Generation of TWO G51D SNCA missense mutation iPSC lines (CRICKi011-A, CRICKi012-A) from two individuals at risk of Parkinson's disease.

Mutations or multiplications of the SNCA (Synuclein Alpha) gene cause rare autosomal dominant Parkinson's disease (PD). The SNCA G51D missense mutation is associated with a synucleinopathy that shares PD and multiple system atrophy (MSA) characteristics. We generated induced pluripotent stem cell (iPSC) lines from two individuals with SNCA G51D missense mutations at risk of PD. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. The resulting human iPSCs displayed normal morphology, expressed markers associated with pluripotency, and differentiated into the three germ layers. The iPSC lines could facilitate disease-modelling and therapy development studies for synucleinopathies.

Generation of an MTM1-mutant iPSC line (CRICKi008-A) from an individual with X-linked myotubular myopathy (XLMTM).

Centronuclear myopathies (CNMs) are a group of inherited rare muscle disorders characterised by the abnormal position of the nucleus in the center of the muscle fiber. One of CNM is the X-Linked Myotubular Myopathy, caused by mutations in the myotubularin (MTM1) gene (XLMTM), characterised by profound muscle hypotonia and weakness, severe bulbar and respiratory involvement. Here, we generated an induced pluripotent stem cell (iPSC) line from a patient with a severe form of XLMTM. Dermal fibroblasts were reprogrammed to pluripotency using a non-integrating mRNA-based protocol. This new MTM1-mutant iPSC line could facilitate disease-modelling and therapy development studies for XLMTM.

Generation of FOUR iPSC lines (CRICKi004-A; CRICKi005-A; CRICKi006-A, CRICKi007-A) from Spinal muscle atrophy patients with lower extremity dominant (SMALED) phenotype.

Spinal muscular atrophy with lower extremity dominant (SMALED) is a hereditary neuromuscular disorder characterized by degeneration of spinal cord motor neurons resulting in lower limbs muscle weakness and paralysis. Mutations in DYNC1H1, which encodes BICD2, a multifunctional adaptor for microtubule motor proteins, cause the disorder. Here, we generated four induced pluripotent stem cell (iPSC) lines from patients with SMALED. Dermal fibroblasts were obtained from the MRC neuromuscular disease biobank and reprogrammed using non-integrating mRNA-based protocol. Characterization of the four iPSC lines included karyotyping and Sanger sequencing, while the expression of associated markers confirmed pluripotency and differentiation potential.

Generation of an iPSC line (CRICKi001-A) from an individual with a germline SMARCA4 missense mutation and autism spectrum disorder.

Germline missense mutations in the BAF swi/snf chromatin remodeling subunit SMARCA4 are associated with neurodevelopmental disorders, including Coffin Siris Syndrome (CSS). Here, we generated an induced pluripotent stem cell line from a male patient with atypical CSS features and a de novo heterozygous missense mutation in the SMARCA4 gene (c.3607C>T, p.(Arg1203Cys)). Hair root derived keratinocytes were reprogrammed using non-integrative Sendai virus vector delivery of pluripotency factors. iPSCs generated display normal morphology and molecular karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.

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.

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

Variability among donors, non-standardized methods for isolation, and characterization contribute to mesenchymal stem/stromal cell (MSC) heterogeneity. Induced pluripotent stem cell (iPSCs)-derived MSCs would circumvent many of current issues and enable large-scale production of standardized cellular therapy. To explore differences between native MSCs (nMSCs) and iPSC-derived MSCs (iMSCs), we developed isogeneic lines from Wharton's jelly (WJ) from the umbilical cords of two donors (#12 and #13) under xeno-free conditions. Next, we reprogrammed them into iPSCs (iPSC12 and iPSC13) and subsequently differentiated them back into iMSCs (iMSC12 and iMSC13) using two different protocols, which we named ARG and TEX. We assessed their differentiation capability, transcriptome, immunomodulatory potential, and interferon-γ (IFNG)-induced changes in metabolome. Our data demonstrated that although both differentiation protocols yield iMSCs similar to their parental nMSCs, there are substantial differences. The ARG protocol resulted in iMSCs with a strong immunomodulatory potential and lower plasticity and proliferation rate, whereas the TEX protocol raised iMSCs with a higher proliferation rate, better differentiation potential, though weak immunomodulatory response. Our data suggest that, following a careful selection and screening of donors, nMSCs from umbilical's cord WJ can be easily reprogrammed into iPSCs, providing an unlimited source of material for differentiation into iMSCs. However, the differentiation protocol should be chosen depending on their clinical use.

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.

Erratum: Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.

This corrects the article DOI: 10.1038/nature24675.

Pluripotent state transitions coordinate morphogenesis in mouse and human embryos.

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.

Induced Pluripotent Stem Cell Differentiation and Three-Dimensional Tissue Formation Attenuate Clonal Epigenetic Differences in Trichohyalin.

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study, we used exome sequencing, array comparative genomic hybridization (CGH), miRNA array, DNA methylation array, three-dimensional (3D) tissue engineering, and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE), which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected, ≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex, a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin, TCHH), found that in one of the iPSC clones (iKCL004), TCHH retained a DNA methylation signature characteristic of the original somatic cells, whereas in other iPSC line (iKCL011), the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE, suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome.

Generation of KCL039 clinical grade human embryonic stem cell line.

The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Generation of KCL034 clinical grade human embryonic stem cell line.

The KCL034 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility, specific and non-specific human pathogens.

Generation of KCL033 clinical grade human embryonic stem cell line.

The KCL033 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. The line was also validated for sterility and specific and non-specific human pathogens.

Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene.

The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

Self-organization of the human embryo in the absence of maternal tissues.

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.

Sendai Virus-Based Reprogramming of Mesenchymal Stromal/Stem Cells from Umbilical Cord Wharton's Jelly into Induced Pluripotent Stem Cells.

In an attempt to bring pluripotent stem cell biology closer to reaching its full potential, many groups have focused on improving reprogramming protocols over the past several years. The episomal modified Sendai virus-based vector has emerged as one of the most practical ones. Here we describe reprogramming of mesenchymal stromal/stem cells (MSC) derived from umbilical cord Wharton's Jelly into induced pluripotent stem cells (iPSC) using genome non-integrating Sendai virus-based vectors. The detailed protocols of iPSC colony cryopreservation (vitrification) and adaption to feeder-free culture conditions are also included.

The Molecular Karyotype of 25 Clinical-Grade Human Embryonic Stem Cell Lines.

The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson's, Huntington's, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.