Constitutive expression of MN1 immortalizes human CB cells in vitro. (A) Outline of stromal feeder-containing LTC-IC assay or LTC with human CB cells. (B) Western blot showing protein expression of MN1 in MN1-transduced mouse BM and CB cells and FLAG protein (FLAG-ND13) in ND13-transduced CB cells. β-actin was probed on the same blot as loading control. (C) Proportion of GFP⁺ cells 2 days after the end of transduction and after culture on feeder cells for 56 days (at the time of CFC plating) for CTL- and MN1-transduced CB cells (CTL, n = 9; MN1, n = 14). (D) Total yield of colonies from primary LTC assay of CTL- or MN1-transduced CB cells (CTL, n = 9; MN1, n = 14). (E) Outline of serial stromal feeder-containing LTC assay. After each LTC, cells were placed in CFC media and a proportion of them were replated on fresh-feeder layers for the next round of LTC assay. (F) Cumulative total cell output from primary to quaternary LTC assays (CTL, n = 2; MN1, 2nd n = 6, 3rd n = 4, 4th n = 2). (G) Cumulative CD34⁺ cell output from primary to quaternary LTC assays (CTL, 1st n = 5; MN1, 1st n = 10, 2nd n = 5, 3rd n = 2, 4th n = 1). (H) Number of CFCs recovered from primary to quaternary LTC assays (CTL, first n = 2, second n = 2; MN1, first n = 6, second n = 6, third n = 4, fourth n = 2). (I) Proportion of hematopoietic progenitor cells in LTCs of CTL- and MN1-transduced CB cells (day 60 on feeder cells, a representative fluorescence-activated cell sorter [FACS} blot is shown). (J) Morphology of CTL- and MN1-transduced CB cells at the end of the 60-day culture period. Micrographs were acquired using an Axioplan2 Zeiss microscope and images were captured using OpenLab software (Perkin Elmer); original magnification ×400 for CTL and ×630 for MN1. Data represent mean ± standard deviation. *P < .05, **P < .01, ***P < .001. CMP, common myeloid progenitor CD34⁺CD38⁺CD123^loCD45RA⁻; GMP, granulocyte-macrophage progenitor CD34⁺CD38⁺CD123^loCD45RA⁺; MEP, megakaryocyte-erythrocyte progenitor CD34⁺CD38⁺CD123⁻CD45RA⁻.