Deciphering "Immaturity-Stemness" in Human Epidermal Stem Cells at the Levels of Protein-Coding and Non-Coding Genomes: A Prospective Computational Approach.

The epidermis hosts populations of epithelial stem cells endowed with well-documented renewal and regenerative functions. This tissue thus constitutes a model for exploring the molecular characteristics of stem cells, which remain to date partially characterized at the molecular level in human skin. Our group has investigated the regulatory functions of the KLF4/TGFB1 and the MAD4/MAX/MYC signaling pathways in the control of the immaturity-stemness versus differentiation fate of keratinocyte stem and precursor cells from human interfollicular epidermis. We described that down-modulation of either KLF4 or MXD4/MAD4 using RNA interference tools promoted an augmented stemness cellular status; an effect which was associated with significant transcriptional changes, as assessed by RNA-sequencing. Here, we have implemented a computational approach aimed at integrating the level of the coding genome, comprising the transcripts encoding conventional proteins, and the non-coding genome, with a focus on long non-coding RNAs (lncRNAs). In addition, datasets of micro-RNAs (miRNAs) with validated functions were interrogated in view of identifying miRNAs that could make the link between protein-coding and non-coding transcripts. Putative regulons comprising both coding and long non-coding transcripts were built, which are expected to contain original pro-stemness candidate effectors available for functional validation approaches. In summary, interpretation of our basic functional data together with in silico biomodeling gave rise to a prospective picture of the complex constellation of transcripts regulating the keratinocyte stemness status.

Generation of heterozygous and homozygous NF1 lines from human-induced pluripotent stem cells using CRISPR/Cas9 to investigate bone defects associated with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders caused by heterozygous germline NF1 mutations. NF1 affects many systems, including the skeletal system. To date, no curative therapies are available for skeletal manifestations such as scoliosis and tibial dysplasia, mainly due to the lack of knowledge about the mechanisms that underlie this process. By using CRISPR/Cas9-mediated gene editing in human-induced pluripotent stem cells (hiPSCs) to minimize the variability due to genetic background and epigenetic factors, we generated isogenic heterozygous and homozygous NF1-deficient hiPSC lines to investigate the consequences of neurofibromin inactivation on osteoblastic differentiation. Here, we demonstrate that loss of one or both copies of NF1 does not alter the potential of isogenic hiPSCs to differentiate into mesenchymal stem cells (hiPSC-MSCs). However, NF1 (+/-) and NF1 (-/-) hiPSC-MSCs show a defect in osteogenic differentiation and mineralization. In addition, we show that a mono-allelic deletion in NF1 in an isogenic context is sufficient to impair cell differentiation into osteoblasts. Overall, this study highlights the relevance of generating isogenic lines, which may help in genotype-phenotype correlation and provide a human cellular model to understand the molecular mechanisms underlying NF1 and, thus, discover new therapeutic strategies.

Generating Functional and Highly Proliferative Melanocytes Derived from Human Pluripotent Stem Cells: A Promising Tool for Biotherapeutic Approaches to Treat Skin Pigmentation Disorders.

Melanocytes are essential for skin homeostasis and protection, and their loss or misfunction leads to a wide spectrum of diseases. Cell therapy utilizing autologous melanocytes has been used for years as an adjunct treatment for hypopigmentary disorders such as vitiligo. However, these approaches are hindered by the poor proliferative capacity of melanocytes obtained from skin biopsies. Recent advances in the field of human pluripotent stem cells have fueled the prospect of generating melanocytes. Here, we have developed a well-characterized method to produce a pure and homogenous population of functional and proliferative melanocytes. The genetic stability and potential transformation of melanocytes from pluripotent stem cells have been evaluated over time during the in vitro culture process. Thanks to transcriptomic analysis, the molecular signatures all along the differentiation protocol have been characterized, providing a solid basis for standardizing the protocol. Altogether, our results promise meaningful, broadly applicable, and longer-lasting advances for pigmentation disorders and open perspectives for innovative biotherapies for pigment disorders.

MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate.

Deciphering the pathways that regulate human epidermal precursor cell fate is necessary for future developments in skin repair and graft bioengineering. Among them, characterization of pathways regulating the keratinocyte (KC) precursor immaturity versus differentiation balance is required for improving the efficiency of KC precursor ex vivo expansion. In this study, we show that the transcription factor MXD4/MAD4 is expressed at a higher level in quiescent KC stem/progenitor cells located in the basal layer of human epidermis than in cycling progenitors. In holoclone KCs, stable short hairpin-RNA‒mediated decreased expression of MXD4/MAD4 increases MYC expression, whose modulation increases the proliferation of KC precursors and maintenance of their clonogenic potential and preserves the functionality of these precursors in three-dimensional epidermis organoid generation. Altogether, these results characterize MXD4/MAD4 as a major piece of the stemness puzzle in the human epidermis KC lineage and pinpoint an original avenue for ex vivo expansion of human KC precursors.

Human-Induced Pluripotent Stem Cell‒Derived Keratinocytes, a Useful Model to Identify and Explore the Pathological Phenotype of Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS), an autosomal dominant skin disorder, is characterized by skin fragility. Genetically, the majority of cases are related to missense sequence variations in two keratin genes K5 or K14, leading to cytolysis of basal keratinocytes (KCs) and intraepidermal blistering. Progress toward the identification of treatments has been hampered by an incomplete understanding of the mechanisms underlying this disease and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome, thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSCs). In this study, we generated hiPSC-derived KCs from patients carrying keratin gene K5-dominant sequence variations and compared them with nonaffected hiPSC-derived KCs as well as their primary counterparts. Our results showed that EBS hiPSC-derived KCs displayed proliferative defects, increased capacity to migrate, alteration of extracellular signal‒regulated kinase signaling pathway, and cytoplasmic keratin filament aggregates as observed in primary EBS KCs. Of interest, EBS hiPSC-derived KCs exhibited downregulation of hemidesmosomal proteins, revealing the different effects of keratin gene K5 sequence variations on keratin cytoskeletal organization. With a combination of culture miniaturization and treatment with the chaperone molecule 4-phenylbutyric acid, our results showed that hiPSC-derived KCs represent a suitable model for identifying novel therapies for EBS.

Clinical Grade Human Pluripotent Stem Cell-Derived Engineered Skin Substitutes Promote Keratinocytes Wound Closure In Vitro.

Chronic wounds, such as leg ulcers associated with sickle cell disease, occur as a consequence of a prolonged inflammatory phase during the healing process. They are extremely hard to heal and persist as a significant health care problem due to the absence of effective treatment and the uprising number of patients. Indeed, there is a critical need to develop novel cell- and tissue-based therapies to treat these chronic wounds. Development in skin engineering leads to a small catalogue of available substitutes manufactured in Good Manufacturing Practices compliant (GMPc) conditions. Those substitutes are produced using primary cells that could limit their use due to restricted sourcing. Here, we propose GMPc protocols to produce functional populations of keratinocytes and fibroblasts derived from pluripotent stem cells to reconstruct the associated dermo-epidermal substitute with plasma-based fibrin matrix. In addition, this manufactured composite skin is biologically active and enhances in vitro wounding of keratinocytes. The proposed composite skin opens new perspectives for skin replacement using allogeneic substitute.

KLF4 inhibition promotes the expansion of keratinocyte precursors from adult human skin and of embryonic-stem-cell-derived keratinocytes.

