Method of immortalizing human keratinocytes and immortalized keratinocyte cell lines

Abstract

A method of immortalizing a human keratinocyte. Also provided are immortalized human keratinocytes, for example immortalized human keratinocytes that have been immortalized using the presently disclosed method. Further provided are organotypic cultures, co-cultures, assays and kits comprising one or more immortalized human keratinocytes. In one embodiment, the immortalized human keratinocyte is produced by sequentially deleting the p16 locus, using CRISPR-Cas9 based gene editing, followed by lentiviral-based introduction of the gene encoding human telomerase reverse transcriptase (hTERT).

Description

[0001] METHOD OF IMMORTALIZING HUMAN KERATINOCYTES AND IMMORTALIZED KERATINOCYTE CELL LINES

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to a method of immortalizing a human keratinocyte. The disclosure further relates to immortalized human keratinocytes, for example immortalized human keratinocytes that have been immortalized using the presently disclosed method. Also disclosed are organotypic cultures, co-cultures, assays and kits comprising one or more immortalized human keratinocytes.

[0004] BACKGROUND

[0005] Human primary cells undergo a finite number of divisions before entering irreversible growth arrest, known as cellular senescence (or in some cases, apoptosis). This limitation, termed the “end replication problem,” is circumvented in 85-90% of cancers, stem cells, and germ cells through activation of telomerase, which is a complex composed of the catalytic subunit telomerase reverse transcriptase (hTERT) and an RNA moiety (TERC). Telomerase uses its RNA moiety as a template to elongate / maintain telomere length, thereby circumventing replicative aging of cells (Allsopp et al., PNAS 1992). hTERT has thus been used to immortalize various human primary cell types (e.g., Bodnar et al., Science 1998; Ouellette et al., Hum Mol Gen 2004).

[0006] However, keratinocytes have proven more difficult to immortalize. To facilitate the study of human epithelial cells, James Rheinwald’s group at Harvard attempted to immortalize primary human keratinocytes using a retroviral pBABE-hTERT construct (from Robert Weinberg’s lab). They found that hTERT expression alone was insufficient. Instead, they found that at low frequency, immortalized colonies emerged that expressed hTERT and also had homozygous deletions in the p16 locus — these became known as N / TERTs (Dickson et al., MCB 2000).

[0007] N / TERTs, along with HaCaT cells (which immortalized spontaneously unlike N / TERTs), are the only 2 widely used immortalized keratinocyte lines. Both have now been cultured for over two decades and are derived from male, Caucasian donors. However, due to their long-term culture, these cell lines may have accumulated mutations that affect their behaviour, for example mutations that select for rapid growth. Moreover, N / TERTs are restricted for commercial and industry-based applications due to the patented pBABE-hTERT vector.

[0008] Given the limited genetic diversity in these cell lines, there is an urgent need to generate new immortalized keratinocyte lines from diverse ethnicities and from both genders. These new lines would support general research, CRISPR-based genome editing, and drug screening, all of which require clonal expansion and extended replicative capacity.

[0009] Additionally, skin pigmentation and tanning, regulated by melanocyte- keratinocyte interactions, vary across ethnic groups. To better understand and genetically modulate these processes (through knockout / overexpression studies), the research community requires ethnically diverse immortalized keratinocyte lines.

[0010] SUMMARY

[0011] In one aspect, there is provided a method of immortalizing a human keratinocyte, comprising: inactivating p16 or reducing p16 expression levels in the keratinocyte; and overexpressing human Telomerase Reverse Transcriptase (hTERT) or increasing hTERT expression levels in the keratinocyte.

[0012] In one embodiment, the p16 is inactivated or the p16 expression levels are reduced prior to overexpressing hTERT or increasing the hTERT expression levels.

[0013] In one embodiment, hTERT is overexpressed in the keratinocyte.

[0014] In one embodiment, hTERT is overexpressed by introducing an hTERT transgene into the keratinocyte.

[0015] In one aspect, there is provided an immortalized human keratinocyte, wherein the keratinocyte is immortalized by the method as disclosed herein.

[0016] In another aspect, there is provided an immortalized human keratinocyte, comprising: a p16 gene which is inactivated or has reduced expression levels, and a human telomerase reverse transcriptase (hTERT) transgene.

[0017] In one embodiment, the keratinocyte is immortalized by the method as described herein. In one embodiment, the p16 expression levels are reduced and / or the hTERT expression levels are increased relative to a non-immortalized keratinocyte.

