Limits...
Immortalization of Primary Keratinocytes and Its Application to Skin Research.

Choi M, Lee C - Biomol Ther (Seoul) (2015)

Bottom Line: Therefore, a number of immortalization attempts have been made with success to overcome this limitation.Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained.Finally, its application to a three-dimensional skin culture system will be described.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea.

ABSTRACT
As a major component of the epidermal tissue, a primary keratinocyte has served as an essential tool not only for the study of pathogenesis of skin-related diseases but also for the assessment of potential toxicities of various chemicals used in cosmetics. However, its short lifespan in ex vivo setting has been a great hurdle for many practical applications. Therefore, a number of immortalization attempts have been made with success to overcome this limitation. In order to understand the immortalization process of a primary keratinocyte, several key biological phenomena governing its lifespan will be reviewed first. Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained. Finally, its application to a three-dimensional skin culture system will be described.

No MeSH data available.


Related in: MedlinePlus

Step-wise transformation of normal primary keratinocytes into immortalized ones via bypass of mortality stage 1 (M1, replicative senescence) and 2 (M2, cellular crisis). Typical cellular changes, which are associated with M1 and M2, are also described.
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f2-bt-23-391: Step-wise transformation of normal primary keratinocytes into immortalized ones via bypass of mortality stage 1 (M1, replicative senescence) and 2 (M2, cellular crisis). Typical cellular changes, which are associated with M1 and M2, are also described.

Mentions: Unlike germline and stem cells, somatic cells have a limited lifespan. They grow slowly and stop cell division when cultured in vitro for a certain period of time. Leonard Hayflick first described this finite replicative potential of normal cells in culture a half century ago (Hayflick, 1965). Therefore, this phenomenon has been often referred to as the “Hayflick limit” (Ohtani et al., 2009). Typical human primary keratinocytes possess an in vitro lifespan of around 15–20 population doublings (PDs) in serum-free and chemically defined media (Stoppler et al., 1997; Kiyono et al., 1998). When normal cells encounter the so-called “Hayflick limit”, they enter a viable state of permanent quiescence, which is often termed as cellular senescence (Hayflick and Moorhead, 1961). Continuous replication of typical primary human cells appears to be prevented by two discrete events: mortality stage 1 (M1) and mortality stage 2 (M2) (Fig. 2). M1 and M2 are often designated as “replicative senescence” and “cellular crisis” stages, respectively (Dimri et al., 1995; Coates, 2002; Cong et al., 2002). Cells entering replicative senescence first cease to respond to exogenous mitogenic stimuli and acquire increased cellular adhesion to the extracellular matrix while losing cell-cell contacts. They also shows the enlarged and flattened cell morphology, the increased lysosomal biogenesis (Shelton et al., 1999; Serrano and Blasco, 2001; Narita et al., 2003; Ben-Porath and Weinberg, 2004, 2005), the development of multiple nuclei (Stewart and Weinberg, 2002), and the formation of heterochromatic foci (Fridman and Tainsky, 2008). In addition to prolonged in vitro culture of primary cells, various types of cellular stresses including telomere erosion, DNA damage, overexpression of tumor suppressor genes or oncogenes, oxidative stress, continuous mitogenic stimuli, and a variety of chemicals can also induce senescence (Drayton and Peters, 2002; Ben-Porath and Weinberg, 2005). Despite different nature of each stress, replicative and stress-induced senescence seem to share at least some similar molecular pathways to execute their final outcome, which is a blockage of cell division.


Immortalization of Primary Keratinocytes and Its Application to Skin Research.

Choi M, Lee C - Biomol Ther (Seoul) (2015)

Step-wise transformation of normal primary keratinocytes into immortalized ones via bypass of mortality stage 1 (M1, replicative senescence) and 2 (M2, cellular crisis). Typical cellular changes, which are associated with M1 and M2, are also described.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556197&req=5

f2-bt-23-391: Step-wise transformation of normal primary keratinocytes into immortalized ones via bypass of mortality stage 1 (M1, replicative senescence) and 2 (M2, cellular crisis). Typical cellular changes, which are associated with M1 and M2, are also described.
Mentions: Unlike germline and stem cells, somatic cells have a limited lifespan. They grow slowly and stop cell division when cultured in vitro for a certain period of time. Leonard Hayflick first described this finite replicative potential of normal cells in culture a half century ago (Hayflick, 1965). Therefore, this phenomenon has been often referred to as the “Hayflick limit” (Ohtani et al., 2009). Typical human primary keratinocytes possess an in vitro lifespan of around 15–20 population doublings (PDs) in serum-free and chemically defined media (Stoppler et al., 1997; Kiyono et al., 1998). When normal cells encounter the so-called “Hayflick limit”, they enter a viable state of permanent quiescence, which is often termed as cellular senescence (Hayflick and Moorhead, 1961). Continuous replication of typical primary human cells appears to be prevented by two discrete events: mortality stage 1 (M1) and mortality stage 2 (M2) (Fig. 2). M1 and M2 are often designated as “replicative senescence” and “cellular crisis” stages, respectively (Dimri et al., 1995; Coates, 2002; Cong et al., 2002). Cells entering replicative senescence first cease to respond to exogenous mitogenic stimuli and acquire increased cellular adhesion to the extracellular matrix while losing cell-cell contacts. They also shows the enlarged and flattened cell morphology, the increased lysosomal biogenesis (Shelton et al., 1999; Serrano and Blasco, 2001; Narita et al., 2003; Ben-Porath and Weinberg, 2004, 2005), the development of multiple nuclei (Stewart and Weinberg, 2002), and the formation of heterochromatic foci (Fridman and Tainsky, 2008). In addition to prolonged in vitro culture of primary cells, various types of cellular stresses including telomere erosion, DNA damage, overexpression of tumor suppressor genes or oncogenes, oxidative stress, continuous mitogenic stimuli, and a variety of chemicals can also induce senescence (Drayton and Peters, 2002; Ben-Porath and Weinberg, 2005). Despite different nature of each stress, replicative and stress-induced senescence seem to share at least some similar molecular pathways to execute their final outcome, which is a blockage of cell division.

Bottom Line: Therefore, a number of immortalization attempts have been made with success to overcome this limitation.Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained.Finally, its application to a three-dimensional skin culture system will be described.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea.

ABSTRACT
As a major component of the epidermal tissue, a primary keratinocyte has served as an essential tool not only for the study of pathogenesis of skin-related diseases but also for the assessment of potential toxicities of various chemicals used in cosmetics. However, its short lifespan in ex vivo setting has been a great hurdle for many practical applications. Therefore, a number of immortalization attempts have been made with success to overcome this limitation. In order to understand the immortalization process of a primary keratinocyte, several key biological phenomena governing its lifespan will be reviewed first. Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained. Finally, its application to a three-dimensional skin culture system will be described.

No MeSH data available.


Related in: MedlinePlus