Lineage analysis of clones generated from individual CD34⁺lin⁻CD7⁻ thymocytes. Clones A and B were generated from different single CD34⁺lin⁻CD7⁻ cells isolated by FACS, and cultured on OP9 stroma. (A) FACS of each clone harvested from OP9 stroma analyzing B (CD19) and erythroid (glycophorin; top) and NK (CD56 vs CD7) potential (middle). The same clones were replated into methylcellulose medium and generated CFU, which were analyzed by FACS for myeloid and erythroid potential (CD66b vs glycophorin; bottom). (B) RT-PCR analysis of clones A and B (at a different time point to FACS analysis) reveals the expression of T lymphoid (pTα), erythroid (glycophorin), and/or B lymphoid (Pax 5) genes. In these assays, the lineages produced from each clones fluctuate over time in culture (eg, clone B has CD19⁺ cells at one time but is negative for Pax 5 later in culture). Thus, lineage potential is assigned based on either FACS or RT-PCR marker expression at any time during the life of the clone.