Scientific Resources

Human fibroblast cells collected from a 3-year old,female Rett Syndrome patient with a 32bp deletion in the X-linked MECP2 gene were obtained from the Coriell Institute. Fibroblasts were reprogrammed to iPSC cells using a Sendai-virus delivery system expressing human KOSM transcription factors. Cell-line pluripotency was demonstrated by gene expression,immunocytochemistry,in-vitro differentiation trilineage capacity and was of normal karyotype. Interestingly,subsequent clones retained the epigenetic memory of the parent fibroblasts allowing for the segregation of wild-type and mutant expressing clones. This MECP2 mutant expressing clone may serve as a model for investigating MECP2 reactivation in Rett's Syndrome. View Publication

Loeys-Dietz syndrome (LDS) is an autosomal-dominant connective tissue disorder,commonly caused by genetic mutation of transforming growth factor-beta receptor (TGFBR)-1 or TGFBR2. This study describes the generation of human induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells obtained from an LDS patient with TGFBR2 mutation (R193W). Analysis confirmed the cells had a normal karyotype,expressed typical pluripotency markers,had the ability to differentiate into all three germ layers in vivo,and retained the TGFBR2 mutation from the derived hiPSCs. This iPSC line represents a potentially useful tool for investigating LDS disease mechanisms. View Publication

In the field of hematopoietic regeneration,deriving hematopoietic stem cells (HSCs) from pluripotent stem cells with engraftment potential is the central mission. Unstable hematopoietic differentiation protocol due to variation factors such as serums and feeder cells,remains a major technical issue impeding the screening of key factors for the derivation of HSCs. In combination with hematopoietic cytokines,UM171 has the capacity to facilitate the maintenance and expansion of human primary HSCs in vitro. Here,using a serum-free,feeder-free,and chemically defined induction protocol,we observed that UM171 enhanced hematopoietic derivation through the entire process of hematopoietic induction in vitro. UM171 facilitated generation of robust CD34(+)CD45(+) derivatives that formed more and larger sized CFU-GM as well as larger sized CFU-Mix. In our protocol,the derived hematopoietic progenitors failed to engraft in NOG mice,indicating the absence of long-term HSC from these progenitors. In combination with other factors and protocols,UM171 might be broadly used for hematopoietic derivation from human pluripotent stem cells in vitro. View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting,but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific,but not primed-specific,proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus,identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting. View Publication

The developmental pathway for human megakaryocytes remains unclear and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis,we have identified a cluster of cells within immature hematopoietic stem and progenitor cell populations that specifically express genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34(+)CD38(+)IL-3Rα(dim)CD45RA(-) common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte/macrophage potential,but exhibited robust differentiation into the megakaryocyte lineage at a high frequency,both in vivo and in vitro The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly,the CD41(+) CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis,independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia,the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus,the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis. View Publication

Brain-derived microvascular endothelial cells (BMECs),which play a central role in blood brain barrier (BBB),can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported,they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy,drug screening,and pathological study. In the recent study,an induction method of BMECs from PSCs has been established,making it possible to more precisely study the in vitro human BBB function. Here,using induced pluripotent stem (iPS) cell-derived BMECs,we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function,and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly,TNF-α,which is known to be secreted from astrocytes and microglia in the cerebral ischemia,prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus,we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs. View Publication

A fast track Hot Start" process was implemented to launch the European Bank for Induced Pluripotent Stem Cells (EBiSC) to provide early release of a range of established control and disease linked human induced pluripotent stem cell (hiPSC) lines. Established practice amongst consortium members was surveyed to arrive at harmonised and publically accessible Standard Operations Procedures (SOPs) for tissue procurement View Publication

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently,the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique,we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen,we further describe strategies for confirming the screening phenotype,as well as genetic perturbation,through analysis of indel rate and transcriptional activation. Beginning with library design,a genome-scale screen can be completed in 9-15 weeks,followed by 4-5 weeks of validation. View Publication

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity,and prepared them in formations of single chain,single chain with joint segment,dual chain with joint segment,and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain,which has a storage modulus of 25 kPa,supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. View Publication

