技术资料

Owing to ongoing recognition of pathogen-associated molecular patterns,immune activation and upregulation of IFN-stimulated genes (ISGs) are sustained in the chronically infected host. Albeit most ISGs are important effectors for containing viral replication,some might exert compensatory immune suppression to limit pathological dysfunctions,although the mechanisms are not fully understood. In this study,we report that the ISG lymphocyte Ag 6 complex,locus E (LY6E) is a negative immune regulator of monocytes. LY6E in monocytes negatively modulated CD14 expression and subsequently dampened the responsiveness to LPS stimulation in vitro. In the setting of chronic HIV infection,the upregulation of LY6E was correlated with reduced CD14 level on monocytes; however,the immunosuppressive effect of LY6E was not adequate to remedy the hyperresponsiveness of activated monocytes. Taken together,the regulatory LY6E pathway in monocytes represents one of negative feedback mechanisms that counterbalance monocyte activation,which might be caused by LPS translocation through the compromised gastrointestinal tract during persistent HIV-1 infection and may serve as a potential target for immune intervention. View Publication

Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics,the MB-based method enables the isolation of a wide variety of cells. For example,the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design,validation and nucleofection into cells,we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol,we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity,as confirmed by electrophysiology and immunocytochemistry. After designing MBs,their ordering and validation requires 2 weeks,and the isolation process requires 3 h. View Publication

The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds,UM171 being the prototype,is independent of suppression of the aryl hydrocarbon receptor,which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy. 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

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast,induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs,which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology,immunophenotype,in vitro differentiation potential,and gene expression profiles as primary MSCs. However,iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics,whereas tissue-specific,senescence-associated,and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion,but they remained rejuvenated with regard to age-related DNAm. Overall,iPS-MSCs are similar to MSCs,but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns - particularly of DNAm patterns associated with tissue type and aging. View Publication

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture,the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study,we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells,including cardiomyocytes and fibroblasts,using a methionine-free culture condition. When cultured in the methionine-free condition,human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition,gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore,in fabricated cardiac cell sheets,spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination. View Publication

The acquisition of innate immune response is requisite to having bona fide differentiation of airway epithelium. Procedures developed to differentiate lung airway from human pluripotent stem cells (hPSCs) have demonstrated anecdotal evidence for innate immune response,but an in-depth exploration of response levels is lacking. Herein,using an established method of airway epithelial generation from hPSCs,we show that hPSC-derived epithelial cells are able to up-regulate expression of TNF$\$,IL8 and IL1$\$ response to challenge with bacterial endotoxin LPS,but lack response from genes associated with innate immune response in other cell types. Further,stimulation of cells with TNF-$\$ in auto-induction of TNF$\$,as well as cytokine responses of IL8 and IL1$\$ The demonstration of innate immune induction in hPSC-derived airway epithelia gives further strength to the functionality of in vitro protocols aimed at generating differentiated airway cells that can potentially be used in a translational setting. Finally,we propose that innate immune challenge of airway epithelium from human pluripotent stem cell sources be used as a robust validation of functional in vitro differentiation. View Publication

Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor,Y15,was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines,profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878,and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines,TPC1,BCPAP and K1,and 380,109,and 74 genes were significantly textgreater2-fold changed with Y15 treatment,respectively. Common up-regulated genes were involved in apoptosis,cell cycle,transcription and heat shock; down-regulated genes were involved in cell cycle,cell-to-cell interactions,and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes textgreater4-fold (ptextless0.05). Among genes changed 4 fold,11 were shared between the treatments,including those involved in metabolism,cell cycle,migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib,Sorafenib,Pazopanib,and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor,detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines,demonstrated differential response changes with Y15 and PF-04554878 treatment,and showed the synergy of Y15 with PF-04554878,Cabozantinib,Sorafenib,Pazopanib,and Sunitinib. View Publication

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson's disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development,A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here,we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons,which mimics embryonic DA neuron development. In our protocol,we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method,and then convert the FP cells to A9 DA neurons,which could be maintained in vitro for several months. This efficient,repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients,in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD. 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

We have previously demonstrated that the Src family kinase Yes,the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition,we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation,YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI,is demonstrated to be responsible for this effect. Moreover,IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion,we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal. View Publication

Embryonic stem (ES) cells are characterized by pluripotency,defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade,great progress has been made on the cell culture conditions,transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions,responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein-tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells,and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal,while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation,the role of this kinase family in human ES cells is largely unknown. Here,we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome,Fyn,c-Yes,c-Src,Lyn,Lck and Hck were expressed in H1,H7 and H9 hES cells,while Fgr,Blk,Srm,Brk,and Frk transcripts were not detected. Of these,c-Yes,Lyn,and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs,while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast,Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation,cultures were treated with inhibitors specific for the Src kinase family. Remarkably,human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed,pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together,these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate,and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program. View Publication