(a) Hematoxylin and eosin staining (upper left panel) and immunohistochemical detection of the indicated proteins (brown) in paraffin sections of tumours generated by in vitro-transformed fibroblasts. Cells immunopositive for hCD34 and tubulin-β-III are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bar: 100 μm. (b) Semiquantitative RT-PCR analysis of expression levels of the indicated germ layer markers in transformed fibroblasts and 4 primary tumours induced by two independent transformed fibroblast cell lines (tumour 3 and 4 from Transformed-1 cell line and tumour 1 and 2 from Transformed-2 cell line). ACTB was used as control for RNA amount. Full scans of the gels, including molecular weight markers, are shown in Supplementary Fig. S7. (c, e) In vitro differentiation of Transformed-1 and Transformed-2 cell lines and the corresponding hTERT-immortalized control cells after (c) 5 d of adipogenic differentiation or (e) 7 d of osteogenic differentiation. Transformed cells were plated at densities of 1 × 10⁴cells /cm² for adipogenesis and 3 × 10³cells /cm² for osteogenesis. Scale bar: 100 μm. (d,f) Quantitative analysis of incorporated Oil red O after elution with isopropanol (d) or Alkaline Phosphatase (ALP) activity by luminescent analysis (f). Values represent means ± s. d. from three biological replicates. Statistical significance of the differences between uninduced and induced transformed cells is indicated.

(a) Relative abundance of cells immunopositive for the indicated antigens in two independent cell lines of in vitro-transformed fibroblasts compared to the corresponding hTERT-immortalized control cell lines. The average percentage of positive cells in the two transformed cell lines for each antigen measured by flow cytometric analysis is indicated. The red line indicates a relative abundance of 1 and values above the line indicate enrichment of the immunopositive cells in transformed fibroblasts compared to hTERT-immortalized cells. (b,c) Immunodetection of SSEA-1 in the Transformed-2 cell line and the corresponding hTERT-immortalized control cell line by flow cytometry (b) and fluorescence microscopy (c). The percentage of SSEA-1⁺ cells and the significance of the difference between the two populations determined by K-S test are indicated. The signal intensities and the percentage of SSEA-1⁺ cells in the hTERT-immortalized cell lines were not different (p > 0.05) from the background measured in unstained cells or cells stained with an isotype control antibody (Supplementary Fig.S2a). Scale bar: 40 μm. (d) Flow cytometric analysis of SSEA-1 in primary and in vitro-transformed mammary epithelial cells. The percentage of SSEA-1⁺ cells is indicated. (e) Immunohistochemical detection of SSEA-1 in clinical samples from one inflammatory myofibroblastoma (top left panel) and three malignant fibrohistiocytomas. Representative images of varying abundance and distribution of SSEA-1⁺ cells observed in the tissue microarray are shown. No SSEA-1⁺ positive cells were detected in normal control tissues (bottom panels). Scale bar: 150 μm. (f) Abundance of SSEA-1⁺ cells in a polyclonal population of primary fibroblasts transformed by serial introduction of transforming factors under conditions of high infection efficiency or in four clones obtained by simultaneous introduction of the transforming factors under conditions of low infection efficiency. Injection of the clones marked by an asterisk induced tumour formation in mice.

(a) Abundance of SSEA-1⁺ cells in populations of sorted SSEA-1⁺ (top) and SSEA-1⁻ fibroblasts (bottom) over time in culture in a representative time course experiment. About 1% of SSEA-1⁺ cells were present in the unsorted population. (b,c) Immunodetection of SSEA-1 in untreated cells (b) or cells treated with 25mM blebbistatin for 24h (c) by fluorescence microscopy. Pairs of daughter cells were identified as described in Supplementary Fig. S3 and Methods. Scale bar: 20 μm. The percentage of cells undergone each type of cell division is indicated. (d) Immunodetection of SSEA-1 in an isolated single SSEA-1⁻ -sorted fibroblast and in its daughter cells generated after 3 weeks of culture by fluorescence microscopy. Scale bar: 80 μm. (e) Flow cytometric analysis of SSEA-1 in three independent clonal populations resulting from single SSEA-1⁻ fibroblasts after three weeks of culture. Variable percentages of SSEA-1⁺ cells in each clone suggest that conversion to SSEA-1⁺ fibroblasts occurs in a stochastic manner. (f) Heatmap representing relative expression levels of genes showing a difference of at least 1.5-fold between SSEA-1⁺ and SSEA-1⁻ fibroblasts with a p value < 0.05. Two biological replicates for each subpopulation were analyzed. Red and blue represents the highest and lowest value of each gene among all samples, respectively.

(a) Experimental design to track cells responsible for tumour initiation among in vitro-transformed fibroblasts and possible outcomes. Higher percentage of GFP⁺ cells in the tumours compared to that in the injected cells indicates greater tumorigenicity of labelled SSEA-1⁺ cells than competing unlabeled SSEA-1⁻ cells. (b) Percentages of GFP⁺ cells in tumours induced by the indicated combinations of unlabeled and labelled cells measured by flow cytometric analysis. Dots represent individual tumours at 6 weeks after injection. Crosses are injections that failed to form tumours. Solid lines indicate median values of GFP⁺ cells for each group. Statistical significance of the differences between the experimental values and the expected values under the hypothesis of equal tumorigenicity of the injected unlabeled and labelled cells (see Methods) is indicated for each group. (c) Immunohistochemical detection of GFP (brown) in tumours resulting from injection of the indicated cells. Pictures of four adjacent fields were combined for each tumour. Scale bar: 150 μm.

