stem cell factor (SCF), and the cytokine, interleukin-3 (IL-3), to induce primitive and definitive haematopoiesis from mesodermal derivatives. Subsequently, mature erythroid cells (i.e. eryP) are released into the supernatant medium, and these may be readily harvested.

• Adherent cells in the cocultures are periodically harvested and reseeded onto fresh monolayers of OP9 cells, to avoid overgrowth of mES-cell derivatives by the proliferating OP9 cells.

• A population of definitive erythroid progenitor cells remains attached to the OP9 monolayer, and may be harvested with relative facility. The efficiency of production of erythroid progenitor cells is evaluated by colony assay (Protocol 3).

The sequential stages of haematopoietic cell differentiation in this system are described in the following sections.

Mouse ES cells maintained on feeder cells

Mouse ES cells maintained on feeder cells

Figure 1 Schematic of mES/OP9-cell coculture system for haematopoietic differentiation.
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