Regeneration of the entire human epidermis using transgenic stem cells.

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

The role of oncogenic Ras in human skin tumorigenesis depends on the clonogenic potential of the founding keratinocytes.

The role of Ras in human skin tumorigenesis induction is still ambiguous. Overexpression of oncogenic Ras causes premature senescence in cultured human cells and hyperplasia in transgenic mice. Here, we investigated whether the oncogenic insult outcome might depend on the nature of the founding keratinocyte. We demonstrate that overexpression of the constitutively active Ras-V12 induces senescence in primary human keratinocyte cultures, but that some cells escape senescence and proliferate indefinitely. Ras overexpression in transient-amplifying- or stem-cell-enriched cultures shows that p16 (encoded by CDKN2A) levels are crucial for the final result. Indeed, transient-amplifying keratinocytes expressing high levels of p16 are sensitive to Ras-V12-induced senescence, whereas cells with high proliferative potential, but that do not display p16, are resistant. The subpopulation that sustains the indefinite culture growth exhibits stem cell features. Bypass of senescence correlates with inhibition of the pRb (also known as RB1) pathway and resumption of telomerase reverse transcriptase (TERT) activity. Immortalization is also sustained by activation of the ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1) and Akt pathways. Moreover, only transduced cultures originating from cultures bearing stem cells induce tumors in nude mice. Our findings demonstrate that the Ras overexpression outcome depends on the clonogenic potential of the recipient keratinocyte and that only the stem cell compartment is competent to initiate tumorigenesis.

Clonal analysis of human clonogenic keratinocytes.

Regenerative medicine has its roots in harnessing stem cells for permanent restoration of damaged or diseased tissues. The first procedure for the transplantation of epidermal cultures in massive full-thickness burns was established in the 1980s. Since then, epithelial stem cell-based therapies have been further developed in cell and gene therapy protocols aimed at restoring visual acuity in severe ocular burns and treating patients affected by genetic skin diseases, as Epidermolysis Bullosa. The clinical success of these Advanced Therapy Medicinal Products (ATMPs) requires the presence of a defined number of epithelial stem cells in the grafts, detected as holoclone-forming cells. To date, the most trustworthy method to identify and measure holoclones in a culture is the clonal analysis of clonogenic keratinocytes. Here we describe in detail how to perform such a clonal analysis and identify each epidermal clonal type.

RSPO1-mutated keratinocytes from palmoplantar keratoderma display impaired differentiation, alteration of cell-cell adhesion, EMT-like phenotype and invasiveness properties: implications for squamous cell carcinoma susceptibility in patients with 46XX disorder of sexual development.

BACKGROUND: Secreted R-spondin (RSPO) proteins play a key role in reproductive organ development, epithelial stem cell renewal and cancer induction by reinforcing canonical Wnt signaling. We have previously reported that palmoplantar keratoderma (PPK), predisposition to cutaneous squamous cell carcinoma (SCC) development and sex reversal segregate as autosomal recessive trait in patients carrying RSPO1-mutations. Although our previous findings suggested that RSPO1 secreted from fibroblasts regulates keratinocyte growth or differentiation, the role of this protein in the epidermis remains largely unexplored. Our study was aimed at expanding the phenotypic, molecular and functional characterization of RSPO1-mutated skin and keratinocytes. RESULTS: Cultured primary keratinocytes from PPK skin of a RSPO1-mutated XX-sex reversed patient displayed highly impaired differentiation and epithelial-mesenchymal transition (EMT)-like phenotype. Interestingly, RSPO1-mutated PPK skin expressed markers of increased proliferation, dedifferentiation and altered cell-cell adhesion. Furthermore, all these signs were more evident in SCC specimens of the patient. Cultured PPK patient's keratinocytes exhibited increased expression of cell‒matrix adhesion proteins and extracellular matrix remodeling enzymes. Moreover, they showed invasiveness properties in an organotypic skin model in presence of PPK fibroblasts, which behave like cancer-associated fibroblasts. However, the co-culture with normal fibroblasts or treatment with the recombinant RSPO1 protein did not revert or reduce the EMT-like phenotype and invasion capability of PPK keratinocytes. Notably, RSPO1-mutated PPK fibroblasts induced a hyperproliferative and dedifferentiated phenotype of age-matched normal control plantar keratinocytes. Wnt signaling has a key role in both PPK promotion and SCC development. Accordingly, Wnt mediators were differentially expressed in both PPK keratinocytes and skin specimens of RSPO1-mutated patient compared to control. CONCLUSIONS: Altogether our data indicate that the absence of RSPO1 in patients with 46XX disorder of sexual development affects the skin microenvironment and epidermal integrity, thus contributing to the risk of SCC tumorigenesis in palmoplantar regions exposed to major frictional stresses.

Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

Nonirradiated human fibroblasts and irradiated 3T3-J2 murine fibroblasts as a feeder layer for keratinocyte growth and differentiation in vitro on a fibrin substrate.

The standard method for producing graftable epithelia relies on the presence of a feeder layer of lethally irradiated 3T3-J2 murine fibroblasts (Rheinwald and Green technique). Here, we studied a new keratinocyte culture system, which envisages the utilization of nonirradiated human fibroblasts embedded into a fibrin substrate, in cultures destined for a future clinical application. We tested this culture system using keratinocytes grown on a fibrin gel precoated with 3T3-J2 murine fibroblasts as a control. In order to evaluate the new technology, we compared the clonogenic potential and the proliferative, differentiative and metabolic characteristics of keratinocytes cultured on the fibrin gel under the two culture conditions. The results demonstrated that the proposed technology did not impair the behavior of cultured keratinocytes and revealed that cells maintained their proliferative potential and phenotype under the experimental conditions. In particular, the demonstration of stem cell maintenance under the adopted culture conditions is very important for acute burn treatment with skin substitutes. This work is a first step in the evaluation of a new keratinocyte culture system, which has been studied in order to take advantage of an additional human cell population (i.e. nonirradiated, growing fibroblasts) for future transplantation purposes in acute and chronic wounds. Additional research will allow us to attain (1) the removal of murine cells in the initial phase of keratinocyte cultures, and (2) the removal of other potentially dangerous animal-derived materials from the entire culture system.

Toward Combined Cell and Gene Therapy for Genodermatoses.

To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.

Genotype-phenotype relationships in trichothiodystrophy patients with novel splicing mutations in the XPD gene.

Trichothiodystrophy (TTD) is a rare, autosomal recessive neurodevelopmental disorder most commonly caused by mutations in ERCC2 (XPD), a gene that encodes a subunit of the transcription/repair factor IIH (TFIIH). Here, we describe two TTD cases in which detailed biochemical and molecular investigations offered a clue to explain their moderately affected phenotype. Patient TTD22PV showed new mutated XPD alleles: one contains a nonsense mutation (c.1984C>T) encoding a nonfunctional truncated product (p.Gln662X) whereas the second carries a genomic deletion (c.2191-18_c.2213del) that affects the splicing of intron 22 and generates multiple out-of-frame transcripts from codon 731. XPD mRNA from the second allele corresponds to 20% of the total. The predicted proteins, which are longer than normal, affect the cellular repair activity but only partially interfere with TFIIH stability, suggesting that the observed changes in the C-ter region of XPD cause minor structural changes that do not drastically compromise the transcriptional activity of TFIIH. Patient TTD24PV was compound heterozygous for a typical TTD allele (c.2164C>T, p.Arg722Trp) and for a new XPD allele with a mutation that partially affects intron 10 splicing, resulting in both mutated and normal XPD transcripts (that together represent 15% of the total XPD mRNA). Compared to the previously described TTD compound heterozygotes for the Arg722Trp change, Patient TTD24PV's cells show similar level of TFIIH but increased repair activity, suggesting that even low amounts of normal XPD subunits are able to partially rescue the functionality of TFIIH complexes.

Altered expression of selected microRNAs in melanoma: antiproliferative and proapoptotic activity of miRNA-155.

