RESUMEN
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.
Asunto(s)
MicroARNs , ARN Largo no Codificante , Humanos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estudios Prospectivos , Transducción de Señal , Células Madre/metabolismo , MicroARNs/metabolismo , Proteínas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
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.
Asunto(s)
Trastornos de la Pigmentación , Células Madre Pluripotentes , Vitíligo , Humanos , Trastornos de la Pigmentación/terapia , Melanocitos/patología , Piel/patología , Vitíligo/terapia , Vitíligo/patología , Pigmentación de la PielRESUMEN
"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.
Asunto(s)
Células Madre Embrionarias/citología , Hiperpigmentación/patología , Melanocitos/patología , Modelos Biológicos , Neurofibromatosis 1/patología , Proliferación Celular , AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Melaninas/metabolismo , Melanocitos/enzimología , Melanocitos/metabolismo , Melanocitos/ultraestructura , Mutación/genética , Neurofibromina 1/genética , Fenotipo , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
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.
Asunto(s)
Técnicas In Vitro/normas , Células Madre Pluripotentes/citología , Pruebas de Toxicidad/métodos , Diferenciación Celular , Proliferación Celular , Humanos , Control de CalidadRESUMEN
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.
Asunto(s)
Células Madre Adultas/citología , Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Células Epidérmicas , Melanocitos/citología , Células Madre Pluripotentes/citología , Células Madre Adultas/metabolismo , Línea Celular , Epidermis/metabolismo , Humanos , Hipopigmentación/metabolismo , Hipopigmentación/terapia , Melanocitos/metabolismo , Células Madre Pluripotentes/metabolismo , Trasplante de Células MadreRESUMEN
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.
RESUMEN
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-RNAâmediated 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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Epidérmicas , Queratinocitos , Humanos , Diferenciación Celular , Epidermis/metabolismo , Queratinocitos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
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.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Epidermis/embriología , MicroARNs/fisiología , Linaje de la Célula , Células Cultivadas , Humanos , Queratinocitos/citología , Especificidad de ÓrganosRESUMEN
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.
Asunto(s)
Células Madre Pluripotentes/trasplante , Trasplante de Piel/métodos , Animales , Diferenciación Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Humanos , Queratinocitos/trasplante , Células Madre Pluripotentes/citología , Trasplante Homólogo/efectos adversos , Trasplante Homólogo/métodosRESUMEN
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 signalâregulated 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.
Asunto(s)
Epidermólisis Ampollosa Simple , Células Madre Pluripotentes Inducidas , Epidermólisis Ampollosa Simple/metabolismo , Quinasas MAP Reguladas por Señal Extracelular , Humanos , Queratina-14/genética , Queratina-14/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Queratinocitos/metabolismo , Queratinas/genética , Queratinas/metabolismo , Mutación , FenotipoRESUMEN
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.
Asunto(s)
Células Madre Pluripotentes , Piel Artificial , Humanos , Queratinocitos , Piel , Ingeniería de Tejidos/métodosRESUMEN
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.
Asunto(s)
Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Reparación del ADN , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Queratinocitos/metabolismo , Transducción de Señal , Células Madre/metabolismo , Anticuerpos Monoclonales/farmacología , Butadienos/farmacología , Ciclo Celular , Células Cultivadas , Ensamble y Desensamble de Cromatina , Ensayo Cometa , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factor 2 de Crecimiento de Fibroblastos/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Inestabilidad Genómica , Histonas/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , Nitrilos/farmacología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/metabolismo , Serina , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/efectos de la radiación , Células Madre/efectos de los fármacos , Células Madre/efectos de la radiación , Factores de TiempoRESUMEN
BACKGROUND: Cell therapy for large burns is dependent upon autologous epidermis reconstructed in vitro. However, the effectiveness of current procedures is limited by the delay needed to culture the patient's own keratinocytes. To assess whether the keratinocyte progeny of human embryonic stem cells (hESCs) could be used to form a temporary skin substitute for use in patients awaiting autologous grafts, we investigated the cells' capability of constructing a pluristratified epidermis. METHODS: hESCs from lines H9 and SA01 were seeded at least in triplicate on fibroblast feeder cells for 40 days in a medium supplemented with bone morphogenetic protein 4 and ascorbic acid. Molecular characterisation of cell differentiation was done throughout the process by quantitative PCR, fluorescence-activated cell sorting, and immunocytochemical techniques. Keratinocyte molecular differentiation and functional capacity to construct a human epidermis were assessed in vitro and in vivo. FINDINGS: From hESCs, we generated a homogeneous population of cells that showed phenotypic characteristics of basal keratinocytes. Expression levels of genes encoding keratin 14, keratin 5, integrin alpha6, integrin beta4, collagen VII, and laminin 5 in these cells were similar to those in basal keratinocytes. After seeding on an artificial matrix, keratinocytes derived from hESCs (K-hESCs) formed a pluristratified epidermis. Keratin-14 immunostaining was seen in the basal compartment, with keratin 10 present in layers overlying the basal layer. Involucrin and filaggrin, late markers of epidermal differentiation, were detected in the uppermost layers only. 12 weeks after grafting onto five immunodeficient mice, epidermis derived from K-hESCs had a structure consistent with that of mature human skin. Human involucrin was appropriately located in spinous and granular layers and few Ki67-positive cells were detected in the basal layer. INTERPRETATION: hESCs can be differentiated into basal keratinocytes that are fully functional--ie, able to construct a pluristratified epidermis. This resource could be developed to provide temporary skin substitutes for patients awaiting autologous grafts. FUNDING: Institut National de la Santé et de la Recherche Médicale, University Evry Val d'Essonne, Association Française contre les Myopathies, Fondation René Touraine, and Genopole.
