Human epithelial hair follicle stem cells and their progeny: current state of knowledge, the widening gap in translational research and future challenges.

Epithelial hair follicle stem cells (eHFSCs) are required to generate, maintain and renew the continuously cycling hair follicle (HF), supply cells that produce the keratinized hair shaft and aid in the reepithelialization of injured skin. Therefore, their study is biologically and clinically important, from alopecia to carcinogenesis and regenerative medicine. However, human eHFSCs remain ill defined compared to their murine counterparts, and it is unclear which murine eHFSC markers really apply to the human HF. We address this by reviewing current concepts on human eHFSC biology, their immediate progeny and their molecular markers, focusing on Keratin 15 and 19, CD200, CD34, PHLDA1, and EpCAM/Ber-EP4. After delineating how human eHFSCs may be selectively targeted experimentally, we close by defining as yet unmet key challenges in human eHFSC research. The ultimate goal is to transfer emerging concepts from murine epithelial stem cell biology to human HF physiology and pathology.

Cycling up the epidermis: reconciling 100 years of debate.

There is likely general consensus within the skin research community that cell cycle control is critical to epidermal homeostasis and disease. The current predominant model proposes that keratinocytes switch off DNA replication and undergo cell cycle and cell growth arrest as they initiate terminal differentiation. However, this model cannot explain key physiological features of the skin, mainly why squamous differentiation prevails over proliferation in benign hyperproliferative disorders. In recent years, we have proposed an alternative model that involves mitotic slippage and endoreplication. This new model is controversial and has encountered resistance within the field. However, looking back at history, the epidermal cell cycle has been a matter of controversy and debate for around 100 years now. The accumulated data are confusing and contradictory. Our present model can explain and reconcile both old and new paradoxical observations. Here, we explain and discuss the endoreplicative cell cycle, the evidence for and against its existence in human epidermis and the important implications for skin homeostasis and disease. We show that regardless of the strengths or weaknesses of the Endoreplication Model, the existing evidence in support of the Cell Cycle Arrest Model is very weak.

DNA damage signalling histone H2AX is required for tumour growth.

Cancer most frequently develops in self-renewal tissues that are the target of genetic alterations due to mutagens or intrinsic DNA replication errors. Histone γH2AX has a critical role in the cellular DNA repair pathway cascade and contributes to genomic stability. However, the role of γH2AX in the ontology of cancer is unclear. We have investigated this issue in the epidermis, a self-renewal epithelium continuously exposed to genetic hazard and replication stress. Silencing H2AX caused cell cycle hyperactivation, impaired DNA repair and epidermal hyperplasia in the skin. However, mutagen-induced carcinogenesis was strikingly reduced in the absence of H2AX. KO tumours appeared significantly later than controls and were fewer, smaller and more benign. The stem cell marker Δp63 drastically diminished in the KO epidermis. We conclude that H2AX is required for tissue-making during both homoeostasis and tumourigenesis, possibly by contributing to the control and repair of stem cells. Therefore, although H2AX is thought to act as a tumour suppressor and our results show that it contributes to homeostasis, they also indicate that it is required for the development of cancer.

Myc is required for ß-catenin-mediated mammary stem cell amplification and tumorigenesis.

BACKGROUND: Basal-like breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Recent studies have linked activation of the Wnt/ß-catenin pathway, and its downstream target, Myc, to basal-like breast cancer. Transgenic mice K5ΔNßcat previously generated by our team present a constitutive activation of Wnt/ß-catenin signaling in the basal myoepithelial cell layer, resulting in focal mammary hyperplasias that progress to invasive carcinomas. Mammary lesions developed by K5ΔNßcat mice consist essentially of basal epithelial cells that, in contrast to mammary myoepithelium, do not express smooth muscle markers. METHODS: Microarray analysis was used to compare K5ΔNßcat mouse tumors to human breast tumors, mammary cancer cell lines and the tumors developed in other mouse models. Cre-Lox approach was employed to delete Myc from the mammary basal cell layer of K5ΔNßcat mice. Stem cell amplification in K5ΔNßcat mouse mammary epithelium was assessed with 3D-culture and transplantation assays. RESULTS: Histological and microarray analyses of the mammary lesions of K5ΔNßcat females revealed their high similarity to a subset of basal-like human breast tumors with squamous differentiation. As in human basal-like carcinomas, the Myc pathway appeared to be activated in the mammary lesions of K5ΔNßcat mice. We found that a basal cell population with stem/progenitor characteristics was amplified in K5ΔNßcat mouse preneoplastic glands. Finally, the deletion of Myc from the mammary basal layer of K5ΔNßcat mice not only abolished the regenerative capacity of basal epithelial cells, but, in addition, completely prevented the tumorigenesis. CONCLUSIONS: These results strongly indicate that ß-catenin-induced stem cell amplification and tumorigenesis rely ultimately on the Myc pathway activation and reinforce the hypothesis that basal stem/progenitor cells may be at the origin of a subset of basal-like breast tumors.

