Induced pluripotent stem cell (iPSC) line MLi005-A derived from a patient with dominant dystrophic epidermolysis bullosa (DDEB).

We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). 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 MLi005-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-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types.

Epidermal Basement Membrane Substitutes for Bioengineering of Human Epidermal Equivalents.

Epidermal basement membrane, a tightly packed network of extracellular matrix (ECM) components, is a source of physical, chemical, and biological factors required for the structural and functional homeostasis of the epidermis. Variations within the ECM create distinct environments, which can affect the property of cells in the basal layer of the epidermis and subsequently affect keratinocyte differentiation and stratification. Very little attention has been paid to mimicking basement membrane in organotypic cultures. In this study, using parameters outlined in a consensus on the quality standard of organotypic models suitable for dermatological research, we have evaluated three basement membrane substitutes. We compared fibronectin with three complex three-dimensional matrices: Matrigel, decellularized dermal fibroblast‒produced and ‒assembled ECM, and a dry human amniotic membrane. Our results suggest that Matrigel is not a suitable substrate for human epidermal equivalent culture, whereas the two other complex three-dimensional substitutes, decellularized dermal fibroblast‒produced and ‒assembled ECM and dry human amniotic membrane, were superior to single layer fibronectin coating. Human epidermal equivalents cultured on either decellularized dermal fibroblast‒produced and ‒assembled ECM or on dry human amniotic membrane generated hemidesmosomes, whereas those on fibronectin did not. In addition, human epidermal equivalent cultured on decellularized dermal fibroblast‒produced and ‒assembled ECM and on dry human amniotic membrane can be maintained in culture 4 days longer than human epidermal equivalent cultured on fibronectin without compromising the barrier function.

mRNA-Based Reprogramming Under Xeno-Free and Feeder-Free Conditions.

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).

Human pluripotent stem cells: An alternative for 3D in vitro modelling of skin disease.

To effectively study the skin and its pathology, various platforms have been used to date, with in vitro 3D skin models being considered the future gold standard. These models have generally been engineered from primary cell lines. However, their short life span leading to the use of various donors, imposes issues with genetic variation. Human pluripotent stem cell (hPSC)-technology holds great prospects as an alternative to the use of primary cell lines to study the pathophysiology of human skin diseases. This is due to their potential to generate an unlimited number of genetically identical skin models that closely mimic the complexity of in vivo human skin. During the past decade, researchers have therefore started to use human embryonic and induced pluripotent stem cells (hESC/iPSC) to derive skin resident-like cells and components. These have subsequently been used to engineer hPSC-derived 3D skin models. In this review, we focus on the advantages, recent developments, and future perspectives in using hPSCs as an alternative cell source for modelling human skin diseases in vitro.

Markers for Ca++ -induced terminal differentiation of keratinocytes in vitro under defined conditions.

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.

Stem Cell Research Lab Resource: Stem Cell LineInduced pluripotent stem cell (iPSC) line MLi-003A derived from an individual with the maximum number of filaggrin (FLG) tandem repeats.

We have generated MLi003-A, a new induced pluripotent stem cell (iPSC) line derived from hair follicle keratinocytes of a healthy male characterized with a maximum number of filaggrin tandem repeats, making this iPSC line the best control for studies on skin barrier function. The characterization of the MLi003-A cell line consisted of molecular karyotyping, high-throughput array-based sequencing composed of Fluidigm microfluidics technology and next-generation sequencing of the filaggrin alleles, and pluripotency and differentiation potentials testing by immunofluorescence of associated markers both in vitro and in vivo. The MLi-003A line has been also tested for ability to differentiate into keratinocytes.

Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.

Induced pluripotent stem cell line heterozygous for p.R501X mutation in filaggrin: KCLi003-A.

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.

Induced pluripotent stem cell line heterozygous for p.R2447X mutation in filaggrin: KCLi002-A.

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.

Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype.

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

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

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.

Mapping human genetic diversity in Asia.

Asia harbors substantial cultural and linguistic diversity, but the geographic structure of genetic variation across the continent remains enigmatic. Here we report a large-scale survey of autosomal variation from a broad geographic sample of Asian human populations. Our results show that genetic ancestry is strongly correlated with linguistic affiliations as well as geography. Most populations show relatedness within ethnic/linguistic groups, despite prevalent gene flow among populations. More than 90% of East Asian (EA) haplotypes could be found in either Southeast Asian (SEA) or Central-South Asian (CSA) populations and show clinal structure with haplotype diversity decreasing from south to north. Furthermore, 50% of EA haplotypes were found in SEA only and 5% were found in CSA only, indicating that SEA was a major geographic source of EA populations.