Analysis of donor-derived cell frequency and telomerase activity during serial transplantation of HSCs. (A) FACS-purified HSCs (n = 150) from donor mTR^–/– mice (n = 4) and mTR^+/+ mice (n = 4) were serially transplanted until exhaustion of transplantation capacity. The fraction of donor-type (Ly5.1⁺) cells in the bone marrow of long-term (> 4 months) reconstituted mice (n ≥ 5) was determined by FACS analysis at each stage of serial transplantation. The decrease in frequency of donor-derived BM cells during the first and second rounds of serial transplantation of mTR^+/+ HSCs and mTR^–/– HSCs was statistically significant (P < .05; Student t test). Error bars (standard deviation) are shown. (B) HSCs (n = 250) from adult mice and recipient mice at each stage of serial transplantation were sorted directly into lysis buffer. Telomerase activity was measured for duplicate sample extracts at each stage of serial transplantation by the TRAP assay. A telomerase extract from NIH3T3 cells (n = 2500) was included as a positive control. The internal control for PCR efficiency is indicated by the arrow on the right. (C) Telomerase activity was measured for HSC samples from 3 or more mice at each stage of serial transplantation and averaged for all samples. The mean level of activity and error bars (standard deviation) are shown. We did not observe a significant change in telomerase activity level in HSCs during serial transplantation (P = .26).