The proto-oncogene Myc is essential for mammary stem cell function.

The mammary epithelium comprises two major cell lineages: basal and luminal. Basal cells (BCs) isolated from the mammary epithelium and transplanted into the mouse mammary fat pad cleared from the endogenous epithelium regenerate the mammary gland, strongly suggesting that the basal epithelial compartment harbors a long-lived cell population with multipotent stem cell potential. The luminal cell layer is devoid of the regenerative potential, but it contains cells with clonogenic capacity, the luminal progenitors. Mammary BCs and luminal progenitors express high levels of the transcription factor Myc. Here, we show that deletion of Myc from mammary basal epithelial cells led to impaired stem cell self-renewal as evaluated by limiting dilution and serial transplantation assays. Luminal progenitor population was significantly diminished in mutant epithelium suggesting control by the BC layer. Colony formation assay performed with isolated BCs showed that clonogenic capacity was abolished by Myc deletion. Moreover, transplanted BCs depleted of Myc failed to produce epithelial outgrowths. Stimulation with ovarian hormones estrogen (E) and progesterone (P) partially rescued the repopulation capacity of Myc-depleted BCs; however, the Myc-deficient mammary epithelium developed in response to E/P treatment lacked stem and progenitor cells. This study provides the first evidence that in the mammary gland, Myc has an essential nonredundant function in the maintenance of the self-renewing multipotent stem cell population responsible for the regenerative capacity of the mammary epithelium and is required downstream from ovarian hormones, for the control of mammary stem and progenitor cell functions.

DNA damage triggers squamous metaplasia in human lung and mammary cells via mitotic checkpoints.

Epithelial transdifferentiation is frequent in tissue hyperplasia and contributes to disease in various degrees. Squamous metaplasia (SQM) precedes epidermoid lung cancer, an aggressive and frequent malignancy, but it is rare in the epithelium of the mammary gland. The mechanisms leading to SQM in the lung have been very poorly investigated. We have studied this issue on human freshly isolated cells and organoids. Here we show that human lung or mammary cells strikingly undergo SQM with polyploidisation when they are exposed to genotoxic or mitotic drugs, such as Doxorubicin or the cigarette carcinogen DMBA, Nocodazole, Taxol or inhibitors of Aurora-B kinase or Polo-like kinase. To note, the epidermoid response was attenuated when DNA repair was enhanced by Enoxacin or when mitotic checkpoints where abrogated by inhibition of Chk1 and Chk2. The results show that DNA damage has the potential to drive SQM via mitotic checkpoints, thus providing novel molecular candidate targets to tackle lung SCC. Our findings might also explain why SCC is frequent in the lung, but not in the mammary gland and why chemotherapy often causes complicating skin toxicity.

