Transcriptional profiling of putative human epithelial stem cells

Abstract

Background: Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC) class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells.

Results: Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-kappaB are downregulated/inhibited in MHC negative basal cells.

Conclusion: This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial stem cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

Figure 1

MHCI negative cells express low levels of Ki67. (A) A representative flow cytometric analysis of the expression of proliferation antigen Ki67 in MHCI positive, and MHCI negative cells. Gates were set using isotype control antibodies and single color control antibodies. In this experiment 3.9% of MHCI positive cells express Ki67, while only 0.9% of MHCI negative cells express Ki67. Although the exact values of proliferating population may vary from experiment to experiment the ratio of MHCI positive and MHCI negative proliferating populations stay constant. This result demonstrate quiescent nature of MHCI negative cells. (B) Single positive isotype control for PE. (C) Single positive control for FITC. (D) Secondary control.

Figure 2

Comparison of colony forming efficiencies of α6⁺/MHCI⁺ and α6⁺/MHCI^- cells. A representative experiment of the colony forming efficiency (CFE) of sorted α6⁺/MHCI⁺ cells and α6⁺/MHCI^- cells cultured on 3T3 fibroblast feeder layer. Primary culture of α6⁺/MHCI^- cells exhibits lower CFE than α6⁺/MHCI⁺ cells. However, secondary culture of α6⁺/MHCI^- cells exhibits higher CFE than α6⁺/MHCI⁺ cells (*P < 0.001). The CFE of α6⁺/MHCI⁺ cells increased ~3.6× from primary to secondary culture, while it increased ~38× for α6⁺/MHCI^-. These results indicate higher proliferative potential of α6⁺/MHCI^- cells compared to α6⁺/MHCI⁺ cells. Cells directly sorted by FACS (from niche, i.e. primary cultures) were seeded at the following concentrations: α6⁺/MHCI⁺ cells, 3,000 cells per plate; α6⁺/MHCI^- cells, 10,000 cells per plate. It must be noted that even though higher cell number was plated for α6⁺/MHCI^- cells in the primary cultures, the CFE of these cells was lower than the CFE of α6⁺/MHCI⁺ cells. In secondary cultures equal numbers of cells were plated (100 cells per plate) for both, α6⁺/MHCI⁺ cells, and α6⁺/MHCI^- cells.

Figure 3

Expression of interfollicular epidermal stem cell markers in MHCI negative cells. (A) Differentially expressed genes identified by microarray analysis that were discussed in the text. The numbers shown are in fold change in log2 scale and are the highest score for the gene. (B) The differentially expressed genes identified by microarray analysis and/or known epidermal SC markers were confirmed using semi-quantitative RT-PCR. PCR was run for 25, 30, and 35 cycles. β-catenin mRNA, which did not show significant change of expression in the two cell populations was used as a control.

Figure 4

CD71 expression in MHCI negative and MHCI positive cells. (A) showing A representative flow cytometry analysis of the expression of MHCI and CD71 indicating that epidermal cells that exhibit lack/low expression of MHCI also exhibit lower expression of CD71 than cells that express high level of MHCI. Gates were set using isotype control antibodies and single color antibodies. The geometrical mean channel fluorescence of the populations is indicated. (B) Single positive control for PE. (C) Single positive control for FITC. (D) Isotype control.

Figure 5

Lack of Notch1 activity in MHCI negative cells. Figure demonstrating lack of cleaved/active Notch1 expression in α6⁺/MHCI^- cells and its presence in α6⁺/MHCI⁺ The data are obtained by flow cytometry analysis using an antibody specific for cleaved/active Notch1.

Figure 6

NF-κB activity in MHCI negative and MHCI positive cells. (A) A representative flow cytometry analysis of the expression of MHCI and NF-κB subunit RelA/p65 proteins showing that epidermal cells that exhibit low expression of RelA/p65 also exhibit lack/low expression of MHCI. The geometrical mean channel fluorescence of the populations is indicated. (B) Single positive control for PE. (C) Single positive control for FITC. (D) Secondary control.

Figure 7

NF-κB activity in α6⁺/MHCI negative and MHCI positive cells. Nuclear extracts of sorted cells were analyzed for NF-κB p50-binding activity; data were expressed in optical density (O.D.) units obtained with the TransAM ELISA NF-κB assay for phosopho-p50 (**P < 0.017).

Figure 8

Changes in the gene expression during epidermal differentiation. (A) Genes that are gradually downregulated, or upregulated during epidermal differentiation. The EST* represents the Human clone 23933 mRNA (B) Genes that are upregulated in α6⁺/MHCI⁺ cells (TA cells), and subsequently downregulated in suprabasal cells.