技术资料

Surface ectoderm (SE) cells give rise to structures including the epidermis and ectodermal associated appendages such as hair,eye,and the mammary gland. In this study,we validate a protocol that utilizes BMP4 and the $$-secretase inhibitor DAPT to induce SE differentiation from human induced pluripotent stem cells (hiPSCs). hiPSC-differentiated SE cells expressed markers suggesting their commitment to the SE lineage. Computational analyses using integrated quantitative transcriptomic and proteomic profiling reveal that TGF$$ superfamily signaling pathways are preferentially activated in SE cells compared with hiPSCs. SE differentiation can be enhanced by selectively blocking TGF$$-RI signaling. We also show that SE cells and neural ectoderm cells possess distinct gene expression patterns and signaling networks as indicated by functional Ingenuity Pathway Analysis. Our findings advance current understanding of early human SE cell development and pave the way for modeling of SE-derived tissue development,studying disease pathogenesis,and development of regenerative medicine approaches. View Publication

The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX),an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37,CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX,whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90,robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells,while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile,by RNA-seq,of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX,including phototransduction genes,exhibit a significant delay in expression. We report on temporal changes in gene signatures,including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors,providing a reference map for functional studies in retinal cultures. View Publication

Fibroblasts from patients with Type I bipolar disorder (BPD) and their unaffected siblings were obtained from an Old Order Amish pedigree with a high incidence of BPD and reprogrammed to induced pluripotent stem cells (iPSCs). Established iPSCs were subsequently differentiated into neuroprogenitors (NPs) and then to neurons. Transcriptomic microarray analysis was conducted on RNA samples from iPSCs,NPs and neurons matured in culture for either 2 weeks (termed early neurons,E) or 4 weeks (termed late neurons,L). Global RNA profiling indicated that BPD and control iPSCs differentiated into NPs and neurons at a similar rate,enabling studies of differentially expressed genes in neurons from controls and BPD cases. Significant disease-associated differences in gene expression were observed only in L neurons. Specifically,328 genes were differentially expressed between BPD and control L neurons including GAD1,glutamate decarboxylase 1 (2.5 fold) and SCN4B,the voltage gated type IV sodium channel beta subunit (-14.6 fold). Quantitative RT-PCR confirmed the up-regulation of GAD1 in BPD compared to control L neurons. Gene Ontology,GeneGo and Ingenuity Pathway Analysis of differentially regulated genes in L neurons suggest that alterations in RNA biosynthesis and metabolism,protein trafficking as well as receptor signaling pathways may play an important role in the pathophysiology of BPD. View Publication

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles,we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform,mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred,thus increasing screening throughput by textgreater30-fold. Compared to conventional methods,micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. View Publication

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with non-integrative constructs. Numerous studies,however,including those describing disease models,are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs,but in mesenchymal and endothelial iPSC derivatives,the transgenes experienced random up-regulation of Nanog and c-Myc. Additionally,we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies,which utilize cellular products derived from iPSCs generated with retro- or lentiviruses,should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work,however,is to communicate the possibility of transgene reactivation in retro- or lenti- iPSC derivatives and the associated loss of cellular fidelity in vitro,which may impact the outcomes of disease modeling and related experimentation. View Publication

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

Optimization of pluripotent stem cell expansion and differentiation is facilitated by biological tools that permit non-invasive and dynamic monitoring of pluripotency,and the ability to select for an undifferentiated input cell population. Here we report on the generation and characterisation of clonal human embryonic stem (HES3,H9) and human induced pluripotent stem cell lines (UQEW01i-epifibC11) that have been stably modified with an artificial EOS(C3+) promoter driving expression of EGFP and puromycin resistance-conferring proteins. We show that EGFP expression faithfully reports on the pluripotency status of the cells in these lines and that antibiotic selection allows for an efficient elimination of differentiated cells from the cultures. We demonstrate that the extinction of the expression of the pluripotency reporter during differentiation closely correlates with the decrease in expression of conventional pluripotency markers,such as OCT4 (POU5F1),TRA-1-60 and SSEA4 when screening across conditions with various levels of pluripotency-maintaining or differentiation-inducing signals. We further illustrate the utility of these lines for real-time monitoring of pluripotency in embryoid bodies and microfluidic bioreactors. View Publication

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells,but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors,we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation,reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a(+)CD42b(+) platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b(+) platelets were present in thrombi after laser-induced vessel wall injury. In contrast,sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression,decreased GATA1 expression,and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression,particularly its later decline,is key to producing functional platelets from selected iPSC clones. 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

Down's syndrome is a common disorder with enormous medical and social costs,caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene,XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases,we inserted a large,inducible XIST transgene into the DYRK1A locus on chromosome 21,in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications,chromosome-wide transcriptional silencing and DNA methylation to form a ‘chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21,free from genetic and epigenetic noise. Notably,deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of ‘chromosome therapy'. View Publication

Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study,we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519CtextgreaterT (p.R120X) into induced pluripotent stem cells (iPSC),and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells,suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization,Golgi cohesion and G$\$1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren),we were able to restore up to 20% of endogenous,full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients. View Publication

In Huntington's disease (HD),whether transneuronal spreading of mutant huntingtin (mHTT) occurs and its contribution to non-cell autonomous damage in brain networks is largely unknown. We found mHTT spreading in three different neural network models: human neurons integrated in the neural network of organotypic brain slices of HD mouse model,an ex vivo corticostriatal slice model and the corticostriatal pathway in vivo. Transneuronal propagation of mHTT was blocked by two different botulinum neurotoxins,each known for specifically inactivating a single critical component of the synaptic vesicle fusion machinery. Moreover,healthy human neurons in HD mouse model brain slices displayed non-cell autonomous changes in morphological integrity that were more pronounced when these neurons bore mHTT aggregates. Altogether,our findings suggest that transneuronal propagation of mHTT might be an important and underestimated contributor to the pathophysiology of HD. View Publication