Asunto(s)
Células Madre Embrionarias/citología , Células Epidérmicas , Queratinocitos/citología , Células Madre Pluripotentes/citología , Piel Artificial , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Proteínas Filagrina , Humanos , Queratinocitos/metabolismo , Queratinas/metabolismo , Ratones , Ingeniería de TejidosRESUMEN
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.
Asunto(s)
Queratinocitos/inmunología , Queratinocitos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Piel/metabolismo , Adulto , Animales , Diferenciación Celular , Regulación hacia Abajo , Células Epidérmicas/metabolismo , Células Epidérmicas/patología , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Xenoinjertos , Humanos , Queratinocitos/patología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Desnudos , Piel/patología , Células MadreRESUMEN
PURPOSE: Radiosensitivity of stem cells is a matter of debate. For mouse somatic stem cells, both radiosensitive and radioresistant stem cells have been described. By contrast, the response of human stem cells to radiation has been poorly studied. As epidermis is a radiosensitive tissue, we evaluated in the present work the radiosensitivity of cell populations enriched for epithelial stem cells of human epidermis. METHODS AND MATERIALS: The total keratinocyte population was enzymatically isolated from normal human skin. We used flow cytometry and antibodies against cell surface markers to isolate basal cell populations from human foreskin. Cell survival was measured after a dose of 2Gy with the XTT assay at 72h after exposure and with a clonogenic assay at 2 weeks. Transcriptome analysis using oligonucleotide microarrays was performed to assess the genomic cell responses to radiation. RESULTS: Cell sorting based on two membrane proteins, alpha6 integrin and the transferrin receptor CD71, allowed isolation of keratinocyte populations enriched for the two types of cells found in the basal layer of epidermis: stem cells and progenitors. Both the XTT assay and the clonogenic assay showed that the stem cells were radioresistant whereas the progenitors were radiosensitive. We made the hypothesis that upstream DNA damage signalling might be different in the stem cells and used microarray technology to test this hypothesis. The stem cells exhibited a much more reduced gene response to a dose of 2Gy than the progenitors, as we found that 6% of the spotted genes were regulated in the stem cells and 20% in the progenitors. Using Ingenuity Pathway Analysis software, we found that radiation exposure induced very specific pathways in the stem cells. The most striking responses were the repression of a network of genes involved in apoptosis and the induction of a network of cytokines and growth factors. CONCLUSION: These results show for the first time that keratinocyte populations enriched for stem cells from human epidermis are radioresistant. Based on both repressed and induced genes, we found that the major response of the irradiated stem cell population was the regulation of genes functionally related to cell death, cell survival and apoptosis.
Asunto(s)
Queratinocitos/citología , Queratinocitos/efectos de la radiación , Células Madre/efectos de la radiación , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/efectos de la radiación , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , Tolerancia a Radiación/genéticaRESUMEN
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.
Asunto(s)
Queratinocitos/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular , Haplorrinos , Queratinocitos/citologíaAsunto(s)
Diferenciación Celular , Melanocitos/fisiología , Trastornos de la Pigmentación/terapia , Células Madre Pluripotentes/fisiología , Trasplante de Células Madre/métodos , Fenómenos Cronobiológicos/fisiología , Desarrollo Embrionario/fisiología , Humanos , Melanocitos/citología , Modelos Biológicos , Piel/citología , Piel/embriología , Pigmentación de la Piel/fisiología , Trasplante de Células Madre/estadística & datos numéricosAsunto(s)
Técnicas de Cultivo de Célula , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Animales , Ácido Ascórbico/farmacología , Diferenciación Celular , Linaje de la Célula , Separación Celular/métodos , Células Cultivadas/trasplante , Fenómenos Cronobiológicos , Técnicas de Cocultivo , Células Madre Embrionarias/efectos de los fármacos , Células Epidérmicas , Humanos , Queratinocitos/citología , Queratinocitos/trasplante , Ratones , Ratones SCID , Neuronas/citología , Células Madre Pluripotentes/efectos de los fármacos , Trasplante HeterólogoRESUMEN
BACKGROUND: MALDI-TOF-MS has become an important analytical tool in the identification of proteins and evaluation of their role in biological processes. A typical protocol consists of sample purification, separation of proteins by 2D-PAGE, enzymatic digestion and identification of proteins by peptide mass fingerprint. Unfortunately, this approach is not appropriate for the identification of membrane or low or high pI proteins. An alternative technique uses 1D-PAGE, which results in a mixture of proteins in each gel band. The direct analysis of the proteolytic digestion of this mixture is often problematic because of poor peptide detection and consequent poor sequence coverage in databases. Sequence coverage can be improved through the combination of several matrices. RESULTS: The aim of this study was to trust the MALDI analysis of complex biological samples, in order to identify proteins that interact with the membrane network of keratinocytes. Peptides obtained from protein trypsin digestions may have either hydrophobic or hydrophilic sections, in which case, the direct analysis of such a mixture by MALDI does not allow desorbing of all peptides. In this work, MALDI/MS experiments were thus performed using four different matrices in concert. The data were analysed with three algorithms in order to test each of them. We observed that the use of at least two matrices in concert leads to a twofold increase of the coverage of each protein. Considering data obtained in this study, we recommend the use of HCCA in concert with the SA matrix in order to obtain a good coverage of hydrophilic proteins, and DHB in concert with the SA matrix to obtain a good coverage of hydrophobic proteins. CONCLUSION: In this work, experiments were performed directly on complex biological samples, in order to see systematic comparison between different matrices for real-life samples and to show a correlation that will be applicable to similar studies. When 1D gel is needed, each band may contain a great number of proteins, each present in small amounts. To improve the proteins coverage, we have performed experiments with some matrices in concert. These experiments enabled reliable identification of proteins, without the use of Nanospray MS/MS experiments.
RESUMEN
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.