MYC accelerates p21CIP-induced megakaryocytic differentiation involving early mitosis arrest in leukemia cells.

p21(CIP) is a potent cell cycle inhibitor often up-regulated in differentiation. Protooncogene MYC induces cell growth and proliferation, inhibits differentiation and represses p21(CIP). However, both molecules are involved in processes of polyploidisation, cell size increase, differentiation and senescence. It is unclear why MYC has a dual role in differentiation. We have previously shown that overexpression of p21(CIP) in K562 myeloid cells induces megakaryocytic differentiation with polyploidy. We have now investigated the requirements for p21(CIP) to block mitosis and induce differentiation in the presence of overactivated MYC. Silencing and over-expression studies showed that p21(CIP) is required to induce differentiation. However, the expression of p21(CIP) needs to be transient to irreversibly inhibit mitosis but not DNA replication, what leads to polyploidy. Transient overexpression of p21(CIP) caused early down-regulation of mitotic Cyclins and up-regulation of G1/S Cyclins D and E, changes typical of endoreplication. Interestingly, over-activation of MYC did not release the proliferative block imposed by p21(CIP) and instead, accelerated cell size increase, megakaryocytic differentiation and polyploidisation. Our data suggests that in some systems p21(CIP) takes part in a mitosis control driving MYC-induced cellular growth into differentiation.

D2-40 immunohistochemical overexpression in cutaneous squamous cell carcinomas: a marker of metastatic risk.

BACKGROUND: Approximately 4% of cutaneous squamous cell carcinomas (cSCCs) develop lymphatic metastases. The value of lymphatic endothelial markers to enhance the detection of lymphatic tumor invasion in cSCC has not been assessed previously. OBJECTIVE: We sought to evaluate the use of the antibody D2-40, a podoplanin immunohistochemical marker, to identify tumor lymph vessel invasion in cSCC and to assess its expression in tumor cells. METHODS: This was a retrospective case-control study. A series of 101 cSCC, including 51 cases that developed lymphatic metastatic spread (metastasizing cSCC [MSCC]) and 50 cases that resolved definitely after surgical excision (non-MSCC) were included in the study. Lymph vessel invasion using D2-40 was evaluated on all primary biopsy specimens. The percentage of tumor cells showing D2-40 positivity and intensity scoring were recorded. All the immunohistochemical findings were correlated with the clinicopathological features. RESULTS: Lymph vessel invasion was observed in 8% of non-MSCCs and in 25.5% of MSCCs (P = .031). D2-40 expression was significantly increased, both in intensity (odds ratio 4.42 for intensity ++/+++) and in area (odds ratio 2.29 for area >10%), in MSCC when compared with non-MSCC. Interestingly, almost half (49%) of the MSCC had moderate to intense D2-40 positivity compared with 16% of non-MSCC. D2-40 immunohistochemical expression was increased in tumors with an infiltrative pattern of extension. In the multivariate analysis, histologically poorly differentiated tumors, recurrent lesions, and cSCC showing D2-40 overexpression (in intensity) were significantly associated with lymphatic metastases development (odds ratios 15.67, 14.72, and 6.07, respectively). LIMITATIONS: This was a retrospective study. CONCLUSION: The expression of podoplanin associates with high metastatic risk in cSCC.

Protooncogene MYC drives human melanocyte melanogenesis and senescence.

In spite of extensive research and advances on the molecular biology of melanoma, the process of melanocytic differentiation or its relationship with proliferation is poorly understood. The role of proto-oncogenes in normal melanocyte biology is also intriguing. Proto-oncogene MYC is overexpressed in 40% of melanomas. It has been suggested that MYC can mediate senescence bypass in malignant melanocytes, an important event in melanoma development, likely in cooperation with other oncogenic pathways. However, despite the apparent importance of MYC in melanoma, its functions in normal melanocytes are unknown. We have overexpressed MYC in freshly isolated human primary melanocytes and studied the effects on melanocytic proliferation and differentiation. MYC promoted a transient activation of melanocytes including cell cycle entry, DNA damage and cell migration. Subsequently, MYC induced melanogenesis, increased cellular size and complexity and senescence. Interestingly, we also found strong expression of MYC in regions of human nevi displaying high pigmentation and high expression of senescence marker p16. The results altogether show that MYC drives melanocytic differentiation and suggest that senescence is associated with differentiation. We discuss the implications into the mechanisms governing melanocytic differentiation and the development of melanoma.

A novel loss-of-function mutation of the voltage-gated potassium channel Kv10.2 involved in epilepsy and autism.