[0018] In one embodiment, the p16 is inactivated.

[0019] In one embodiment, the p16 is inactivated by knocking out the p16 gene.

[0020] In one embodiment, the p16 is inactivated or the p16 expression levels are reduced due to one or more deletions in the p16 locus.

[0021] In one embodiment, the one or more deletions result in nonsense mutations.

[0022] In one embodiment, the p16 gene is inactivated or the p16 expression levels are reduced using CRISPR-Cas gene editing.

[0023] In one embodiment, the CRISPR-Cas gene editing is CRISPR-Cas9 gene editing.

[0024] In one embodiment, the CRISPR-Cas gene editing comprises transducing the keratinocyte with a p16-specific sgRNA plasmid comprising the nucleic acid sequence as set forth in SEQ ID NO: 2.

[0025] In one embodiment, the hTERT transgene comprises a nucleic acid as set forth in SEQ ID NO: 1.

[0026] In one embodiment, the hTERT transgene is introduced into the keratinocyte via a lentiviral vector comprising the hTERT transgene.

[0027] In one embodiment, the lentiviral vector further comprises a blasticidin resistance marker.

[0028] In one embodiment, the lentiviral vector is an inducible expression vector that allows the p16 gene to be re-activated or the p16 expression levels to be increased.

[0029] In one embodiment, the p16 gene re-activation or increase in p16 expression levels is doxycycline-inducible.

[0030] In one embodiment, the lentiviral vector is a pTRIPZ vector.

[0031] In one embodiment, the lentiviral vector comprises the nucleic acid sequence as set forth in SEQ ID NO: 3.

[0032] In one embodiment, the keratinocyte is derived from a donor having an ethnicity selected from the group comprising: Chinese, Indian, Malay and African.

[0033] In one embodiment, the keratinocyte is derived from a female donor.

[0034] In one embodiment, the keratinocyte further comprises a detectable label fused to a histone protein.

[0035] In one embodiment, the detectable label is a fluorescent label. In one embodiment, the histone protein is H2B.

[0036] In one embodiment, the keratinocyte further comprises mRFP-H2B.

[0037] In one aspect, there is provided an organotypic culture comprising one or more keratinocytes as described herein.

[0038] In one aspect, there is provided a co-culture comprising one or more keratinocytes as described herein.

[0039] In one aspect, there is provided a kit for an assay comprising one or more keratinocytes as described herein.

[0040] In one embodiment, the assay is selected from the group comprising: a pigmentation assay, a wound healing assay, a cell migration assay, a drug screening assay, and a DNA damage and repair assay.

[0041] In one aspect, there is provided a kit for a genetic screen to identify modulators of keratinocyte differentiation / senescence / stemness / pigmentation, comprising one or more keratinocytes as described herein.

[0042] DEFINITIONS

[0043] The term “immortalizing” as used herein refers to a process of modifying a cell so that it proliferates indefinitely, thereby bypassing normal cellular senescence. Thus, an “immortalized keratinocyte” is a keratinocyte which is able to proliferate indefinitely.

[0044] The term “keratinocyte” as used herein refers to an epithelial cell type predominantly located in the epidermis of mammalian skin. Keratinocytes are responsible for the synthesis of keratin proteins and play a central role in the formation of the skin barrier. These cells originate in the basal layer of the epidermis and undergo a regulated process of differentiation and migration toward the surface, culminating in the formation of the stratum corneum. Unless otherwise specified, the term “keratinocytes” encompasses primary keratinocytes and genetically modified keratinocytes derived from human or non-human sources.

[0045] The term “p16” or “CDKN2A” (cyclin-dependent kinase inhibitor 2A), used interchangeably herein, refers a protein which functions as a tumor suppressor by regulating the cell cycle and inhibiting cell division. p16 inhibits the activity of cyclin- dependent kinases CDK4 and CDK6, thereby preventing phosphorylation of the retinoblastoma protein (pRb) and halting progression from the G1 to the S phase of the cell cycle. Loss, mutation, or epigenetic silencing of CDKN2A is frequently associated with uncontrolled cellular proliferation and is implicated in various human cancers. Unless otherwise specified, the term encompasses wild-type and mutant forms of p16, as well as recombinant or synthetic variants thereof.

