The expansion of Stat3-C–transduced CRUs in recipients that underwent transplantation is markedly enhanced. (A) Experimental design for assessing the expansion of HSCs in mice that underwent serial transplantation. From the CRU frequencies obtained before and after the primary and secondary recipients underwent transplantation, input and output GFP⁺ CRU numbers were determined and then used to calculate the extent of CRU expansion obtained in each group. (Detailed results from which the CRU frequencies shown were determined are provided in Table S3). (B) Expansion of GFP⁺ CRU numbers in primary and secondary recipients of MIG-transduced (▨) and Stat3-C–transduced (▪) bone marrow cells from the experiments shown in panel C. Values shown are the mean ± SEM for each group. (*P < .05 in a Student t test ANOVA by comparison with the value for MIG-transduced cells). (C) Cumulative expansion of transduced (GFP⁺) CRUs during the course of 2 serial transplantations. The number of Stat3-C– or MIG-transduced CRUs injected into primary recipients (Input) was determined from a separate analysis of their frequency. The primary (1°) output indicates the total number of GFP⁺ CRUs determined to be present in the marrow of the primary mice (mean ± 2 SEM) 40 weeks later. Marrow cells from the primary recipients of STAT3-C–transduced cells (ie, SC no. 21, SC no. 22, and SC no. 23) were assessed individually. For the 4 primary recipients of MIG-transduced cells, the marrow cells were pooled and then assayed. The further expansion of GFP⁺ CRUs attained after 48 weeks in secondary recipients was similarly determined from assays in tertiary recipients. Cumulative values (2° outputs) were calculated by multiplying the numbers obtained from the primary mice (1° output) by the further expansion calculated to have occurred in the secondary mice (for details, see Table S3). (D) Southern blot analysis of proviral integrations in the cells regenerated from Stat3-C–transduced HSCs. Analyses of the bone marrow from a recipient of a limiting number of Stat3-C–transduced CRUs (SC no. 23: 0.3-2.2 CRUs, 95% CI; and SC nos. 21-22: 1.6-11 CRUs, 95% CI [from panel C]) and from their secondary and tertiary recipients of these cells are shown. Genomic DNA was digested with Hin dlll and electrophoresed, and blots were hybridized with a GFP probe. M denotes molecular weight markers, and vertical lines designate noncontiguous lanes in the same autoradiography. Each lane contained DNA from an individual mouse.