Coordinating energy metabolism and signaling pathways in epithelial self-renewal and differentiation.

Epidermal stem cells (EPSCs) are essential for maintaining skin homeostasis and ensuring a proper wound healing. During in vitro cultivations, EPSCs give rise to transient amplifying progenitors and differentiated cells, finally forming a stratified epithelium that can be grafted onto patients. Epithelial grafts have been used in clinics to cure burned patients or patients affected by genetic diseases. The long-term success of these advanced therapies relies on the presence of a correct amount of EPSCs that guarantees long-term epithelial regeneration. For this reason, a deeper understanding of self-renewal and differentiation is fundamental to fostering their clinical applications.The coordination between energetic metabolism (e.g., glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and amino acid synthesis pathways), molecular signalling pathways (e.g., p63, YAP, FOXM1, AMPK/mTOR), and epigenetic modifications controls fundamental biological processes as proliferation, self-renewal, and differentiation. This review explores how these signalling and metabolic pathways are interconnected in the epithelial cells, highlighting the distinct metabolic demands and regulatory mechanisms involved in skin physiology.

Chromosome 9p trisomy increases stem cells clonogenic potential and fosters T-cell exhaustion in JAK2-mutant myeloproliferative neoplasms.

JAK2V617F is the most recurrent genetic mutation in Philadelphia-negative chronic Myeloproliferative Neoplasms (MPNs). Since the JAK2 locus is located on Chromosome 9, we hypothesized that Chromosome 9 copy number abnormalities may be a disease modifier in JAK2V617F-mutant MPN patients. In this study, we identified a subset of MPN patients with partial or complete Chromosome 9 trisomy (+9p patients), who differ from JAK2V617F-homozygous MPN patients as they carry three JAK2 alleles as well as three copies of all neighboring gene loci, including CD274, encoding immunosuppressive Programmed death-ligand 1 (PD-L1) protein. Investigation of the clonal hierarchy revealed that the JAK2V617F occurs first, followed by +9p. Functionally, CD34+ cells from +9p MPN patients demonstrated increased clonogenicity, generating a greater number of primitive colonies, due to high OCT4 and NANOG expression, with knock-down of these genes leading to a genotype-specific decrease in colony numbers. Moreover, our analysis revealed increased PD-L1 surface expression in malignant monocytes from +9p patients, while analysis of the T cell compartment unveiled elevated levels of exhausted cytotoxic T cells. Overall, here we identify a distinct novel subgroup of MPN patients, who feature a synergistic interplay between +9p and JAK2V617F that shapes immune escape characteristics and increased stemness in CD34+ cells.

Biochemical role of FOXM1-dependent histone linker H1B in human epidermal stem cells.

Epidermal stem cells orchestrate epidermal renewal and timely wound repair through a tight regulation of self-renewal, proliferation, and differentiation. In culture, human epidermal stem cells generate a clonal type referred to as holoclone, which give rise to transient amplifying progenitors (meroclone and paraclone-forming cells) eventually generating terminally differentiated cells. Leveraging single-cell transcriptomic data, we explored the FOXM1-dependent biochemical signals controlling self-renewal and differentiation in epidermal stem cells aimed at improving regenerative medicine applications. We report that the expression of H1 linker histone subtypes decrease during serial cultivation. At clonal level we observed that H1B is the most expressed isoform, particularly in epidermal stem cells, as compared to transient amplifying progenitors. Indeed, its expression decreases in primary epithelial culture where stem cells are exhausted due to FOXM1 downregulation. Conversely, H1B expression increases when the stem cells compartment is sustained by enforced FOXM1 expression, both in primary epithelial cultures derived from healthy donors and JEB patient. Moreover, we demonstrated that FOXM1 binds the promotorial region of H1B, hence regulates its expression. We also show that H1B is bound to the promotorial region of differentiation-related genes and negatively regulates their expression in epidermal stem cells. We propose a novel mechanism wherein the H1B acts downstream of FOXM1, contributing to the fine interplay between self-renewal and differentiation in human epidermal stem cells. These findings further define the networks that sustain self-renewal along the previously identified YAP-FOXM1 axis.

Challenges of enzyme therapy: Why two players are better than one.

