RESUMO
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.
Assuntos
Técnicas de Cultura de Células/normas , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Regulamentação Governamental , Humanos , Controle de QualidadeRESUMO
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.
RESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Hibridização Genômica Comparativa , Corpos Embrioides/citologia , Feminino , Citometria de Fluxo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Cromossomos Humanos Par 12 , Hibridização Genômica Comparativa , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Corpos Embrioides/citologia , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Masculino , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TrissomiaRESUMO
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.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Óvulo/citologia , Animais , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Hibridização Genômica Comparativa , Corpos Embrioides/citologia , Feminino , Citometria de Fluxo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/transplante , Humanos , Cariótipo , Camundongos , Camundongos SCID , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Teratoma/metabolismo , Teratoma/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante HeterólogoRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Alelos , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Corpos Embrioides/citologia , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Antígenos CD15/metabolismo , Masculino , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Embrião de Mamíferos/citologia , Células-Tronco Embrionárias Humanas/citologia , Alelos , Diferenciação Celular , Células Cultivadas , Corpos Embrioides/citologia , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Antígenos CD15/metabolismo , Masculino , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Alelos , Diferenciação Celular , Células Cultivadas , Corpos Embrioides/citologia , Feminino , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Antígenos CD15/metabolismo , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Fator 3 de Transcrição de Octâmero/metabolismo , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Cromossomos Humanos Par 12 , Embrião de Mamíferos/citologia , Corpos Embrioides/citologia , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Imuno-Histoquímica , Cariótipo , Masculino , Repetições de Microssatélites/genética , Microscopia de Fluorescência , Oócitos/citologia , Fatores de Transcrição/metabolismo , TrissomiaRESUMO
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.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Blastocisto/citologia , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Repetições de Microssatélites/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Blastocisto/citologia , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Genótipo , Teste de Histocompatibilidade , Humanos , Cariótipo , Repetições de Microssatélites/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Feminino , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Repetições de Microssatélites/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Cromossomos Humanos Par 12 , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Masculino , Repetições de Microssatélites/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TrissomiaRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Cromossomos Humanos Par 8 , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Masculino , Repetições de Microssatélites/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TrissomiaRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Masculino , Repetições de Microssatélites/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Blastocisto/citologia , Células-Tronco Embrionárias Humanas/citologia , Antígenos de Grupos Sanguíneos/metabolismo , Diferenciação Celular , Células Cultivadas , Reprogramação Celular , Feminino , Genótipo , Teste de Histocompatibilidade , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cariótipo , Repetições de Microssatélites/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Revascularisation of ischaemic tissue remains an area of substantial unmet clinical need in cardiovascular disease. Strategies to induce therapeutic angiogenesis are therefore attractive. Our recent focus has been on human embryonic stem cell (hESC) strategies since hESC can be maintained in a pluripotent state or differentiated into any desired cell type, including endothelial cells (EC), under defined differentiation culture conditions. We recently published a protocol for non-good manufacturing practice (GMP) feeder- and serum-free hESC-EC-directed monolayer differentiation to vascular EC demonstrating the potential to generate hESC-derived EC in a GMP-compliant manner suitable for use in clinical trials. In this study we modified that laboratory protocol to GMP compliance. EC production was confirmed by flow cytometry, qRT-PCR and production of vascular structures in Matrigel®, yielding approximately 30 % mature VE-cadherin(+)/PECAM-1(+) cells using the GMP-compliant hESC line RC13. In conclusion, we have successfully demonstrated the production of vascular EC under GMP-compliant conditions suitable for clinical evaluation.
Assuntos
Biotecnologia/normas , Técnicas de Cultura de Células/normas , Diferenciação Celular , Células-Tronco Embrionárias/fisiologia , Células Endoteliais/fisiologia , Fidelidade a Diretrizes/normas , Guias como Assunto/normas , Isquemia , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Caderinas/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/transplante , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Citometria de Fluxo/normas , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isquemia/patologia , Isquemia/fisiopatologia , Isquemia/cirurgia , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Fatores de TempoRESUMO
Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.