Altered expression of microRNAs (miRNAs) has been detected in cancer, suggesting that these small non-coding RNAs can act as oncogenes or tumor suppressor genes. In the present study, we investigated the expression of miRNA-17-5p, miRNA-18a, miRNA-20a, miRNA-92a, miRNA-146a, miRNA-146b and miRNA-155 by real-time quantitative RT-PCR in a panel of melanocyte cultures and melanoma cell lines and explored the possible role of miRNA-155 in melanoma cell proliferation and survival. The analyzed miRNAs were selected on the basis of previous studies strongly supporting their involvement in cancer development and/or progression. We found that miRNA-17-5p, miRNA-18a, miRNA-20a, and miRNA-92a were overexpressed, whereas miRNA-146a, miRNA-146b and miRNA-155 were down-regulated in the majority of melanoma cell lines with respect to melanocytes. Ectopic expression of miRNA-155 significantly inhibited proliferation in 12 of 13 melanoma cell lines with reduced levels of this miRNA and induced apoptosis in 4 out of 4 cell lines analyzed. In conclusion, our data further support the finding of altered miRNA expression in melanoma cells and establish for the first time that miRNA-155 is a negative regulator of melanoma cell proliferation and survival.

Inactivation of p16INK4a (inhibitor of cyclin-dependent kinase 4A) immortalizes primary human keratinocytes by maintaining cells in the stem cell compartment.

Replicative senescence of human keratinocytes is determined by a progressive decline of clonogenic and dividing cells, and its timing is controlled by clonal evolution (i.e., the transition from stem cells to transient amplifying and postmitotic cells). Progressive increase of p16INK4a (inhibitor of cyclin-dependent kinase 4A) expression has been shown to correlate with keratinocyte clonal evolution. Thus, the aim of our study is to understand whether p16INK4a accumulation is a triggering mechanism of epidermal clonal evolution or a secondary event. We show that inactivation of p16INK4a, by an antisense strategy, allows primary human keratinocytes to escape replicative senescence. Specifically, p16INK4a inactivation alone blocks clonal evolution and maintains keratinocytes in the stem cell compartment. Antisense excision is followed by keratinocyte senescence, confirming that persistent p16INK4a inactivation is required for maintenance of clonal evolution block. Immortalization is accompanied by resumption of B-Cell Specific Moloney murine leukemia virus site 1 (Bmi-1) expression and telomerase activity, hallmarks of tissue regenerative capacity. In turn, Bmi-1 expression is necessary to maintain the impairment of clonal evolution induced by p16INK4a inactivation. Finally, p16INK4a down-regulation in transient amplifying keratinocytes does not affect clonal evolution, and cells undergo senescence. Thus, p16INK4a inactivation appears to selectively prevent clonal conversion in cells endowed with a high proliferative potential. These data indicate that p16INK4a regulates keratinocyte clonal evolution and that inactivation of p16INK4a in epidermal stem cells is necessary for maintaining stemness.

Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

Effective retrovirus-mediated gene transfer in normal and mutant human melanocytes.

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo.

OBJECTIVE: To induce complete and reproducible repigmentation of large "stable" vitiligo lesions by means of autologous cultured epidermal grafts using a rapid, simple, and minimally invasive surgical procedure. DESIGN: Achromic epidermis was removed by means of appropriately settled erbium:YAG laser, and autologous epidermal grafts were applied onto the recipient bed. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. SETTING: A biosafety level 3-type cell culture facility, a surgical ambulatory department, and a dermatological department in a hospital. PATIENTS: Twenty-one patients with different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were failure of at least 2 standard medical approaches; no therapy for at least 12 months; no progression of old lesions or appearance of new lesions; no Koebner phenomenon within the past 18 months; and no autoimmune disorders. RESULTS: The average percentage of repigmentation in 21 patients was 75.9% (1759.7 cm2 repigmented/2315.8 cm2 transplanted). Three patients showed a reactivation of their vitiligo and did not show repigmentation. The remaining 18 patients, with 43 distinct lesions, showed an average percentage of repigmentation of 90% (1759.7 cm2 repigmented/1953.4 cm2 transplanted). CONCLUSIONS: Under appropriate conditions, cultured epidermal grafts induce complete repigmentation of stable vitiligo lesions. Erbium:YAG laser surgery can supply a fast and precise tool for disepithelialization, hence allowing treatment of large vitiligo lesions during a single surgical operation.

MicroRNA-155 targets the SKI gene in human melanoma cell lines.

The SKI protein is a transcriptional coregulator over-expressed in melanoma. Experimentally induced down-regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA-155 (miR-155) is down-regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR-155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR-155 interacted with SKI 3'UTR and impaired gene expression. Transfection of melanoma cells with miR-155 reduced SKI levels, while inhibition of endogenous miR-155 up-regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over-expressing a 3'UTR-deleted SKI were still susceptible to the antiproliferative effect of miR-155. Our data demonstrate for the first time that SKI is a target of miR-155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth-suppressive effect of miR-155 found in this malignancy.