Expanded autologous skin keratinocytes are currently used in cutaneous cell therapy, and embryonic-stem-cell-derived keratinocytes could become a complementary alternative. Regardless of keratinocyte provenance, for efficient therapy it is necessary to preserve immature keratinocyte precursors during cell expansion and graft processing. Here, we show that stable and transient downregulation of the transcription factor Krüppel-like factor 4 (KLF4) in keratinocyte precursors from adult skin, using anti-KLF4 RNA interference or kenpaullone, promotes keratinocyte immaturity and keratinocyte self-renewal in vitro, and enhances the capacity for epidermal regeneration in mice. Both stable and transient KLF4 downregulation had no impact on the genomic integrity of adult keratinocytes. Moreover, transient KLF4 downregulation in human-embryonic-stem-cell-derived keratinocytes increased the efficiency of skin-orientated differentiation and of keratinocyte immaturity, and was associated with improved generation of epidermis. As a regulator of the cell fate of keratinocyte precursors, KLF4 could be used for promoting the ex vivo expansion and maintenance of functional immature keratinocyte precursors.

Differentiation of nonhuman primate pluripotent stem cells into functional keratinocytes.

BACKGROUND: Epidermal grafting using cells derived from pluripotent stem cells will change the face of this side of regenerative cutaneous medicine. To date, the safety of the graft would be the major unmet deal in order to implement long-term skin grafting. In this context, experiments on large animals appear unavoidable to assess this question and possible rejection. Cellular tools for large animal models should be constructed. METHODS: In this study, we generated monkey pluripotent stem cell-derived keratinocytes and evaluated their capacities to reconstruct an epidermis, in vitro as well as in vivo. RESULTS: Monkey pluripotent stem cells were differentiated efficiently into keratinocytes able to reconstruct fully epidermis presenting a low level of major histocompatibility complex class-I antigens, opening the way for autologous or allogeneic epidermal long-term grafting. CONCLUSIONS: Functional keratinocytes generated from nonhuman primate embryonic stem cells and induced pluripotent stem cells reproduce an in-vitro and in-vivo stratified epidermis. These monkey skin grafts will be considered to model autologous or allogeneic epidermal grafting using either embryonic stem cells or induced pluripotent stem cells. This graft model will allow us to further investigate the safety, efficacy and immunogenicity of nonhuman primate PSC-derived epidermis in the perspective of human skin cell therapy.

Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.

Quality control of cell cultures used in new in vitro toxicology assays is crucial to the provision of reliable, reproducible and accurate toxicity data on new drugs or constituents of new consumer products. This chapter explores the key scientific and ethical criteria that must be addressed at the earliest stages of developing toxicology assays based on human pluripotent stem cell (hPSC) lines. It also identifies key considerations for such assays to be acceptable for regulatory, laboratory safety and commercial purposes. Also addressed is the development of hPSC-based assays for the tissue and cell types of greatest interest in drug toxicology. The chapter draws on a range of expert opinion within the European Commission/Cosmetics Europe-funded alternative testing cluster SEURAT-1 and consensus from international groups delivering this guidance such as the International Stem Cell Banking Initiative. Accordingly, the chapter summarizes the most up-date best practices in the use and quality control of human Pluripotent Stem Cell lines in the development of in vitro toxicity assays from leading experts in the field.

In vitro modeling of hyperpigmentation associated to neurofibromatosis type 1 using melanocytes derived from human embryonic stem cells.

"Café-au-lait" macules (CALMs) and overall skin hyperpigmentation are early hallmarks of neurofibromatosis type 1 (NF1). One of the most frequent monogenic diseases, NF1 has subsequently been characterized with numerous benign Schwann cell-derived tumors. It is well established that neurofibromin, the NF1 gene product, is an antioncogene that down-regulates the RAS oncogene. In contrast, the molecular mechanisms associated with alteration of skin pigmentation have remained elusive. We have reassessed this issue by differentiating human embryonic stem cells into melanocytes. In the present study, we demonstrate that NF1 melanocytes reproduce the hyperpigmentation phenotype in vitro, and further characterize the link between loss of heterozygosity and the typical CALMs that appear over the general hyperpigmentation. Molecular mechanisms associated with these pathological phenotypes correlate with an increased activity of cAMP-mediated PKA and ERK1/2 signaling pathways, leading to overexpression of the transcription factor MITF and of the melanogenic enzymes tyrosinase and dopachrome tautomerase, all major players in melanogenesis. Finally, the hyperpigmentation phenotype can be rescued using specific inhibitors of these signaling pathways. These results open avenues for deciphering the pathological mechanisms involved in pigmentation diseases, and provide a robust assay for the development of new strategies for treating these diseases.