[0046] The term “inactivating” as used herein refers to a process by which a gene’s normal function is disrupted or switched off, preventing it from producing its target protein or preventing it from producing a functional protein. Thus, a p16 gene which is inactivated is unable to produce the p16 protein or produce a functional p16 protein. There are several ways a gene can be inactivated, including but not limited to: naturally occurring or targeted mutations, which alter the normal gene sequence; epigenetic modifications, which can silence gene expression, gene knockout whereby a gene is deliberately deleted or disrupted, and RNA interference (RNAi), whereby small RNA molecules are used to block gene expression.

[0047] The term “expression level” as used herein refers to the amount of a gene product, such as the gene’s mRNA (transcript) levels, protein levels or both mRNA and protein levels.

[0048] The term “reducing expression levels” or “reduced expression levels” as used herein refers to a reduction in expression levels relative to the expression levels of a nonimmortalized keratinocyte, for example a wildtype keratinocyte or an engineered or genetically modified keratinocyte which is not immortalized. The reduction can occur at the transcriptional, post-transcriptional, translational, or post-translational level, and may be achieved through various means including, but not limited to, RNA interference (RNAi), antisense oligonucleotides, CRISPR-based gene repression, small molecule inhibitors, or epigenetic modifications.

[0049] Hence, an immortalized keratinocyte comprising a p16 gene with “reduced expression levels” may have reduced p16 mRNA levels, reduced p16 protein levels, or reduced p16 mRNA and reduced p16 protein levels relative to a non-immortalized keratinocyte.

[0050] The term “increasing expression levels” as used herein refers to an increase in expression levels relative to the expression levels of a non-immortalized keratinocyte, for example a wildtype keratinocyte or an engineered or genetically modified keratinocyte which is not immortalized. The increase can occur at the transcriptional, post- transcriptional, translational, or post-translational level, and may be achieved through various means including transducing a plasmid / vector with a transgene or CRISPR activation (CRISPRa).

[0051] Hence, an immortalized keratinocyte comprising a p16 gene with “increased hTERT expression levels” may have increased hTERT mRNA levels, increase hTERT protein levels, or increased hTERT mRNA and increased hTERT protein levels relative to a non-immortalized keratinocyte.

[0052] The term “hTERT” or “human Telomerase Reverse Transcriptase” as used herein refers to is the catalytic subunit of the human telomerase enzyme complex, which is responsible for the elongation of telomeric DNA sequences at the ends of linear chromosomes. hTERT exhibits reverse transcriptase activity, utilizing an intrinsic RNA template (TERC) to synthesize telomeric repeats (TTAGGG) and thereby maintain chromosomal integrity and replicative capacity. In normal somatic cells, hTERT expression is tightly regulated and typically repressed, resulting in progressive telomere shortening and cellular senescence. In contrast, hTERT is frequently upregulated in immortalized and cancerous cells, contributing to sustained proliferative potential. Unless otherwise specified, the term “hTERT” includes full-length human hTERT protein, functional variants, and biologically active fragments thereof.

[0053] The term “overexpressing” as used herein refers to increasing expression levels such that they are higher than the expression levels in a non-immortalized keratinocyte, for example a wildtype keratinocyte or an engineered keratinocyte which is not immortalized. Gene overexpression may be achieved through various molecular biology techniques, including but not limited to the use of strong or inducible promoters, gene amplification, vector-based expression systems, or modifications to regulatory sequences that enhance transcriptional efficiency.

[0054] Hence, “overexpressing hTERT” in a keratinocyte refers to increasing the hTERT levels beyond the hTERT levels normally found in a non-immortalized keratinocyte.

[0055] The term “Clustered Regularly Interspaced Short Palindromic Repeats” or “CRISPR” as used herein refers to a family of DNA sequences found within the genomes of prokaryotic organisms, such as bacteria and archaea, which function as part of an adaptive immune system. These sequences are associated with CRISPR-associated (Cas) proteins that enable targeted cleavage of foreign nucleic acids. The CRISPR-Cas system has been repurposed as a versatile genome editing tool, allowing site-specific modification of DNA sequences in a wide range of organisms. In the context of the present disclosure, the term encompasses engineered CRISPR-Cas systems, including but not limited to CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13, as well as variants and derivatives thereof. These systems may be used to introduce targeted insertions, deletions, substitutions, or epigenetic modifications at specific genomic loci. CRISPR-Cas systems typically involve the use of guide RNAs (gRNAs) or single-guide RNAs (sgRNAs) designed to direct the Cas protein to a target nucleic acid sequence, enabling precise sequence manipulation.

