Transition toward Human Cytomegalovirus Susceptibility in Early Human Embryonic Stem Cell-Derived Neural Precursors.

Congenital human cytomegalovirus (HCMV) infection is associated with neurodevelopmental disabilities. To dissect the earliest events of infection in the developing human brain, we studied HCMV infection during controlled differentiation of human embryonic stem cells (hESC) into neural precursors. We traced a transition from viral restriction in hESC, mediated by a block in viral binding, toward HCMV susceptibility in early hESC-derived neural precursors. We further revealed the role of platelet-derived growth factor receptor alpha (PDGFRα) as a determinant of the developmentally acquired HCMV susceptibility.

Modeling sex differences in humans using isogenic induced pluripotent stem cells.

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.

Laminin111-based defined culture promoting self-renewing human pluripotent stem cells with properties of the early post-implantation epiblast.

In the mammalian embryo, a formative pluripotent phase is proposed to exist at the early post-implantation period, during the transition from the pre-implantation naive-to the post-implantation primed-epiblast. By recapitulating a laminin component of the extracellular matrix niche during embryonic formative transition, and defined culture conditions, we generated cultures highly enriched for self-renewing human pluripotent stem cells (hPSCs), exhibiting properties of early post-implantation epiblast cells. These hPSCs display post-implantation-epiblast gene expression profiles. FGF and TGF-ß signaling maintain their self-renewal for multiple passages. They have inactive canonical Wnt signaling, do not express primitive streak markers, and are competent to initiate differentiation toward germline and somatic fates. hPSCs exhibiting early post-implantation epiblast properties may shed light on human embryonic PSCs development and may serve for initiating somatic and germ cell specification.

Hadassah, provider of "Regulatory-Ready" pluripotent clinical-grade stem cell banks.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines.

Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. METHODS: We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. FINDINGS: We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. INTERPRETATION: Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.

Somatic mutations and activation-induced cytidine deaminase (AID) expression in established rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), allowing the production of specific antibody-secreting cell lines. We and others have previously found that in contrast to peripheral blood B cells, EBV-transformed lymphoblastoid cell lines express the activation-induced cytidine deaminase (AID) gene. The opposite is true for the germinal center-specific BCL6 gene: it is expressed in adult peripheral blood B cells and is no longer expressed in LCLs. The present work extends our findings and shows that whereas AID expression is rapidly induced following EBV infection, BCL6 expression is gradually down-regulated and is fully extinguished in already established LCLs. The question of whether AID activation induces the process of somatic hypermutation (SHM) was investigated in adult-derived LCLs. It was found that the VH gene from the rheumatoid factor-producing RF LCL (derived from a rheumatoid arthritis patient), accumulated somatic point mutations in culture. Overall, nine unique mutations have accumulated in the rearranged VH gene since the generation of the RF cell line. Four additional intraclonal mutations were found among 10 cellular clones of the RF cells. One out of the four was in CDR1 and could be correlated with loss of antigen-binding activity in three out of the 10 clones. Altogether, these 13 mutations were preferentially targeted to the DGYW motifs and showed preference for CG nucleotides, indicating that they were AID-mediated. By contrast, mutations were not detected among 3700-4000 nucleotides each of the Vlambda, Cmu and GAPDH genes derived from the same RF cell cultures and the cellular clones. Our results thus show that AID may generate point mutations in the rearranged Ig VH during in vitro cell culture of adult-LCLs and that these mutations may be responsible, at least in part, for the known instability and occasional loss of antigen-binding activity of antibody-secreting LCLs.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation.

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

The knockdown of H19lncRNA reveals its regulatory role in pluripotency and tumorigenesis of human embryonic carcinoma cells.

The function of imprinted H19 long non-coding RNA is still controversial. It is highly expressed in early embryogenesis and decreases after birth and re-expressed in cancer. To study the role of H19 in oncogenesis and pluripotency, we down-regulated H19 expression in vitro and in vivo in pluripotent human embryonic carcinoma (hEC) and embryonic stem (hES) cells. H19 knockdown resulted in a decrease in the expression of the pluripotency markers Oct4, Nanog, TRA-1-60 and TRA-1-81, and in the up-regulation of SSEA1; it further attenuated cell proliferation, decreased cell-matrix attachment, and up-regulated E-Cadherin expression. SCID-Beige mice transplanted with H19 down-regulated hEC cells exhibited slower kinetics of tumor formation, resulting in an increased animal survival. Tumors derived from H19 down-regulated cells showed a decrease in the expression of pluripotency markers and up-regulation of SSEA-1 and E-cadherin. Our results suggest that H19 oncogenicity in hEC cells is mediated through the regulation of the pluripotency state.

Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny.

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension.

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.