技术资料

Conventional methods for quantification of undifferentiated pluripotent stem cells such as fluorescence-activated cell sorting and real-time PCR analysis have technical limitations in terms of their sensitivity and recyclability. Herein,we designed a real-time in situ label-free monitoring system on the basis of a specific electrochemical signature of human pluripotent stem cells in vitro. The intensity of the signal of hPSCs highly corresponded to the cell number and remained consistent in a mixed population with differentiated cells. The electrical charge used for monitoring did not markedly affect the proliferation rate or molecular characteristics of differentiated human aortic smooth muscle cells. After YM155 treatment to ablate undifferentiated hPSCs,their specific signal was significantly reduced. This suggests that detection of the specific electrochemical signature of hPSCs would be a valid approach to monitor potential contamination of undifferentiated hPSCs,which can assess the risk of teratoma formation efficiently and economically. View Publication

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress proneuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs,miR-375,was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally,miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly,motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly,SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons. View Publication

BACKGROUND Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore,the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence,we focus on optimizing hESCs for clinic application through gene modification. RESULTS Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-$\$ expression of HLA II. CONCLUSION We generated HLA II defected hESCs via deleting CIITA,a master regulator of constitutive and IFN-$\$ expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g.,T cells and DCs) and escape the attack of receptors' CD4(+) T cells,which are the main effector cells of cellular immunity in allograft. View Publication

Human-pluripotent-stem-cell-derived kidney cells (hPSC-KCs) have important potential for disease modelling and regeneration. Whether the hPSC-KCs can reconstitute tissue-specific phenotypes is currently unknown. Here we show that hPSC-KCs self-organize into kidney organoids that functionally recapitulate tissue-specific epithelial physiology,including disease phenotypes after genome editing. In three-dimensional cultures,epiblast-stage hPSCs form spheroids surrounding hollow,amniotic-like cavities. GSK3β inhibition differentiates spheroids into segmented,nephron-like kidney organoids containing cell populations with characteristics of proximal tubules,podocytes and endothelium. Tubules accumulate dextran and methotrexate transport cargoes,and express kidney injury molecule-1 after nephrotoxic chemical injury. CRISPR/Cas9 knockout of podocalyxin causes junctional organization defects in podocyte-like cells. Knockout of the polycystic kidney disease genes PKD1 or PKD2 induces cyst formation from kidney tubules. All of these functional phenotypes are distinct from effects in epiblast spheroids,indicating that they are tissue specific. Our findings establish a reproducible,versatile three-dimensional framework for human epithelial disease modelling and regenerative medicine applications. View Publication

Targeted nucleases,including zinc-finger nucleases (ZFNs),transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However,realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids,the direct delivery of nuclease proteins to cells provides rapid action and fast turnover,leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression,purification and delivery of ZFN proteins,which are intrinsically cell-permeable; TALEN proteins,which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein,whose nucleofection into cells facilitates rapid induction of multiplexed modifications,are described,along with procedures for evaluating nuclease protein activity. Once they are constructed,nuclease proteins can be expressed and purified within 6 d,and they can be used to induce genomic modifications in human cells within 2 d. View Publication

BACKGROUND Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions,and its targets are enriched with ASD-associated genes. METHODS To further understand the molecular links between CHD8 functions and ASD,we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs. RESULTS Transcriptome profiling revealed that CHD8 hemizygosity (CHD8 (+/-)) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development,$$-catenin/Wnt signaling,extracellular matrix,and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia,as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly,we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g.,HGMA2) were dysregulated in CHD8 (+/-) neural progenitors or neurons. CONCLUSIONS We have established a renewable source of CHD8 (+/-) iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume. View Publication

