Systematic optimization of human pluripotent stem cells media using Design of Experiments.

Human pluripotent stem cells (hPSC) are used to study the early stages of human development in vitro and, increasingly due to somatic cell reprogramming, cellular and molecular mechanisms of disease. Cell culture medium is a critical factor for hPSC to maintain pluripotency and self-renewal. Numerous defined culture media have been empirically developed but never systematically optimized for culturing hPSC. We applied design of experiments (DOE), a powerful statistical tool, to improve the medium formulation for hPSC. Using pluripotency and cell growth as read-outs, we determined the optimal concentration of both basic fibroblast growth factor (bFGF) and neuregulin-1 beta 1 (NRG1β1). The resulting formulation, named iDEAL, improved the maintenance and passage of hPSC in both normal and stressful conditions, and affected trimethylated histone 3 lysine 27 (H3K27me3) epigenetic status after genetic reprogramming. It also enhances efficient hPSC plating as single cells. Altogether, iDEAL potentially allows scalable and controllable hPSC culture routine in translational research. Our DOE strategy could also be applied to hPSC differentiation protocols, which often require numerous and complex cell culture media

PEDV and PDCoV Pathogenesis: The Interplay Between Host Innate Immune Responses and Porcine Enteric Coronaviruses

Enteropathogenic porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV), members of the coronavirus family, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. These two viruses pose significant economic and public health burdens, even as both continue to emerge and reemerge worldwide. The ability to evade, circumvent or subvert the host’s first line of defense, namely the innate immune system, is the key determinant for pathogen virulence, survival, and the establishment of successful infection. Unfortunately, we have only started to unravel the underlying viral mechanisms used to manipulate host innate immune responses. In this review, we gather current knowledge concerning the interplay between these viruses and components of host innate immunity, focusing on type I interferon induction and signaling in particular, and the mechanisms by which virus-encoded gene products antagonize and subvert host innate immune responses. Finally, we provide some perspectives on the advantages gained from a better understanding of host-pathogen interactions. This includes their implications for the future development of PEDV and PDCoV vaccines and how we can further our knowledge of the molecular mechanisms underlying virus pathogenesis, virulence, and host coevolution

Revealing the neuronal phenotypes of Williams syndrome in a dish

The invention of induced pluripotent stem cells (iPSCs) like never before has opened novel opportunity to study diseases in relevant cell types. Within less than 10 years, a variety of diseases such as Rett syndrome, long QT syndrome and spinal muscular atrophy, have been successfully modeled. In this study, Williams syndrome (WS), a rare genetic neurodevelopmental disorder, that is caused by hemizygous deletion of 25 genes on chromosome 7, is of interest because of its unique cognitive and social profiles which are opposite to those of autism spectrum disorders. Despite the extensive studies on WS hypersociability, little is known about haploinsufficiency effect of those genes on molecular and cellular phenotypes in neuronal level due to the lack of relevant human cellular model. Using reprogramming approach, we found that WS iPSC-derived neural progenitor cells has increased apoptosis and therefore increased doubling time. The phenotypes could be rescued by complementation of frizzled9, one of 25 genes typically deleted in WS. Moreover, WS iPSC-derived CTIP2 positive (cortical layer V /VI) pyramidal neurons exhibit morphology alterations including longer total dendrites and increasing dendritic spine number per neuron, which are similarly observed in postmortem layer V/VI neurons. In addition, WS iPSC- derived neurons show an increase in calcium transient frequency likely due to an increase in number of dendritic spines. While we demonstrated that this promising iPSC system could be used to model such multigenic neurodevelopmental disorder, the technology itself still needs to be optimized. Culture medium, one of the most important factors required for maintenance of self-renewal and pluripotency of iPSC, has been developed empirically and claimed as an optimal formulation. We applied design of experiments, a statistic and mathematic tool widely used in engineering and chemical research, to systematically determine the most efficient concentration of final 2 components (basic fibroblast growth factor and neuregulin1) in the culture medium. Compared to commercial medium (mTeSR1), our optimized formulation (iDEAL) for iPSC decreases apoptotic cells, improves pluripotency maintenance, supports no-weekend medium change routine, enhances survival of single-cell passaging as well as reactivates inactive X chromosome upon reprogramming, providing more efficient and suitable condition for iPSC

