技术资料

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8,which is ˜99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD,both have been reported to migrate,on Western blotting (WB),at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore,detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies,immunoprecipitation,mass spectrometry,and studies using recombinant proteins,here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ˜22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate,on denaturing SDS-PAGE,at ˜28 kD to 180 kD. Interestingly,different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins,which can spontaneously form high M.W protein species. Finally,we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether,the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells. View Publication

Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless,the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however,conflicting data generated from different biological sources prevent conclusions being drawn. In this work,we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate,an adjunctive medication for schizophrenia,brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders. ?? 2014 Elsevier B.V. View Publication

Human induced pluripotent stem cells (hiPSCs) offer the potential to study otherwise inaccessible cell types. Critical to this is the directed differentiation of hiPSCs into functional cell lineages. This is of particular relevance to research into neurological disease,such as Parkinson's disease (PD),in which midbrain dopaminergic neurons degenerate during disease progression but are unobtainable until post-mortem. Here we report a detailed study into the physiological maturation over time of human dopaminergic neurons in vitro. We first generated and differentiated hiPSC lines into midbrain dopaminergic neurons and performed a comprehensive characterisation to confirm dopaminergic functionality by demonstrating dopamine synthesis,release,and re-uptake. The neuronal cultures include cells positive for both tyrosine hydroxylase (TH) and G protein-activated inward rectifier potassium channel 2 (Kir3.2,henceforth referred to as GIRK2),representative of the A9 population of substantia nigra pars compacta (SNc) neurons vulnerable in PD. We observed for the first time the maturation of the slow autonomous pace-making (textless10 Hz) and spontaneous synaptic activity typical of mature SNc dopaminergic neurons using a combination of calcium imaging and electrophysiology. hiPSC-derived neurons exhibited inositol tri-phosphate (IP3) receptor-dependent release of intracellular calcium from the endoplasmic reticulum in neuronal processes as calcium waves propagating from apical and distal dendrites,and in the soma. Finally,neurons were susceptible to the dopamine neuron-specific toxin 1-methyl-4-phenylpyridinium (MPP+) which reduced mitochondrial membrane potential and altered mitochondrial morphology. Mature hiPSC-derived dopaminergic neurons provide a neurophysiologically-defined model of previously inaccessible vulnerable SNc dopaminergic neurons to bridge the gap between clinical PD and animal models. View Publication

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence,protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation. The integrity of cell:cell contacts has been shown to influence lineage choice during pluripotent cell differentiation,with disruption of cell:cell contacts promoting mesendoderm formation and maintenance of cell:cell contacts resulting in the preferential formation of neurectoderm. Staurosporine is a broad spectrum inhibitor of serine/threonine kinases which has several effects on cell function,including interruption of cell:cell contacts,decreasing focal contact size,inducing epithelial to mesenchyme transition (EMT) and promoting cell differentiation. The possibility that staurosporine could influence lineage choice from pluripotent cells in culture was investigated. The addition of staurosporine to differentiating mouse EPL resulted in preferential formation of mesendoderm and mesoderm populations,and inhibited the formation of neurectoderm. Addition of staurosporine to human ES cells similarly induced primitive streak marker gene expression. These data demonstrate the ability of staurosporine to influence lineage choice during pluripotent cell differentiation and to mimic the effect of disrupting cell:cell contacts. Staurosporine induced mesendoderm in the absence of known inducers of formation,such as serum and BMP4. Staurosporine induced the expression of mesendoderm markers,including markers that were not induced by BMP4,suggesting it acted as a broad spectrum inducer of molecular gastrulation. This approach has identified a small molecule regulator of lineage choice with potential applications in the commercial development of ES cell derivatives,specifically as a method for forming mesendoderm progenitors or as a culture adjunct to prevent the formation of ectoderm progenitors during pluripotent cell differentiation. ?? 2014. View Publication

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development,several issues regarding their pluripotency,differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study,we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that,upon spontaneous differentiation,iPS-MBMC present high amounts of terminal $\$-galactopyranoside residues,pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation,prompting an ectoderm commitment. Exposed $\$-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface,which could modulate intercellular or intracellular interactions. Together,our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and,therefore,the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation. View Publication