Human embryonic stem cells (hESCs) are predicted to be an unlimited source of hepatocytes which can pave the way for applications such as cell replacement therapies or as a model of human development or even to predict the hepatotoxicity of drug compounds. We have optimized a 23-d differentiation protocol to generate hepatocyte-like cells (HLCs) from hESCs,obtaining a relatively pure population which expresses the major hepatic markers and is functional and mature. The stability of the HLCs in terms of hepato-specific marker expression and functionality was found to be intact even after an extended period of in vitro culture and cryopreservation. The hESC-derived HLCs have shown the capability to display sensitivity and an alteration in the level of CYP enzyme upon drug induction. This illustrates the potential of such assays in predicting the hepatotoxicity of a drug compound leading to advancement of pharmacology View Publication

WNT/$\$-CATENIN signaling promotes the hematopoietic/endothelial differentiation of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs). The transient addition of a GSK3$\$ (GSKi) has been found to facilitate in vitro endothelial cell differentiation from hESCs/hiPSCs. Because hematopoietic and endothelial cells are derived from common progenitors (hemogenic endothelial progenitors [HEPs]),we examined the effect of transient GSKi treatment on hematopoietic cell differentiation from hiPSCs. We found that transient GSKi treatment at the start of hiPSC differentiation induction altered the gene expression profile of the cells. Multiple CDX/HOX genes,which are expressed in the posterior mesoderm of developing embryos,were significantly upregulated by GSKi treatment. Further,inclusion of the GSKi in a serum- and stroma-free culture with chemically defined medium efficiently induced HEPs,and the HEPs gave rise to various lineages of hematopoietic and endothelial cells. Therefore,transient WNT/$\$-CATENIN signaling triggers activation of the CDX/HOX pathway,which in turn confers hemogenic posterior mesoderm identity to differentiating hiPSCs. These data enhance our understanding of human embryonic hematopoietic/endothelial cell development and provide a novel in vitro system for inducing the differentiation of hematopoietic cells from hiPSCs. View Publication

Primary dilated cardiomyopathy (DCM) is a non-ischemic heart disease with impaired pumping function of the heart. In this study,we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a healthy volunteer and a primary DCM patient to investigate the impact of DCM on iPSC-CMs' responses to different types of anisotropic strain. A bioreactor system was established that generates cardiac-mimetic forces of 150 kPa at 5% anisotropic cyclic strain and 1. Hz frequency. After confirming cardiac induction of the iPSCs,it was determined that fibronectin was favorable to other extracellular matrix protein coatings (gelatin,laminin,vitronectin) in terms of viable cell area and density,and was therefore selected as the coating for further study. When iPSC-CMs were exposed to three strain conditions (no strain,5% static strain,and 5% cyclic strain),the static strain elicited significant induction of sarcomere components in comparison to other strain conditions. However,this induction occurred only in iPSC-CMs from a healthy volunteer (control iPSC-CMs")� View Publication

Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving,such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes,new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs),called human intestinal organoids (HIOs),have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However,given that HIOs are small three-dimensional structures grown in vitro,methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds,and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro,the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast,HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine,which need to be explored further to develop them into fully functional tissue. View Publication

The generation of liquefied poly-ɛ-caprolactone (PCL) droplets by means of a microfluidic device results in uniform-sized microspheres,which are validated as microcarriers for human embryonic stem cell culture. Formed droplet size and size distribution,as well as the resulting PCL microsphere size,are correlated with the viscosity and flow rate ratio of the dispersed (Q d) and continuous (Q c) phases. PCL in dichloromethane increases its viscosity with concentration and molecular weight. Higher viscosity and Q d/Q c lead to the formation of larger droplets,within two observed formation modes: dripping and jetting. At low viscosity of dispersed phase and Q d/Q c,the microfluidic device is operated in dripping mode,which generates droplets and microspheres with greater size uniformity. Solutions with lower molecular weight PCL have lower viscosity,resulting in a wider concentration range for the dripping mode. When coated with extracellular matrix (ECM) proteins,the fabricated PCL microspheres are demonstrated capable of supporting the expansion of human embryonic stem cells. View Publication

Tools for rapid and efficient transgenesis in safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE� View Publication