[0056] The term “derived from a donor” is intended to refer to a keratinocyte that has been obtained directly or indirectly from the defined donor’s tissue, including through isolation, culture, or expansion. It therefore includes keratinocytes that have been freshly obtained from a donor and also keratinocytes that were previously obtained from the donor but have since undergone multiple rounds of culture over the short or long term, and also keratinocytes that have since undergone minimal or substantial manipulation, including but not limited to genetic modification, or differentiation, provided the keratinocyte retains phenotypic or genotypic characteristics attributable to the donor source.

[0057] The term “organotypic culture” as used herein refers to a culture system which allows for the in vitro growth of complex biological tissues which replicates part of their normal function and physiology. Hence, an organotypic skin culture would replicate the structure and function of native skin, typically by culturing human keratinocytes and dermal fibroblasts at an air-liquid interface, thereby creating an epidermal-like sheet with similarities to human epidermis.

[0058] The term "and / or", e.g., "X and / or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning.

[0059] Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements / components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of + / - 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value.

[0060] Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed sub-ranges 1 % to 2%, 1 % to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1 %, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed the ranges 1 .00% to 5.00% and also 1 .0% to 5.0% and all their intermediate values (such as 1.01 %, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1 %, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth / breadth of a range.

[0061] Additionally, when describing some embodiments, the disclosure may have disclosed a method and / or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and / or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure. Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features / characteristics discussed herein, one or more of these features / characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

[0062] DESCRIPTION OF EMBODIMENTS

[0063] It will be appreciated by a person skilled in the art that other variations and / or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.

[0064] In one aspect, there is provided a method of immortalizing a human keratinocyte, comprising:

[0065] • inactivating p16 or reducing p16 expression levels in the keratinocyte; and

[0066] • overexpressing hTERT or increasing hTERT expression levels in the keratinocyte.

[0067] Surprisingly, the presently claimed method is able to efficiently and reliably immortalize human keratinocytes. Briefly, CRISPR-Cas9 technology is first used to delete the p16 locus in a non-immortalized keratinocyte. Next, an hTERT transgene was transduced using an in-house lentiviral vector to enhance transduction efficiency and to avoid restrictions associated with using patented pBABE-based vectors.

[0068] In one embodiment, the p16 is inactivated or the p16 expression levels are reduced prior to overexpressing hTERT or increasing hTERT expression levels. Advantageously, this two-step approach of first inactivating p16 or reducing p16 expression levels, followed by overexpressing hTERT or increasing hTERT expression levels enables the controlled and reproducible immortalization of keratinocytes. Without wishing to be bound by theory, the present inventors believe that performing p16 inactivation first helps to alleviates the stress of the subsequent hTERT overexpression. In one embodiment, hTERT is overexpressed by introducing an hTERT transgene into the keratinocyte.

[0069] In another embodiment, the hTERT is overexpressed using CRISPR activation (CRISPRa). This typically involves a transcriptional activator domain fused to the CRISPR-Cas9 protein, which, when guided to the hTERT's promoter by a hTERT single guide RNA, enhances transcription.

[0070] In one aspect, there is provided an immortalized human keratinocyte, wherein the keratinocyte is immortalized by the method of any preceding claim.

[0071] Accordingly, in one aspect, there is provided an immortalized human keratinocyte, comprising:

[0072] • a p16 gene which is inactivated or has reduced expression levels, and

[0073] • a human telomerase reverse transcriptase (hTERT) transgene.

[0074] In one embodiment, the keratinocyte is immortalized by the method as disclosed herein.

[0075] In one embodiment, the p16 expression levels are reduced and / or the hTERT expression levels are increased relative to a non-immortalized keratinocyte, for example a wildtype keratinocyte or an engineered keratinocyte which is not immortalized.

[0076] Thus, in one embodiment, the p16 expression levels are reduced relative to the p16 expression levels in a non-immortalized keratinocyte.

[0077] In one embodiment, the hTERT expression levels are increased relative to the p16 expression levels in a non-immortalized keratinocyte.

[0078] In one embodiment, the non-immortalized keratinocyte is a wildtype keratinocyte.

[0079] In one embodiment, the non-immortalized keratinocyte is an engineered keratinocyte which is not immortalized.

[0080] In one embodiment, the expression levels are mRNA levels. Thus, in one embodiment the p16 mRNA levels are reduced. In another embodiment, the hTERT mRNA levels are increased.