The implementation of novel collaborative structures for the identification and resolution of barriers to pluripotent stem cell translation.

Increased global connectivity has catalyzed technological development in almost all industries, in part through the facilitation of novel collaborative structures. Notably, open innovation and crowd-sourcing-of expertise and/or funding-has tremendous potential to increase the efficiency with which biomedical ecosystems interact to deliver safe, efficacious and affordable therapies to patients. Consequently, such practices offer tremendous potential in advancing development of cellular therapies. In this vein, the CASMI Translational Stem Cell Consortium (CTSCC) was formed to unite global thought-leaders, producing academically rigorous and commercially practicable solutions to a range of challenges in pluripotent stem cell translation. Critically, the CTSCC research agenda is defined through continuous consultation with its international funding and research partners. Herein, initial findings for all research focus areas are presented to inform global product development strategies, and to stimulate continued industry interaction around biomanufacturing, strategic partnerships, standards, regulation and intellectual property and clinical adoption.

Functional melanocytes derived from human pluripotent stem cells engraft into pluristratified epidermis.

Melanocytes are essential for skin homeostasis and protection, and their defects in humans lead to a wide array of diseases that are potentially extremely severe. To date, the analysis of molecular mechanisms and the function of human melanocytes have been limited because of the difficulties in accessing large numbers of cells with the specific phenotypes. This issue can now be addressed via a differentiation protocol that allows melanocytes to be obtained from pluripotent stem cell lines, either induced or of embryonic origin, based on the use of moderate concentrations of a single cytokine, bone morphogenic protein 4. Human melanocytes derived from pluripotent stem cells exhibit all the characteristic features of their adult counterparts. This includes the enzymatic machinery required for the production and functional delivery of melanin to keratinocytes. Melanocytes also integrate appropriately into organotypic epidermis reconstructed in vitro. The availability of human cells committed to the melanocytic lineage in vitro will enable the investigation of those mechanisms that guide the developmental processes and will facilitate analysis of the molecular mechanisms responsible for genetic diseases. Access to an unlimited resource may also prove a vital tool for the treatment of hypopigmentation disorders when donors with matching haplotypes become available in clinically relevant banks of pluripotent stem cell lines.

miR-203 modulates epithelial differentiation of human embryonic stem cells towards epidermal stratification.

The molecular mechanisms controlling the differentiation of human basal keratinocyte stem cells towards the epidermis are well characterized, whereas the earliest process leading to the specification of embryonic stem cells into keratinocytes is still not well understood. MicroRNAs are regulators of many cellular events, but evidence for microRNA acting on the differentiation of human embryonic stem cells into a specific lineage has been elusive. By using our recent protocol for obtaining functional keratinocytes from hESC, we attempted to analyze the role of microRNAs in the early stages of epidermal differentiation. Thus, we identified a set of 5 microRNAs, namely miR-200a, miR-200b, miR-203, miR-205 and miR-429, that are specifically overexpressed during the early stages of the differentiation process. Interestingly, our functional analyses revealed an instrumental role of miR-203, which had been previously shown to play a key role during the formation of the pluristratified epidermis by basal keratinocyte stem cells, in the early keratinocyte commitment. These results highlight the determinant and unique role of miR-203 during the entire process of epidermal development by extending its spectrum of action from the early commitment of embryonic stem cells to ultimate differentiation of the organ.

Concise review: Epidermal grafting: the case for pluripotent stem cells.

