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BACKGROUND Although studies have shown that Oct4,Sox2,Klf4,and c-Myc (OKSM)-mediated induced pluripotent stem cell (iPSC) technology sensitizes cancer cells to drugs,the potential risk of inserting c-Myc and random insertions of exogenous sequences into the genome persists. Several authors,including us,have presented microRNA (miRNA)-mediated reprogramming as an alternative approach. Herein,we evaluated the efficacy of miRNA-mediated reprogramming on hepatocellular carcinoma (HCC) cells. METHODS Among three miRNAs (miR-200c,miR-302s,and miR-369s) that were previously presented for miRNA-mediated reprogramming,miR-302 was expressed at low levels in HCC cells. After transfecting three times with miR-302,the cells were incubated in ES medium for 3 weeks and then characterized. RESULTS iPSC-like spheres were obtained after the 3-week incubation. Spheres presented high NANOG and OCT4 expression,low proliferation,high apoptosis,low epithelial-mesenchymal transition marker expression (N-cadherin,TGFBR2),and sensitization to drugs. Several miRNAs were changed (e.g.,low oncomiR miR-21,high miR-29b). cMyc was decreased,and methylation was elevated on histone 3 at lysine 4 (H3K4). Differentiated cells expressed markers of each germ layer (GFAP,FABP4,and ALB). AOF2 (also known as LSD1 or KDM1),one of the targets for miR-302,was repressed in iPSC-like-spheres. Silencing of AOF2 resulted in similar features of iPSC-like-spheres,including cMyc down-regulation and H3K4 methylation. In drug-resistant cells,sensitization was achieved through miR-302-mediated reprogramming. CONCLUSIONS miR-302-mediated iPSC technology reprogrammed HCC cells and improved drug sensitivity through AOF2 down-regulation,which caused H3K4 methylation and c-Myc repression. View Publication

Patients with dyskeratosis congenita (DC),a disorder of telomere maintenance,suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity,which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state,that several telomerase components are targeted by pluripotency-associated transcription factors,and that in autosomal dominant DC,transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase,and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients. View Publication

Human bone marrow multipotent mesenchymal stromal cells are progenitor cells that can be expanded in vitro and differentiate into various cells of mesodermal origin. They contribute to the bone marrow reticular niche,where mature B cells and long-lived plasma cells are maintained. Multipotent mesenchymal stromal cells were recently shown to modulate T- and B-cell proliferation and differentiation,dendritic cell maturation,and natural killer activity. These immunoregulatory properties encouraged a possible use of these cells to modulate autoimmune responses in humans. We studied the influence of bone marrow mesenchymal stem cells on highly purified B-cell subsets isolated from healthy donors and total B cells from pediatric systemic lupus erythematosus patients. Bone marrow mesenchymal stem cells promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B cells stimulated with an agonist of Toll-like receptor 9,in the absence of B cell receptor triggering. They strongly enhanced proliferation and differentiation into plasma cells of memory B-cell populations. A similar effect was observed in response to polyclonal stimulation of B cells isolated from pediatric patients with systemic lupus erythematosus. This study casts important questions on bone marrow mesenchymal stem cells as a therapeutic tool in autoimmune diseases in which B-cell activation is crucially implicated in the pathogenesis of the disease. View Publication

Long non-coding RNAs (lncRNAs) are known players in the regulatory circuitry of the self-renewal in human embryonic stem cells (hESCs). However,most hESC-specific lncRNAs remain uncharacterized. Here we demonstrate that growth-arrest-specific transcript 5 (GAS5),a known tumour suppressor and growth arrest-related lncRNA,is highly expressed and directly regulated by pluripotency factors OCT4 and SOX2 in hESCs. Phenotypic analysis shows that GAS5 knockdown significantly impairs hESC self-renewal,but its overexpression significantly promotes hESC self-renewal. Using RNA sequencing and functional analysis,we demonstrate that GAS5 maintains NODAL signalling by protecting NODAL expression from miRNA-mediated degradation. Therefore,we propose that the above pluripotency factors,GAS5 and NODAL form a feed-forward signalling loop that maintains hESC self-renewal. As this regulatory function of GAS5 is stem cell specific,our findings also indicate that the functions of lncRNAs may vary in different cell types due to competing endogenous mechanisms. View Publication