Bmi-1 reduction plays a key role in physiological and premature aging of primary human keratinocytes.

Accumulation of senescent cells contributes to the reduced regenerative capacity in aged tissues. By evaluating the molecular pathways of senescence in relation to proliferative potential of primary keratinocyte cultures from young and old healthy donors, and from young patients with inherited defects leading to premature aging, we demonstrated that p16(INK4a) is a reliable marker of both physiological and premature epidermal aging. Analysis of the expression and activity of p16(INK4a) regulators showed that stem cell depletion, reduced proliferation, and p16(INK4a) upregulation in keratinocytes derived from the chronologically and prematurely aged epidermis strongly correlate with Bmi-1 downregulation. In highly proliferative tissues, replicative and premature senescence participate in determining senescent cell accumulation. Our findings demonstrated that Bmi-1 is downregulated in human keratinocytes during both in vitro processes, in parallel with p16(INK4a) upregulation and accomplishment of clonal conversion. When premature senescence was induced by specific exogenous stimuli, concomitant Ets-1 upregulation was also observed. Moreover, Bmi-1 inhibited Ets-1-mediated p16(INK4a) upregulation. Finally, Bmi-1 overexpression reduced p16(INK4a) promoter activity and decreased protein expression in aged and diseased keratinocytes, inducing a delay of clonal conversion and an increase of cell clonogenic ability. Altogether these findings underline a key role of Bmi-1 downregulation in enforcing aging in primary human keratinocytes.

Keratinocyte cultures from involved skin in vitiligo patients show an impaired in vitro behaviour.

Vitiligo depigmentation is considered a consequence of either melanocyte disappearance or loss of functioning melanocytes in the involved areas. However, it has been reported that keratinocytes in involved vitiligo skin are damaged too. Based on this evidence, we evaluated the in vitro behaviour, in life span cultures, of involved and uninvolved vitiligo keratinocytes and their expression of proliferation, differentiation and senescence markers. An additional purpose was to investigate whether vitiligo keratinocytes from depigmented skin are able to sustain survival and growth of normal melanocytes (when added in co-culture experiments), as normal human keratinocytes manage to do. Our results demonstrate that almost all involved vitiligo keratinocytes have a shorter life span in vitro than the uninvolved cells and all of them do not maintain melanocytes in culture in a physiological ratio. Modification of proliferation and senescence marker expression also occurs. Indeed, we detected low initial expression levels of the senescence marker p16 in involved vitiligo keratinocytes, despite their shorter in vitro life span, and increased expression of proliferating cell nuclear antigen and p53. This preliminary analysis of a small number of in vitro cultured vitiligo keratinocytes suggests an impaired senescence process in lesional vitiligo keratinocytes and attempts to regulate it.

Long-term engraftment of single genetically modified human epidermal holoclones enables safety pre-assessment of cutaneous gene therapy.

Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy.

Piebald trait: implication of kit mutation on in vitro melanocyte survival and on the clinical application of cultured epidermal autografts.

Piebald trait leukoderma results from "loss-of-function" mutations in the kit gene. Correlations between mutation type and clinical phenotype have been reported. However, mutation classification has been mainly based on the clinical features of patients. The aim of this study was to get a better understanding of the pathogenesis of human piebaldism by establishing whether the kit mutation type may affect the in vitro survival/proliferation of patient melanocytes. Overall, the research was finalized to implement the clinical application of the autologous cultured epidermis in the treatment of piebald patients. Seven patients, who were transplanted with autologous in vitro reconstituted epidermis, showed an average percentage of repigmentation of 90.7. Six novel and one previously reported mutations were found and their postulated effects discussed in relation to the clinical phenotype and in vitro behavior of epidermal cells. Although mutation type did not impair repigmentation given by autotransplantation, it was shown to influence the survival/proliferation of co-cultured melanocytes and keratinocytes. In particular, tyrosine kinase domain mutations were found with melanocyte loss and keratinocyte senescence during expansion of epidermal cultures. Results indicate that the clinical application of cultured epidermis in piebald patients may be optimized by investigating mutation functional effects before planning surgical operations.