BACKGROUND: Novel developmental mutations associated with disease are a continuous challenge in medicine. Clinical consequences caused by these mutations include neuron and cognitive alterations that can lead to epilepsy or autism spectrum disorders. Often, it is difficult to identify the physiological defects and the appropriate treatments. RESULTS: We have isolated and cultured primary cells from the skin of a patient with combined epilepsy and autism syndrome. A mutation in the potassium channel protein Kv10.2 was identified. We have characterised the alteration of the mutant channel and found that it causes loss of function (LOF). Primary cells from the skin displayed a very striking growth defect and increased differentiation. In vitro treatment with various carbonic anhydrase inhibitors with various degrees of specificity for potassium channels, (Brinzolamide, Acetazolamide, Retigabine) restored the activation capacity of the mutated channel. Interestingly, the drugs also recovered in vitro the expansion capacity of the mutated skin cells. Furthermore, treatment with Acetazolamide clearly improved the patient regarding epilepsy and cognitive skills. When the treatment was temporarily halted the syndrome worsened again. CONCLUSIONS: By in vitro studying primary cells from the patient and the activation capacity of the mutated protein, we could first, find a readout for the cellular defects and second, test pharmaceutical treatments that proved to be beneficial. The results show the involvement of a novel LOF mutation of a Potassium channel in autism syndrome with epilepsy and the great potential of in vitro cultures of primary cells in personalised medicine of rare diseases.

Shiga Toxin-B Targeted Gold Nanorods for Local Photothermal Treatment in Oral Cancer Clinical Samples.

Introduction: A great challenge in nanomedicine, and more specifically in theranostics, is to improve the specificity, selectivity, and targeting of nanomaterials towards target tissues or cells. The topical use of nanomedicines as adjuvants to systemic chemotherapy can significantly improve the survival of patients affected by localized carcinomas, reducing the side effects of traditional drugs and preventing local recurrences. Methods: Here, we have used the Shiga toxin, to design a safe, high-affinity protein-ligand (ShTxB) to bind the globotriaosylceramide receptor (GB3) that is overexpressed on the surfaces of preneoplastic and malignant cancer cells in the head and neck tumors. Results: We find that ShTxB functionalized gold nanorods are efficiently retrotranslocated to the GB3-positive cell cytoplasms. After 3 minutes of laser radiation with a wavelength resonant with the AuNR longitudinal localized surface plasmon, the death of the targeted cancer cells is activated. Both preclinical murine models and patient biopsy cells show the non-cytotoxic nature of these functionalized nanoparticles before light activation and their treatment selectivity. Discussion: These results show how the use of nanomedicines directed by natural ligands can represent an effective treatment for aggressive localized cancers, such as squamous cell carcinoma of the oral cavity.

p21CIP1 controls the squamous differentiation response to replication stress.

The control of cell fate is critical to homeostasis and cancer. Cell cycle cdk inhibitor p21CIP1 has a central and paradoxical role in the regulatory crossroads leading to senescence, apoptosis, or differentiation. p21 is an essential target of tumor suppressor p53, but it also is regulated independently. In squamous self-renewal epithelia continuously exposed to mutagenesis, p21 controls cell fate by mechanisms still intriguing. We previously identified a novel epidermoid DNA damage-differentiation response. We here show that p21 intervenes in the mitosis block that is required for the squamous differentiation response to cell cycle deregulation and replication stress. The inactivation of endogenous p21 in human primary keratinocytes alleviated the differentiation response to oncogenic loss of p53 or overexpression of the DNA replication major regulator Cyclin E. The bypass of p21-induced mitotic block involving upregulation of Cyclin B allowed DNA damaged cells to escape differentiation and continue to proliferate. In addition, loss of p21 drove keratinocytes from differentiation to apoptosis upon moderate UV irradiation. The results show that p21 is required to drive keratinocytes towards differentiation in response to genomic stress and shed light into its dual and paradoxical role in carcinogenesis.

Allergenicity to worldwide invasive grass Cortaderia selloana as environmental risk to public health.

