We examined the effect of using ALDH as an additional marker to enrich for CSCs by sorting two CD44v-expressing adenocarcinoma cell lines, H1650 and HCC827, into CD44v high /ALDH high , CD44v high /ALDH low , CD44v low /ALDH high and CD44v low /ALDH low populations. Gates were set to sort the highest and lowest 10-20% of the CD44/ALDH-expressing cells from each cell line (Figure S1 C, D) for subsequent comparisons by various CSC assays. In both cell lines in the in vitro cell proliferation MTS assay, CD44v high /ALDH low cells showed the highest proliferation rates followed by CD44v high /ALDH high cells, compared with the other two populations; with the effect tending to be more pronounced in the H1650 cell line (Figure 3 A, B) . In the chemotherapeutic-resistance test, in the H1650 cell line only, CD44v high /ALDH low showed higher percentages of viable cells compared with the other three populations, whereas in the HCC827 cell line, CD44v low /ALDH low showed the lowest viability (Figure 3 C) . In vivo tumor-formation ability was also compared among the four populations from both cell lines, using the more immunocompromised NOD/SCID mice as recipients and by injecting 100,000 or 1,000 cells from each sorted population in these mice. When the number of injected cells was 100,000, CD44v high /ALDH high cells produced the largest tumor nodules, followed by CD44v high /ALDH low , whereas the CD44v low /ALDH high and CD44v low /ALDH low formed significantly smaller nodules in both the H1650 and HCC827 cell lines (Figure S2 A, B) . When the number of injected cells was reduced to 1000, only CD44v high /ALDH high cells, followed by CD44v high /ALDH low cells, were still able to form large tumor nodules, whereas the CD44v low /ALDH high and CD44v low /ALDH low cells formed no, or much smaller, nodules in both H1650 and HCC827 cell lines (Figure 3 D, E) .
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First, he collected the 24 candidate factors. When all 24 genes encoding these transcription factors were introduced into skin fibroblasts, few actually generated colonies that were remarkably similar to ES cells. Secondly, further experiments were conducted with smaller numbers of transcription factors added to identify the key factors, through a very simple and yet sensitive assay system. Lastly, he identified the four key factors. They found that 4 transcriptional factors (Myc, Oct3/4, Sox2 and Klf4) were sufficient to convert mouse embryonic or adult fibroblasts to pluripotent stem cells (capable of producing teratomas in vivo and contributing to chimeric mice).