ABSTRACT
Endothelial dysfunction is widely implicated in cardiovascular pathological changes and development of vascular disease. In view of the fact that the spontaneous endothelial cell (EC) regeneration is a slow and insufficient process, it is of great interest to explore alternative cell sources capable of generating functional ECs. Vascular smooth muscle cell (SMC) composes the majority of the vascular wall and retains phenotypic plasticity in response to various stimuli. The aim of this study is to test the feasibility of the conversion of SMC into functional EC through the use of reprogramming factors. Human SMCs are first dedifferentiated for 4 days to achieve a vascular progenitor state expressing CD34, by introducing transcription factors OCT4, SOX2, KLF4 and c-MYC. These SMC-derived progenitors are then differentiated along the endothelial lineage. The SMC-converted ECs exhibit typical endothelial markers expression and endothelial functions in vitro, in vivo and in disease model. Further comprehensive analysis indicates that mesenchymal-to-epithelial transition is requisite to initiate SMCs reprogramming into vascular progenitors and that members of the Notch signalling pathway regulate further differentiation of the progenitors into endothelial lineage. Together, we provide the first evidence of the feasibility of the conversion of human SMCs towards endothelial lineage through an intermediate vascular progenitor state induced by reprogramming.
Subject(s)
Cell Transdifferentiation , Endothelium, Vascular/cytology , Hindlimb , Ischemia/therapy , Muscle, Smooth, Vascular/cytology , Regeneration , Animals , Cells, Cultured , Coculture Techniques , Endothelium, Vascular/metabolism , Humans , Ischemia/metabolism , Kruppel-Like Factor 4 , Mice , Mice, SCID , Muscle, Smooth, Vascular/metabolism , Signal TransductionABSTRACT
BACKGROUND: The majority of children with eczema in the UK are looked after in primary care yet we know little about their care in this setting. AIM: To compare the diagnosis, assessment, and treatment of eczema in primary care with published diagnostic criteria and management guidelines. DESIGN & SETTING: Cross-sectional study using data from a randomised controlled feasibility study. General practices, UK. METHOD: Baseline data from children aged 1 month to 5 years recruited 'in-consultation' for the Choice of Moisturiser in Eczema Treatment (COMET) feasibility study was used. These included clinician diagnosis and global severity assessment; the parent-completed Patient Orientated Eczema Measure (POEM); a questionnaire about eczema treatments, including use of topical corticosteroid (TCS); and, the Eczema Area Severity Index (EASI) carried out by trained researchers. Descriptive analyses were undertaken to compare diagnoses with UK diagnostic criteria, severity assessments, and treatment with the National Institute for Health and Care Excellent (NICE) guidance. RESULTS: Data were available for 90 participants. Only 46% of participants labelled as having eczema met the UK diagnostic criteria. Agreement between the global severity assessment by a healthcare practitioner with the EASI and POEM measures of eczema severity were 44% and 48% respectively. Emollients and TCSs were underused with 44% of participants not using any emollient and 46% using one or more TCSs. The 'match' between eczema severity and TCSs potency was poor. CONCLUSION: Discrepancies were found between the diagnosis, assessment, and treatment of children with eczema in primary care, and UK diagnostic criteria and guidelines. Further investigation to explore the reasons for this discordance, and whether it matters, is needed.
ABSTRACT
The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study, we used exome sequencing, array comparative genomic hybridization (CGH), miRNA array, DNA methylation array, three-dimensional (3D) tissue engineering, and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE), which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected, ≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex, a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin, TCHH), found that in one of the iPSC clones (iKCL004), TCHH retained a DNA methylation signature characteristic of the original somatic cells, whereas in other iPSC line (iKCL011), the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE, suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome.
