Alice Pébay

Efficacy and dynamics of self-targeting CRISPR/Cas constructs for gene editing in the retina

Publisher: Cold Spring Harbor Laboratory

Date: 05-01-2018

DOI: 10.1101/243683

Abstract: Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of in vivo genome editing including the anticipatory treatment of monogenic retinal diseases. We previously reported the utility of adeno-associated virus (AAV)-mediated CRISPR/Cas genome editing in the retina however, with this type of viral delivery system, active endonucleases will remain in the retina for an extended period, making genotoxicity a significant consideration in clinical applications. To address this issue, we have designed a self-destructing “kamikaze” CRISPR/Cas system that disrupts the Cas enzyme itself following expression. Four guide RNAs (sgRNAs) were designed to target Streptococcus pyogenes Cas9 (SpCas9), after in situ validation, the selected sgRNAs were cloned into a dual AAV vector. One construct was used to deliver SpCas9 and the other delivered sgRNAs directed against SpCas9 and the target locus (yellow fluorescent protein, YFP), in the presence of mCherry. Both constructs were packaged into AAV2 vector and intravitreally administered in C57BL/6 and Thy1-YFP transgenic mice. After 8 weeks the expression of SpCas9, the efficacy of YFP gene disruption was quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated-YFP/SpCas9 targeting CRISPR/Cas compared to those treated with YFP targeting CRISPR/Cas alone. We also show that AAV2-mediated delivery of YFP/SpCas9 targeting CRISPR/Cas significantly reduced the number of YFP fluorescent cells among mCherry-expressing cells (~85.5% reduction compared to LacZ/SpCas9 targeting CRISPR/Cas) in transfected retina of Thy1-YFP transgenic mice. In conclusion, our data suggest that a self-destructive “kamikaze” CRISPR/Cas system can be used as a robust tool for refined genome editing in the retina, without compromising on-target efficiency.

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease patient-derived cortical neurons

Publisher: The Company of Biologists

Date: 12-2022

DOI: 10.1242/DMM.049651

Abstract: CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3, with most affected in iduals carrying at least one allele with a 966 bp deletion. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). We differentiated these isogenic iPSCs, and iPSCs from an unrelated healthy control donor, to neurons and identified disease-related changes relating to protein synthesis, trafficking and degradation, and in neuronal activity, which were not apparent in CLN3-corrected or healthy control neurons. CLN3 neurons showed numerous membrane-bound vacuoles containing erse storage material and hyperglycosylation of the lysosomal LAMP1 protein. Proteomic analysis showed increase in lysosomal-related proteins and many ribosomal subunit proteins in CLN3 neurons, accompanied by downregulation of proteins related to axon guidance and endocytosis. CLN3 neurons also had lower electrophysical activity as recorded using microelectrode arrays. These data implicate inter-related pathways in protein homeostasis and neurite arborization as contributing to CLN3 disease, and which could be potential targets for therapy.

G-protein Coupled Receptors in Stem Cell Self-Renewal and Differentiation

Publisher: Springer Science and Business Media LLC

Date: 13-07-2010

DOI: 10.1007/S12015-010-9167-9

Abstract: Stem cells have great potential for understanding early development, treating human disease, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidence now exists demonstrating the potent effects of various G-protein coupled receptor (GPCR) ligands on the biology of stem cells. This review aims to give an overview of the current knowledge of the regulation of embryonic and somatic stem cell maintenance and differentiation by GPCR ligands.

A new design to enhance the conductive and convective heat transfer of latent heat thermal energy storage units

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.APPLTHERMALENG.2022.118955

Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids

Publisher: MDPI AG

Date: 30-12-2021

DOI: 10.3390/CELLS11010124

Abstract: Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-cl electrophysiological recordings show that the microglia-like population supported the emergence of more mature and ersified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

Participant understanding and recall of informed consent for induced pluripotent stem cell biobanking

