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Abstract Background Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement. Results Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed. Conclusion Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions.

Abstract Background We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons. HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII). Results A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7–10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons. Conclusion This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.

The emergence of brain organoids has revolutionized our understanding of neurodevelopment and neurological diseases by providing an in vitro model system that recapitulates key aspects of human brain development. However, conventional organoid protocols often overlook the role of microglia, the resident immune cells of the central nervous system. Microglia dysfunction is implicated in various neurological disorders, highlighting the need for their inclusion in organoid models. Here, we present a novel method for generating neuroimmune assembloids using human-induced pluripotent stem cell (iPSC)-derived cortical organoids and microglia. Building upon our previous work generating myelinating cortical organoids, we extend our methodology to include the integration of microglia, ensuring their long-term survival and maturation within the organoids. We describe two integration methods: one involving direct addition of microglia progenitors to the organoids and an alternative approach where microglia and dissociated neuronal progenitors are aggregated together in a defined ratio. To facilitate downstream analysis, we also describe a dissociation protocol for single-cell RNA sequencing (scRNA-seq) and provide guidance on fixation, cryosectioning, and immunostaining of assembloid structures. Overall, our protocol provides a comprehensive framework for generating neuroimmune assembloids, offering researchers a valuable tool for studying the interactions between neural cell types and immune cells in the context of neurological diseases., (© 2024. Springer Science+Business Media, LLC.)

Species of Phytophthora , Phytopythium , and Pythium affect soybean seed and seedlings each year, primarily through reduced plant populations and yield. Oxathiapiprolin is effective at managing several foliar diseases caused by some oomycetes. The objectives of these studies were to evaluate oxathiapiprolin in a discriminatory dose assay in vitro; evaluate oxathiapiprolin as a soybean seed treatment on a moderately susceptible cultivar in 10 environments; compare the impact of seed treatment on plant populations and yields in environments with low and high precipitation; and compare a seed treatment mixture on cultivars with different levels of resistance in four environments. There was no reduction in growth in vitro among 13 species of Pythium at 0.1 µg ml -1 . Soybean seed treated with the base fungicide plus oxathiapiprolin (12 and 24 µg a.i. seed -1 ) alone, oxathiapiprolin (12 µg a.i. seed -1 ) plus mefenoxam (6 µg a.i. seed -1 ), or oxathiapiprolin (24 µg a.i. seed -1 ) plus ethaboxam (12.1 µg a.i. seed -1 ) had greater yields in environments that received ≥50 mm of precipitation within 14 days after planting compared with those that received less. Early plant population and yield were significantly higher for seed treated with oxathiapiprolin (24 µg a.i. seed -1 ) + metalaxyl (13.2 µg a.i. seed -1 ) compared with nontreated for six of seven cultivars in at least one of four environments. Oxathiapiprolin combined with another Oomycota fungicide applied to seed has the potential to be used to protect soybean plant establishment and yield in regions prone to poor drainage after high levels of precipitation.

We have generated a controlled and manipulable resource that captures genetic risk for Alzheimer's disease: iPSC lines from 53 individuals coupled with RNA and proteomic profiling of both iPSC-derived neurons and brain tissue of the same individuals. Data collected for each person include genome sequencing, longitudinal cognitive scores, and quantitative neuropathology. The utility of this resource is exemplified here by analyses of neurons derived from these lines, revealing significant associations between specific Aβ and tau species and the levels of plaque and tangle deposition in the brain and, more importantly, with the trajectory of cognitive decline. Proteins and networks are identified that are associated with AD phenotypes in iPSC neurons, and relevant associations are validated in brain. The data presented establish this iPSC collection as a resource for investigating person-specific processes in the brain that can aid in identifying and validating molecular pathways underlying AD., Competing Interests: Declaration of interests D.J.S. is a director and consultant for Prothena Biosciences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We have reported previously that retinal pigment epithelium (RPE) differentiated from induced pluripotent stem cells (iPSC) generated from fibroblasts of patients with age-related macular degeneration (AMD) exhibit a retinal degenerative disease phenotype and a distinct transcriptome compared to age-matched controls. Since the genetic composition of the iPSC and RPE are inherited from fibroblasts, we investigated whether differential behavior was present in the parental fibroblasts and iPSC prior to differentiation of the cell lines into RPE. Principal component analyses revealed significant overlap (essentially no differences) in the transcriptome of fibroblasts between AMD and controls. After reprogramming, there was no significant difference in the transcriptome of iPSC generated from AMD versus normal donors. In contrast, the transcriptome of RPE derived from iPSC segregated into two distinct clusters of AMD-derived cells versus controls. Interestingly, mitochondrial dysfunction in AMD-derived RPE was evident after approximately two months in culture. Moreover, these differences in mitochondrial dysfunction were not evident in the parental fibroblasts and iPSC. This study demonstrates an altered transcriptome and impaired mitochondrial function in RPE derived from AMD patients versus controls, and demonstrates these differences are not present in the original fibroblasts or iPSC. These results suggest that pathology in AMD is triggered upon differentiation of parent cells into RPE. More study of this phenomenon could advance the current understandings of the etiology of AMD and the development of novel therapeutic targets., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

