SynQuant: an automatic tool to quantify synapses from microscopy images.

MOTIVATION: Synapses are essential to neural signal transmission. Therefore, quantification of synapses and related neurites from images is vital to gain insights into the underlying pathways of brain functionality and diseases. Despite the wide availability of synaptic punctum imaging data, several issues are impeding satisfactory quantification of these structures by current tools. First, the antibodies used for labeling synapses are not perfectly specific to synapses. These antibodies may exist in neurites or other cell compartments. Second, the brightness of different neurites and synaptic puncta is heterogeneous due to the variation of antibody concentration and synapse-intrinsic differences. Third, images often have low signal to noise ratio due to constraints of experiment facilities and availability of sensitive antibodies. These issues make the detection of synapses challenging and necessitates developing a new tool to easily and accurately quantify synapses. RESULTS: We present an automatic probability-principled synapse detection algorithm and integrate it into our synapse quantification tool SynQuant. Derived from the theory of order statistics, our method controls the false discovery rate and improves the power of detecting synapses. SynQuant is unsupervised, works for both 2D and 3D data, and can handle multiple staining channels. Through extensive experiments on one synthetic and three real datasets with ground truth annotation or manually labeling, SynQuant was demonstrated to outperform peer specialized unsupervised synapse detection tools as well as generic spot detection methods. AVAILABILITY AND IMPLEMENTATION: Java source code, Fiji plug-in, and test data are available at https://github.com/yu-lab-vt/SynQuant. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aberrant Calcium Signaling in Astrocytes Inhibits Neuronal Excitability in a Human Down Syndrome Stem Cell Model.

Down syndrome (DS) is a genetic disorder that causes cognitive impairment. The staggering effects associated with an extra copy of human chromosome 21 (HSA21) complicates mechanistic understanding of DS pathophysiology. We examined the neuron-astrocyte interplay in a fully recapitulated HSA21 trisomy cellular model differentiated from DS-patient-derived induced pluripotent stem cells (iPSCs). By combining calcium imaging with genetic approaches, we discovered the functional defects of DS astroglia and their effects on neuronal excitability. Compared with control isogenic astroglia, DS astroglia exhibited more-frequent spontaneous calcium fluctuations, which reduced the excitability of co-cultured neurons. Furthermore, suppressed neuronal activity could be rescued by abolishing astrocytic spontaneous calcium activity either chemically by blocking adenosine-mediated signaling or genetically by knockdown of inositol triphosphate (IP3) receptors or S100B, a calcium binding protein coded on HSA21. Our results suggest a mechanism by which DS alters the function of astrocytes, which subsequently disturbs neuronal excitability.

Automated Functional Analysis of Astrocytes from Chronic Time-Lapse Calcium Imaging Data.

Recent discoveries that astrocytes exert proactive regulatory effects on neural information processing and that they are deeply involved in normal brain development and disease pathology have stimulated broad interest in understanding astrocyte functional roles in brain circuit. Measuring astrocyte functional status is now technically feasible, due to recent advances in modern microscopy and ultrasensitive cell-type specific genetically encoded Ca2+ indicators for chronic imaging. However, there is a big gap between the capability of generating large dataset via calcium imaging and the availability of sophisticated analytical tools for decoding the astrocyte function. Current practice is essentially manual, which not only limits analysis throughput but also risks introducing bias and missing important information latent in complex, dynamic big data. Here, we report a suite of computational tools, called Functional AStrocyte Phenotyping (FASP), for automatically quantifying the functional status of astrocytes. Considering the complex nature of Ca2+ signaling in astrocytes and low signal to noise ratio, FASP is designed with data-driven and probabilistic principles, to flexibly account for various patterns and to perform robustly with noisy data. In particular, FASP explicitly models signal propagation, which rules out the applicability of tools designed for other types of data. We demonstrate the effectiveness of FASP using extensive synthetic and real data sets. The findings by FASP were verified by manual inspection. FASP also detected signals that were missed by purely manual analysis but could be confirmed by more careful manual examination under the guidance of automatic analysis. All algorithms and the analysis pipeline are packaged into a plugin for Fiji (ImageJ), with the source code freely available online at https://github.com/VTcbil/FASP.

Early pathogenic event of Alzheimer's disease documented in iPSCs from patients with PSEN1 mutations.

