Epithelial cells, including those isolated from the epidermis consist of a family of cells: stem cells, progenitor or transitional cells and differentiated cells. Stem cells can be defined as cells that have the potential to divide and to produce a replica cell as well as differentiated progeny and are thought to last the lifetime of the organism [15
]. In the interfollicular epidermis (non-hair follicle) and in the oral mucosa and other epithelia, these cells are reported to have specific expression patterns of several cell surface markers [21
], and in vitro
are reported to be “small’ in size (15–20 microns in diameter when attached to the growth surface) [15
]. Progenitor or transitional cells are dividing cells committed to differentiation, are larger (30–40 microns) and the differentiated cells are greater than 40 microns and have a distinctive “differentiated” appearance in culture.
In routine tissue culture of epithelia cells, it is thought that some stem cells are in the initial culture, along with progenitor or transitional cells but that the stem cells are “lost” during culture growth and passage, with progenitor cells forming the primary cultures, with limited life-span.
The routine culture of primary keratinocytes from skin and other epithelia involves changing the spent medium over the cell monolayer every second day with standard amount of medium (T-25 flask uses 5
ml of medium; a T-75 flask holds 15
ml of medium, etc.). At about 70% confluence, the monolayer is passed or split, using trypsin or dispase and if treated with care, a primary culture can be passed 7–12 times. In this type of “routine” keratinocyte culture, monolayer confluence induces differentiation and eventual cell death if the cells are not passed soon after reaching 100%.
As presented in this report, a technique has been developed that produced epithelial cell strains with a high percentage of small diameter cells. The cells have proliferative potential and grow in a coordinated monolayer/suspension. The technology has a number of unusual manipulations: the cells were fed once a day, with 2-3X the amount of medium, which is serum and fatty acid free and contains low calcium levels. The cells maintained active cell proliferation at 100% confluence so that the progeny cells were pushed or popped into the overlying medium. The spent medium cell suspension, containing about 80,000 cells/ml, at about 80% viability, was then poured into a new flask (Figure ), where a majority of the cells then attached, forming a new monolayer. The new flask became confluent, the monolayer producing ePUK cells, which formed a new monolayer upon transfer to another flask, expanding the cell strain over 1–2
months’ time. The original flask stopped producing ePUKs, and the monolayer consisted of large, aged cells and “died” after 10–12
The ePUKs were more fragile than their traditionally cultured counterparts, most notably being sensitive to centrifugation. It was thought that the cells might grow as spheroid cells, [27
], since epithelial cells with enhanced growth potential can grow as bundles of cells in suspension, but no evidence of this type of cells growth was seen.
The ePUK producing monolayers appeared to have a high nutrient requirement, as indexed by high glucose utilization. It is possible that the rapid growth of the cells, and growth of the ePUKs in suspension after monolayer confluence, was simply because of frequent feeding of the primary epithelial cells with larger than routine amounts of medium. Beside the use of larger medium volumes, feeding the cells every day was also essential to the technology; failure to do either of these protocol requirements stopped the growth of the cells as ePUK cultures. Thus, the ePUK culture technique may, in part be providing the nutrients necessary for growth. This concept would suggest that constant feeding of the cells, as in a continuous feed bioreactor, may allow the cells to expand for a limitless number of passages allowing creation of master cell banks for use in cell based therapies.
The importance of nutrient levels in ePUK proliferation implicates cell signaling pathways such as mTOR as control points in this type of in vitro
epithelial cell growth [28
]. This pathway is also implicated by recent studies by us demonstrating that rapamycin, an inhibitor of mTOR(C1) allows for greatly expanded growth of oral mucosal epithelial cells in monolayer cultures [29
The data indicating that ePUK growth of keratinocytes maintains a smaller diameter cell fraction suggested that an early progenitor or stem-like cell was supported by this culture method since early progenitor/stem-like epithelial cells are theorized to be small diameter cells. The observation that ePUKs did not express the α6β4bri
phenotype reported to indicate true epithelial stem cells [21
], suggested that ePUKs were possibly early progenitor cells maintained by the serum and fatty acid-free low calcium medium that stopped differentiation and a frequent feeding schedule that supplied nutrients for cell growth. Alternately these cells could be “stem” like cells that are not expressing α6β4-integrin since they are in suspension and have no immediate need to express this receptor for laminin [22
Moreover, in a recent publication Chaffer et al [30
] report the spontaneous growth of a cell type from normal and neoplastic nonstem cells that can convert to a stem-like state. These cells are basal-like human mammary epithelial cells that came de novo
from differentiated cells in culture, i.e. transdifferentiation, and float on top of existing cell monolayers. This conversion was observed in normal epithelial breast cells, the cells floated atop of the cell monolayer and it was stated that their presence was a rare occurrence. This report is intriguing because the cells are similar to ePUKs, which in our hands are routinely induced using simple technology, and are produced in large numbers (80,000/ml/day in flasks with 30–40
ml). It appears that ePUKs may be a stem-like cell that can be easily induced from adult epithelial tissues that do not express the α6
The in vitro phenotype of ePUK cells could be maintained because of the 1) extra nutrients provided to the cells by frequent feeding of 2-3X volumes of medium, and/or 2) some product made by the cells, as evidenced by the requirement for spent medium for successful passage of ePUK suspensions to form new monolayers, and/or 3) by the fact that the cells are never trypsinized or scraped for passage, so that the cell surface is never perturbed.
The cells in this report were primary adult cells in culture that were never passed using enzyme digestion and never cyropreserved. Application of this technology to adult human keratinocytes initially grown using traditional protocols, and passed with trypsin and cryopreserved (for as long as 2–3
yrs.) also yielded ePUK cells; These ePUKs grew with a shorter ePUK production period, and less ePUK expansion (data not shown). Neonatal foreskin keratinocytes were not used in these studies.
iPS cells have been induced from human foreskin keratinocytes using specific transcription factors that were introduced using retroviral vectors indicating that the keratinocyte cell type can be reprogrammed [4
]. The large percentage of small diameter cells growing as ePUKs, in suspension, and lack of ePUK monolayer stratification and differentiation suggests that ePUK keratinocytes are more “plastic” in phenotype and that they may be a useful stem-cell like type for epigenetic manipulation with small molecules, as already been reported by other investigators using mouse keratinocyte cultures [9