Publisher: Springer Science and Business Media LLC

Date: 14-06-2016

DOI: 10.1007/S10561-016-9563-8

Abstract: The ability to generate human induced pluripotent stem cells (iPSCs) has opened new avenues for human disease modelling and therapy. The aim of our study was to determine research participants' understanding of the information given when donating skin biopsies for the generation of patient-specific iPSCs. A customised 35-item questionnaire based on previous iPSC consent guidelines was sent to participants who had previously donated s les for iPSC research. The questionnaire asked pertinent demographic details, participants' motivation to take part in iPSC research and their attitudes towards related ethical issues. 234 participants were contacted with 141 (60.3 %) complete responses received. The median duration between recruitment and follow-up questioning was 313 days (range 10-573 days). The majority of participants (n = 129, 91.5 %) believed they understood what a stem cell was however, only 22 (16.1 %) correctly answered questions related to basic stem cell properties. We found no statistically significant difference in responses from participants with different levels of education, or those with a health sciences background. The poor understanding amongst participants of iPSC research is unlikely to be unique to our study and may impact future research if not improved. As such, there is a need to develop an easily understood yet comprehensive consent process to ensure ongoing ethical progress of iPSC biobanking.

Human pluripotent stem cells for the modelling of diseases of the retina and optic nerve: toward a retina in a dish

Publisher: Elsevier BV

Date: 10-2019

DOI: 10.1016/J.COPH.2019.09.003

Abstract: Human pluripotent stem cells can be differentiated into specific, relevant cell types of interest including the cells of the retina and optic nerve. These cells can then be used to study fundamental biology as well as disease modelling and subsequent screening of potential treatments. Many models of differentiation and modelling have relied on two-dimensional monocultures of specific cell types, which are not representative of the complexity of the human retina and optic nerve. Hence, more complex models of the human retina and optic nerve are required. Three-dimensional organoids and emerging cell culture methods may provide more physiologically relevant models to study developmental biology and pathology of the retina and optic nerve.

AAV2-mediated gene therapy for Bietti crystalline dystrophy provides functional CYP4V2 in multiple relevant cell models.

Publisher: Springer Science and Business Media LLC

Date: 09-06-2022

DOI: 10.1038/S41598-022-12210-8

Abstract: Bietti crystalline dystrophy (BCD) is an inherited retinal disease (IRD) caused by mutations in the CYP4V2 gene. It is a relatively common cause of IRD in east Asia. A number of features of this disease make it highly amenable to gene supplementation therapy. This study aims to validate a series of essential precursor in vitro experiments prior to developing a clinical gene therapy for BCD. We demonstrated that HEK293, ARPE19, and patient induced pluripotent stem cell (iPSC)-derived RPE cells transduced with AAV2 vectors encoding codon optimization of CYP4V2 (AAV2.coCYP4V2) resulted in elevated protein expression levels of CYP4V2 compared to those transduced with AAV2 vectors encoding wild type CYP4V2 (AAV2.wtCYP4V2), as assessed by immunocytochemistry and western blot. Similarly, we observed significantly increased CYP4V2 enzyme activity in cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. We also showed CYP4V2 expression in human RPE/choroid explants transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2. These preclinical data support the further development of a gene supplementation therapy for a currently untreatable blinding condition—BCD. Codon-optimized CYP4V2 transgene was superior to wild type in terms of protein expression and enzyme activity. Ex vivo culture of human RPE cells provided an effective approach to test AAV-mediated transgene delivery.

Non-classical Signalling Mechanisms in Stem Cells

Publisher: InTech

Date: 26-04-2011

DOI: 10.5772/13820

Functionalised Collagen I Membranes as a Bruch's Membrane Mimetic for Outer Retinal <i>In Vitro</i> Models

Publisher: Elsevier BV

Date: 2023

DOI: 10.2139/SSRN.4498484

Microglia Orchestrate Neuronal Activity in Brain Organoids

Publisher: Elsevier BV

Date: 2021

DOI: 10.2139/SSRN.3773789

Microglial amyloid beta clearance is driven by PIEZO1 channels

Publisher: Cold Spring Harbor Laboratory

Date: 19-03-2022

DOI: 10.1101/2022.03.18.484831

Abstract: Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer’s disease (AD), toxic amyloid beta (Aβ) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aβ. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia have been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC) -derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo , 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aβ pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. We show that PIEZO1 orchestrates Aβ clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aβ inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aβ clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single cell datasets. These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aβ burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

Cell type-specific manifestations of cortical thickness heterogeneity in schizophrenia