Enzyme-based therapy has garnered significant attention for its current applications in various diseases. Despite the notable advantages associated with the use of enzymes as therapeutic agents, that could have high selectivity, affinity, and specificity for the target, their application faces challenges linked to physico-chemical and pharmacological properties. These limitations can be addressed through the encapsulation of enzymes in nanoplatforms as a comprehensive solution to mitigate their degradation, loss of activity, off-target accumulation, and immunogenicity, thus enhancing bioavailability, therapeutic efficacy, and circulation time, thereby reducing the number of administrations, and ameliorating patient compliance. The exploration of novel nanomedicine-based enzyme therapeutics for the treatment of challenging diseases stands as a paramount goal in the contemporary scientific landscape, but even then it is often not enough. Combining an enzyme with another therapeutic (e.g., a small molecule, another enzyme or protein, a monoclonal antibody, or a nucleic acid) within a single nanocarrier provides innovative multidrug-integrated therapy and ensures that both the actives arrive at the target site and exert their therapeutic effect, leading to synergistic action and superior therapeutic efficacy. Moreover, this strategic approach could be extended to gene therapy, a field that nowadays has gained increasing attention, as enzymes acting at genomic level and nucleic acids may be combined for synergistic therapy. This multicomponent therapeutic approach opens opportunities for promising future developments. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Neurological Disease Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery > Emerging Technologies.

AlPaCas: allele-specific CRISPR gene editing through a protospacer-adjacent-motif (PAM) approach.

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas.

Decoding the Human Epidermal Complexity at Single-Cell Resolution.

The epidermis is one of the largest tissues in the human body, serving as a protective barrier. The basal layer of the epidermis, which consists of epithelial stem cells and transient amplifying progenitors, represents its proliferative compartment. As keratinocytes migrate from the basal layer to the skin surface, they exit the cell cycle and initiate terminal differentiation, ultimately generating the suprabasal epidermal layers. A deeper understanding of the molecular mechanisms and pathways driving keratinocytes' organization and regeneration is essential for successful therapeutic approaches. Single-cell techniques are valuable tools for studying molecular heterogeneity. The high-resolution characterization obtained with these technologies has identified disease-specific drivers and new therapeutic targets, further promoting the advancement of personalized therapies. This review summarizes the latest findings on the transcriptomic and epigenetic profiling of human epidermal cells, analyzed from human biopsy or after in vitro cultivation, focusing on physiological, wound healing, and inflammatory skin conditions.

Stairways to Advanced Therapies for Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a devastating genetic skin disease typified by a plethora of different phenotypes and ranking from severe, early lethal, to mild localized forms. Although there is no cure for EB, recent progress in pharmacology and molecular and cellular biology is boosting the development of new advanced therapeutic strategies. Here we will focus on two main categories of such therapies: (1) those aimed at controlling inflammation and inducing reepithelialization of the wounds, and (2) those, perhaps more challenging and ambitious, that aim to permanently regenerate a fully functional epidermis, which requires targeting of epidermal stem cells. In both cases, the genetic variants underlying the different EB forms and factors, such as genetic background, modifier genes, comorbidities, and lifestyle, all of which impinge on EB genotype-phenotype correlation, need to be defined.

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.

Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

Hologene 5: A Phase II/III Clinical Trial of Combined Cell and Gene Therapy of Junctional Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients' skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm2). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses. Clinical Trial Registration: EudraCT Number: 2018-000261-36.

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.

Calreticulin Ins5 and Del52 mutations impair unfolded protein and oxidative stress responses in K562 cells expressing CALR mutants.

Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.

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.

Aerobic glycolysis tunes YAP/TAZ transcriptional activity.

Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ.

YAP/TAZ incorporation in the ß-catenin destruction complex orchestrates the Wnt response.

The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the ß-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for ß-TrCP recruitment to the complex and ß-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/ß-catenin signaling. In line, the ß-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.

Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

BMP signaling controls muscle mass.

Cell size is determined by the balance between protein synthesis and degradation. This equilibrium is affected by hormones, nutrients, energy levels, mechanical stress and cytokines. Mutations that inactivate myostatin lead to excessive muscle growth in animals and humans, but the signals and pathways responsible for this hypertrophy remain largely unknown. Here we show that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is the fundamental hypertrophic signal in mice. Inhibition of BMP signaling causes muscle atrophy, abolishes the hypertrophic phenotype of myostatin-deficient mice and strongly exacerbates the effects of denervation and fasting. BMP-Smad1/5/8 signaling negatively regulates a gene (Fbxo30) that encodes a ubiquitin ligase required for muscle loss, which we named muscle ubiquitin ligase of the SCF complex in atrophy-1 (MUSA1). Collectively, these data identify a critical role for the BMP pathway in adult muscle maintenance, growth and atrophy.

p63, Sharp1, and HIFs: master regulators of metastasis in triple-negative breast cancer.

Metastasis is the most significant cause of cancer-associated morbidity and mortality but remains poorly understood. Recent work revealed that metastasis of aggressive triple-negative breast cancers is suppressed by Sharp1, a factor that promotes degradation of hypoxia-inducible factors (HIF) and blunts HIF-induced malignant cell behavior.