Scientific Resources

Human pluripotent stem cells derived cardiomyocytes (PSC-CMs) have been widely used for disease modeling,drug safety screening,and preclinical cell therapy to regenerate myocardium. Most studies have utilized PSC-CM grown in vitro for a relatively short period after differentiation. These PSC-CMs demonstrated structural,electrophysiological,and mechanical features of primitive cardiomyocytes. A few studies have extended in vitro PSC-CM culture time and reported improved maturation of structural and electromechanical properties. The degree of mitochondrial maturation,however,remains unclear. This study characterized the development of mitochondria during prolonged in vitro culture. PSC-CM demonstrated an improved mitochondrial maturation with prolonged culture,in terms of increased mitochondrial relative abundance,enhanced membrane potential,and increased activity of several mitochondrial respiratory complexes. These are in parallel with the maturation of other cellular components. However,the maturation of mitochondria in PSC-CMs grown for extended in vitro culture exhibits suboptimal maturation when compared with the maturation of mitochondria observed in the human fetal heart during similar time interval. View Publication

BACKGROUND Fabry disease (FD) is a lysosomal storage disease in which glycosphingolipids (GB3) accumulate in organs of the human body,leading to idiopathic hypertrophic cardiomyopathy and target organ damage. Its pathophysiology is still poorly understood. OBJECTIVES We aimed to generate patient-specific induced pluripotent stem cells (iPSC) from FD patients presenting cardiomyopathy to determine whether the model could recapitulate key features of the disease phenotype and to investigate the energy metabolism in Fabry disease. METHODS Peripheral blood mononuclear cells from a 30-year-old Chinese man with a diagnosis of Fabry disease,GLA gene (IVS4+919G>A) mutation were reprogrammed into iPSCs and differentiated into iPSC-CMs and energy metabolism was analyzed in iPSC-CMs. RESULTS The FD-iPSC-CMs recapitulated numerous aspects of the FD phenotype including reduced GLA activity,cellular hypertrophy,GB3 accumulation and impaired contractility. Decreased energy metabolism with energy utilization shift to glycolysis was observed,but the decreased energy metabolism was not modified by enzyme rescue replacement (ERT) in FD-iPSCs-CMs. CONCLUSION This model provided a promising in vitro model for the investigation of the underlying disease mechanism and development of novel therapeutic strategies for FD. This potential remedy for enhancing the energetic network and utility efficiency warrants further study to identify novel therapies for the disease. View Publication

Tissue-resident macrophages,such as microglia,Kupffer cells,and Langerhans cells,derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally,independently of circulating monocytes and HSCs. In contrast,adult blood monocytes,as well as infiltrating,gut,and dermal macrophages,derive from Myb-dependent HSCs. These findings are derived from the mouse,using gene knockouts and lineage tracing,but their applicability to human development has not been formally demonstrated. Here,we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy,we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent,RUNX1-,and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages,such as alveolar and kidney macrophages,microglia,Kupffer cells,and Langerhans cells. View Publication

Classical lissencephaly is a genetic neurological disorder associated with mental retardation and intractable epilepsy,and Miller-Dieker syndrome (MDS) is the most severe form of the disease. In this study,to investigate the effects of MDS on human progenitor subtypes that control neuronal output and influence brain topology,we analyzed cerebral organoids derived from control and MDS-induced pluripotent stem cells (iPSCs) using time-lapse imaging,immunostaining,and single-cell RNA sequencing. We saw a cell migration defect that was rescued when we corrected the MDS causative chromosomal deletion and severe apoptosis of the founder neuroepithelial stem cells,accompanied by increased horizontal cell divisions. We also identified a mitotic defect in outer radial glia,a progenitor subtype that is largely absent from lissencephalic rodents but critical for human neocortical expansion. Our study,therefore,deepens our understanding of MDS cellular pathogenesis and highlights the broad utility of cerebral organoids for modeling human neurodevelopmental disorders. View Publication

Induced pluripotent stem cells have great potential as a human model system in regenerative medicine,disease modeling,and drug screening. However,their use in medical research is hampered by laborious reprogramming procedures that yield low numbers of induced pluripotent stem cells. For further applications in research,only the best,competent clones should be used. The standard assays for pluripotency are based on genomic approaches,which take up to 1 week to perform and incur significant cost. Therefore,there is a need for a rapid and cost-effective assay able to distinguish between pluripotent and nonpluripotent cells. Here,we describe a novel multiplexed,high-throughput,and sensitive peptide-based multiple reaction monitoring mass spectrometry assay,allowing for the identification and absolute quantitation of multiple core transcription factors and pluripotency markers. This assay provides simpler and high-throughput classification into either pluripotent or nonpluripotent cells in 7 min analysis while being more cost-effective than conventional genomic tests. View Publication

