技术资料

Organs-on-chips are microengineered in vitro tissue structures that can be used as platforms for physiological and pathological research. They provide tissue-like microenvironments in which different cell types can be co-cultured in a controlled manner to create synthetic organ mimics. Blood vessels are an integral part of all tissues in the human body. Development of vascular structures is therefore an important research topic for advancing the field of organs-on-chips since generated tissues will require a blood or nutrient supply. Here,we have engineered three-dimensional constructs of vascular tissue inside microchannels by injecting a mixture of human umbilical vein endothelial cells,human embryonic stem cell-derived pericytes (the precursors of vascular smooth muscle cells) and rat tail collagen I into a polydimethylsiloxane microfluidic channel with dimensions 500 μm × 120 μm × 1 cm (w × h × l). Over the course of 12 h,the cells organized themselves into a single long tube resembling a blood vessel that followed the contours of the channel. Detailed examination of tube morphology by confocal microscopy revealed a mature endothelial monolayer with complete PECAM-1 staining at cell–cell contacts and pericytes incorporated inside the tubular structures. We also demonstrated that tube formation was disrupted in the presence of a neutralizing antibody against transforming growth factor-beta (TGF-β). The TGF-β signaling pathway is essential for normal vascular development; deletion of any of its components in mouse development results in defective vasculogenesis and angiogenesis and mutations in humans have been linked to multiple vascular genetic diseases. In the engineered microvessels,inhibition of TGF-β signaling resulted in tubes with smaller diameters and higher tortuosity,highly reminiscent of the abnormal vessels observed in patients with one particular vascular disease known as hereditary hemorrhagic telangiectasia (HHT). In summary,we have developed microengineered three-dimensional vascular structures that can be used as a model to test the effects of drugs and study the interaction between different human vascular cell types. In the future,the model may be integrated into larger tissue constructs to advance the development of organs-on-chips. View Publication

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA),known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts,the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA,resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones,improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus,mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny,and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases. View Publication

Human embryonic stem cells (hESCs) and induced pluripotent cells have the potential to provide an unlimited source of tissues for regenerative medicine. For this purpose,development of defined/xeno-free culture systems under feeder-free conditions is essential for the expansion of hESCs. Most defined/xeno-free media for the culture of hESCs contain basic fibroblast growth factor (bFGF). Therefore,bFGF is thought to have an almost essential role for the expansion of hESCs in an undifferentiated state. Here,we report identification of small molecules,some of which were neurotransmitter antagonists (trimipramine and ethopropazine),which promote long-term hESC self-renewal without bFGF in the medium. The hESCs maintained high expression levels of pluripotency markers,had a normal karyotype after 20 passages,and could differentiate into all three germ layers. ?? 2013 Elsevier Inc. View Publication

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs,the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8),which are expressed in simple epithelial cells,while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Hepatocyte transplantation has been used to treat liver disease. The availability of cells for these procedures is quite limited. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be a useful source of hepatocytes for basic research and transplantation if efficient and effective differentiation protocols were developed and problems with tumorigenicity could be overcome. Recent evidence suggests that the cell of origin may affect hiPSC differentiation. Thus,hiPSCs generated from hepatocytes may differentiate back to hepatocytes more efficiently than hiPSCs from other cell types. We examined the efficiency of reprogramming adult and fetal human hepatocytes. The present studies report the generation of 40 hiPSC lines from primary human hepatocytes under feeder-free conditions. Of these,37 hiPSC lines were generated from fetal hepatocytes,2 hiPSC lines from normal hepatocytes,and 1 hiPSC line from hepatocytes of a patient with Crigler-Najjar syndrome,type 1. All lines were confirmed reprogrammed and expressed markers of pluripotency by gene expression,flow cytometry,immunocytochemistry,and teratoma formation. Fetal hepatocytes were reprogrammed at a frequency over 50-fold higher than adult hepatocytes. Adult hepatocytes were only reprogrammed with six factors,while fetal hepatocytes could be reprogrammed with three (OCT4,SOX2,NANOG) or four factors (OCT4,SOX2,NANOG,LIN28 or OCT4,SOX2,KLF4,C-MYC). The increased reprogramming efficiency of fetal cells was not due to increased transduction efficiency or vector toxicity. These studies confirm that hiPSCs can be generated from adult and fetal hepatocytes including those with genetic diseases. Fetal hepatocytes reprogram much more efficiently than adult hepatocytes,although both could serve as useful sources of hiPSC-derived hepatocytes for basic research or transplantation. View Publication

In metazoans,the nuclear lamina is thought to play an important role in the spatial organization of interphase chromosomes,by providing anchoring sites for large genomic segments named lamina-associated domains (LADs). Some of these LADs are cell-type specific,while many others appear constitutively associated with the lamina. Constitutive LADs (cLADs) may contribute to a basal chromosome architecture. By comparison of mouse and human lamina interaction maps,we find that the sizes and genomic positions of cLADs are strongly conserved. Moreover,cLADs are depleted of synteny breakpoints,pointing to evolutionary selective pressure to keep cLADs intact. Paradoxically,the overall sequence conservation is low for cLADs. Instead,cLADs are universally characterized by long stretches of DNA of high A/T content. Cell-type specific LADs also tend to adhere to this A/T rule" in embryonic stem cells� View Publication

Activin/Nodal signaling via SMAD2/3 maintains human embryonic stem cell (hESC) pluripotency by direct transcriptional regulation of NANOG or,alternatively,induces mesoderm and definitive endoderm (DE) formation. In search of an explanation for these contrasting effects,we focused on SNON (SKIL),a potent SMAD2/3 corepressor that is expressed in hESCs but rapidly down-regulated upon differentiation. We show that SNON predominantly associates with SMAD2 at the promoters of primitive streak (PS) and early DE marker genes. Knockdown of SNON results in premature activation of PS and DE genes and loss of hESC morphology. In contrast,enforced SNON expression inhibits DE formation and diverts hESCs toward an extraembryonic fate. Thus,our findings provide novel mechanistic insight into how a single signaling pathway both regulates pluripotency and directs lineage commitment. View Publication

Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet,acquiring donor cells is,in most instances,an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances,as the isolation of urinary cells is simple (30 ml of urine are sufficient),cost-effective and universal (can be applied to any age,gender and race). Moreover,the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential,and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases,including those affecting the kidney. View Publication

Retinitis pigmentosa,other inherited retinal diseases,and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here,we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx+ photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells,facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. STEM CELLS2013;31:408–414 View Publication

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death,but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis,or of its underlying transcriptome network. Therefore,the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes,whereas MeHg altered fewer transcripts. To attenuate batch effects,analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (backslashtextless20 % overlap). Moreover,within one test system,little overlap between the PS changed by the two compounds has been observed. However,using TFBS enrichment,a relatively large ‘common response' to VPA and MeHg could be distinguished from ‘compound-specific' responses. In conclusion,the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles. View Publication

Human U1 small nuclear (sn)RNA,required for splicing of pre-mRNA,is encoded by genes on chromosome 1 (1p36). Imperfect copies of these U1 snRNA genes,also located on chromosome 1 (1q12-21),were thought to be pseudogenes. However,many of these variant" (v)U1 snRNA genes produce fully processed transcripts. Using antisense oligonucleotides to block the activity of a specific vU1 snRNA in HeLa cells� View Publication