Subject(s)
Cell Differentiation/genetics , Epigenesis, Genetic , Induced Pluripotent Stem Cells/cytology , Intermediate Filament Proteins/genetics , Tissue Engineering/methods , Adult , Cell Line , Cellular Reprogramming/genetics , Clone Cells , DNA Methylation/genetics , Epidermis/metabolism , Gene Expression Profiling , Genomic Instability , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Infant, Newborn , Intermediate Filament Proteins/metabolism , Keratinocytes/cytology , Keratinocytes/metabolism , Male , MicroRNAs/genetics , MicroRNAs/metabolism , Mutation Rate , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
The KCL012 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (46 trinucleotide repeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Huntingtin Protein/genetics , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Fertilization in Vitro , Genotype , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Polymorphism, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Trinucleotide Repeats/geneticsABSTRACT
The KCL027 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Huntingtin Protein/genetics , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Fertilization in Vitro , Genotype , Histocompatibility Testing , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Pedigree , Polymorphism, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Trinucleotide Repeats/geneticsABSTRACT
The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Huntingtin Protein/genetics , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Fertilization in Vitro , Genotype , Histocompatibility Testing , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Pedigree , Polymorphism, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Trinucleotide Repeats/geneticsABSTRACT
The KCL013 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (42 trinucleotide repeats; 17 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Huntingtin Protein/genetics , Alleles , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Fertilization in Vitro , Genotype , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Pedigree , Polymorphism, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Trinucleotide Repeats/geneticsABSTRACT
The KCL036 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (38 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Huntingtin Protein/genetics , Alleles , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Fertilization in Vitro , Genotype , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Pedigree , Polymorphism, Genetic , Transcription Factors/genetics , Transcription Factors/metabolism , Trinucleotide Repeats/genetics , Troponin T/metabolismABSTRACT
The KCL040 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Cell Differentiation , Cell Line , Comparative Genomic Hybridization , Fibroblasts/cytology , Genotype , Histocompatibility Testing , Human Embryonic Stem Cells/metabolism , Humans , Male , Microscopy, Fluorescence , Middle Aged , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The KCL034 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility, specific and non-specific human pathogens.
Subject(s)
Human Embryonic Stem Cells/cytology , Cell Differentiation , Cell Line , Comparative Genomic Hybridization , Embryo, Mammalian/cytology , Genotype , Histocompatibility Testing , Human Embryonic Stem Cells/metabolism , Humans , Male , Microscopy, Fluorescence , Middle Aged , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.
Subject(s)
Human Embryonic Stem Cells/cytology , Alkaline Phosphatase/metabolism , Blastocyst/cytology , Cell Differentiation , Cell Line , Comparative Genomic Hybridization , Genotype , Histocompatibility Testing , Human Embryonic Stem Cells/metabolism , Humans , Karyotype , Male , Microscopy, Fluorescence , Middle AgedABSTRACT
Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature.
Subject(s)
Cell Differentiation , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Animals , Humans , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
Huntington disease (HD; OMIM 143100), a progressive neurodegenerative disorder, is caused by an expanded trinucleotide CAG (polyQ) motif in the HTT gene. Cardiovascular symptoms, often present in early stage HD patients, are, in general, ascribed to dysautonomia. However, cardio-specific expression of polyQ peptides caused pathological response in murine models, suggesting the presence of a nervous system-independent heart phenotype in HD patients. A positive correlation between the CAG repeat size and severity of symptoms observed in HD patients has also been observed in in vitro HD cellular models. Here, we test the suitability of human embryonic stem cell (hESC) lines carrying HD-specific mutation as in vitro models for understanding molecular mechanisms of cardiac pathology seen in HD patients. We have differentiated three HD-hESC lines into cardiomyocytes and investigated CAG stability up to 60 days after starting differentiation. To assess CAG stability in other tissues, the lines were also subjected to in vivo differentiation into teratomas for 10 weeks. Neither directed differentiation into cardiomyocytes in vitro nor in vivo differentiation into teratomas, rich in immature neuronal tissue, led to an increase in the number of CAG repeats. Although the CAG stability might be cell line-dependent, induced pluripotent stem cells generated from patients with larger numbers of CAG repeats could have an advantage as a research tool for understanding cardiac symptoms of HD patients.
Subject(s)
Cell Differentiation/genetics , Human Embryonic Stem Cells/metabolism , Huntington Disease/pathology , Mutation/genetics , Myocytes, Cardiac/cytology , Trinucleotide Repeat Expansion/genetics , Cell Line , Humans , Pluripotent Stem Cells/cytologyABSTRACT
Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening.
