Radiation Induces Diffusible Feeder Cell Factor(s) That Cooperate with ROCK Inhibitor to Conditionally Reprogram and Immortalize Epithelial Cells

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction. Consistent with these experiments, conditioned medium was shown to induce and maintain conditionally immortalized cells, which was accompanied by increased telomerase expression. The activity of conditioned medium directly correlated with radiation-induced apoptosis of the feeder cells. Thus, the induction of conditionally reprogrammed cells is mediated by a combination of Y-27632 and a diffusible factor (or factors) released by apoptotic feeder cells

Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis

BackgroundSkin injuries in horses frequently lead to chronic wounds that lack a keratinocyte cover essential for healing. The limited proliferation of equine keratinocytes using current protocols has limited their use for regenerative medicine. Previously, equine induced pluripotent stem cells (eiPSCs) have been produced, and eiPSCs could be differentiated into equine keratinocytes suitable for stem cell-based skin constructs. However, the procedure is technically challenging and time-consuming. The present study was designed to evaluate whether conditional reprogramming (CR) could expand primary equine keratinocytes rapidly in an undifferentiated state but retain their ability to differentiate normally and form stratified epithelium.MethodsConditional reprogramming was used to isolate and propagate two equine keratinocyte cultures. PCR and FISH were employed to evaluate the equine origin of the cells and karyotyping to perform a chromosomal count. FACS analysis and immunofluorescence were used to determine the purity of equine keratinocytes and their proliferative state. Three-dimensional air-liquid interphase method was used to test the ability of cells to differentiate and form stratified squamous epithelium.ResultsConditional reprogramming was an efficient method to isolate and propagate two equine keratinocyte cultures. Cells were propagated at the rate of 2.39 days/doubling for more than 40 population doublings. A feeder-free culture method was also developed for long-term expansion. Rock-inhibitor is critical for both feeder and feeder-free conditions and for maintaining the proliferating cells in a stem-like state. PCR and FISH validated equine-specific markers in the cultures. Karyotyping showed normal equine 64, XY chromosomes. FACS using pan-cytokeratin antibodies showed a pure population of keratinocytes. When ROCK inhibitor was withdrawn and the cells were transferred to a three-dimensional air-liquid culture, they formed a well-differentiated stratified squamous epithelium, which was positive for terminal differentiation markers.ConclusionsOur results prove that conditional reprogramming is the first method that allows for the rapid and continued in vitro propagation of primary equine keratinocytes. These unlimited supplies of autologous cells could be used to generate transplants without the risk of immune rejection. This offers the opportunity for treating recalcitrant horse wounds using autologous transplantation

Catalytic-defective hTERT mutants cooperate with HPV E7 to immortalize HFKs.

<p><u>(A) Growth curves.</u> Primary HFKs were transduced with the indicated pBABE-puro based retroviruses containing wild-type hTERT, hTERT N+T, or hTERT-D868A and pLXSN-based retroviruses containing E7 or empty vector and then doubly selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro and growth curves were plotted with population doubling over time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. Wild-type hTERT, hTERT-D868A, and hTERT N+T are all able to immortalize HFKs in combination with E7. <u>(B) Telomerase activity in early passage of the transduced cells.</u> CHAP lysates were harvested from early (p5) and telomerase activity was measured by quantitative real-time TRAP. <u>(C) Telomerase activity in late passage of the transduced cells.</u> Telomerase activity in late passage of cells was measured by quantitative real- time TRAP.</p

HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes

<div><p>Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.</p> </div

Bmi1 protein expression is increased in immortalized and tumorigenic cervical cell lines and positively correlates with disease stage in cervical dysplasia and neoplasia <i>in vivo</i>.

<p>(<b>A</b>) Bmi1 protein levels were quantified by Western blot in primary HFKs and primary human ectocervical cells (HECs) expressing E6 or immortalized by E6/E7 and the cervical cancer cell line HeLa. Lysates were separated by 4–20% gradient SDS-PAGE. Antibodies were used to detect hTERT (1∶1000, Origene), Bmi1 (1∶200, F6, Millipore) and GAPDH (1∶2000, FL-335, Santa Cruz). (<b>B</b>)Tissue from a case of invasive cervical cancer was acquired. Representative images are shown containing cancerous lesions and adjacent normal, intact epithelium. Tissue staining with hematoxylin and eosin (<b>i</b>) and immunohistochemical stain with Bmi1 (1∶200, F6, Millipore) (<b>ii</b>) are shown. (<b>C</b>)Tissues of Cervical Intraepithelial Neoplasia Stage 1 (CIN1), CIN2, CIN3 or carcinoma <i>in situ</i>, and invasive cervical carcinoma were acquired. To quantify Bmi1 expression, stained slides were subjected to a randomized, blinded review by a board-certified clinical pathologist. A subset of slides was scored multiple times to demonstrate reproducibility. For each sample, the case number and diagnosis is provided with the corresponding an intensity score, the percentage of positive cells, the corresponding positivity score, and the combined score. Each case received an intensity score from 0–3 (0 = negative, 1 = weak, 2 = moderate, 3 = intense) and the percentage of positive cells was recorded, which was converted to a positivity score (0 = less than 10%, 1 = 11–49%, 2 = 50–74%, 3 = 75–100%). Combined scores were calculated by adding the intensity score and positivity scores. (<b>D</b>) Immunohistochemical staining with hematoxylin and eosin (<b>i, iii, v, vii, ix</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii, iv, vi, viii, x</b>) was performed. Representative images are shown. Relevant controls are shown, staining with hematoxylin and eosin (<b>i</b>) and for Bmi1 protein (1∶100, F6, Millipore) (<b>ii</b>). Scale bar = 50 µm. (<b>E</b>) Mean and standard deviation of combined scores are shown.</p

A telomere elongation-defective hTERT mutant cooperates with HPV E7 for immortalizing human keratinocytes (HFKs).