Publisher: Springer Science and Business Media LLC

Date: 10-02-2022

DOI: 10.1038/S41380-022-01460-7

Abstract: Brain morphology differs markedly between in iduals with schizophrenia, but the cellular and genetic basis of this heterogeneity is poorly understood. Here, we sought to determine whether cortical thickness (CTh) heterogeneity in schizophrenia relates to interregional variation in distinct neural cell types, as inferred from established gene expression data and person-specific genomic variation. This study comprised 1849 participants in total, including a discovery (140 cases and 1267 controls) and a validation cohort (335 cases and 185 controls). To characterize CTh heterogeneity, normative ranges were established for 34 cortical regions and the extent of deviation from these ranges was measured for each in idual with schizophrenia. CTh deviations were explained by interregional gene expression levels of five out of seven neural cell types examined: (1) astrocytes (2) endothelial cells (3) oligodendrocyte progenitor cells (OPCs) (4) excitatory neurons and (5) inhibitory neurons. Regional alignment between CTh alterations with cell type transcriptional maps distinguished broad patient subtypes, which were validated against genomic data drawn from the same in iduals. In a predominantly neuronal/endothelial subtype (22% of patients), CTh deviations covaried with polygenic risk for schizophrenia (sczPRS) calculated specifically from genes marking neuronal and endothelial cells ( r = −0.40, p = 0.010). Whereas, in a predominantly glia/OPC subtype (43% of patients), CTh deviations covaried with sczPRS calculated from glia and OPC-linked genes ( r = −0.30, p = 0.028). This multi-scale analysis of genomic, transcriptomic, and brain phenotypic data may indicate that CTh heterogeneity in schizophrenia relates to inter-in idual variation in cell-type specific functions. Decomposing heterogeneity in relation to cortical cell types enables prioritization of schizophrenia subsets for future disease modeling efforts.

Pleiotropic effects of lysophosphatidic acid on striatal astrocytes

Publisher: Wiley

Date: 10-1999

DOI: 10.1002/(SICI)1098-1136(199910)28:1<25::AID-GLIA3>3.0.CO;2-D

Abstract: Lysophosphatidic acid (LPA) is a potent lipid mediator that is likely involved in erse functions in the brain. Several recent studies have suggested that astrocytes are important target cells for LPA. In the present study, we have identified the signal transduction pathways activated following LPA stimulation in mouse striatal astrocytes in primary culture. In cells prelabeled with myo-[3H]inositol, LPA stimulated the formation of [3H]inositol phosphates (EC50 = 0.7 microM). This effect was reproduced neither by other lysophospholipids nor by phosphatidic acid. Astrocyte pretreatment with pertussis toxin partially abolished this LPA response indicating the involvement of a Gi/Go protein. In [3H]adenine-prelabeled cells, LPA strongly inhibited the formation of [3H]cyclic AMP induced by forskolin (EC(50) = 0.3 microM) and by isoproterenol and PACAP-38. These inhibitory effects were strongly reduced by pertussis toxin treatment. Although with a lesser potency (EC50 = 5 microM), LPA also stimulated the release of [3H]arachidonic acid from [3H]arachidonic acid-prelabeled astrocytes. This latter effect was totally inhibited by mepacrine, did not involve a pertussis toxin-sensitive G protein, and was highly dependent on external calcium. LPA also stimulated the activity of both extracellular signal-regulated kinases (Erk) Erk1 and Erk2 by a mechanism involving a Gi/Go protein. Surprisingly, in contrast to that observed in fibroblasts, LPA was totally ineffective in stimulating DNA synthesis. These results provide additional evidence in favor of an important physiological role of LPA in the astrocytic functions.

A sporadic Alzheimer's blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies

Publisher: Ivyspring International Publisher

Date: 2022

DOI: 10.7150/THNO.72685

Growth Factors and the Serum-Free Culture of Human Pluripotent Stem Cells

Publisher: Elsevier

Date: 2013

DOI: 10.1016/B978-0-12-385942-6.00029-9

ALPK3-deficient cardiomyocytes generated from patient-derived induced pluripotent stem cells and mutant human embryonic stem cells display abnormal calcium handling and establish that ALPK3 deficiency underlies familial cardiomyopathy

Publisher: Oxford University Press (OUP)

Date: 22-04-2016

DOI: 10.1093/EURHEARTJ/EHW160

Abstract: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.