Subject(s)
Embryonic Stem Cells/metabolism , Epidermis/metabolism , Induced Pluripotent Stem Cells/metabolism , Models, Biological , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Cellular Reprogramming , DNA Methylation , Embryonic Stem Cells/cytology , Epithelial-Mesenchymal Transition , Humans , Induced Pluripotent Stem Cells/cytology , Keratin-14/genetics , Keratin-14/metabolism , Keratinocytes/cytology , Keratinocytes/metabolism , Permeability , Principal Component Analysis , Teratoma/pathology , Transcription Factors/genetics , Transcription Factors/metabolism , TranscriptomeABSTRACT
Here, we describe a pre-derivation embryo haplotyping strategy that we developed in order to maximize the efficiency and minimize the costs of establishing banks of clinical grade hESC lines in which human leukocyte antigen (HLA) haplotypes match a significant proportion of the population. Using whole genome amplification followed by medium resolution HLA typing using PCR amplification with sequence-specific primers (PCR-SSP), we have typed the parents, embryos and hESC lines from three families as well as our eight clinical grade hESC lines and shown that this technical approach is rapid, reliable and accurate. By employing this pre-derivation strategy where, based on HLA match, embryos are selected for a GMP route on day 3-4 of development, we would have drastically reduced our cGMP laboratory running costs.
Subject(s)
Embryonic Stem Cells/cytology , Embryonic Stem Cells/immunology , Histocompatibility Testing , Cell Culture Techniques/methods , Cell Culture Techniques/standards , Cell Line , Female , HLA Antigens/genetics , Haplotypes , Histocompatibility Testing/methods , Humans , Huntington Disease/diagnosis , Huntington Disease/genetics , Male , Polymerase Chain Reaction , Pregnancy , Preimplantation Diagnosis/methods , Tissue BanksABSTRACT
The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.
Subject(s)
Blastocyst/cytology , Cell Culture Techniques/methods , Cell Culture Techniques/standards , Cryopreservation/methods , Embryonic Stem Cells/cytology , Cell Line , Humans , Laser Capture Microdissection , United KingdomABSTRACT
BACKGROUND AIMS: Human embryonic stem (hES) cells hold great potential for cell therapy and regenerative medicine because of their pluripotency and capacity for self-renewal. The conditions used to derive and culture hES cells vary between and within laboratories depending on the desired use of the cells. Until recently, stem cell culture has been carried out using feeder cells, and culture media, that contain animal products. Recent advances in technology have opened up the possibility of both xeno-free and feeder-free culture of stem cells, essential conditions for the use of stem cells for clinical purposes. To date, however, there has been limited success in achieving this aim. METHODS, RESULTS AND CONCLUSIONS: Protocols were developed for the successful derivation of two normal and three specific mutation-carrying (SMC) (Huntington's disease and myotonic dystrophy 1) genomically stable hES cell lines, and their adaptation to feeder-free culture, all under xeno-free conditions.
Subject(s)
Cell Differentiation , Coculture Techniques , Embryonic Stem Cells/physiology , Animals , Cell Survival , Cells, Cultured , Culture Media, Serum-Free , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/transplantation , Feeder Cells/cytology , Feeder Cells/physiology , Humans , Huntington Disease/genetics , Huntington Disease/therapy , Myotonic Dystrophy/genetics , Myotonic Dystrophy/therapyABSTRACT
The use of stem cells for regenerative medicine has captured the imagination of the public, with media attention contributing to rising expectations of clinical benefits. Human embryonic stem cells (hESCs) are the best model for capital investment in stem cell therapy and there is a clear need for their robust genetic characterization before scaling-up cell expansion for that purpose. We have to be certain that the genome of the starting material is stable and normal, but the limited resolution of conventional karyotyping is unable to give us such assurance. Advanced molecular cytogenetic technologies such as array comparative genomic hybridization for identifying chromosomal imbalances, and single nucleotide polymorphism analysis for identifying ethnic background and loss of heterozygosity should be introduced as obligatory diagnostic tests for each newly derived hESC line before it is deposited in national stem cell banks. If this new quality standard becomes a requirement, as we are proposing here, it would facilitate and accelerate the banking process, since end-users would be able to select the most appropriate line for their particular application, thus improving efficiency and streamlining the route to manufacturing therapeutics. The pharmaceutical industry, which may use hESC-derived cells for drug screening, should not ignore their genomic profile as this may risk misinterpretation of results and significant waste of resources.