title: Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-beta1 concentrations.
abstract: We have previously introduced the concept of high proliferative potential-quiescent (HPP-Q) cells to refer to primitive human hematopoietic progenitors, on which transforming growth factor-beta1 (TGF-beta1) exerts a pleiotropic effect. TGF-beta1 confers to these slow-dividing cells a mitogenic receptor(low) phenotype and maintains immature properties by preventing differentiation and apoptosis. However, the effect of TGF-beta1 on long-term expansion has not yet been clearly demonstrated. Here, we describe the characterization of a human skin keratinocyte subpopulation, highly enriched for primitive epidermal precursors, on the basis of high adhesion capacity (Adh+++) and low expression of the epidermal growth factor receptor (Adh+++EGF-Rlow). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10(7)-10(8), increasing to 10(12)-10(13) in cultures initiated with selected Adh+++EGF-Rlow precursors. Characterization of these cells revealed a hitherto unknown property of TGF-beta1: its addition at a very low concentration (10 pg/ml) in long-term cultures induces a very significant additional increase of expansion. In this optimized system, outputs obtained in cultures initiated with Adh+++EGF-Rlow cells repeatedly reached 10(16)-10(17) ( approximately 60 population doublings, approximately 4 x 10(18) keratinocytes produced per clonogenic cell present in the initial population). At the molecular level, this effect is associated with an increase in Smad1, Smad2 and Smad3 phosphorylation and an increase in alpha6 and beta1 integrin expression. No such effect could be observed on mature keratinocytes with low adhesion capacity (Adh-/+). We finally demonstrated that the progeny of Adh+++EGF-Rlow precursors after long-term expansion is still capable of generating a pluristratified epidermis in a model for skin reconstruction. In conclusion, after further characterizing the phenotype of primitive epidermal precursors, we demonstrated a new function of TGF-beta1, which is to promote undifferentiated keratinocyte amplification.
authors.affiliation: Laboratoire de Biologie des Cellules Souches Humaines, CNRS-UPR 9045, Institut André Lwoff, 94800 Villejuif, France.
authors.forename: Nicolas O
authors.initials: NO
authors.lastname: Fortunel
authors.forename: Jacques A
authors.initials: JA
authors.lastname: Hatzfeld
authors.forename: Pierre-Antoine
authors.initials: PA
authors.lastname: Rosemary
authors.forename: Corinne
authors.initials: C
authors.lastname: Ferraris
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Monier
authors.forename: Valérie
authors.initials: V
authors.lastname: Haydont
authors.forename: Joanna
authors.initials: J
authors.lastname: Longuet
authors.forename: Benoit
authors.initials: B
authors.lastname: Brethon
authors.forename: Bing
authors.initials: B
authors.lastname: Lim
authors.forename: Isabelle
authors.initials: I
authors.lastname: Castiel
authors.forename: Rainer
authors.initials: R
authors.lastname: Schmidt
authors.forename: Antoinette
authors.initials: A
authors.lastname: Hatzfeld
mesh_terms: D002448:Cell Adhesion / Q000502:physiology*; D002455:Cell Division; D004268:DNA-Binding Proteins / Q000378:metabolism; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D039503:Integrin alpha6 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D051785:Smad Proteins; D051898:Smad1 Protein; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
chemical_list: D004268:DNA-Binding Proteins; D039503:Integrin alpha6; D019012:Integrin beta1; C497399:SMAD1 protein, human; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051785:Smad Proteins; D051898:Smad1 Protein; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D066246:ErbB Receptors