Allergies to grass pollen affects about 20% of the population worldwide. In the last few decades, the South American grass Cortaderia selloana (CS, Pampas grass) has expanded worldwide in a variety of countries including the USA, Australia and Western Europe. In many of these locations, CS has strikingly spread and has now been classified an invasive species. Many pernicious consequences of CS have been reported for local biodiversity, landscape and structures. However, the effect on human health has not been studied. To investigate this issue, we have chosen a European region on the northern cost of Spain where CS spread is overwhelming, Cantabria. We obtained CS pollen extract and analysed the allergenic reaction of 98 patients that were allergic to pollen of local grasses. We determined the skin reaction and the presence of specific IgE antibodies (sIgE) to CS or to a typical autochthonous grass, Phleum pratense. We also compared the seasonal symptoms with reported grass pollen counts in the area. The results strongly suggest that CS can cause respiratory allergies at a similar extent to the local grasses. Given that CS pollinises later than the local grasses, this would extend the period of grass allergies in the region for about three months every year, as stated by most of the patients. This is the first study reported on the effects of the striking expansion of CS on human health. Considering the strong impact that respiratory allergies have on the population, our results suggest that CS can currently constitute a relevant environmental health issue.

Keratinocyte Differentiation by Flow Cytometry.

The epidermis is continuously exposed to environmental hazard and undergoes continuous cell renewal. The maintenance of the epidermal balance between proliferation and differentiation is essential for the homeostasis of the skin. Proliferation and terminal differentiation are compartmentalized in basal and suprabasal layers, respectively. These compartments can be identified by different patterns of protein expression that can be used as differentiation markers. For instance, components of the intermediate filament cytoskeleton keratins K5 and K14 are confined to the proliferative basal layer, while keratins K1 and K10, keratins K6 and K16, or precursors of the cornified envelope such as involucrin are expressed by suprabasal terminally differentiating cells. The analysis of the expression of these markers allows studying the imbalance typical of disease. Although these markers have been traditionally analyzed on skin microsections, on attached cells by immunostaining or by western blotting, it is possible and advantageous to quantify them by flow cytometry. We have extensively applied this technology onto human and mouse keratinocytes. Here we describe detailed flow cytometry methods to determine the differentiation status of keratinocyte populations.

Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors.

Keratinocytes are hard to transfect. Viral vectors are a good alternative to genetically modify primary keratinocytes. A classical method is the use of retroviral vectors by co-culture of keratinocytes with virus-producer cells. This method is efficient in high-calcium conditions with feeder cells. However, sometimes co-culture is not possible and is more laborious as producer cells need to be replaced by feeder cells. Our solution is the use of lentiviral vectors, far more efficient as supernatant on keratinocytes. In this chapter we describe improved detailed protocols for stable genetic modification of human primary keratinocytes of the skin or head and neck, in both low- and high-calcium conditions by lentiviral vectors.

The DNA damage response links human squamous proliferation with differentiation.

How rapid cell multiplication leads to cell differentiation in developing tissues is still enigmatic. This question is central to morphogenesis, cell number control, and homeostasis. Self-renewal epidermoid epithelia are continuously exposed to mutagens and are the most common target of cancer. Unknown mechanisms commit rapidly proliferating cells to post-mitotic terminal differentiation. We have over-activated or inhibited the endogenous DNA damage response (DDR) pathways by combinations of activating TopBP1 protein, specific shRNAs, or chemical inhibitors for ATR, ATM, and/or DNA-PK. The results dissect and demonstrate that these signals control keratinocyte differentiation in proliferating cells independently of actual DNA damage. The DDR limits keratinocyte multiplication upon hyperproliferative stimuli. Moreover, knocking down H2AX, a common target of the DDR pathways, inhibits the epidermoid phenotype. The results altogether show that the DDR is required to maintain the balance proliferation differentiation and suggest that is part of the squamous program. We propose a homeostatic model where genetic damage is automatically and continuously cleansed by cell-autonomous mechanisms.

Squamous differentiation requires G2/mitosis slippage to avoid apoptosis.

