技术资料

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

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-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

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here,we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids,which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung,organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing,we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles,suggesting that HLOs are an excellent model to study human lung development,maturation and disease. View Publication

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) $$$$ and $$$$ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells. View Publication

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability,with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia,coarsened facial features and intellectual disability,due to truncating mutations in the sorting nexin gene SNX14,encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate,a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma,accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction. View Publication

BACKGROUND Anagrelide is a cytoreductive agent used to lower platelet counts in essential thrombocythemia. Although the drug has been known to selectively inhibit megakaryopoiesis for many years,the molecular mechanism accounting for this activity is still unclear. OBJECTIVES AND METHODS To address this issue we have compared the global gene expression profiles of human hematopoietic cells treated ex-vivo with and without anagrelide while growing under megakaryocyte differentiation conditions,using high-density oligonucleotide microarrays. Gene expression data were validated by the quantitative polymerase chain reaction and mined to identify functional subsets and regulatory pathways. RESULTS We identified 328 annotated genes differentially regulated by anagrelide,including many genes associated with platelet functions and with the control of gene transcription. Prominent among the latter was TRIB3,whose expression increased in the presence of anagrelide. Pathway analysis revealed that anagrelide up-regulated genes that are under the control of the transcription factor ATF4,a known TRIB3 inducer. Notably,immunoblot analysis demonstrated that anagrelide induced the phosphorylation of eIF2α,which is an upstream regulator of ATF4,and increased ATF4 protein levels. Furthermore,salubrinal,an inhibitor of eIF2α dephosphorylation,increased the expression of ATF4-regulated genes and blocked megakaryocyte growth. CONCLUSIONS These findings link signaling through eIF2α/ATF4 to the anti-megakaryopoietic activity of anagrelide and identify new potential modulators of megakaryopoiesis. View Publication

The application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously,we identified a human pluripotent stem-cell-specific lectin probe,called rBC2LCN,by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A,termed rBC2LCN-PE23,which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells,followed by its internalization,allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus,rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies. View Publication

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Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However,the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632),a small molecule that inhibits the rho-associated protein kinase pathway,is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection,cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple,effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation. View Publication

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) assert a great future for the cardiovascular diseases,both to study them and to explore therapies. However,a comprehensive assessment of the viral vectors used to modify these cells is lacking. In this study,we aimed to compare the transduction efficiency of recombinant adeno-associated vectors (AAV),adenoviruses and lentiviral vectors in hESC,hiPSC,and the derived cardiomyocytes. In undifferentiated cells,adenoviral and lentiviral vectors were superior,whereas in differentiated cells AAV surpassed at least lentiviral vectors. We also tested four AAV serotypes,1,2,6,and 9,of which 2 and 6 were superior in their transduction efficiency. Interestingly,we observed that AAVs severely diminished the viability of undifferentiated cells,an effect mediated by induction of cell cycle arrest genes and apoptosis. Furthermore,we show that the transduction efficiency of the different viral vectors correlates with the abundance of their respective receptors. Finally,adenoviral delivery of the calcium-transporting ATPase SERCA2a to hESC and hiPSC-derived cardiomyocytes successfully resulted in faster calcium reuptake. In conclusion,adenoviral vectors prove to be efficient for both differentiated and undifferentiated lines,whereas lentiviral vectors are more applicable to undifferentiated cells and AAVs to differentiated cells. View Publication

Reprogramming of patient cells to human induced pluripotent stem cells (hiPSC) has facilitated in vitro disease modeling studies aiming at deciphering the molecular and cellular mechanisms that contribute to disease pathogenesis and progression. To fully exploit the potential of hiPSC for biomedical applications,technologies that enable the standardized generation and expansion of hiPSC from large numbers of donors are required. Paralleled automated processes for the expansion of hiPSC could provide an opportunity to maximize the generation of hiPSC collections from patient cohorts while minimizing hands-on time and costs. In order to develop a simple method for the parallel expansion of human pluripotent stem cells (hPSC) we established a protocol for their cultivation as undifferentiated aggregates in a bench-top bioreactor system (BioLevitator™). We show that long-term expansion (10 passages) of hPSCs either in mTeSR or E8 medium preserved a normal karyotype,three-germ-layer differentiation potential and high expression of pluripotency-associated markers. The system enables the expansion from low inoculation densities (0.3 × 10(5) cells/mL) and provides a simplified,cost-efficient and time-saving method for the provision of hiPSC at midi-scale. Implementation of this protocol in cell production schemes has the potential to advance cell manufacturing in many areas of hiPSC-based medical research. View Publication