RESUMEN
From 2006 to 2011, Roslin Cells Ltd derived 17 human embryonic stem cells (hESC) while developing (RCM1, RC-2 to -8, -10) and implementing (RC-9, -11 to -17) quality assured standards of operation in a facility operating in compliance with European Union (EU) directives and United Kingdom (UK) licensure for procurement, processing and storage of human cells as source material for clinical application, and targeted to comply with an EU Good Manufacturing Practice specification. Here we describe the evolution and specification of the facility, its operation and outputs, complementing hESC resource details communicated in Stem Cell Research Lab Resources.
Asunto(s)
Técnicas de Cultivo de Célula/normas , Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Células Cultivadas , Regulación Gubernamental , Humanos , Control de CalidadRESUMEN
The human embryonic stem cell line RCe021-A (RC-17) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a day 3 embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe021-A (RC-17) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
RESUMEN
The human embryonic stem cell line RCe013-A (RC-9) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following informed consent. RCe013-A (RC-9) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
RESUMEN
The human embryonic stem cell line RCe015-A (RC-11) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a fragmented cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe015-A (RC-11) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
RESUMEN
The human embryonic stem cell line RCe009-A (RC-5) was derived from a frozen and thawed Day 2 embryo voluntarily donated as unsuitable and surplus to requirement for fertility treatment following informed consent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic fibrosis trans-membrane regulator gene associated with the disease cystic fibrosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Hibridación Genómica Comparativa , Cuerpos Embrioides/citología , Femenino , Citometría de Flujo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Microsatellite DNA marker identity and HLA and blood group typing data are available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cromosomas Humanos Par 12 , Hibridación Genómica Comparativa , Síndrome de Down/metabolismo , Síndrome de Down/patología , Cuerpos Embrioides/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Masculino , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TrisomíaRESUMEN
The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Óvulo/citología , Animales , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Hibridación Genómica Comparativa , Cuerpos Embrioides/citología , Femenino , Citometría de Flujo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/trasplante , Humanos , Cariotipo , Ratones , Ratones SCID , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Teratoma/metabolismo , Teratoma/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
The human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Alelos , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cuerpos Embrioides/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Antígeno Lewis X/metabolismo , Masculino , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe011-A (RC-7) was derived from a failed to fertilise oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
Asunto(s)
Embrión de Mamíferos/citología , Células Madre Embrionarias Humanas/citología , Alelos , Diferenciación Celular , Células Cultivadas , Cuerpos Embrioides/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Antígeno Lewis X/metabolismo , Masculino , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Alelos , Diferenciación Celular , Células Cultivadas , Cuerpos Embrioides/citología , Femenino , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Antígeno Lewis X/metabolismo , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
The human embryonic stem cell line RCe014-A (RC-10) was derived from a fresh oocyte voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XY and 47XY +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Células Cultivadas , Cromosomas Humanos Par 12 , Embrión de Mamíferos/citología , Cuerpos Embrioides/citología , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Inmunohistoquímica , Cariotipo , Masculino , Repeticiones de Microsatélite/genética , Microscopía Fluorescente , Oocitos/citología , Factores de Transcripción/metabolismo , TrisomíaRESUMEN
The human embryonic stem cell line RCe007-A (RC-3) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Blastocisto/citología , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Femenino , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Repeticiones de Microsatélite/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe008-A (RC-4) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to ectoderm and mesoderm in vitro. It has a mixed 46XX/45X female karyotype and microsatellite PCR identity and blood group typing data is available.
Asunto(s)
Células Madre Embrionarias Humanas/citología , Blastocisto/citología , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Femenino , Genotipo , Prueba de Histocompatibilidad , Humanos , Cariotipo , Repeticiones de Microsatélite/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe019-A (RC-15) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a cleavage stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe019-A (RC-15) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 46XX/47XX, +8 female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Femenino , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Repeticiones de Microsatélite/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe017-A (RC-13) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a frozen and thawed blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe017-A (RC-13) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a mixed 47XY, +12/48XY, +1, +12 male karyotype and microsatellite PCR identity, HLA and blood group typing data are available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cromosomas Humanos Par 12 , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Masculino , Repeticiones de Microsatélite/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TrisomíaRESUMEN
The human embryonic stem cell line RCe018-A (RC-14) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a blastocyst stage embryo voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe018-A (RC-14) shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a male karyotype with an extra copy of chromosome 8 (47XY, +8). Microsatellite PCR identity, HLA and blood group typing data are available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Cromosomas Humanos Par 8 , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Masculino , Repeticiones de Microsatélite/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TrisomíaRESUMEN
The human embryonic stem cell line RCe016-A (RC-12) was derived under quality assured compliance with UK regulations, EU Directives and International guidance for tissue procurement, processing and storage according to good manufacturing practice (GMP) standards. The cell line was derived from a cryopreserved blastocyst stage embryo voluntarily donated as surplus to fertility requirements following informed consent. RCe016-A (RC-12) shows normal pluripotency marker expression and differentiation to three germ layers in vitro. Karyology revealed a mixed male karyotype at early passage (P15), which resolved as normal 46XY by passage 33. Microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Masculino , Repeticiones de Microsatélite/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The human embryonic stem cell line RCe020-A (RC-16) was derived under quality assured compliance with UK regulation, European Union Directives and International guidance for tissue procurement, processing and storage according to Good Manufacturing Practice (GMP) standards. The cell line was derived from a failed to fertilise oocyte voluntarily donated as unsuitable or surplus to fertility requirements following informed consent. RCe020-A (RC-16) shows normal pluripotency marker expression and differentiates to mesoderm and potentially ectoderm in vitro. It has an abnormal 47XX, +14, i(20)(q10) female karyotype and microsatellite PCR identity, HLA and blood group typing data is available.
Asunto(s)
Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciación Celular , Células Cultivadas , Reprogramación Celular , Femenino , Genotipo , Prueba de Histocompatibilidad , Células Madre Embrionarias Humanas/metabolismo , Humanos , Cariotipo , Repeticiones de Microsatélite/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The broad capacity of pluripotent human embryonic stem cells (hESC) to grow and differentiate demands the development of rapid, scalable, and label-free methods to separate living cell populations for clinical and industrial applications. Here, we identify differences in cell stiffness, expressed as cell elastic modulus (CEM), for hESC versus mesenchymal progenitors, osteoblast-like derivatives, and fibroblasts using atomic force microscopy and data processing algorithms to characterize the stiffness of cell populations. Undifferentiated hESC exhibited a range of CEMs whose median was nearly three-fold lower than those of differentiated cells, information we exploited to develop a label-free separation device based on the principles of tangential flow filtration. To test the device's utility, we segregated hESC mixed with fibroblasts and hESC-mesenchymal progenitors induced to undergo osteogenic differentiation. The device permitted a throughput of 10(6)-10(7) cells per min and up to 50% removal of specific cell types per single pass. The level of enrichment and depletion of soft, pluripotent hESC in the respective channels was found to rise with increasing stiffness of the differentiating cells, suggesting CEM can serve as a major discriminator. Our results demonstrate the principle of a scalable, label-free, solution for separation of heterogeneous cell populations deriving from human pluripotent stem cells.
