RESUMO
Reprogramming somatic cells into induced pluripotent stem cells (iPSC) has provided a gateway for many novel discoveries in the field of tissue engineering, regenerative medicine and cell therapy. The need for an efficient, less laborious and fast reprogramming protocol under xeno-free, feeder-free and chemically defined conditions has never been greater. Here we describe a novel approach to reprogramming using the StemRNA 3rd Gen Reprogramming Kit (ReproCELL) which encompasses non-modified microRNAs (NM-miRNA), non-modified E3, K3, B18R RNAs (EKB NM-RNA) and non-modified mRNAs for six crucial transcription factors (OSKMNL NM-RNA).
Assuntos
Reprogramação Celular , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Reprogramação Celular/genética , Fibroblastos , RNA Mensageiro/genética , Medicina RegenerativaRESUMO
We have generated MLi004-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a female patient with recessive dystrophic epidermolysis bullosa (RDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi004-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-004A line has been also tested for ability to differentiate into fibroblasts.
Assuntos
Epidermólise Bolhosa Distrófica , Células-Tronco Pluripotentes Induzidas , Colágeno Tipo VII/genética , Epidermólise Bolhosa Distrófica/genética , Feminino , Fibroblastos , Humanos , Mutação , PeleRESUMO
Differentiation of normal human keratinocytes (NHK) grown in vitro as a monolayer to confluency can be triggered with an acute increase in concentration of extracellular Ca++ . Over several days, induced by Ca++ , the cells form pseudostratified sheets that somewhat resemble the basic organization of the intact skin. This experimental system is widely used in studies of keratinocyte biology and skin pathology. However, expression pattern of the genes considered as markers for cells in specific layers of epidermis in vivo does not always match the specific pattern observed in vitro and might lead to misinterpretation of data. Here, we demonstrate that among 18 markers of terminally differentiated keratinocytes of stratum granulosum (SG) and stratum corneum (SC) in vivo, only four (CDSN, KPRP, LCE1C and SPRR4) have reproduced their expression pattern in vitro. Our data suggest that findings based on two-dimensional (2D) Ca++ -induced terminal differentiation of NHK in vitro should be subjected to additional scrutiny before conclusions could be made and, if possible, verified in other experimental system that might more faithfully represent the in vivo microenvironment.
Assuntos
Cálcio/farmacologia , Diferenciação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Queratinócitos/fisiologia , Biomarcadores/metabolismo , Células Cultivadas , Proteínas Ricas em Prolina do Estrato Córneo/genética , Epiderme/metabolismo , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Queratinócitos/metabolismo , Proteínas/genética , RNA Mensageiro/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas de Filamentos Intermediários/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Proteínas Filagrinas , Imunofluorescência , Heterozigoto , Humanos , Repetições de Microssatélites/genética , Mycoplasma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus Sendai/genéticaRESUMO
We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.