During human brain development,multiple signaling pathways generate diverse cell types with varied regional identities. Here,we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor,neuronal,and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together,these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. View Publication

Specifically ablating genes in human induced pluripotent stem cells (iPSCs) allows for studies of gene function as well as disease mechanisms in disorders caused by loss-of-function (LOF) mutations. While techniques exist for engineering such lines,we have developed and rigorously validated a method of simultaneous iPSC reprogramming while generating CRISPR/Cas9-dependent insertions/deletions (indels). This approach allows for the efficient and rapid formation of genetic LOF human disease cell models with isogenic controls. The rate of mutagenized lines was strikingly consistent across experiments targeting four different human epileptic encephalopathy genes and a metabolic enzyme-encoding gene,and was more efficient and consistent than using CRISPR gene editing of established iPSC lines. The ability of our streamlined method to reproducibly generate heterozygous and homozygous LOF iPSC lines with passage-matched isogenic controls in a single step provides for the rapid development of LOF disease models with ideal control lines,even in the absence of patient tissue. View Publication

The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently,knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led to genomic instability. Here,we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies,due to downstream suppression of p21,without affecting apoptosis and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns,displayed normal karyotypes,contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion,transient p53 suppression increases reprogramming efficiency without affecting genomic stability,rendering the method suitable for in vitro mechanistic studies with the possibility for future clinical translation. View Publication

Cell replacement therapy with human pluripotent stem cell-derived neurons has the potential to ameliorate neurodegenerative dysfunction and central nervous system injuries,but reprogrammed neurons are dissociated and spatially disorganized during transplantation,rendering poor cell survival,functionality and engraftment in vivo. Here,we present the design of three-dimensional (3D) microtopographic scaffolds,using tunable electrospun microfibrous polymeric substrates that promote in situ stem cell neuronal reprogramming,neural network establishment and support neuronal engraftment into the brain. Scaffold-supported,reprogrammed neuronal networks were successfully grafted into organotypic hippocampal brain slices,showing an ∼3.5-fold improvement in neurite outgrowth and increased action potential firing relative to injected isolated cells. Transplantation of scaffold-supported neuronal networks into mouse brain striatum improved survival ∼38-fold at the injection site relative to injected isolated cells,and allowed delivery of multiple neuronal subtypes. Thus,3D microscale biomaterials represent a promising platform for the transplantation of therapeutic human neurons with broad neuro-regenerative relevance. View Publication

Human induced pluripotent stem cells (iPSCs) hold promise as a source of adult-derived,patient-specific pluripotent cells for use in cell-based regenerative therapies. However,current methods of cell culture are tedious and expensive,and the mechanisms underlying cell proliferation are not understood. In this study,we investigated expression and function of iPSC integrin extracellular matrix receptors to better understand the molecular mechanisms of cell adhesion,survival,and proliferation. We show that iPSC lines generated using Oct-3/4,Sox-2,Nanog,and Lin-28 express a repertoire of integrins similar to that of hESCs,with prominent expression of subunits alpha5,alpha6,alphav,beta1,and beta5. Integrin function was investigated in iPSCs cultured without feeder layers on Matrigel or vitronectin,in comparison to human embryonic stem cells. beta1 integrins were required for adhesion and proliferation on Matrigel,as shown by immunological blockade experiments. On vitronectin,the integrin alphavbeta5 was required for initial attachment,but inhibition of both alphavbeta5 and beta1 was required to significantly decrease iPSC proliferation. Furthermore,iPSCs cultured on vitronectin for 9 passages retained normal karyotype,pluripotency marker expression,and capacity to differentiate in vitro. These studies suggest that vitronectin,or derivatives thereof,might substitute for Matrigel in a more defined system for iPSC culture. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2),because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures,e.g. -80 °C. This instability leads to ice recrystallization,causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (˜-80 °C) of laboratory deep freezers. Moreover,a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at -80 °C for periods that extend up to at least one year,with the post-thaw viability,plating efficiency,and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2. View Publication

We mutated,by gene targeting,the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies,1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r,6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous,neor-containing,Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene. View Publication