To identify the molecular mechanisms and novel therapeutic targets of late-onset Alzheimer's Disease (LOAD), we performed an integrative network analysis of multi-omics profiling of four cortical areas across 364 donors with varying cognitive and neuropathological phenotypes. Our analyses revealed thousands of molecular changes and uncovered neuronal gene subnetworks as the most dysregulated in LOAD. ATP6V1A was identified as a key regulator of a top-ranked neuronal subnetwork, and its role in disease-related processes was evaluated through CRISPR-based manipulation in human induced pluripotent stem cell-derived neurons and RNAi-based knockdown in Drosophila models. Neuronal impairment and neurodegeneration caused by ATP6V1A deficit were improved by a repositioned compound, NCH-51. This study provides not only a global landscape but also detailed signaling circuits of complex molecular interactions in key brain regions affected by LOAD, and the resulting network models will serve as a blueprint for developing next-generation therapeutic agents against LOAD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD), the most common form of dementia, is recognized as a heterogeneous disease with diverse pathophysiologic mechanisms. In this study, we interrogate the molecular heterogeneity of AD by analyzing 1543 transcriptomes across five brain regions in two AD cohorts using an integrative network approach. We identify three major molecular subtypes of AD corresponding to different combinations of multiple dysregulated pathways, such as susceptibility to tau-mediated neurodegeneration, amyloid-β neuroinflammation, synaptic signaling, immune activity, mitochondria organization, and myelination. Multiscale network analysis reveals subtype-specific drivers such as GABRB2 , LRP10 , MSN , PLP1 , and ATP6V1A We further demonstrate that variations between existing AD mouse models recapitulate a certain degree of subtype heterogeneity, which may partially explain why a vast majority of drugs that succeeded in specific mouse models do not align with generalized human trials across all AD subtypes. Therefore, subtyping patients with AD is a critical step toward precision medicine for this devastating disease., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Modeling age-related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch's membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch's membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age-matched (normal) controls. RPE derived from iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell attachment and viability was tested on nitrite-modified ECM, a typical modification of aged Bruch's membrane. DNA microarrays with hierarchical clustering and analysis of mitochondrial function were used to elucidate possible mechanisms for the observed phenotypes. Differentiated RPE displayed cell-specific morphology and markers. The TER and phagocytic capacity were similar among iPSC-derived RPE cultures. However, distinct clusters were found for the transcriptomes of AMD and control iPSC-derived RPE. AMD-derived iPSC-RPE downregulated genes responsible for metabolic-related pathways and cell attachment. AMD-derived iPSC-RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite-modified ECM. Cells that did attach induced the expression of complement genes. Despite reprogramming, iPSC derived from AMD patients yielded RPE with a transcriptome that is distinct from that of age-matched controls. When challenged with an AMD-like modification of Bruch's membrane, AMD-derived iPSC-RPE activated the complement immune system., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Adult neurogenesis defects have been demonstrated in the brains of Alzheimer's disease (AD) patients. The neurogenesis impairment is an early critical event in the course of familiar AD (FAD) associated with neuronal loss. It was suggested that neurologic dysfunction in AD may be caused by impaired functioning of hippocampal neural stem cells (NSCs). Multiple metabolic and structural abnormalities in neural mitochondria have long been suspected to play a critical role in AD pathophysiology. We hypothesize that the cause of such abnormalities could be defective elimination of damaged mitochondria. In the present study, we evaluated mitophagy efficacy in a cellular AD model, hiPSC-derived NSCs harboring the FAD-associated PS1 M146L mutation. We found several mitochondrial respiratory chain defects such as lower expression levels of cytochrome c oxidase (complex IV), cytochrome c reductase (complex III), succinate dehydrogenase (complex II), NADH:CoQ reductase (complex I), and also ATP synthase (complex V), most of which had been previously associated with AD. The mitochondrial network morphology and abundance in these cells was aberrant. This was associated with a marked mitophagy failure stemming from autophagy induction blockage, and deregulation of the expression of proteins involved in mitochondrial dynamics. We show that treating these cells with autophagy-stimulating drug bexarotene restored autophagy and compensated mitochondrial anomalies in PS1 M146L NSCs, by enhancing the clearance of mitochondria. Our data support the hypothesis that pharmacologically induced mitophagy enhancement is a relevant and novel therapeutic strategy for the treatment of AD.