[0081] In one embodiment, the expression levels are protein levels. Thus, in one embodiment the p16 protein levels are decreased. In another embodiment, the hTERT protein levels are increased. In one embodiment, the expression levels are both mRNA and protein levels. Thus, in one embodiment the p16 mRNA and p16 protein levels are decreased. In another embodiment, the hTERT mRNA and hTERT protein levels are increased.

[0082] In one embodiment, the p16 is inactivated.

[0083] In one embodiment, the p16 is inactivated by knocking out the p16 gene.

[0084] In one embodiment, the p16 is inactivated or the p16 expression levels are reduced due to one or more deletions in the p16 locus.

[0085] In one embodiment, the one or more deletions result in nonsense mutations, for example in one or both p16 alleles. In one embodiment, the keratinocyte has nonsense mutations in both p16 alleles.

[0086] In one embodiment, the p16 gene is inactivated or the p16 expression levels are reduced using CRISPR-Cas gene editing. Advantageously, the CRIPSR-Cas system allows the p16 gene to be accurately and reliably inactivated.

[0087] In one embodiment, the CRISPR-Cas gene editing is CRISPR-Cas9 gene editing.

[0088] In one embodiment, the CRISPR-Cas gene editing comprises transducing the keratinocyte with a p16-specific sgRNA plasmid comprising the nucleic acid sequence as set forth in SEQ ID NO: 2.

[0089] In one embodiment, the hTERT transgene comprises a nucleic acid as set forth in SEQ ID NO: 1.

[0090] In one embodiment, the hTERT transgene is introduced into the keratinocyte via a vector comprising the hTERT transgene, for example a viral vector comprising the hTERT transgene.

[0091] Hence, in one embodiment, the hTERT transgene is introduced into the keratinocyte via a lentiviral vector comprising the hTERT transgene. Advantageously, lentiviruses are able to transduce both replicating and non-replicating cells (in contrast, retroviruses can only transduce replicating cells). Thus, the use of lentiviral vectors increases the transduction efficiency for non-immortalized keratinocyte cell lines, which have limited proliferative capacity.

[0092] In one embodiment, the lentiviral vector further comprises a blasticidin resistance marker.

[0093] In one embodiment, the lentiviral vector is an inducible expression vector that allows the p16 gene to be re-activated or the p16 expression levels to be increased. The advantage of the inducible expression vector is that it allows p16 to be re-expressed in the presently disclosed immortalized keratinocytes which have inactivated p16 genes or p16 genes with reduced expression levels. This can be useful for example to conduct CRISPR-based screens in immortalized keratinocytes, followed by re-activating p16 expression and selecting for non-senescent cells or cells with an altered senescence- associated secretory phenotype.

[0094] In one embodiment, the p16 gene re-activation or increase in p16 expression levels is selected from the group comprising: doxycycline-inducible, tetracyclineinducible, ecdysone-inducible, tamoxifen-inducible and IPTG-inducible.

[0095] In one embodiment, the p16 gene re-activation or increase in p16 expression levels is doxycycline-inducible.

[0096] In one embodiment, the lentiviral vector is a pTRIPZ vector.

[0097] In one embodiment, the lentiviral vector comprises the nucleic acid sequence as set forth in SEQ ID NO: 3.

[0098] In one embodiment, the keratinocyte is derived from a donor having an ethnicity selected from the group comprising: Chinese, Indian, Malay, African and Caucasian.

[0099] In one embodiment, the keratinocyte is derived from a donor having an ethnicity selected from the group comprising: Chinese, Indian, Malay, and African. Advantageously, the present inventors have successfully generated immortalized keratinocytes that represent a wide range of different ethnicities. This provides immortalized keratinocytes that are ethnically diverse in contrast to existing immortalized keratinocyte cell lines which are derived from only Caucasians or have unknown ethnicity.

[0100] In one embodiment, the keratinocyte is derived from a male or female donor.

[0101] In one embodiment, the keratinocyte is derived from a female donor. Advantageously, the present inventors have successfully generated immortalized keratinocytes that have been derived from female donors. This provides a useful alternative to existing immortalized keratinocyte cell lines which are derived from only male donors.

[0102] In one embodiment, the keratinocyte further comprises a detectable label fused to a histone protein. In one embodiment, the detectable label is a fluorescent label, for example GFP, eGFP, RFP, mRFP, mCherry, YFP, blue fluorescent proteins such as mTagBFP2, and far red fluorescent proteins such as miRFP670nano3.

[0103] In one embodiment, the detectable label is mRFP.