Revealing the neuronal phenotypes of Williams syndrome in a dish

The invention of induced pluripotent stem cells (iPSCs) like never before has opened novel opportunity to study diseases in relevant cell types. Within less than 10 years, a variety of diseases such as Rett syndrome, long QT syndrome and spinal muscular atrophy, have been successfully modeled. In this study, Williams syndrome (WS), a rare genetic neurodevelopmental disorder, that is caused by hemizygous deletion of 25 genes on chromosome 7, is of interest because of its unique cognitive and social profiles which are opposite to those of autism spectrum disorders. Despite the extensive studies on WS hypersociability, little is known about haploinsufficiency effect of those genes on molecular and cellular phenotypes in neuronal level due to the lack of relevant human cellular model. Using reprogramming approach, we found that WS iPSC-derived neural progenitor cells has increased apoptosis and therefore increased doubling time. The phenotypes could be rescued by complementation of frizzled9, one of 25 genes typically deleted in WS. Moreover, WS iPSC-derived CTIP2 positive (cortical layer V /VI) pyramidal neurons exhibit morphology alterations including longer total dendrites and increasing dendritic spine number per neuron, which are similarly observed in postmortem layer V/VI neurons. In addition, WS iPSC- derived neurons show an increase in calcium transient frequency likely due to an increase in number of dendritic spines. While we demonstrated that this promising iPSC system could be used to model such multigenic neurodevelopmental disorder, the technology itself still needs to be optimized. Culture medium, one of the most important factors required for maintenance of self-renewal and pluripotency of iPSC, has been developed empirically and claimed as an optimal formulation. We applied design of experiments, a statistic and mathematic tool widely used in engineering and chemical research, to systematically determine the most efficient concentration of final 2 components (basic fibroblast growth factor and neuregulin1) in the culture medium. Compared to commercial medium (mTeSR1), our optimized formulation (iDEAL) for iPSC decreases apoptotic cells, improves pluripotency maintenance, supports no-weekend medium change routine, enhances survival of single-cell passaging as well as reactivates inactive X chromosome upon reprogramming, providing more efficient and suitable condition for iPSC

Modeling Williams syndrome with induced pluripotent stem cells

The development of induced pluripotent stem cells (iPSCs) like never before has opened novel opportunity to study diseases in relevant cell types. In our recent study, Williams syndrome (WS), a rare genetic neurodevelopmental disorder, that is caused by hemizygous deletion of 25-28 genes on chromosome 7, is of interest because of its unique cognitive and social profiles. Little is known about haploinsufficiency effect of those deleted genes on molecular and cellular phenotypes at the neural level due to the lack of relevant human cellular model. Using the cellular reprogramming approach, we reported that WS iPSC-derived neural progenitor cells (NPCs) has increased apoptosis and therefore increased doubling time, which could be rescued by complementation of frizzled 9, one of the genes typically deleted in WS. Moreover, WS iPSC-derived CTIP2-positive pyramidal neurons exhibit morphologic alterations including longer total dendrites and increasing dendritic spine number. In addition, WS iPSC-derived neurons show an increase in calcium transient frequency and synchronized activity likely due to increased number of dendritic spines and synapses. Our work integrated cross-level data from genetics to behavior of WS individuals and revealed altered cellular phenotypes in WS human NPCs and neurons that could be validated in other model systems such as magnetic resonance imaging (MRI) in live subjects and postmortem brain tissues

The contribution of GTF2I haploinsufficiency to Williams syndrome

Williams syndrome (WS) is a neurodevelopmental disorder involving hemideletion of as many as 26-28 genes, resulting in a constellation of unique physical, cognitive and behavior phenotypes. The haploinsufficiency effect of each gene has been studied and correlated with phenotype(s) using several models including WS subjects, animal models, and peripheral cell lines. However, links for most of the genes to WS phenotypes remains unclear. Among those genes, general transcription factor 2I (GTF2I) is of particular interest as its haploinsufficiency is possibly associated with hypersociability in WS. Here, we describe studies of atypical WS cases as well as mouse models focusing on GTF2I that support a role for this protein in the neurocognitive and behavioral profiles of WS individuals. We also review collective studies on diverse molecular functions of GTF2I that may provide mechanistic explanation for phenotypes recently reported in our relevant cellular model, namely WS induced pluripotent stem cell (iPSC)-derived neurons. Finally, in light of the progress in gene-manipulating approaches, we suggest their uses in revealing the neural functions of GTF2I in the context of WS

Micro RNA detection in long-term fixed tissue of cortical glutamatergic pyramidal neurons after targeted laser-capture neuroanatomical microdissection

BackgroundFormalin fixation (FF) is the standard and most common method for preserving postmortem brain tissue. FF stabilizes cellular morphology and tissue architecture, and can be used to study the distinct morphologic and genetic signatures of different cell types. Although the procedure involved in FF degrades messenger RNA over time, an alternative approach is to use small RNAs (sRNAs) for genetic analysis associated with cell morphology. Although genetic analysis is carried out on fresh or frozen tissue, there is limited availability or impossibility on targeting specific cell populations, respectively.New methodThe goal of this study is to detect miRNA and other classes of sRNA stored in formalin or in paraffin embedded for over decades. Two brain samples, one formed by a mixed population of cortical and subcortical cells, and one formed by pyramidal shaped cells collected by laser-capture microdissection, were subjected to sRNA sequencing.ResultsPerforming bioinformatics analysis over the sequenced sRNA from brain tissue, we detected several classes of sRNA, such as miRNAs that play key roles in brain neurodevelopmental and maintenance pathways, and hsa-mir-155 expression in neurons. Comparison with existing method: Our method is the first to combine the approaches for: laser-capture of pyramidal neurons from long-term formalin-fixed brain; extract sRNA from laser-captured pyramidal neurons; apply a suite of bioinformatics tools to detect miRNA and other classes of sRNAs on sequenced samples having high levels of RNA degradation.ConclusionThis is the first study to show that sRNA can be rescued from laser-captured FF pyramidal neurons