BACKGROUND CHD8 (chromodomain helicase DNA-binding protein 8),which codes for a member of the CHD family of ATP-dependent chromatin-remodeling factors,is one of the most commonly mutated genes in autism spectrum disorders (ASD) identified in exome-sequencing studies. Loss of function mutations in the gene have also been found in schizophrenia (SZ) and intellectual disabilities and influence cancer cell proliferation. We previously reported an RNA-seq analysis carried out on neural progenitor cells (NPCs) and monolayer neurons derived from induced pluripotent stem (iPS) cells that were heterozygous for CHD8 knockout (KO) alleles generated using CRISPR-Cas9 gene editing. A significant number of ASD and SZ candidate genes were among those that were differentially expressed in a comparison of heterozygous KO lines (CHD8(+/-)) vs isogenic controls (CHD8(+/-)),including the SZ and bipolar disorder (BD) candidate gene TCF4,which was markedly upregulated in CHD8(+/-) neuronal cells. METHODS In the current study,RNA-seq was carried out on CHD8(+/-) and isogenic control (CHD8(+/+)) cerebral organoids,which are 3-dimensional structures derived from iPS cells that model the developing human telencephalon. RESULTS TCF4 expression was,again,significantly upregulated. Pathway analysis carried out on differentially expressed genes (DEGs) revealed an enrichment of genes involved in neurogenesis,neuronal differentiation,forebrain development,Wnt/β-catenin signaling,and axonal guidance,similar to our previous study on NPCs and monolayer neurons. There was also significant overlap in our CHD8(+/-) DEGs with those found in a transcriptome analysis carried out by another group using cerebral organoids derived from a family with idiopathic ASD. Remarkably,the top DEG in our respective studies was the non-coding RNA DLX6-AS1,which was markedly upregulated in both studies; DLX6-AS1 regulates the expression of members of the DLX (distal-less homeobox) gene family. DLX1 was also upregulated in both studies. DLX genes code for transcription factors that play a key role in GABAergic interneuron differentiation. Significant overlap was also found in a transcriptome study carried out by another group using iPS cell-derived neurons from patients with BD,a condition characterized by dysregulated WNT/β-catenin signaling in a subgroup of affected individuals. CONCLUSIONS Overall,the findings show that distinct ASD,SZ,and BD candidate genes converge on common molecular targets-an important consideration for developing novel therapeutics in genetically heterogeneous complex traits. View Publication

Neural cultures derived from Huntington's disease (HD) patient-derived induced pluripotent stem cells were used for 'omics' analyses to identify mechanisms underlying neurodegeneration. RNA-seq analysis identified genes in glutamate and GABA signaling,axonal guidance and calcium influx whose expression was decreased in HD cultures. One-third of gene changes were in pathways regulating neuronal development and maturation. When mapped to stages of mouse striatal development,the profiles aligned with earlier embryonic stages of neuronal differentiation. We observed a strong correlation between HD-related histone marks,gene expression and unique peak profiles associated with dysregulated genes,suggesting a coordinated epigenetic program. Treatment with isoxazole-9,which targets key dysregulated pathways,led to amelioration of expanded polyglutamine repeat-associated phenotypes in neural cells and of cognitive impairment and synaptic pathology in HD model R6/2 mice. These data suggest that mutant huntingtin impairs neurodevelopmental pathways that could disrupt synaptic homeostasis and increase vulnerability to the pathologic consequence of expanded polyglutamine repeats over time. View Publication

BACKGROUND The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research,including mechanistic studies of hematopoiesis,the development of cellular therapies for autoimmune diseases,induced transplant tolerance,anticancer immunotherapies,disease modeling,and drug/toxicity screening. Over the past years,significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal,endothelial,and hematopoietic specification. Thus,it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free,defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. METHODS The hemogenic endothelium differentiation was accomplished in an adherent,serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial,myeloid,and lymphoid potential. RESULTS Monolayer induction based on GSK3 inhibition,described here,yielded a large number of CD31(+)CD34(+) hemogenic endothelium cells. When isolated and propagated in adherent conditions,these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells,these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid,T-lymphoid,and B-lymphoid cells. CONCLUSION The results of this study substantiate a method that significantly reduces the complexity of current protocols for hematopoietic induction,offers a defined system to study the factors that affect the early stages of hematopoiesis,and provides a new route of lymphoid and myeloid cell derivation from human pluripotent stem cells,thus enhancing their use in translational medicine. View Publication