Derivation of Lymphoid Lineage Cells from Human Embryonic Stem Cells on Co-Culture in OP9 and OP9DL1 Stromal Cells.

We have been able to direct the differentiation of CD34^pos cells derived from H9 and H7 hESCs toward lymphoid lineages using OP9 or OP9DL1 feeder layers. OP9 and OP9DL1 stromal cell lines are consistently better than S17 or OP9-jagged in inducing hESC-derived CD34^pos cells at different time points in culture. By day 14 in culture, about 8.7% and 7.9% of differentiated H9 hES cells harvested from co-cultures in OP9 or OP9DL1 respectively were CD34^pos. The CD34^pos cells in these cultures were enriched for by magnetic sorting. The clonogenic potential of differentiated hES cell suspensions and of sorted CD34^pos cells were determined using methylcellulose colony assays. The highest clonogenic potential was noted after 18 days in co-culture when we obtained 142 +/− 31 and 218 +/− 38 colonies per million cells plated from OP9 and OP9DL1. Selection for CD34^pos cells enriched the clonogenic activity 27-fold. Morphologic analyses of representative colonies from the methylcellulose cultures showed cells of the erythroid, myeloid, lymphoid and megakaryocytic lineages were generated. Directed differentiation of CD34^pos cells towards lymphoid lineages was initiated by replating the sorted CD34^pos cells onto irradiated OP9 or OP9DL1 stromal cells supplemented with stem cell factor (SCF), IL3, IL7 and Flt3L. Under these conditions, B lineage (CD19^pos) and myeloid lineage (CD14^pos or CD15^pos) cells were observed when CD34^pos cells were grown in OP9, and T lineage cells were observed when CD34^pos cells were grown in OP9DL1. CD4^posCD8^pos cells were evident after 12 days of secondary culture in OP9DL1 and were prominent by day 20. Only a few of the CD4^pos, CD8^pos and CD4^posCD8^pos cells, however, were noted to be CD3^pos. Addition of SCF + IL3, SCF + Flt3L, or SCF + Flt3L + IL7 at initiation of hESC differentiation increased the percentage of CD34^pos cells and/ or increased the hematopoietic (clonogenic) potential of sorted CD34^pos cells. CD34^pos cells derived from hESCs grown in OP9DL1 for 14 days and replated in OP9DL1 supplemented with Flt3L + IL7 +/− SCF generated the highest percentages of CD4^posCD8^pos cells. Additional phenotypic analyses and functional studies of sorted CD4^pos, CD8^pos and CD4^posCD8^pos cells generated from these cultures are needed to further characterize their lymphoid properties.