[0104] In one embodiment, the histone protein is selected from the group comprising H2A, H2B, H3 and H4.

[0105] In one embodiment, the histone protein is H2B.

[0106] In one embodiment, the keratinocyte further comprises mRFP-H2B.

[0107] In one aspect, there is provided an organotypic culture comprising one or more keratinocytes as described herein. Thus, in one embodiment, the organotypic culture is an organotypic skin culture.

[0108] In one aspect, there is provided a co-culture comprising one or more keratinocytes as described herein.

[0109] In one aspect, there is provided a kit for an assay comprising one or more keratinocytes as described herein.

[0110] In one embodiment, the assay is selected from the group comprising: a pigmentation assay, a wound healing assay, a cell migration assay, a drug screening assay, and a DNA damage and repair assay.

[0111] In one aspect, there is provided a kit for a CRISPR-based genetic screen to identify modulators of keratinocyte differentiation / senescence / stemness / pigmentation, comprising one or more keratinocytes as described herein.

[0112] In one aspect, there is provided a method substantially as described herein.

[0113] In one aspect, there is provided an immortalized keratinocyte substantially as described herein.

[0114] BRIEF DESCRIPTION OF FIGURES

[0115] Figure 1 shows the sequencing result of the immortalised cell lines Kera 1 , 6, 10 and 13.

[0116] Figure 2 shows representative western blots of the primary and immortalised (imrtl) cell lines of Kera 2, 4 and 6.

[0117] Figure 3 shows growth curves of the immortalised keratinocytes against their corresponding primary keratinocytes. All primary cell lines growth were tracked until they stopped growing whereas the immortalised keratinocytes continued to grow past the end of the experiment. Kera 8, 9, and 12 are not shown.

[0118] Figure 4 shows graphs depicting the drop in Ki67 positive immortalised keratinocytes upon etoposide (ETOP) exposure. The keratinocytes were incubated with 20pM of ETOP for 2 days and fixed 3 days later. (n=3 for Kera 1 , 3, 4, 6, 7, 11 , 13. n=4 for the rest. Two-tailed unpaired t-test was used for statistics. ** = p<0.01 , *** = p<0.001 , **** = p<0.0001 ).

[0119] Figure 5 shows graphs depicting the increase in LB1 negative immortalised keratinocytes upon etoposide (ETOP) exposure. The keratinocytes were incubated with 20pM of ETOP for 2 days and fixed 3 days after. (n=3 for Kera 1 , 3, 4, 6, 7, 11 , 13. n=4 for the rest. Two-tailed unpaired t-test was used for statistics. ** = p<0.01 , *** = p<0.001 , **** = p<0.0001 ).

[0120] Figure 6 shows a graph demonstrating that CaCh exposure differentiated the immortalised keratinocytes. The cells were seeded sparse and incubated in + / - 1.2mM CaCh for 3 days before fixation and immunofluorescence microscopy against K10. The K10-positive area against nuclei-positive area per frame were calculated. Kera 12 has not been experimented on yet. The averages of each set was used to calculate statistics. (n=3, two-tailed unpaired t-test was used. * = p<0.05, ** = p<0.01 , *** = p<0.001 ).

[0121] SEQUENCES

[0122] SEQ ID NO: 1 - hTERT transgene nucleic acid sequence

[0123] SEQ ID NO: 2 - p16 specific single guide RNA (sgRNA) plasmid nucleic acid sequence

[0124] SEQ ID NO: 3 - In-house lentiviral vector + hTERT transgene nucleic acid sequence

[0125] EXAMPLES

[0126] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0127] Example 1 - Immortalisation process for keratinocytes

[0128] To immortalize keratinocytes, the present inventors performed a targeted knockout of the p16 locus and overexpressed hTERT in keratinocytes. By transfecting both the CRISPR / Cas9 and p16 specific sgRNA plasmid, the present inventors directly mutated the p16 gene in the primary keratinocyes.

[0129] The resulting keratinocytes were sequenced and the sequencing results are shown in Figure 1. The results indicate that Kera 1 has a base pair (bp) addition after the cut site in one chromosome, and a 19 bp deletion in the other (14 bp before and 5 bp after the cut site). Kera 6 has 3 peaks at times and as such the specific mutation could not be distinguished. Kera 10 has an 8 bp deletion (4 bp before and after the cut site) in one chromosome and a bp deletion after the cut site in the other. Kera 13 also has a bp deletion in one chromosome but has 32 bp deletion in the other (8 bp before and 24 bp after the cut site). All identified mutations lead to a nonsense mutation in both alleles of p16. The corresponding primary cell lines were also sequenced but do not display such mutations (data not shown).