Alzheimer's disease (AD) is the most common age-related dementia characterized by progressive neuronal loss. However, the molecular mechanisms for the neuronal loss is still debated. Here, we used induced pluripotent stem cells (iPSCs) derived from somatic cells of familial AD patients carrying PSEN1 mutations to study the early pathogenic event of AD. We found that premature neuronal differentiation with decreased proliferation and increased apoptosis occured in AD-iPSC-derived neural progenitor cells (AD-NPCs) once neuronal differentiation was initiated, together with higher levels of Aß42 and phosphorylated tau. Premature neuronal differentiation in AD-NPCs was caused by PSEN1 mutations and might be correlated to multiple dysregulated processes including but not limited to Wnt-Notch pathway. Our study documented previously unappreciated early NPC dysfunction in AD-NPCs, providing valuable new insights into the early mechanisms underlying AD pathogenesis.

Conversion of human fibroblasts into functional cardiomyocytes by small molecules.

Reprogramming somatic fibroblasts into alternative lineages would provide a promising source of cells for regenerative therapy. However, transdifferentiating human cells into specific homogeneous, functional cell types is challenging. Here we show that cardiomyocyte-like cells can be generated by treating human fibroblasts with a combination of nine compounds that we term 9C. The chemically induced cardiomyocyte-like cells uniformly contracted and resembled human cardiomyocytes in their transcriptome, epigenetic, and electrophysiological properties. 9C treatment of human fibroblasts resulted in a more open-chromatin conformation at key heart developmental genes, enabling their promoters and enhancers to bind effectors of major cardiogenic signals. When transplanted into infarcted mouse hearts, 9C-treated fibroblasts were efficiently converted to chemically induced cardiomyocyte-like cells. This pharmacological approach to lineage-specific reprogramming may have many important therapeutic implications after further optimization to generate mature cardiac cells.

Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells.

INTRODUCTION: Embryonic stem cells (ESCs) provide an attractive cell source for basic research and disease treatment. Currently, the common culture system for mouse ESC requires mouse embryonic fibroblast (MEF) as a feeder layer supplemented with leukemia inhibitory factor (LIF). The drawbacks associated with MEF and the cost of LIF have motivated exploration of new feeder cell types to maintain self-renewal of mouse ESCs without the need of exogenous LIF. However, why these feeder cells could maintain ESCs at the undifferentiated state independent of exogenous LIF is unclear. METHODS: We derived mouse ESC lines using human foreskin fibroblast (HFF) in the absence of exogenous LIF. We also examined the dependence of HFF on the JAK-Stat3 pathway to maintain ESC identities and explored the potential molecular basis for HFF to support self-renewal of ESCs. RESULTS: HFF supported mouse ESC self-renewal superiorly to MEFs. Using the HFF system, multiple lines of mouse ESCs were successfully derived without addition of exogenous LIF and any small molecular inhibitors. These ESCs had capacities to self-renew for a long period of time and to differentiate into various cell types of the three germ layers both in vitro and in vivo. Moreover, the ESCs participated in embryonic development and contributed to germ cell lineages in the chimeric mouse. At a molecular level, HFF was dependent on the JAK-Stat3 pathway to maintain ESC self-renewal. The high level of interleukin-6 (IL-6) produced by HFF might be responsible for the exogenous LIF-independent effect. CONCLUSION: This study describes an efficient, convenient and economic system to establish and maintain mouse ESC lines, and provides insights into the functional difference in the support of ESC culture between MEF and HFF.

Nucleolin maintains embryonic stem cell self-renewal by suppression of p53 protein-dependent pathway.

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain pluripotent developmental potential. The unique characteristics of ESCs, including a distinct transcriptional network, a poised epigenetic state, and a specific cell cycle profile, distinguish them from somatic cells. However, the molecular mechanisms underlying these special properties of ESCs are not fully understood. Here, we report that nucleolin, a nucleolar protein highly expressed in undifferentiated ESCs, plays an essential role for the maintenance of ESC self-renewal. When nucleolin is knocked down by specific short hairpin RNA (shRNA), ESCs display dramatically reduced cell proliferation rate, increased cell apoptosis, and G(1) phase accumulation. Down-regulation of nucleolin also leads to evident ESC differentiation as well as decreased self-renewal ability. Interestingly, expression of pluripotency markers (Oct4 and Nanog) is unaltered in these differentiated cells. Mechanistically, depletion of nucleolin up-regulates the p53 protein level and activates the p53-dependent pathway, at least in part, via increasing p53 protein stability. Silencing of p53 rescues G(1) phase accumulation and apoptosis caused by nucleolin deficiency entirely, although it partially blocks abnormal differentiation in nucleolin-depleted ESCs. It is noteworthy that knocking down nucleolin in NIH3T3 cells affected cell survival and proliferation in a much milder way, despite the comparable silencing efficiency obtained in ESCs and NIH3T3 cells. Collectively, our data demonstrate that nucleolin is a critical regulator of ESC self-renewal and that suppression of the p53-dependent pathway is the major molecular mechanism underlying functions of nucleolin in ESCs.

