Single-cell expression profiling of human epidermal stem and transit-amplifying cells: Lrig1 is a regulator of stem cell quiescence

Considerable progress has been made in characterizing epidermal stem cells by microarray analysis of FACS-selected populations. One limitation of this approach is that the gene expression profiles represent the average of the cell population, potentially masking cellular heterogeneity of functional significance. To overcome this problem, we have performed single-cell expression profiling. We have generated cDNA libraries from single human epidermal cells, designated as stem or transit-amplifying cells on the basis of Delta1 and melanoma-associated chondroitin sulfate proteoglycan expression. Of the 14 putative stem cell markers identified, we selected one, the EGF receptor antagonist leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1), for further study. Lrig1 was expressed in groups of basal cells in human interfollicular epidermis previously identified as enriched for stem cells. Overexpression of Lrig1 decreased keratinocyte proliferation but did not affect the proportion of stem and transit-amplifying cells, as judged by clonal growth characteristics. Down-regulation of Lrig1 using siRNA increased cell-surface EGF receptor levels, enhanced activation of downstream pathways, and stimulated proliferation. Lrig1 acted in part by negatively regulating the Myc promoter. We propose that Lrig1 maintains epidermal stem cells in a quiescent nondividing state, and that Lrig1 down-regulation triggers proliferation.

[1]  F. Watt,et al.  Stem Cell Depletion Through Epidermal Deletion of Rac1 , 2005, Science.

[2]  Amy Li,et al.  Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[3]  D. Roop,et al.  Deregulated expression of c-Myc depletes epidermal stem cells , 2001, Nature Genetics.

[4]  B. Morgan,et al.  Distinct stem cell populations regenerate the follicle and interfollicular epidermis. , 2005, Developmental cell.

[5]  E. Fuchs,et al.  AP-2α: a regulator of EGF receptor signaling and proliferation in skin epidermis , 2006, The Journal of cell biology.

[6]  T. Sun,et al.  Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis , 1990, Cell.

[7]  Yann Barrandon,et al.  Long-term renewal of hair follicles from clonogenic multipotent stem cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[8]  F. Watt,et al.  Designer skin: lineage commitment in postnatal epidermis. , 2002, Trends in cell biology.

[9]  E. Fuchs,et al.  Defining the Epithelial Stem Cell Niche in Skin , 2004, Science.

[10]  N. Perrimon,et al.  The Transmembrane Molecule Kekkon 1 Acts in a Feedback Loop to Negatively Regulate the Activity of the Drosophila EGF Receptor during Oogenesis , 1999, Cell.

[11]  S. Inui,et al.  Targeted disruption of LIG‐1 gene results in psoriasiform epidermal hyperplasia 1 , 2002, FEBS letters.

[12]  F. Watt,et al.  Evidence that Myc activation depletes the epidermal stem cell compartment by modulating adhesive interactions with the local microenvironment , 2003, Development.

[13]  M. Kashimata,et al.  Epidermal growth factor system is a physiological regulator of development of the mouse fetal submandibular gland and regulates expression of the α6‐integrin subunit , 1997 .

[14]  J. Rihel,et al.  Single-Cell Transcriptional Analysis of Neuronal Progenitors , 2003, Neuron.

[15]  E. Ziff,et al.  Mxi1 Is a Repressor of the c-myc Promoter and Reverses Activation by USF* , 1999, The Journal of Biological Chemistry.

[16]  M. Loeffler,et al.  Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. , 1990, Development.

[17]  F. Watt,et al.  Stimulation of human epidermal differentiation by Delta–Notch signalling at the boundaries of stem-cell clusters , 2000, Current Biology.

[18]  B. Lim,et al.  Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-β1 concentrations , 2003, Journal of Cell Science.

[19]  Fiona M. Watt,et al.  Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression , 1993, Cell.

[20]  I. Amit,et al.  LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation , 2004, The EMBO journal.

[21]  F M Watt,et al.  The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis. , 1999, Development.

[22]  R. Eady,et al.  Desmosomal proteins, including desmoglein 3, serve as novel negative markers for epidermal stem cell-containing population of keratinocytes , 2003, Journal of Cell Science.

[23]  F M Watt,et al.  c-Myc promotes differentiation of human epidermal stem cells. , 1997, Genes & development.

[24]  Yaping Liu,et al.  Capturing and profiling adult hair follicle stem cells , 2004, Nature Biotechnology.

[25]  Fiona M Watt,et al.  c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny , 2001, Current Biology.

[26]  S. Nishikawa,et al.  Molecular characterization of melanocyte stem cells in their niche , 2005, Development.

[27]  R. Henriksson,et al.  LRIG1 protein in human cells and tissues , 2003, Cell and Tissue Research.

[28]  N. Çilingiroǧlu.  [Health services and health economics]. , 1988, Nufusbilim dergisi.

[29]  F. Watt,et al.  Role of LIM kinases in normal and psoriatic human epidermis. , 2006, Molecular biology of the cell.

[30]  Neil Winegarden,et al.  Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA , 2002, Nature Biotechnology.

[31]  C. Chuang,et al.  Functional divergence of the MAP kinase pathway ERK1 and ERK2 activate specific transcription factors , 1994, FEBS letters.

[32]  Rüdiger Klein,et al.  Mig6 is a negative regulator of EGF receptor–mediated skin morphogenesis and tumor formation , 2006, Nature Medicine.

[33]  Crispin J. Miller,et al.  Amplification protocols introduce systematic but reproducible errors into gene expression studies. , 2004, BioTechniques.

[34]  F. Watt,et al.  PA‐FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation , 2000, FEBS letters.

[35]  S. Inui,et al.  LRIG‐1 Provides a Novel Prognostic Predictor in Squamous Cell Carcinoma of the Skin: Immunohistochemical Analysis for 38 Cases , 2005, Dermatologic surgery : official publication for American Society for Dermatologic Surgery [et al.].

[36]  F. Watt,et al.  Contribution of stem cells and differentiated cells to epidermal tumours , 2003, Nature Reviews Cancer.

[37]  L. Taichman,et al.  Multiple classes of stem cells in cutaneous epithelium: a lineage analysis of adult mouse skin , 2001, The EMBO journal.

[38]  Elaine Fuchs,et al.  Stem cells of the skin epithelium , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[39]  R. Fonseca,et al.  Ectopic expression of VAV1 reveals an unexpected role in pancreatic cancer tumorigenesis. , 2005, Cancer cell.

[40]  F. Watt,et al.  Stem cell patterning and fate in human epidermis , 1995, Cell.

[41]  Mark C Udey,et al.  Characterization and isolation of stem cell-enriched human hair follicle bulge cells. , 2005, The Journal of clinical investigation.

[42]  H. Green,et al.  Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes , 1977, Nature.

[43]  J. Zanet,et al.  Endogenous Myc controls mammalian epidermal cell size, hyperproliferation, endoreplication and stem cell amplification , 2005, Journal of Cell Science.

[44]  F. Watt,et al.  Signaling via beta1 integrins and mitogen-activated protein kinase determines human epidermal stem cell fate in vitro. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[45]  F. Watt,et al.  Role of melanoma chondroitin sulphate proteoglycan in patterning stem cells in human interfollicular epidermis , 2003, Development.