Induced pluripotent stem cell line from an atopic dermatitis patient heterozygous for c.2282del4 mutation in filaggrin: KCLi001-A

Abstract

We have generated an induced pluripotent stem cell (iPSC) line KCLi001-A (iOP118) from a female atopic dermatitis (AD) patient, heterozygous for the loss-of-function mutation c.2282del4 in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of AD-iPSCs were performed under xeno-free culture conditions. Characterization of KCLi001-A line included molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

Comparison of human isogeneic Wharton’s jelly MSCs and iPSC-derived MSCs reveals differentiation-dependent metabolic responses to IFNG stimulation

“…Characterization of iPSC lines has been described in detail previously 26–29 . Once the lines are confirmed negative for SeV 26,27 , they are subjected to further characterization:Morphology—iPSC lines have to (i) propagate for > 3 passages, (ii) have well-defined colony edges, (iii) form a tightly packed colony, and (iv) have an absence of differentiated cells.Detection of pluripotency-associated markers—immunodetection of pluripotency-associated markers TRA-1-81, POU class 5 homeobox 1 (POU5F1)/octamer-binding transcription factor 4 (OCT4), TRA-1-60, and NANOG 27,28 .Determination of genomic stability—we use array-comparative genomic hybridization to determine genomic stability of the iPSC lines every three months of continuous culture 27,28 .Genotyping—amplification of polymorphic microsatellite markers is used for cell line identification at the molecular level 27,28 .Differentiation into three germ layers in vitro—iPSC colonies were left to differentiate without passage over a period of at least 3 weeks in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS (Hyclone). The cells are then fixed and permeabilized in the dish.…”

“…The cells are then fixed and permeabilized in the dish. Markers representing the three germ layers, mesoderm, ectoderm, and endoderm, are detected with immunostaining 27,28 .Differentiation into three germ layers in vivo—2 × 10 6 iPSC suspended in Matrigel were injected subcutaneously into flanks of NOD/SCID mice. The mice were killed 6–10 weeks later, depending on size of the tumor.…”

“…Characterization of iPSC lines has been described in detail previously 26 – 29 . Once the lines are confirmed negative for SeV 26 , 27 , they are subjected to further characterization: Morphology—iPSC lines have to (i) propagate for > 3 passages, (ii) have well-defined colony edges, (iii) form a tightly packed colony, and (iv) have an absence of differentiated cells. Detection of pluripotency-associated markers—immunodetection of pluripotency-associated markers TRA-1-81, POU class 5 homeobox 1 (POU5F1)/octamer-binding transcription factor 4 (OCT4), TRA-1-60, and NANOG 27 , 28 .…”

“…Once the lines are confirmed negative for SeV 26 , 27 , they are subjected to further characterization: Morphology—iPSC lines have to (i) propagate for > 3 passages, (ii) have well-defined colony edges, (iii) form a tightly packed colony, and (iv) have an absence of differentiated cells. Detection of pluripotency-associated markers—immunodetection of pluripotency-associated markers TRA-1-81, POU class 5 homeobox 1 (POU5F1)/octamer-binding transcription factor 4 (OCT4), TRA-1-60, and NANOG 27 , 28 . Determination of genomic stability—we use array-comparative genomic hybridization to determine genomic stability of the iPSC lines every three months of continuous culture 27 , 28 .…”

“… Detection of pluripotency-associated markers—immunodetection of pluripotency-associated markers TRA-1-81, POU class 5 homeobox 1 (POU5F1)/octamer-binding transcription factor 4 (OCT4), TRA-1-60, and NANOG 27 , 28 . Determination of genomic stability—we use array-comparative genomic hybridization to determine genomic stability of the iPSC lines every three months of continuous culture 27 , 28 . Genotyping—amplification of polymorphic microsatellite markers is used for cell line identification at the molecular level 27 , 28 .…”