Efficient Procurement of Epithelial Stem Cells from Human Tissue Specimens Using a Rho-Associated Protein Kinase Inhibitor Y-27632

Terunuma, Atsushi; Limgala, Renuka P.; Park, Christine J.; Choudhary, Isha; Vogel, Jonathan

doi:10.1089/ten.tea.2009.0339

Cited by 70 publications

(66 citation statements)

References 23 publications

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“…It has recently been shown that inhibition of ROCK greatly increases the cloning efficiency of human embryonic stem cells (13) and human keratinocytes (14). In the current study, we demonstrate that treatment with Y-27632 greatly increased long-term proliferation of primary human keratinocytes and, unexpectedly, enabled them to efficiently bypass senescence and become immortal without detectable cell crisis.…”

Section: Introductionsupporting

confidence: 62%

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Chapman

Meyers³

et al. 2010

J. Clin. Invest.

284

314

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Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications. IntroductionSomatic cells have a limited lifespan, gradually slow in growth, and stop dividing, a process known as cellular senescence. This process is thought to limit the vulnerability of aging cells to disease. Human keratinocytes are invaluable for the study of skin biology and the pathogenesis of skin-related diseases, but their short lifespan in culture is a limitation. Different conditions have been developed to optimize the culture of keratinocytes; for example, the presence of fibroblast feeder cells increases the proliferative capacity of primary keratinocytes from approximately 20 to 40-60 population doublings (1, 2). Spontaneously immortalized keratinocyte lines, for example HaCaT (3) and NIKS (4), have been used for skin-related research. However, these cell lines have genetic abnormalities, such as mutations in p53 (5) or isochromosomes (4).Continuous replication of primary human cells is blocked by 2 separate events: mortality stage 1 (M1; replicative senescence) and mortality stage 2 (M2; crisis). At M1, signaling by shortened telomeres results in activation of the p53 and pRB pathways. M2 represents a critical period of genomic instability, with extremely eroded telomeres, resulting in chromosomal fusions and translocations. In retinal pigment epithelial cells and foreskin fibroblasts (which have decreased expression of components of the p16INK4A/pRB pathway), telomerase expression is sufficient to bypass both M1 and M2 and stabilize and elongate chromosome ends (6). However, telomerase expression is not sufficient for immortalization of keratinocytes, and p16INK4A function must also be disrupted (7,8).Keratinocytes expressing the E6 and E7 proteins encoded by high-risk human papillomavirus (HPV) types bypass both M1 and M2 blocks and become immortal (reviewed in ref. 9). E7 inactivates and degrades the pRB retinoblastoma tumor suppressor protein to induce G 1 /S phase progression of th...

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Section: Introductionsupporting

confidence: 62%

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Chapman

Meyers³

et al. 2010

J. Clin. Invest.

284

314

View full text Add to dashboard Cite

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“…Additional studies suggest that the CRC technique promotes clone formation rather than selecting for a tissue stem cell subpopulation (7,46,47). We find that nasal airway basal cells exhibit an eightfold increase in CFCF when cultured using the CRC method.…”

Section: Discussionmentioning

confidence: 72%

Airway Progenitor Clone Formation Is Enhanced by Y-27632–Dependent Changes in the Transcriptome

Reynolds

Rios²,

Wesolowska-Andersen³

et al. 2016

Am J Respir Cell Mol Biol

100

124

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The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more widespread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 3 10 10 cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Wholetranscriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

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“…ROCK kinase, central mediator of the signals from Ras homolog gene family, member A (RhoA), appears to play a critical role in the regulation of the balance between human KCs (hKCs) proliferation and differentiation [14]. It has been reported that blocking ROCK signaling in the initial 6 days of primary culture of epithelial cells from different sources, with the pharmacological inhibitor Y-27632, leads to an increased number of KCs with higher clonogenic capacity, in a concentration-dependent manner [15].…”

mentioning

confidence: 99%

“…propose innovative methodologies to obtain purified stemlike epidermal cells for further use in skin regeneration purposes has been a continuous challenge and a few works [7,12,15] have addressed this need. The current work envisioned to achieve a superior purified epidermal cell fraction by boosting KC stem-like cells among freshly isolated human skin KCs having as rationale the available knowledge regarding the EpSCs interfollicular basal niche.…”

mentioning

confidence: 99%

Boosting and Rescuing Epidermal Superior Population from Fresh Keratinocyte Cultures

Cerqueira

Frias

Reis

et al. 2014

Stem Cells and Development

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Epidermal stem cells (EpSCs) hold great expectations in a regenerative medicine context, but innovative methods that permit to obtain a significant yield of EpSCs or stem-like epidermal cells are still required. We propose a twostep strategy to obtain a superior epidermal stem-like cell fraction among primary keratinocytes (KCs) isolated from adult human skin. The approach is based on the combination of rapid adherence to collagen IV with the rockassociated kinase inhibitor (ROCKi) treatment, and the subsequent immunomagnetic separation of the a6 high / CD71 dim cell subset. The combined collagen IV and ROCKi treatment showed not only to enhance cells clonogenic capacity, but also to induce an early epidermal phenotypic signature, along with the diminished expression of late differentiation-associated markers. More importantly, collagen IV and the ROCKi efficiently promoted a synergized effect over a6 high /CD71 dim expression, boosting the number of highly proliferative KCs stem-like cells as demonstrated by the expression of ki67. This cell fraction showed a superior ability to generate a 3D stratified epithelium formed by cells with successive differentiation phenotypes. Overall, this strategy indulged the possibility to uncover, among adult KCs, a superior epidermal cell population with stem-like proliferation capacity and early differentiation degree to be used in numerous skin regeneration approaches.

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Efficient Procurement of Epithelial Stem Cells from Human Tissue Specimens Using a Rho-Associated Protein Kinase Inhibitor Y-27632

Cited by 70 publications

References 23 publications

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

Airway Progenitor Clone Formation Is Enhanced by Y-27632–Dependent Changes in the Transcriptome

Boosting and Rescuing Epidermal Superior Population from Fresh Keratinocyte Cultures

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