PB CD34⁻Lin⁻CD45⁻ cells give rise to hematopoietic and endothelial-lineage cells on LTC-HE. (A) Sorting criteria used to separate fresh adult human CD34⁻Lin⁻CD45⁻ and CD34⁻Lin⁻CD45⁺ cells from PB. (B) RT-PCR showing CD34 and CD45 mRNA expression in freshly sorted CD34⁻Lin⁻CD45⁻ and CD34⁻Lin⁻CD45⁺ cells, and hematopoietic stem cells (HSCs) as a control. (C) Time-course of immunophenotypic changes in CD34⁻Lin⁻CD45⁻ cells during LTC-HE: progressive appearance of CD34⁺CD45⁺ and CD34⁻CD45⁺ cells. Data shown as mean ± SE of 6 independent experiments. (D) Representative plots showing the expression of hematopoietic markers by CD34⁻Lin⁻CD45⁻ compared with CD34⁻Lin⁻CD45⁺ cells after 6 weeks in LTC-HEs. The mean ± SE of 20 independent experiments is shown in each quadrant. (E) After 6 weeks in LTC-HEs, both cell populations showed similar CFU capacity. Represenative bright-field microscopy images (original magnification 40×). (F) Time-course of immunophenotypic changes in CD34⁻Lin⁻CD45⁻ cells during LTC-HE: progressive appearance of CD45⁻KDR⁺, CD45⁺KDR⁺, and CD45⁺KDR⁻ cells. Data shown as mean ± SE of 6 independent experiments. (G) Representative plots showing that after 6 weeks of culture (under LTC-HE conditions) CD34⁻Lin⁻CD45⁻ but not CD34⁻Lin⁻CD45⁺ cells gave rise to CD45⁺ cells and to CD45⁻ cells coexpressing molecules linked to endothelial development. The mean ± SE of 9 independent experiments is shown in each quadrant. (H) Percentage of CD45⁻KDR⁺, CD45⁺KDR⁺, and CD45⁺KDR⁻ cells in LTC-HEs of CD34⁻Lin⁻CD45⁻ cells after 6 weeks. The graph shows the results obtained in each experiment. FSC indicates forward scatter.