RESUMEN
The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.
Asunto(s)
Neoplasias de la Mama , Mama , Embarazo , Humanos , Femenino , Adulto , Paridad , Neoplasias de la Mama/genética , Mutación , Células EpitelialesRESUMEN
We investigated whether maternal metabolic environment affects mesenchymal stromal/stem cells (MSCs) from umbilical cord's Wharton's Jelly (WJ) on a molecular level, and potentially render them unsuitable for clinical use in multiple recipients. In this pilot study on umbilical cords post partum from healthy non-obese (BMI = 19-25; n = 7) and obese (BMI ≥ 30; n = 7) donors undergoing elective Cesarean section, we found that WJ MSC from obese donors showed slower population doubling and a stronger immunosuppressive activity. Genome-wide DNA methylation of triple positive (CD73+CD90+CD105+) WJ MSCs found 67 genes with at least one CpG site where the methylation difference was ≥0.2 in four or more obese donors. Only one gene, PNPLA7, demonstrated significant difference on methylome, transcriptome and protein level. Although the number of analysed donors is limited, our data suggest that the altered metabolic environment related to excessive body weight might bear consequences on the WJ MSCs.
Asunto(s)
Células Madre Mesenquimatosas/patología , Madres , Obesidad/patología , Gelatina de Wharton/patología , Adulto , Antígeno CD56/metabolismo , Estudios de Casos y Controles , Diferenciación Celular , Metilación de ADN , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunomodulación , Lipasa/genética , Lisofosfolipasa , Células Madre Mesenquimatosas/metabolismo , Obesidad/genética , Obesidad/inmunología , Obesidad/metabolismo , Proyectos Piloto , EmbarazoRESUMEN
The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 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 assays.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Proteína Quinasa de Distrofia Miotónica/genética , Alelos , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Hibridación Genómica Comparativa , Embrión de Mamíferos/citología , Cuerpos Embrioides/citología , Femenino , Fertilización In Vitro , Genotipo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Microscopía Fluorescente , Distrofia Miotónica/genética , Distrofia Miotónica/patología , Linaje , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The KCL032 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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/citología , Células Madre Embrionarias Humanas/citología , Biomarcadores/metabolismo , Diferenciación Celular , HumanosRESUMEN
The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). 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.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Línea Celular/citología , Células Madre Embrionarias Humanas/citología , Mutación/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Biomarcadores/metabolismo , Diferenciación Celular , Femenino , Humanos , Masculino , LinajeRESUMEN
The KCL038 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.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Fosfatasa Alcalina/metabolismo , Blastocisto/citología , Línea Celular , Supervivencia Celular , Hibridación Genómica Comparativa , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana EdadRESUMEN
The KCL037 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.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Fosfatasa Alcalina/metabolismo , Blastocisto/citología , Línea Celular , Supervivencia Celular , Hibridación Genómica Comparativa , Genotipo , Prueba de Histocompatibilidad , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana EdadRESUMEN
The KCL021 human embryonic stem cell line was derived from an embryo donated for research that carried a ΔF508 mutation affecting the CFTR gene encoding the cystic fibrosis transmembrane conductance regulator. 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.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Madre Embrionarias Humanas/citología , Fosfatasa Alcalina/metabolismo , Diferenciación Celular , Línea Celular , Hibridación Genómica Comparativa , Embrión de Mamíferos/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Mutación , LinajeRESUMEN
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.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Línea Celular , Hibridación Genómica Comparativa , Embrión de Mamíferos/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The KCL029 human embryonic stem cell line was derived from an embryo donated for research that carried a c.814T>C mutation in the WAS gene, which is linked to the Wiskott-Aldrich syndrome, a rare, inherited, X-linked, recessive disease characterized by immune dysregulation and microthrombocytopenia. The line is also carrier for a mutation p.N1152H in the gene encoding the cystic fibrosis transmembrane conductance regulator CFTR. 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.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Proteína del Síndrome de Wiskott-Aldrich/genética , Fosfatasa Alcalina/metabolismo , Diferenciación Celular , Línea Celular , Hibridación Genómica Comparativa , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Embrión de Mamíferos/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