The regulatory role of histone deacetylase inhibitors in Fgf4 expression is dependent on the differentiation state of pluripotent stem cells.

The identity of embryonic stem cells (ESCs) is controlled by a set of pluripotency genes, including Oct4, Sox2, Nanog, and Fgf4. How their expression is repressed during differentiation and reactivated during reprogramming is largely unknown. Here, using mouse ESCs as well as F9 and P19 cells (mouse embryonal carcinoma cell lines, P19 being considered further differentiated than F9 cells) as models, we found that HDAC inhibitors elevated Fgf4 expression in P19 cells, but reduced it in F9 cells. We also observed that HDAC inhibitors enhanced the expression of Fgf4 and a subset of pluripotency genes in differentiated ESCs, but reduced their expression in undifferentiated and less differentiated ESCs. Mechanistically, we observed more HDAC1 recruitment and a weaker association of histone 4 lysine 5 acetylation at the Fgf4 enhancer in P19 cells compared to F9 cells. Additionally, we demonstrated the interaction between Sox2 and HDAC1 both in vitro and in vivo, implicating a possible role for Sox2 in the recruitment of HDAC1 to the Fgf4 enhancer. We also found that Nanog bound to the Fgf4 enhancer, and this binding was stronger in F9 cells, indicating the involvement of Nanog in the regulation of Fgf4 expression in undifferentiated and less differentiated pluripotent stem cells. This study uncovers an important role of HDAC1 and histone modifications in the repression of Fgf4 and perhaps other pluripotency genes during ESC differentiation. Our results also suggest that HDAC inhibitors may promote reprogramming partially through activating pluripotency genes at some intermediate stages.

Role of Oct4 in maintaining and regaining stem cell pluripotency.

Pluripotency, a characteristic of cells in the inner cell mass of the mammalian preimplantation blastocyst as well as of embryonic stem cells, is defined as the ability of a cell to generate all of the cell types of an organism. A group of transcription factors is essential for the establishment and maintenance of the pluripotent state. Recent studies have demonstrated that differentiated somatic cells could be reverted to a pluripotent state by the overexpression of a set of transcription factors, further highlighting the significance of transcription factors in the control of pluripotency. Among these factors, a member of the POU transcription factor family, Oct4, is central to the machinery governing pluripotency. Oct4 is highly expressed in pluripotent cells and becomes silenced upon differentiation. Interestingly, the precise expression level of Oct4 determines the fate of embryonic stem cells. Therefore, to control the expression of Oct4 precisely, a variety of regulators function at multiple levels, including transcription, translation of mRNA and post-translational modification. Additionally, in cooperation with Sox2, Nanog and other members of the core transcriptional regulatory circuitry, Oct4 activates both protein-coding genes and noncoding RNAs necessary for pluripotency. Simultaneously, in association with transcriptional repressive complexes, Oct4 represses another set of targets involved in developmental processes. Importantly, Oct4 can re-establish pluripotency in somatic cells, and proper reprogramming of Oct4 expression is indispensable for deriving genuine induced pluripotent stem cell lines. In the past several years, genome-wide identification of Oct4 target genes and Oct4-centered protein interactomes has been reported, indicating that Oct4 exerts tight control over pluripotency regulator expression and protects embryonic stem cells in an undifferentiated state. Nevertheless, further investigation is required to fully elucidate the underlying molecular mechanisms through which Oct4 maintains and reinitiates pluripotency. Systemic and dynamic exploration of the protein complexes and target genes associated with Oct4 will help to elucidate the role of Oct4 more comprehensively.

Germline-competent mouse-induced pluripotent stem cell lines generated on human fibroblasts without exogenous leukemia inhibitory factor.