<p>Primary HFKs were transduced with pBABE-puro-based retroviruses containing hTERT or hTERT-HA and pLXSN-based retroviruses with HPV E7 or empty vector and selected with puromycin and G418 as previously described. Cultures were passed continuously in vitro, and growth curves were plotted. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than three times with similar results. <u>(A) Telomerase activity.</u> Quantitative TRAP assays were done as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a>. Similar levels of telomerase activity were observed among cells expressing an hTERT construct. <u>(B) Telomere length.</u> Telomeres lengthened in hTERT/E7 cells, but shortened in hTERT-HA/E7 cells. Both wild-type hTERT and hTERT-HA immortalize HFKs in combination with HPV E7.</p

Bmi1 cooperates with E7 in cell immortalization.

<p>HFKs were doubly infected with pX-Bmi1 and pLXSN-E7, pLXSN-E7 alone, or empty vector. (A) Growth curve. Cells were passaged as described in the Methods section to determine the growth rate and lifespan of the cell populations. Bmi1, in cooperation with E7, induced cell immortalization equivalent to E6 or hTERT and E7. (B) Telomere length. A quantitative PCR-based technique (see Methods) was used to quantify the average telomere length in the indicated cell cultures.</p

Telomerase activity does not correlate with telomere length during immortalization of human genital keratinocytes by the HPV E6 and E7 oncoproteins.

<p>Primary human foreskin keratinocytes (HFKs) and human ectocervical keratinocytes (HECs) were transduced with pLXSN-based retroviruses containing HPV E6, E7, E6/E7, or empty vector and selected as previously described. Cultures were passed continuously in vitro as described in the text and the number of cell doublings calculated and plotted versus time in culture. Cultures that did not proliferate and expand in 20 days were considered senescent and were terminated. This experiment was repeated more than five times with similar results. <u>(A) Immortalized cells exhibit similar levels of telomerase activity as in cervical cancer cells.</u> Quantitative TRAP assays as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#s4" target="_blank">Materials and Methods</a> were used to measure telomerase activity in E6/E7 immortalized HFKs, E6/E7 immortalized HECs, and SiHa (HPV-16 positive), HeLa (HPV-18 positive), and C33A (HPV negative) cervical cancer cell lines. <u>(B) Telomere length stabilizes in E6/E7 immortalized cells and cervical cancer cell lines.</u> Cellular DNAs were isolated from HFKs at indicated passages and cervical cancer cells, and relative telomere length (T/S ratio = telomere/single copy gene) was measured using real-time PCR, as described in the Material and Methods. Immortalized cells and cancer cells have relatively shorter telomeres. <u>(C) Telomere length shortens over cell passages during immortalization.</u> Cellular DNAs were isolated and subjected to real-time PCR-based telomere length measurement.</p

E6, hTERTwt, and hTERT-D868A alter the expression of overlapping gene sets, including chromatin remodeling genes such as <i>Bmi1</i>.

<p>Primary HFKs were stably transduced with either E6, hTERTwt, hTERT-D868A or puro babe control vector. Samples were submitted for whole genome expression array analysis (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003284#ppat.1003284.s005" target="_blank">Figure S3</a></b>). Expression profile changes shown by hTERTwt are compared to expression profile changes in E6 (<b>A</b>) and hTERT-D868A mutant (<b>B</b>). As expected, a significant amount of the expression changes seen in E6 (6991 total changes) were also altered by hTERT wt (1379, representing 20% of the E6 changes). Conversely, more than half of the hTERT changes (58%, 1379 of 2359 changes) were also seen by E6. Interestingly, of the 2359 genes altered by hTERTwt, 2077 of them (88%) were also altered by the hTERT^ci mutant (fold change >1.33 and <i>p</i> value <0.01). (<b>C</b>) 1258 changes were shared by E6, hTERTwt, and hTERT-D868A, including Bmi1. (<b>D</b>) Numerical fold change values are shown for all arrays as they correspond to two probes, NM_005180 and L13689. These accession numbers represent Bmi1 mRNA sequences that are 99% identical. Quantitative RT-PCR was performed on the E6 and hTERTwt or hTERT-D868A (<b>E</b>) with gene-specific primers for Bmi1 to validate the array results, normalized to GAPDH. n = 3. Bars represent mean ± SD.</p

hTERT wt and hTERT-D868A do not activate the hTERT promoter.

<p>Keratinocytes were transfected with either wt hTERT core promoter or the cyclin D1 promoter and either HPV16E6, hTERT wt, or hTERT-D8686A. The pRL-CMV <i>R. reniformis</i> reporter plasmid was also transfected into the cells to standardize for transfection efficiency. Luciferase activity was measured 24 hours after transfection using the Dual luciferase reporter assay system (Promega). Relative fold activation reflects the normalized luciferase activity induced by E6 and hTERT compared to the normalized activity of vector control. The value of pGL3B-hTERT activity with empty was set to 1. Error bars show the standard deviation for at least three independent experiments. Neither hTERT wt nor hTERT-D868A induce hTERT core promoter (A), while they are able to activate cyclin D1 promoter (B). HPV16 E6 was as a positive control for induction of hTERT promoter.</p