[0130] The p16' / _keratinocytes were subsequently transduced with an in-house hTERT lentiviral vector. This vector was modified from the Ebla construct described in Chojnowski et al. eLIFE 2015 to facilitate cloning of hTERT into the vector. Western blots were then performed to determine the levels of hTERT and lamin B1 , which is a senescence marker.

[0131] The results are shown in Figure 2 and indicate that the hTERT-positive Kera 2, 4 and 6 did not drop in senescence marker lamin B1 expression as the passage number increases, unlike their primary counterparts. This suggests that the engineered keratinocytes were not entering a permanent growth arrest (or senescence) and were immortalized. This is further supported by their cumulative population doubling rate (see Figure 3). The immortalized keratinocytes displayed enhanced proliferation potential as compared to their primary counterparts and continued to proliferate far past the end of the experiment. The above results thus clearly demonstrate that the present inventors were able to successfully and reliably immortalize the keratinocytes.

[0132] Example 2 - Experiment to demonstrate that immortalized keratinocytes are able to enter senescence via other pathways besides extensive replication

[0133] As p16 is part of the senescence induction pathway, the present inventors proceeded to determine if the immortalized keratinocytes cells can still enter senescence through other pathways besides extensive replication. To test this, they treated the keratinocytes with the DNA damaging agent etoposide (ETOP) for 2 days before fixing the cells 3 days later. Single cell immunofluorescence was subsequently performed to visualize the senescence marker lamin B1 and the proliferation marker Ki67.

[0134] The results are shown in Figures 4 and 5 and indicate that all cell lines showed a significant increase in lamin B1 -negative senescent cells and a significant drop in Ki67- positive proliferative cell.

[0135] Collectively, these results show that the p16 KO immortalized keratinocytes can still senesce through means besides replication.

[0136] Example 3 - Experiment to demonstrate that immortalized keratinocytes can still differentiate in vitro

[0137] In vivo, keratinocyte differentiation is crucial for forming a functional skin. This differentiation process can be replicated in vitro through exposure to high concentrations of CaCh. To ensure that the immortalized keratinocytes can still differentiate in vitro, the present inventors incubated the keratinocytes with 1.2mM of CaClz for 3 days before single cell immunofluorescence was performed to visualize for the keratinocyte differentiation marker K10.

[0138] The results are shown in Figure 6. Most of the immortalized keratinocytes were able to differentiate in response to the high calcium exposure but Kera 2 and 8 could not. This could be due to differences between cell lines even before the immortalization process or due to the randomness of lentiviral insertions that disrupted the differentiation process. Nevertheless, majority of the cell lines could still differentiate and demonstrate proper keratinocyte function. Taken together, the present disclosure provides a reliable workflow for keratinocyte immortalization and a valuable resource for researchers and clinicians investigating epidermal biology and skin diseases.

[0139] These cell lines are expected to be valuable for both industrial and academic collaborators studying skin biology across ethnicities, including pigmentation. They also support overexpression, knockout studies, and CRISPR-based genetic screens requiring keratinocytes with extended replicative potential.

[0140] REFERENCES

[0141] Allsopp et al, PNAS 1992, 89 (21 ), 10114-10118

[0142] Bodnar et al, Science 1998, vol. 279, no. 534, 349-352

[0143] Chojnowski et al, eLIFE 2015, Aug 27:4:e07759

[0144] Dickson et al, Mol Cell Biol. 2000 Febl 20(4): 1436-47

[0145] Ouellette et al, Hum Mol Gen 2004, Vol 9, No3, 403-411

[0146] APPLICATIONS

[0147] The presently disclosed cells, uses and methods have various applications, such as the following:

[0148] • Organotypic cultures

[0149] • Co-cultures (for example pigmentation assays in 2D or 3D)

[0150] • Wound healing assays

[0151] • Genetic screens, for example CRISPR-based, lentiviral-based or shRNA- based genetic screens to identify modulators of keratinocyte differentiation / senescence / stemness / pigmentation, etc.

[0152] • Cell migration assays

[0153] • Drug screening, DNA damage and repair assays in the presence or absence of anti-aging compounds.

Claims

CLAIMS1 . A method of immortalizing a human keratinocyte, comprising:• inactivating p16 or reducing p16 expression levels in the keratinocyte; and• overexpressing human Telomerase Reverse Transcriptase (hTERT) or increasing hTERT expression levels in the keratinocyte.