In an attempt to bring pluripotent stem cell biology closer to reaching its full potential, many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Vectores Genéticos/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Virus Sendai/genética , Gelatina de Wharton/citología , Diferenciación Celular , Separación Celular/métodos , Células Cultivadas , Criopreservación/métodos , Medios de Cultivo , Fibroblastos/citología , Humanos , Cordón Umbilical/citología , VitrificaciónRESUMEN
BACKGROUND: Human embryonic and induced pluripotent stem cells (hESC and hiPSC) have tremendous potential for clinical implementation. In spite of all hurdles and controversy, clinical trials in treatment of spinal cord injury, macular degeneration of retina, type 1 diabetes and heart failure are already ongoing. SOURCES OF DATA: ClinicalTrials.gov database, International Clinical Trials Registry Platform, PubMed and press releases and websites of companies and institutions working on hESC- and iPSC-based cellular therapy. AREAS OF AGREEMENT: The initial results from multiple clinical trials demonstrate that hESC-based therapies are safe and promising. AREAS OF CONTROVERSY: Are iPSC cells safe in the clinical application? Is there a room for both hESC and iPSC in the future clinical applications? GROWING POINTS: Increasing number of new clinical trials. AREAS TIMELY FOR DEVELOPING RESEARCH: Development of hESC- and/or iPSC-based cellular therapy for other diseases.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Embrionarias/trasplante , Células Madre Pluripotentes Inducidas/trasplante , Ensayos Clínicos como Asunto/métodos , Diabetes Mellitus Tipo 1/terapia , Insuficiencia Cardíaca/terapia , Humanos , Degeneración Macular/terapia , Traumatismos de la Médula Espinal/terapiaRESUMEN
Standardization guidelines for human pluripotent stem cells are still very broadly defined, despite ongoing clinical trials in the U.S., U.K., and Japan. The requirements for validation of human embryonic (hESCs) and induced pluripotent stem cells (iPSCs) in general follow the regulations for other clinically compliant biologics already in place but without addressing key differences between cell types or final products. In order to realize the full potential of stem cell therapy, validation criteria, methodology, and, most importantly, strategy, should address the shortfalls and efficiency of current approaches; without this, hESC- and, especially, iPSC-based therapy will not be able to compete with other technologies in a cost-efficient way. We addressed the protocols for testing cell lines for human viral pathogens and propose a novel strategy that would significantly reduce costs. It is highly unlikely that the multiple cell lines derived in parallel from a tissue sample taken from one donor would have different profiles of endogenous viral pathogens; we therefore argue that samples from the Master Cell Banks of sibling lines could be safely pooled for validation. We illustrate this approach with tiered validation of two sibling clinical-grade hESC lines, KCL033 and KCL034 (stage 1, sterility; stage 2, specific human pathogens; and stage 3, nonspecific human pathogens). The results of all tests were negative. This cost-effective strategy could also be applied for validation of Master Cell Banks of multiple clinical-grade iPSC lines derived from a single donor.
Asunto(s)
Células Madre Pluripotentes , Bancos de Tejidos/normas , Línea Celular , Humanos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
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.
Asunto(s)
Blastocisto/citología , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/normas , Criopreservación/métodos , Células Madre Embrionarias/citología , Línea Celular , Humanos , Captura por Microdisección con Láser , Reino UnidoRESUMEN
BACKGROUND: Umbilical cord blood (UCB) has become the second most common source of stem cells for cell therapy. The recent boom in stem cell research and public fascination with promises of stem cell-based therapies, fueled by the media, have led researchers to explore the potential of UCB stem cells in therapy for non-hematological disorders. SOURCES OF DATA: ClinicalTrials.gov database searched with key words 'cord blood stem cells' on December 28, 2011. AREAS OF AGREEMENT: As a rich source of the most primitive hematopoietic stem cells, UCB has a strong regenerative potential in stem cell-based-therapy for hematological disorders. AREAS OF CONTROVERSY: Potential of UCB stem cells in therapy for non-hematological disorders. GROWING POINTS: Increasing number of clinical trials with UCB stem cell-based therapy for a variety of diseases. AREAS TIMELY FOR DEVELOPING RESEARCH: A need for standardization of criteria for selection of UCB units for stem cell-based therapy, outcome measures and long-term follow-up.