Maintenance of NGFR transgene expression in CAFC progeny. (A) Sorted NGFR-expressing, CD34⁺Lin⁻ cells transduced with either the LINGFR (LIN, NGFR⁺) or MINGFR (MIN, GFR⁺) vectors, or the sorted CD34⁺Lin⁻ cells lacking NGFR expression at day 2 posttransduction with LINGFR (LIN, NGFR⁻) or MINGFR (MIN, NGFR⁻) vectors (see Fig 3) were plated on SyS-1 stromal cell monolayers. Two-fold serial dilution of 100 CD34⁺Lin⁻ input cells were seeded per well and examined for cobblestone area (CA) formation weekly up to 6 weeks. The number of wells lacking any CA at week 5 were plotted as a function of numbers of sorted cells, respectively. The frequencies of CAFC were estimated based on Poisson distribution and the results are indicated. (B) NGFR transgene expression in B cells formed at week 6 of CAFC assays. CD19⁺ B cells (in addition to CD14⁺and CD15⁺ myeloid cells) were generated from sorted CD34⁺Lin⁻NGFR⁺ cells in the presence of SyS-1 stromal cells as shown by the presence of the CD19 marker. Among CD19⁺ B cells, approximately 14% and 50% of the progeny cells derived from LINGFR-transduced (LINGFR⁺) and MINGFR-transduced (MINGFR⁺) CD34⁺Lin⁻NGFR⁺ cells, respectively, expressed the NGFR transgene at the end of CAFC assays.