Although cell therapy has been clinically implemented for several decades, its use is hampered by the difficulty in supplying the amount of epidermal substitute needed to extend the application to all patients who may benefit from it. How human pluripotent stem cells may help meet this challenge is the topic of this review. After reporting on the main current applications and needs of skin grafting, we explore the potential of pluripotent stem cells--either of embryonic origin or produced by genetic reprogramming--to provide the needed clinical-grade keratinocytes, fulfilling industrial scale production, and quality standards. Immunogenicity is clearly an issue, although one may expect cells displaying characteristics of fetal or embryonic skin to have a much better tolerance than adult keratinocytes. The open possibility of a bank of pluripotent stem cell lines selected on the basis of interesting haplotypes may eventually provide a definitive answer. Actually, making the case for pluripotent stem cells in skin grafting goes well beyond that specific cell type. Most cell phenotypes that normally participate to the formation of dermis and epidermis can either already be obtained through in vitro differentiation from pluripotent stem cells or would likely migrate from the host into a graft. However, differentiation protocols for specialized glands and hair follicles remain to be designed. A future can be foreseen when reconstructive medicine will make use of composite grafts integrating several different cell types and biomaterials.

CD98hc (SLC3A2) is a key regulator of keratinocyte adhesion.

BACKGROUND: Adhesion of keratinocytes is crucial for maintaining the integrity of the skin, as demonstrated by the number of dermatological disorders of genetic origin that are associated with a defect of basal keratinocyte adhesion. Integrins are the main component of the molecular networks involved in this phenomenon, but there are many others. In a recent description of proteins associated to caveolae at the plasma membrane of human basal epidermal cells, we demonstrated that CD98hc is localized with ß1 integrin. OBJECTIVES: We investigated the CD98hc proteins interactions and the role of CD98hc in keratinocyte adhesion. METHODS: CD98hc protein interaction was identified following co-immunoprecipitation and proteomic analysis using LTQ-FT mass spectrometer. Extinction of CD98hc gene expression using specific short hairpin RNA or over-expression of CD98hc lacking the ß1 integrin binding site was used to evaluate the role of this protein in keratinocyte fate. RESULTS: We show that CD98hc forms molecular complexes with ß1 and ß4 integrins in primary human keratinocytes and, using immunofluorescence, that these complexes are localized at the plasma membrane, in keeping with a role in adhesion. We confirmed that this protein is a key player of keratinocyte adhesion because in absence of interaction between CD98hc and integrins, ß1 integrin failed to translocate from the cytoplasm to the plasma membrane and keratinocytes expressed epidermal differentiation markers. CONCLUSIONS: All these data strongly suggested that CD98hc is involved in integrin trafficking and by consequence, in keratinocyte adhesion and differentiation.

Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells.

Tissue stem cells must be endowed with superior maintenance and repair systems to ensure genomic stability over multiple generations, which would be less necessary in more differentiated cells. We previously reported that human keratinocyte stem cells were more resistant to ionizing radiation toxicity than their direct progeny, the keratinocyte progenitor cells. In the present study we addressed the mechanisms underlying this difference. Investigations of DNA repair showed that both single and double DNA strand breaks were repaired more rapidly and more efficiently in stem cells than in progenitors. As cell signaling is a key regulatory step in the management of DNA damage, a gene profiling study was performed. Data revealed that several genes of the fibroblast growth factor type 2 (FGF2) signaling pathway were induced by DNA damage in stem cells and not in progenitors. Furthermore, an increased content of the FGF2 protein was found in irradiated stem cells, both for the secreted and the cellular forms of the protein. To examine the role of endogenous FGF2 in DNA repair, stem cells were exposed to FGF2 pathway inhibitors. Blocking the FGF2 receptor (FGF receptor 1) or the kinase (Ras-mitogen-activated protein kinase 1) resulted in a inhibition of single and double DNA strand-break repair in the keratinocyte stem cells. Moreover, supplementing the progenitor cells with exogenous FGF2 activated their DNA repair. We propose that, apart from its well-known role as a strong mitogen and prosurvival factor, FGF2 helps to maintain genomic integrity in stem cells by activating stress-induced DNA repair.