Zika virus (ZIKV) infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the underlying mechanisms remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, at pre- and peri-implantation, because most current ZIKV pregnancy studies have focused on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be infected with ZIKV, and propagate virus causing neural progenitor cell death. These findings are corroborated by the dose-dependent nature of ZIKV susceptibility of hESC-derived trophectoderm cells. Single blastocyst RNA-seq reveals key transcriptional changes upon ZIKV infection, including nervous system development, prior to commitment to the neural lineage. The pregnancy rate of mice is >50% lower in pre-implantation infection than infection at E4.5, demonstrating that pre-implantation ZIKV infection leads to miscarriage. Cumulatively, these data elucidate a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.

Dysfunction and eventual loss of retinal pigment epithelial (RPE) cells is a hallmark of atrophic age-related macular degeneration (AMD), and linked to oxidative and nitrosative damage. Herein, we use a high-throughput screen (HTS) to identify compounds that protect human RPE cells from oxidative stress-induced cell death and elucidate the possible mechanism of action. HTS was used to identify compounds that protect RPE cells from oxidative damage. We tested the identified compound(s) in models of RPE stress, including tert-butyl hydroperoxide (TBHP) exposure, ultraviolet-B (UV-B)-mediated light damage and nitrosative stress to the basement membrane using ARPE-19 cells, primary human RPE cells and induced-pluripotent stem cell (iPSC)-derived RPE cells from patients with AMD. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect gene expression of oxidative stress- and apoptosis-related genes and mitochondrial function was measured using a Seahorse XF96 analyzer to elucidate possible mechanisms of action. Five thousand and sixty-five compounds were screened, and of these, 12 compounds were active based on their ability to improve cell viability after exposure to TBHP. After chemical structure review, we identified ciclopirox olamine as a potent inhibitor of oxidative damage to RPE cells. Ciclopirox olamine increased cell viability in ARPE-19 cells treated with TBHP, UV-B light or on nitrite-modified extracellular matrix (ECM) by 1.68-fold, 1.54-fold and 4.3-fold, respectively (p < 0.01). Treatment with TBHP altered expression of genes related to oxidative stress and apoptosis, which was reversed by pretreatment with ciclopirox olamine. Ciclopirox olamine improved mitochondrial function in TBHP-exposed ARPE-19 cells and iPSC-derived RPE cells. Ciclopirox olamine protected primary human RPE cells and iPSC-derived RPE cells from the oxidative stress or damaged basement membrane. HTS of bioactive Food and Drug Administration (FDA)-approved libraries and follow-up studies can be used to identify small molecules (including ciclopirox olamine) that protect RPE cells exposed to various stressors associated with disease progression of AMD. This strategy can be used to identify potential compounds for treatment and prevention of AMD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Use of clinical-grade human induced pluripotent stem cell (iPSC) lines as a starting material for the generation of cellular therapeutics requires demonstration of comparability of lines derived from different individuals and in different facilities. This requires agreement on the critical quality attributes of such lines and the assays that should be used. Working from established recommendations and guidance from the International Stem Cell Banking Initiative for human embryonic stem cell banking, and concentrating on those issues more relevant to iPSCs, a series of consensus workshops has made initial recommendations on the minimum dataset required to consider an iPSC line of clinical grade, which are outlined in this report. Continued evolution of this field will likely lead to revision of these guidelines on a regular basis.

Background: Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD., Methods: We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aβ and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation., Results: We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aβ42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin., Conclusions: Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aβ42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD.

The human embryonic stem cell line NYSCFe002-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe002-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free. This line is registered as NYSCF101 on the NIH Registry., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The human embryonic stem cell line NYSCFe001-A was derived from a day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe001-A line, registered as NYSCF100 on the NIH registry, presents normal karyotype, is mycoplasma free, expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aβ, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aβ plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aβ and the responsiveness of TAU to Aβ are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The human embryonic stem cell line NYSCFe003-A was derived from a day 5 to day 6 blastocyst in feeder-free and antibiotic free conditions. The blastocyst was voluntarily donated for research as surplus after in vitro fertilization treatment following informed consent. The NYSCFe003-A line expresses all the pluripotency markers and has the potential to differentiate into all three germ layers in vitro. The line presents normal karyotype and is mycoplasma free., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aβ42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology.

