技术资料

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration,Stargardt disease,and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements,such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient,but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study,we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand,administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly,transfection of mRNA encoding a key regulator of RPE gene expression,microphthalmia-associated transcription factor (MITF),confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF,primarily localized in the nucleus. Despite these findings,quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings,therefore,show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe,efficient,and functional. View Publication

Human pluripotent stem (hPS) cells are a potential source of cells for medical therapy and an ideal system to study fate decisions in early development. However,hPS cells cultured in vitro exhibit a high degree of heterogeneity,presenting an obstacle to clinical translation. hPS cells grow in spatially patterned colony structures,necessitating quantitative single-cell image analysis. We offer a tool for analyzing the spatial population context of hPS cells that integrates automated fluorescent microscopy with an analysis pipeline. It enables high-throughput detection of colonies at low resolution,with single-cellular and sub-cellular analysis at high resolutions,generating seamless in situ maps of single-cellular data organized by colony. We demonstrate the tool's utility by analyzing inter- and intra-colony heterogeneity of hPS cell cycle regulation and pluripotency marker expression. We measured the heterogeneity within individual colonies by analyzing cell cycle as a function of distance. Cells loosely associated with the outside of the colony are more likely to be in G1,reflecting a less pluripotent state,while cells within the first pluripotent layer are more likely to be in G2,possibly reflecting a G2/M block. Our multi-scale analysis tool groups colony regions into density classes,and cells belonging to those classes have distinct distributions of pluripotency markers and respond differently to DNA damage induction. Lastly,we demonstrate that our pipeline can robustly handle high-content,high-resolution single molecular mRNA FISH data by using novel image processing techniques. Overall,the imaging informatics pipeline presented offers a novel approach to the analysis of hPS cells that includes not only single cell features but also colony wide,and more generally,multi-scale spatial configuration. View Publication

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However,tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs,especially those in cancer patients. To circumvent these issues,we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable,but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition,invasion,stemness,and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ,downstream protumor factors,and hyaluronan and its cofactor TSG6,which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for off-the-shelf" therapies and bioengineering applications." View Publication

Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs),and their neuronal progeny,will play an important role in disease modeling,drug screening tests,central nervous system development studies,and may even become valuable for regenerative medicine treatments. Nonetheless,it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs,and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here,we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days,and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS),leaving the NP population nearly free of PSCs. View Publication

Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells,which will be useful for experimental application. View Publication

Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells,including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses,such as Japanese encephalitis virus (JEV),provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition,glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast,only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition,functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover,we discover that vimentin intermediate filament,reported as a putative neurovirulent JEV receptor,is highly expressed in NPCs and glial cells,but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally,we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection. View Publication

The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs),subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB),and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However,the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study,we used the latest gene-editing tool,clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9),to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then,we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation,gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly,the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system,and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation. View Publication

Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP),modulating actin-myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells,leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1),the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. View Publication

The Mesenchymal-to-Epithelial Transition (MET) has been recognized as a crucial step for successful reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Thus,it has been demonstrated,that the efficiency of reprogramming can be enhanced by promoting an epithelial expression program in cells,with a concomitant repression of key mesenchymal genes. However,a detailed characterization of the epithelial transition associated with the acquisition of a pluripotent phenotype is still lacking to this date. Here,we integrate a panel of morphological approaches with gene expression analyses to visualize the dynamics of episomal reprogramming of human fibroblasts to iPSCs. We provide the first ultrastructural analysis of human fibroblasts at various stages of episomal iPSC reprogramming,as well as the first real-time live cell visualization of a MET occurring during reprogramming. The results indicate that the MET manifests itself approximately 6-12. days after electroporation,in synchrony with the upregulation of early pluripotency markers,and resembles a reversal of the Epithelial-to-Mesenchymal Transition (EMT) which takes place during mammalian gastrulation. View Publication

In this study,we have identified a novel member of the AMPK family,namely Sucrose non-fermenting related kinase (Snrk),that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart,and brain,and in cell types such as endothelial cells,smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts,and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts,and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally,adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis,and shows an autonomous role for SNRK during mammalian development. View Publication

BACKGROUND Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However,a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to $\$-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in $\$-adrenergic signaling,we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS First,we confirmed that PLB protein was differentially expressed in hESC (HES2,H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant,respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase,Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly,Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally,similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However,Ad-PLB altered neither the global transcriptome nor ICa,L,implicating a PLB-specific effect. CONCLUSIONS Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to $\$-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses. View Publication