Use of CRISPR/Cas ribonucleoproteins for high throughput gene editing of induced pluripotent stem cells

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.YMETH.2021.02.009

Lysosomal alterations and decreased electrophysiological activity in CLN3 disease (966 bp deletion, E295K) patient-derived cortical neurons

Publisher: Cold Spring Harbor Laboratory

Date: 29-04-2022

DOI: 10.1101/2022.04.28.489465

Abstract: CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3 . Most in iduals with CLN3 disease carry at least one allele with a 966 bp deletion in CLN3 which results in the deletion of exons 7 and 8. There is a need for more physiologically relevant human cell-based CLN3 disease models to better understand the cellular changes during the disease process. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient ( CLN3 Δ 966 bp and E295K). The isogenic deletion-corrected and unedited CLN3 patient iPSCs were used for disease modeling. iPSC-derived neurons carrying this particular CLN3 mutation (CLN3 neurons) had lower functional activity as recorded using microelectrode arrays for most of the culture period. Proteomics analysis showed downregulation of proteins related to axon guidance and endocytosis at day in vitro (DIV) 14 and 42 in CLN3 neurons. This was accompanied by an increase in lysosomal-related proteins in CLN3 neurons. Western blot analysis revealed hyperglycosylation of the lysosomal marker, Lysosome Associated Membrane Protein 1 (LAMP1) in CLN3 neurons at DIV 14, 28 and 42, which was not apparent in control neurons. Ultrastructural analysis of CLN3 neurons showed numerous membrane-bound vacuoles containing erse types of storage material, ranging from curvilinear deposits, multilamellar structures to osmiophilic deposits. Our findings suggest alterations in lysosomal function and neurodevelopment involving axon guidance and synaptic transmission in CLN3-deficient neuronal derivatives, which could be potential targets for therapy.

A village in a dish model system for population-scale hiPSC studies

Publisher: Springer Science and Business Media LLC

Date: 09-06-2023

DOI: 10.1038/S41467-023-38704-1

Abstract: The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and different conditions. Human induced pluripotent stem cells are uniquely suited to study these context-dependent effects but cell lines from hundreds or thousands of in iduals are required. Village cultures, where multiple induced pluripotent stem lines are cultured and differentiated in a single dish, provide an elegant solution for scaling induced pluripotent stem experiments to the necessary s le sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned to an induced pluripotent stem line using single-cell sequencing and illustrating that the genetic, epigenetic or induced pluripotent stem line-specific effects explain a large percentage of gene expression variation for many genes. We demonstrate that village methods can effectively detect induced pluripotent stem line-specific effects, including sensitive dynamics of cell states.

Using human pluripotent stem cells to study Friedreich ataxia cardiomyopathy

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.IJCARD.2016.03.040

Abstract: Friedreich ataxia (FRDA) is the most common of the inherited ataxias. It is an autosomal recessive disease characterised by degeneration of peripheral sensory neurons, regions of the central nervous system and cardiomyopathy. FRDA is usually due to homozygosity for trinucleotide GAA repeat expansions found within first intron of the FRATAXIN (FXN) gene, which results in reduced levels of the mitochondrial protein FXN. Reduced FXN protein results in mitochondrial dysfunction and iron accumulation leading to increased oxidative stress and cell death in the nervous system and heart. Yet the precise functions of FXN and the underlying mechanisms leading to disease pathology remain elusive. This is particularly true of the cardiac aspect of FRDA, which remains largely uncharacterized at the cellular level. Here, we summarise current knowledge on experimental models in which to study FRDA cardiomyopathy, with a particular focus on the use of human pluripotent stem cells as a disease model.

Maintenance of Human Embryonic Stem Cells by Sphingosine-1-Phosphate and Platelet-Derived Growth Factor

Publisher: Springer New York

Date: 2017

DOI: 10.1007/7651_2017_4

Abstract: Human embryonic stem cells (hESCs) have historically been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in a medium supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESCs in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a simple alternative chemically defined serum-free system that is relatively inexpensive and advantageous for studying signaling pathways involved in hESC pluripotency.