Familial Alzheimer's disease (FAD) is clearly related with the accumulation of amyloid-beta (Aβ) and its deleterious effect on mitochondrial function is well established. Anomalies in autophagy have also been described in these patients. In the present work, functional analyses have been performed to study mitochondrial recycling process in patient-derived fibroblasts and neurons from induced pluripotent stem cells harboring the presenilin 1 mutation A246E. Mitophagy impairment was observed due to a diminished autophagy degradation phase associated with lysosomal anomalies, thus causing the accumulation of dysfunctional mitochondria labeled by Parkin RBR E3 ubiquitin protein ligase (PARK2). The failure of mitochondrial recycling by autophagy was enhanced in the patient-derived neuronal model. Our previous studies have demonstrated similar mitophagy impairment in sporadic Alzheimer's disease (AD); therefore, our data indicate that mitophagy deficiency should be considered a common nexus between familial and sporadic cases of the disease.

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca 2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical, physiological, cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular, it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species, including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust, allowing for morphological visualization, activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species, thus opening the possibility to study GABAergic function in virtually any vertebrate species.

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.

Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However, dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here, we show that SMAD7, a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling, is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components, and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues, pluripotent cells simply revert to a program of neural conversion. Hence, the "primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians, suggesting an evolutionarily conserved mechanism., (Copyright © 2012 AlphaMed Press.)

The exchange of the oocyte's genome with the genome of a somatic cell, followed by the derivation of pluripotent stem cells, could enable the generation of specific cells affected in degenerative human diseases. Such cells, carrying the patient's genome, might be useful for cell replacement. Here we report that the development of human oocytes after genome exchange arrests at late cleavage stages in association with transcriptional abnormalities. In contrast, if the oocyte genome is not removed and the somatic cell genome is merely added, the resultant triploid cells develop to the blastocyst stage. Stem cell lines derived from these blastocysts differentiate into cell types of all three germ layers, and a pluripotent gene expression program is established on the genome derived from the somatic cell. This result demonstrates the feasibility of reprogramming human cells using oocytes and identifies removal of the oocyte genome as the primary cause of developmental failure after genome exchange.

Two multilaboratory investigations were conducted by SUSTAIN to assess variability in the measurement of vitamin A, the marker used to verify levels of vitamin premix addition to enriched/fortified food aid products, including the widely distributed corn-soy blend (CSB). CSB specifications identify AACC Approved Method 86-06 or equivalent methods for vitamin A analysis, however there is no requirement to demonstrate equivalency. CSB samples with known and blinded levels of vitamin A and a reference standard were analyzed by 16 laboratories using their respective methods. Calculated coefficients of variation across all laboratories and methods for unknown samples and reference standard were 35 and 7.1%, respectively, suggesting the largest source of variation is the vitamin extraction procedure. Laboratories generally overestimated low levels and underestimated high levels of vitamin A within the range of 6000 and 16 000 IU/lb. Only two laboratories demonstrated excellent internal precision (+/- 300 IU vitamin A/lb) and reported values within 95% confidence interval for all blinded samples. Results of this study have implications both for quality control in food aid products (due to the use of vitamin A as a marker) and for regulatory oversight of vitamin A content in commercial food products.

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, alpha fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.

Background: Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement., Results: Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed., Conclusion: Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions.

Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(-)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(-), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain.

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded. After continuous proliferation in vitro for more than 12 months, these ES cell lines maintained their developmental potential to form trophoblast and somatic cells, including cardiac muscle and neuronal tissue.

A rearranged immunoglobulin heavy chain (IgH) transgene-encoded protein is expressed in macrophage lineage cells, in addition to B and T lineages, in transgenic mouse bone marrow. Peripheral macrophages also express transgenic IgH protein. Mature T cells express lower levels than immature thymocytes. Almost all B220+ cells in the bone marrow express transgenic IgH protein, and this early expression in the B lineage is accompanied by a reduction of cell frequency even in the early B220+ CD43+ BP-1- stages, although it is more prominent in BP-1+ pre-B cells. Thus, an IgH transgene can be expressed not only in lymphoid but also in myeloid cells, although its developmental effects are restricted to the B cell lineage.