Recent studies evaluating hematopoietic stem cell (HSC) potential raise the possibility that, in addition to embryonic sources, adult valve fibroblasts may be derived from HSCs. To test this hypothesis, we used methods that allow the potential of a single HSC to be evaluated in vivo. This was achieved by isolation and clonal expansion of single lineage-negative (Lin ⁻ ), c-kit ⁺ , Sca-1 ⁺ , CD34 ⁻ cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) combined with transplantation of individual clonal populations derived from these candidate HSCs into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP ⁺ cells within host valves. A subpopulation of these cells exhibited synthetic properties characteristic of fibroblasts, as evidenced by their expression of mRNA for procollagen 1α1. Further, we show by Y-chromosome–specific fluorescence in situ hybridization analysis of female-to-male transplanted mice that the EGFP ⁺ valve cells are the result of HSC-derived cell differentiation and not the fusion of EGFP ⁺ donor cells with host somatic cells. Together, these findings demonstrate HSC contribution to the adult valve fibroblast population.

Summary. The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34⁺ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1‐expressing cells in CD34⁺ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single‐cell reverse transcriptase polymerase chain reaction (RT–PCR) methods. Using the semiquantitative single‐cell polyA‐PCR + sequence‐specific (SS)‐PCR method, WT1 expression was detected in four (1·3%) out of 319 CD34⁺ BM single cells. To confirm the above results, a single‐cell nested sequence‐specific (NSS)‐RT–PCR method that was less quantitative but more sensitive than the polyA‐PCR + SS‐PCR method was also performed, and WT1 expression was detected in 15 (1·1%) out of 1315 CD34⁺ BM single cells. In total, WT1 expression was found in 19 (1·2%) out of 1634 CD34⁺ BM single cells. No significant differences in the frequencies of WT1‐expressing cells were found between CD34⁺CD38^– and CD34⁺CD38⁺ BM single cells. Furthermore, WT1‐expressing CD34⁺ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage‐specific and cell cycle gene expression in WT1‐expressing CD34⁺ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34⁺CD38^– and lineage‐committed, proliferating CD34⁺CD38⁺ BM cells. Our results could indicate that these WT1‐expressing CD34⁺ BM cells were normal counterparts of leukaemia cells.