Induced pluripotent stem (iPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. Most prior established iPS cell lines were derived and maintained on mouse embryonic fibroblast (MEF) cells supplemented with exogenous leukemia inhibitory factor (LIF). Drawbacks of MEF cells impede optimization as well as dissection of reprogramming events and limit the usage of iPS cell derivatives in therapeutic applications. In this study, we develop a reproducible protocol for efficient reprogramming mouse neural progenitor cells (NPCs) on human foreskin fibroblast (HFF) cells via retroviral transfer of human transcriptional factors OCT4/SOX2/KLF4/C-MYC. Two independent iPS cell lines are derived without exogenous LIF. They display typical undifferentiated morphology and express pluripotency markers Oct4 and Sox2. Transgenes are inactivated and the endogenous Oct4 promoter is completely demethylated in the established iPS cell lines, indicating a fully reprogrammed state. Moreover, the iPS cells can spontaneously differentiate or be induced into various cell types of three embryonic germ layers in vitro and in vivo when they are injected into immunodeficient mice for teratoma formation. Importantly, iPS cells extensively integrate with various host tissues and contribute to the germline when injected into the blastocysts. Interestingly, these two iPS cell lines, while both pluripotent, exhibit distinctive differentiation tendencies towards different lineages. Taken together, the data describe the first genuine mouse iPS cell lines generated on human feeder cells without exogenous LIF, providing a reliable tool for understanding the molecular mechanisms of nuclear reprogramming.

Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells.

Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up 6 days after viral infection with high efficiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas, respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.

Ly-1 antibody reactive clone is an important nucleolar protein for control of self-renewal and differentiation in embryonic stem cells.

Embryonic stem cells (ESCs) possess the capacity to self-renew and differentiate into all cell types of an organism. It is essential to understand how these properties are controlled for the potential usage of their derivatives in clinical settings and reprogramming of differentiated somatic cells. Although transcriptional factors, such as Oct4, Sox2, and Nanog, have been considered as a part of the core regulatory circuitry, a growing body of evidence suggests that additional factors exist and contribute to the control of ESC self-renewal and differentiation. Here, we report that Ly-1 antibody reactive clone (LYAR), a zinc finger nucleolar protein highly expressed in undifferentiated ESCs, plays a critical role in maintaining ESC identity. Its downregulation significantly reduces the rate of ESC growth and increases their apoptosis. Moreover, reduced expression of LYAR in ESCs impairs their differentiation capacity, failing to rapidly silence pluripotency markers and to activate differentiation genes upon differentiation. Mechanistically, LYAR forms a complex with another nucleolar protein, nucleolin, and prevents its self-cleavage, maintaining a normal steady-state level of nucleolin protein in undifferentiated ESCs. Interestingly, the downregulation of nucleolin is detrimental to the growth of ESCs and increases the rate of apoptosis, similarly to the knockdown of LYAR. Thus, our data emphasize the fact that other genes besides Oct4 and Nanog are uniquely required for ESC self-renewal and differentiation and demonstrate that LYAR functions to control the stability of nucleolin protein, which in turn is essential for maintaining the self-renewal of ESCs.

Activation of paternally expressed imprinted genes in newly derived germline-competent mouse parthenogenetic embryonic stem cell lines.

Parthenogenetic embryonic stem (pES) cells provide a valuable in vitro model system for studying the molecular mechanisms that underlie genomic imprinting. However, the pluripotency of pES cells and the expression profiles of paternally expressed imprinted genes have not been fully explored. In this study, three mouse pES cell lines were established and the differentiation potential of these cells in extended culture was evaluated. The undifferentiated cells had a normal karyotype and homozygous genome, and expressed ES-cell-specific molecular markers. The cells remained undifferentiated after more than 50 passages and exhibited pluripotent differentiation capacity. All three lines of the established ES cells produced teratomas; two lines of ES cells produced chimeras and germline transmission. Furthermore, activation of the paternally expressed imprinted genes Snrpn, U2af1-rs1, Peg3, Impact, Zfp127, Dlk1 and Mest in these cells was detected. Some paternally expressed imprinted genes were found to be expressed in the blastocyst stage of parthenogenetically activated embryos in vitro and their expression level increased with extended pES cell culture. Furthermore, our data show that the activation of these paternally expressed imprinted genes in pES cells was associated with a change in the methylation of the related differentially methylated regions. These findings provide direct evidence for the pluripotency of pES cells and demonstrate the association between the DNA methylation pattern and the activation of paternally expressed imprinted genes in pES cells. Thus, the established ES cell lines provide a valuable model for studying epigenetic regulation in mammalian development.