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First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00702

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Long-term expansion of human functional epidermal precursor cells: promotion of extensive amplification by low TGF-ß1 concentrations

¹ Laboratoire de Biologie des Cellules Souches Humaines, CNRS-UPR 9045, Institut André Lwoff, 94800 Villejuif, France
² L'Oréal, Life Sciences Advanced Research, Centre C. Zviak, 92110 Clichy, France
³ Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA

^* Author for correspondence (e-mail: hatzfeld{at}vjf.cnrs.fr)

Accepted 6 June 2003

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-ß1 (TGF-ß1) exerts a pleiotropic effect. TGF-ß1 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-ß1 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-R^low). In our standard culture condition without feeder cells, the mean estimated output for cells from an unfractionated population of primary foreskin keratinocytes was 10⁷-10⁸, increasing to 10¹²-10¹³ in cultures initiated with selected Adh⁺⁺⁺EGF-R^low precursors. Characterization of these cells revealed a hitherto unknown property of TGF-ß1: 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-R^low cells repeatedly reached 10¹⁶-10¹⁷ (60 population doublings, 4x10¹⁸ 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 6 and ß1 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-R^low 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-ß1, which is to promote undifferentiated keratinocyte amplification.

Key words: Human epidermal precursor, Expansion, Feeder layer-free culture, TGF-ß1, High proliferative potential, Reconstructed epidermis, EGF-R

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