The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner,mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However,the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover,we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects,and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells,inducing a dose-dependent increase in SOX2,OCT4 and Nanog proteins,leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. View Publication

Mitochondrial diseases are genetically heterogeneous and present a broad clinical spectrum among patients; in most cases,genetic determinants of mitochondrial diseases are heteroplasmic mitochondrial DNA (mtDNA) mutations. However,it is uncertain whether and how heteroplasmic mtDNA mutations affect particular cellular fate-determination processes,which are closely associated with the cell-type-specific pathophysiology of mitochondrial diseases. In this study,we established two isogenic induced pluripotent stem cell (iPSC) lines each carrying different proportions of a heteroplasmic m.3243A>G mutation from the same patient; one exhibited apparently normal and the other showed most likely impaired mitochondrial respiratory function. Low proportions of m.3243A>G exhibited no apparent molecular pathogenic influence on directed differentiation into neurons and cardiomyocytes,whereas high proportions of m.3243A>G showed both induced neuronal cell death and inhibited cardiac lineage commitment. Such neuronal and cardiac maturation defects were also confirmed using another patient-derived iPSC line carrying quite high proportion of m.3243A>G. In conclusion,mitochondrial respiratory dysfunction strongly inhibits maturation and survival of iPSC-derived neurons and cardiomyocytes; our presenting data also suggest that appropriate mitochondrial maturation actually contributes to cellular fate-determination processes during development. View Publication

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however,the mechanism is mysterious. Here,we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that,conserved across species,stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic,as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner,and many ISGs decrease upon differentiation,at which time cells become IFN responsive,allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly,we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance. View Publication

BACKGROUND Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity,despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive,only cataloging heterogeneity at one point in time,and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity,and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. RESULTS We introduce the programmable Polaris microfluidic lab-on-chip for single-cell sequencing,which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1,such as an altered oxidative stress response,have a large paracrine signaling component. Furthermore,we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation,both associated with the oxidative stress response and altered proteostasis. Interestingly,SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'),with no known co-regulation. CONCLUSION As single-cell methods continue to mature,so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture,live-cell imaging,and single-cell sequencing,we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages,for example,react to inflammation and form treatment resistant HIV reservoirs. View Publication

Despite the high incidence of trophoblast-related diseases,the molecular mechanism of inadequate early trophoblast development is still unclear due to the lack of an appropriate cellular model in vitro. In the present study,we reprogrammed the amniotic cells to be induced pluripotent stem cells (iPSCs) via a non-virus and non-integrated method and subsequently differentiated them into trophoblast-like cells by a modified BMP4 strategy in E6 medium. Compared with the previously studied trophoblast-like cells from ESCs,the iPSCs derived trophoblast-like cells behave similarly in terms of gene expression profiles and biofunctions. Also we confirmed the differentiating tendency from iPSCs to be syncytiotrophoblasts-like cells might be caused by inappropriate differentiating oxygen condition. Additionally,we preliminarily indicated in vitro artificial" differentiation of iPSCs also undergoing a possible trophoblastic stem cell stage as witnessed in vivo. In conclusion we provided an in vitro cellular model to study early trophoblast development for specific individual by using the feasible amnion. View Publication

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka,who first generated iPSCs by retroviral transduction of four reprogramming factors,several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However,the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study,three different strategies,based on retroviral vectors,episomal vectors,and Sendai virus vectors,were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells,including the expression of alkaline phosphatase and stemness maker genes,and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion,the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs. View Publication

AIMS: Circular RNA (circRNA) is a newly validated class of single-stranded RNA,ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types,genome-wide circRNA expression in the heart has yet to be described in detail. METHODS AND RESULTS: We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts,25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools,we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse,respectively. Their abundance generally correlates with the abundance of their cognate linear RNA,but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN),RYR2,and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms,the majority (83%) of which originates from the I-band domain. Finally,we confirmed the expression of selected cardiac circRNA by RT-PCR,Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. CONCLUSIONS: Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA. View Publication