The cellular mechanisms controlling cell fate in self-renewal tissues remain unclear. Cell cycle failure often leads to an apoptosis anti-oncogenic response. We have inactivated Cdk1 or Polo-like-1 kinases, essential targets of the mitotic checkpoints, in the epithelia of skin and oral mucosa. Here, we show that inactivation of the mitotic kinases leading to polyploidy in vivo, produces a fully differentiated epithelium. Cells within the basal layer aberrantly differentiate and contain large or various nuclei. Freshly isolated KO cells were also differentiated and polyploid. However, sustained metaphase arrest downstream of the spindle anaphase checkpoint (SAC) due to abrogation of CDC20 (essential cofactor of anaphase-promoting complex), impaired squamous differentiation and resulted in apoptosis. Therefore, upon prolonged arrest keratinocytes need to slip beyond G2 or mitosis in order to initiate differentiation. The results altogether demonstrate that mitotic checkpoints drive squamous cell fate towards differentiation or apoptosis in response to genetic damage.

Cellular and animal models of skin alterations in the autism-related ADNP syndrome.

Mutations in ADNP have been recently associated with intellectual disability and autism spectrum disorder. However, the clinical features of patients with this syndrome are not fully identified, and no treatment currently exists for these patients. Here, we extended the ADNP syndrome phenotype describing skin abnormalities in both a patient with ADNP syndrome and an Adnp haploinsufficient mice. The patient displayed thin dermis, hyperkeratotic lesions in periarticular areas and delayed wound healing. Patient-derived skin keratinocytes showed reduced proliferation and increased differentiation. Additionally, detection of cell cycle markers indicated that mutant cells exhibited impaired cell cycle progression. Treatment of ADNP-deficient keratinocytes with the ADNP-derived NAP peptide significantly reduced the expression of differentiation markers. Sonography and immunofluorescence staining of epidermal layers revealed that the dermis was thinner in the patient than in a healthy control. Adnp haploinsufficient mice (Adnp+/-) mimicked the human condition showing reduced dermal thickness. Intranasal administration of NAP significantly increased dermal thickness and normalized the levels of cell cycle and differentiation markers. Our observations provide a novel activity of the autism-linked ADNP in the skin that may serve to define the clinical phenotype of patients with ADNP syndrome and provide an attractive therapeutic option for skin alterations in these patients.

Mammalian endoreplication emerges to reveal a potential developmental timer.

Among the most intriguing and relevant questions in physiology is how developing tissues correctly coordinate proliferation with differentiation. Endoreplication, in a broad sense, is a consequence of a cell division block in the presence of an active cell cycle, and it typically occurs as cells differentiate terminally to fulfill a specialised function. Until recently, endoreplication was thought to be a rare variation of the cell cycle in mammals, more common in invertebrates and plants. However, in the last years, endoreplication has been uncovered in various tissues in mammalian organisms, including human. A recent report showing that cells in the mammary gland become binucleate at lactation sheds new insight into the importance of mammalian polyploidisation. We here propose that endoreplication is a widespread phenomenon in mammalian developing tissues that results from an automatic, robust and simple self-limiting mechanism coordinating cell multiplication with differentiation. This mechanism might act as a developmental timer. The model has implications for homeostasis control and carcinogenesis.

Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint.

The epidermis is a self-renewal epithelium continuously exposed to the genotoxic effects of ultraviolet (UV) light, the main cause of skin cancer. Therefore, it needs robust self-protective mechanisms facing genomic damage. p53 has been shown to mediate apoptosis in sunburn cells of the epidermis. However, epidermal cells daily receive sublethal mutagenic doses of UV and massive apoptosis would be deleterious. We have recently unravelled an anti-oncogenic keratinocyte DNA damage-differentiation response to cell cycle stress. We now have studied this response to high or moderate single doses of UV irradiation. Whereas, as expected, high levels of UV induced p53-dependent apoptosis, moderate levels triggered squamous differentiation. UV-induced differentiation was not mediated by endogenous p53. Overexpression of the mitosis global regulator FOXM1 alleviated the proliferative loss caused by UV. Conversely, knocking-down the mitotic checkpoint protein Wee1 drove UV-induced differentiation into apoptosis. Therefore, the results indicate that mitosis checkpoints determine the response to UV irradiation. The differentiation response was also found in cells of head and neck epithelia thus uncovering a common regulation in squamous tissues upon chronic exposure to mutagens, with implications into homeostasis and disease.