2. The method according to claim 1 , wherein the p16 is inactivated or the p16 expression levels are reduced prior to overexpressing hTERT or increasing the hTERT expression levels.

3. The method according to claims 1 or 2, wherein hTERT is overexpressed in the keratinocyte.

4. The method according to claim 3, wherein hTERT is overexpressed by introducing an hTERT transgene into the keratinocyte.

5. An immortalized human keratinocyte, wherein the keratinocyte is immortalized by the method of any preceding claim.

6. An immortalized human keratinocyte, comprising:• a p16 gene which is inactivated or has reduced expression levels, and• a human telomerase reverse transcriptase (hTERT) transgene.

7. The keratinocyte according to claim 6, wherein the keratinocyte is immortalized by the method of any one of claims 1 to 4.

8. The method or keratinocyte according to any preceding claim, wherein the p16 expression levels are reduced and / or the hTERT expression levels are increased relative to a non-immortalized keratinocyte.

9. The method or keratinocyte according to any preceding claim, wherein the p16 is inactivated.

10. The method or keratinocyte according claim 9, wherein the p16 is inactivated by knocking out the p16 gene.

11. The method or keratinocyte according to any preceding claim, wherein the p16 is inactivated or the p16 expression levels are reduced due to one or more deletions in the p16 locus.

12. The method or keratinocyte according to claim 11 , wherein the one or more deletions result in nonsense mutations.

13. The method or keratinocyte according to any preceding claim, wherein the p16 gene is inactivated or the p16 expression levels are reduced using CRISPR-Cas gene editing.

14. The method or keratinocyte according to claim 13, wherein the CRISPR-Cas gene editing is CRISPR-Cas9 gene editing.

15. The method or keratinocyte according to claims 13 or 14, wherein the CRISPR- Cas gene editing comprises transducing the keratinocyte with a p16-specific sgRNA plasmid comprising the nucleic acid sequence as set forth in SEQ ID NO:2.

16. The method or keratinocyte according to any one of claims 4 to 15, wherein the hTERT transgene comprises a nucleic acid as set forth in SEQ ID NO: 1.

17. The method or keratinocyte according to any one of claims 4 to 16, wherein the hTERT transgene is introduced into the keratinocyte via a lentiviral vector comprising the hTERT transgene.

18. The method or keratinocyte according to claim 17, wherein the lentiviral vector further comprises a blasticidin resistance marker.

19. The method or keratinocyte according to claims 17 or 18, wherein the lentiviral vector is an inducible expression vector that allows the p16 gene to be reactivated or the p16 expression levels to be increased.

20. The method or keratinocyte according to claim 19, wherein the p16 gene reactivation or increase in p16 expression levels is doxycycline-inducible.

21. The method or keratinocyte according to any one of claims 17 to 20, wherein the lentiviral vector is a pTRIPZ vector.

22. The method or keratinocyte according to any one of claims 17 to 21 , wherein the lentiviral vector comprises the nucleic acid sequence as set forth in SEQ ID NO:3.

23. The method or keratinocyte according to any preceding claim, wherein the keratinocyte is derived from a donor having an ethnicity selected from the group comprising: Chinese, Indian, Malay and African.

24. The method or keratinocyte according to any preceding claim, wherein the keratinocyte is derived from a female donor.

25. The method or keratinocyte according to any preceding claim, wherein the keratinocyte further comprises a detectable label fused to a histone protein.

26. The method or keratinocyte according to claim 25, wherein the detectable label is a fluorescent label.

27. The method or keratinocyte according to claims 25 or 26, wherein the histone protein is H2B.

28. The method or keratinocyte according to any one of claims 25 to 27, wherein the keratinocyte further comprises mRFP-H2B.

29. An organotypic culture comprising one or more keratinocytes according to any one of claims 5 to 28.

30. A co-culture comprising one or more keratinocytes according to any one of claims 5 to 28.31 . A kit for an assay comprising one or more keratinocytes according to any one of claims 5 to 28.

32. The kit according to claim 31 , wherein the assay is selected from the group comprising: a pigmentation assay, a wound healing assay, a cell migration assay, a drug screening assay, and a DNA damage and repair assay.

33. A kit for a genetic screen to identify modulators of keratinocyte differentiation / senescence / stemness / pigmentation, comprising one or more keratinocytes according to any one of claims 5 to 28.

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