(a,b) Flow cytometric analysis (a) and immunohistochemical detection (brown) (b) of SSEA-1 in primary tumours. Human H-2Kd⁻ cells were gated and plotted. Pictures of four adjacent fields were combined. Scale bar: 150 μm. (c) Immunohistochemical detection (brown) of the indicated proteins in primary tumours resulting from injection of GFP-labeled SSEA-1⁺ fibroblasts mixed with unlabeled SSEA-1⁻ fibroblasts. Cells immunopositive for hCD34 and tubulin-β-III are indicated by arrows. Wide areas of myosin-positive cells were detected. Scale bars: 80 μm (upper and middle panels) and 50 μm (bottom panels). (d) Flow cytometric analysis of CD166 and SSEA-1 in transformed fibroblasts, a primary tumour, and fibroblasts isolated from the tumour. (e) Abundance of cells expressing high levels of the indicated antigens measured by flow cytometric analysis in transformed fibroblasts, three representative primary tumours and two representative tumour fibroblast populations. The gate was set based on the values measured in the transformed fibroblasts. (f) Quantitative RT-PCR measuring expression levels of the mesenchymal markers CDh2 and FGF2 in the indicated samples. Values are normalized to the housekeeping gene PPIA and represent means from two replicates. Statistical significance of the differences compared to the tumour cells (p < 0.001) is indicated by an asterisk. (g) Abundance of fibroblasts in primary tumours resulting from injection of the indicated number of transformed fibroblasts. Values are relative to the number of viable tumour cells after dissociation and represent mean ± s. d. from three tumours. (h) Immunodetection of SSEA-1 in tumour fibroblasts isolated from primary tumours by fluorescence microscopy. Scale bar: 40 μm.

(a) Soft agar assay using unsorted cells form a primary tumour and SSEA-1⁺ tumour fibroblasts. Scale bar: 400 mm. Similar results were obtained with cells from 5 other tumours. (b,c) Quantitative analysis of soft agar assay (b) and non-adherent sphere formation assay (c) using the indicated cells from a primary tumour resulting from injection of 3,000 transformed fibroblasts. The frequency of clonogenic cells is indicated. Values represent means ± s. d. from three replicates. Statistical significance of the differences compared to the unsorted population is indicated by one (p < 0.005) or two (p < 0.001) asterisks. (d) Limiting dilution transplantation assay into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice using the indicated cells from two primary tumours resulting from injection of 3,000 transformed fibroblasts. The frequency of tumorigenic cells (estimate with upper-lower limits) was calculated by limiting dilution analysis as described in the Methods. Statistical significance of the differences compared to the unsorted population is indicated. (e) Summary of the differences in gene expression profiles between SSEA-1⁺ tumour fibroblasts and SSEA-1⁻ cells from three primary tumours. The number of genes differentially-expressed (≥2-fold) in each tumour and of those significantly different in all tumours (≥2-fold, p < 0.05) is indicated. (f) Heatmap representing relative expression levels of the subset (70%) of differentially-expressed genes in all tumours (SSEA-1⁺ tumour fibroblasts vs. SSEA-1⁻ cells, p < 0.05, ≥ 2-fold) which are expressed at similar levels in SSEA-1⁺ tumour fibroblasts and transformed fibroblasts (p > 0.05, < 2-fold). Values from two replicates of transformed fibroblasts and three tumours are represented. Red and blue represents the highest and lowest value of each gene among all samples, respectively.

(a) Serial transplantation of SSEA-1⁺ fibroblasts in BALB/cAnNCr-nu/nu mice. 3 × 10⁶ cells were injected for all passages. (b) Percentages of SSEA-1⁺ cells in the indicated samples measured by flow cytometric analysis. Values represent mean ± s. d. from 2 to 6 tumours. Statistical significance of the differences is indicated. (c) Quantitative analysis of soft agar assay using unsorted cells from primary, secondary and tertiary tumours. The frequency of clonogenic cells is indicated. Values represent mean ± s. d. from three replicates. Statistical significance of the differences is indicated.

Inhibition of tumour suppressor cellular mechanisms and concomitant oncogenic activation convert somatic differentiated cells into pre-malignant SSEA-1⁻ cells. This cellular intermediate is not tumorigenic. Additional, stochastic modifications lead to full reprogramming of somatic cells to SSEA-1⁺ CSCs, conferring self-renewal and differentiation ability. These cellular properties allow reprogrammed somatic cells to initiate and maintain tumours. During tumour growth, SSEA-1⁺ CSCs differentiate into phenotypically diverse, non-tumorigenic cancer cell, thereby generating heterogeneous and hierarchically-organized tumours.