An in vitro outgrowth culture system for normal human keratinocytes

Ura, Hironobu; Takeda, Fujie; Okochi, Hitoshi

doi:10.1016/j.jdermsci.2004.03.005

Cited by 14 publications

(9 citation statements)

References 15 publications

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“…Migration assays Outgrowth migration assays were performed as previously described (Ura et al, 2004). Briefly, early-passage HFK (PD $ 5-7) were suspended in KSFM or KSFM containing different concentrations of either Herbimycin A (Sigma: H6649) or Amiloride (Sigma: A7410).…”

Section: Immunocytochemistrymentioning

confidence: 99%

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Darbro¹,

Schneider²,

Klingelhutz³

2005

Journal of Investigative Dermatology

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Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.

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Section: Immunocytochemistrymentioning

confidence: 99%

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Darbro¹,

Schneider²,

Klingelhutz³

2005

Journal of Investigative Dermatology

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“…The cell population often exhibits a travelling wave-like behaviour. This is true for a variety of cell types studied in vitro: examples include human peritoneal mesothelial cells (Maini et al, 2004a(Maini et al, , 2004b endothelial cells (Takamizawa et al, 1997), airway epithelial cells (Savla et al, 2004;Zahm et al, 1997), fibroblasts (Boucher et al, 1998) and keratinocytes (Ura et al, 2004).…”

Section: Introductionmentioning

confidence: 97%

Travelling Waves of Attached and Detached Cells in a Wound-Healing Cell Migration Assay

2007

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During a wound-healing cell migration assay experiment, cells are observed to detach and undergo mitosis before reattaching as a pair of cells on the substrate. During experiments with mice 3T3 fibroblasts, cell detachment can be confined to the wavefront region or it can occur over the whole wave profile. A multi-species continuum approach to modelling a wound-healing assay is taken to account for this phenomenon. The first cell population is composed of attached motile cells, while the second population is composed of detached immotile cells undergoing mitosis and returning to the migrating population after successful division. The first model describes cell division occurring only in the wavefront region, while a second model describes cell division over the whole of the scrape wound. The first model reverts to the Fisher equation when the reattachment rate relative to the detachment rate is large, while the second case does not revert to the Fisher equation in any limit. The models yield travelling wave solutions. The minimum wave speed is slower than the minimum Fisher wave speed and its dependence on the cell detachment and reattachment rate parameters is analysed. Approximate travelling wave profiles of the two cell populations are determined asymptotically under certain parameter regimes.

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“…[51][52][53] Keratinocytes typically proliferate in low calcium medium and a switch to higher calcium medium induces growth arrest and differentiation. [54][55][56] The hESC-derived keratinocytes exhibited similar behavior; the commercially available media used in this study (SFM and DSFM) contained a low concentration of calcium and thus promoted growth of proliferative K14 expressing keratinocyte precursors. The addition of calcium to the growth medium significantly increased involucrin expression.…”

Section: Hesc-derived Keratinocyte Precursorsmentioning

confidence: 95%

Generation and Differentiation of Human Embryonic Stem Cell-Derived Keratinocyte Precursors

et al. 2006

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Human embryonic stem cells (hESC) hold tremendous potential in the future of tissue engineering, offering promise as a source of virtually unlimited quantities of desired cell and tissue types. We have identified soluble chemical and extracellular matrix factors that permit isolation of keratinocyte precursors from hESCs. Culturing embryoid bodies (EB) formed from hESCs in a defined serum-free keratinocyte growth medium on a gelatin matrix generated keratin 14 (K14) expressing cells with an epithelial morphology. These K14 expressing cells could be subcultured in medium supplemented with hydrocortisone and induced to stratify and terminally differentiate by addition of calcium. Optimum times for obtaining K14 expressing cells were found for EB formation and for differentiation and growth of cultures after EB plating. EB formation was not necessary to generate keratinocyte precursors; direct transfer of hESC colonies to keratinocyte growth medium permitted differentiation into the keratinocyte lineage. With further studies to optimize generation and purification of hESC-derived keratinocyte precursors, these cells could provide a source of epidermal cells for skin tissue engineering applications in vitro or in vivo.

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An in vitro outgrowth culture system for normal human keratinocytes

Cited by 14 publications

References 15 publications

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Travelling Waves of Attached and Detached Cells in a Wound-Healing Cell Migration Assay

Generation and Differentiation of Human Embryonic Stem Cell-Derived Keratinocyte Precursors

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