The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs,how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny,and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d,the in vitro assessment requires another day,and the in vivo assessment requires up to 12 additional weeks. View Publication

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC,these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further,the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial,ventricular (V),and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2,H7,and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4,Rho kinase inhibitor,activin-A,and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16,up to 95% of cells were cTnT(+). Of which,93%,94%,100%,92%,and 92% of cardiac derivatives from HES2,H7,H9,and two iPSC lines,respectively,were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to $\$-adrenergic stimulation. Our simple,cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol,the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors. View Publication

Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics,but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge,we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs),pericytes,astrocytes and neurons derived from renewable cell sources. First,retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs,particularly through adherens junction,tight junction,and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (˜5,000 $\$(2)). Overall,this scalable human BBB model may enable a wide range of neuroscience studies. View Publication

Skeletal dysplasias (SDs) are caused by abnormal chondrogenesis during cartilage growth plate differentiation. To study early stages of aberrant cartilage formation in vitro,we generated the first induced pluripotent stem cells (iPSCs) from fibroblasts of an SD patient with a lethal form of metatropic dysplasia,caused by a dominant mutation (I604M) in the calcium channel gene TRPV4. When micromasses were grown in chondrogenic differentiation conditions and compared with control iPSCs,mutant TRPV4-iPSCs showed significantly (Ptextless0.05) decreased expression by quantitative real-time polymerase chain reaction of COL2A1 (IIA and IIB forms),SOX9,Aggrecan,COL10A1,and RUNX2,all of which are cartilage growth plate markers. We found that stimulation with BMP2,but not TGF$\$1,up-regulated COL2A1 (IIA and IIB) and SOX9 gene expression,only in control iPSCs. COL2A1 (Collagen II) expression data were confirmed at the protein level by western blot and immunofluorescence microscopy. TRPV4-iPSCs showed only focal areas of Alcian blue stain for proteoglycans,while in control iPSCs the stain was seen throughout the micromass sample. Similar staining patterns were found in neonatal cartilage from control and patient samples. We also found that COL1A1 (Collagen I),a marker of osteogenic differentiation,was significantly (Ptextless0.05) up-regulated at the mRNA level in TRPV4-iPSCs when compared with the control,and confirmed at the protein level. Collagen I expression in the TRPV4 model also may correlate with abnormal staining patterns seen in patient tissues. This study demonstrates that an iPSC model can recapitulate normal chondrogenesis and that mutant TRPV4-iPSCs reflect molecular evidence of aberrant chondrogenic developmental processes,which could be used to design therapeutic approaches for disorders of cartilage. View Publication

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems have evolved as an adaptive surveillance and defense mechanism in bacteria and archaea that uses short RNAs to direct degradation of foreign genetic elements. Here,we present our protocol for utilizing the S. pyogenes type II bacterial CRISPR system to achieve sequence-specific genome alterations in human cells. In principle,any genomic sequence of the form N(19)NGG can be targeted with the generation of custom guide RNA (gRNA) which functions to direct the Cas9 protein to genomic targets and induce DNA cleavage. Here,we describe our methods for designing and generating gRNA expression constructs either singly or in a multiplexed manner,as well as optimized protocols for the delivery of Cas9-gRNA components into human cells. Genomic alterations at the target site are then introduced either through nonhomologous end joining (NHEJ) or through homologous recombination (HR) in the presence of an appropriate donor sequence. This RNA-guided editing tool offers greater ease of customization and synthesis in comparison to existing sequence-specific endonucleases and promises to become a highly versatile and multiplexable human genome engineering platform. View Publication

Efficient approaches for the precise genetic engineering of stem cells can enhance both basic and applied stem cell research. Adeno-associated virus (AAV) vectors have demonstrated high-efficiency gene delivery and gene targeting to numerous cell types,and AAV vectors developed specifically for gene delivery to stem cells have further increased gene targeting frequency compared to plasmid construct techniques. This chapter details the production and purification techniques necessary to generate adeno-associated viral vectors for use in high-efficiency gene targeting of adult or pluripotent stem cell applications. Culture conditions used to achieve high gene targeting frequencies in rat neural stem cells and human pluripotent stem cells are also described. View Publication