T cell expansion from umbilical cord blood without thymic stroma cells after stimulation with SCF, IL-7, AND IL-2

We analyzed in vitro expansion and differentiation of T progenitor cells from umbilical cord blood in the absence of thymic epithelium. The expansion setup is performed in the presence of SCF, IL-7, and IL -2 with autologous serum. Using CBMCs as initial source, we compared the growth kinetics of several cell populations in either whole CBMC or CD34+ -enriched-, as well as in CD3CD4CD8-depleted expansion assays by FACS analysis. After 11 days of culture, cell increase values were about 7 fold for CD3+, 6 fold for CD3+CD4+, 7 fold for CD3+CD8+, 4fold for CD3+CD56, 6fold for CD56+, and 0.2 fold for CD34+. We characterized the developmental state of these cell populations by RT –PCR analysis of the lymphoid differentiation markers RAG-1 and pre T-Alpha. In all samples, transcripts of both markers could be detected from day 0 though day 11, however, in case of pre – T-Alpha, nested PCR was always required, indicating lower expression. These findings; therefore, demonstrate that T-cell differentiation events (as opposed to mere expansion) do occur in stroma cell free expansion assays.