Erratum: Genetic variation affects morphological retinal phenotypes extracted from UK Biobank optical coherence tomography images (PLoS Genet (2021) 17:5 (e1009497) DOI: 10.1371/journal.pgen.1009497)

Publisher: Public Library of Science (PLoS)

Date: 18-10-2021

DOI: 10.1371/JOURNAL.PGEN.1009858

Genotype-free demultiplexing of pooled single-cell RNA-seq

Publisher: Cold Spring Harbor Laboratory

Date: 07-03-2019

DOI: 10.1101/570614

Abstract: A variety of experimental and computational methods have been developed to demultiplex s les from pooled in iduals in a single-cell RNA sequencing (scRNA-Seq) experiment which either require adding information (such as hashtag barcodes) or measuring information (such as genotypes) prior to pooling. We introduce scSplit which utilises genetic differences inferred from scRNA-Seq data alone to demultiplex pooled s les. scSplit also extracts a minimal set of high confidence presence/absence genotypes in each cluster which can be used to map clusters to original s les. Using a range of simulated, merged in idual-s le as well as pooled multi-in idual scRNA-Seq datasets, we show that scSplit is highly accurate and concordant with demuxlet predictions. Furthermore, scSplit predictions are highly consistent with the known truth in cell-hashing dataset. We also show that multiplexed-scRNA-Seq can be used to reduce batch effects caused by technical biases. scSplit is ideally suited to s les for which external genome-wide genotype data cannot be obtained (for ex le non-model organisms), or for which it is impossible to obtain unmixed s les directly, such as mixtures of genetically distinct tumour cells, or mixed infections. scSplit is available at: on-xu/scSplit

A Simple Differentiation Protocol for Generation of Induced Pluripotent Stem Cell-Derived Basal Forebrain-Like Cholinergic Neurons for Alzheimer’s Disease and Frontotemporal Dementia Disease Modeling

Publisher: MDPI AG

Date: 02-09-2020

DOI: 10.3390/CELLS9092018

Abstract: The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch cl . We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

Essential Roles of Sphingosine‐1‐Phosphate and Platelet‐Derived Growth Factor in the Maintenance of Human Embryonic Stem Cells

Publisher: Oxford University Press (OUP)

Date: 11-2005

DOI: 10.1634/STEMCELLS.2004-0338

Abstract: Human embryonic stem cells (hESCs) have great potential for use in research and regenerative medicine, but very little is known about the factors that maintain these cells in the pluripotent state. We investigated the role of three major mitogenic agents present in serum--sphingosine-1-phosphate (S1P), lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF)--in maintaining hESCs. We show here that although LPA does not affect hESC growth or differentiation, coincubation of S1P and PDGF in a serum-free culture medium successfully maintains hESCs in an undifferentiated state. Our studies indicate that signaling pathways activated by tyrosine kinase receptors act synergistically with those downstream from lysophospholipid receptors to maintain hESCs in the undifferentiated state. This study is the first demonstration of a role for lysophospholipid receptor signaling in the maintenance of stem cell pluri-potentiality.

A Need for Better Understanding Is the Major Determinant for Public Perceptions of Human Gene Editing

Publisher: Mary Ann Liebert Inc

Date: 2019

DOI: 10.1089/HUM.2018.033

Abstract: The CRISPR/Cas system could provide an efficient and reliable means of editing the human genome and has the potential to revolutionize modern medicine however, rapid developments are raising complex ethical issues. There has been significant scientific debate regarding the acceptability of some applications of CRISPR/Cas, with leaders in the field highlighting the need for the lay public's views to shape expert discussion. As such, we sought to determine the factors that influence public opinion on gene editing. We created a 17-item online survey translated into 11 languages and advertised worldwide. Topic modeling was used to analyze textual responses to determine what factors influenced respondents' opinions toward human somatic or embryonic gene editing, and how this varied among respondents with differing attitudes and demographic backgrounds. A total of 3,988 free-text responses were analyzed. Respondents had a mean age of 32 (range, 11-90) years, and 37% were female. The most prevalent topics cited were Future Generations, Research, Human Editing, Children, and Health. Respondents who disagreed with gene editing for health-related purposes were more likely to cite the topic Better Understanding than those who agreed to both somatic and embryonic gene editing. Respondents from Western backgrounds more frequently discussed Future Generations, compared with participants from Eastern countries. Religious respondents did not cite the topic Religious Beliefs more frequently than did nonreligious respondents, whereas Christian respondents were more likely to cite the topic Future Generations. Our results suggest that public resistance to human somatic or embryonic gene editing does not stem from an inherent mistrust of genome modification, but rather a desire for greater understanding. Furthermore, we demonstrate that factors influencing public opinion vary greatly amongst demographic groups. It is crucial that the determinants of public attitudes toward CRISPR/Cas be well understood so that the technology does not suffer the negative public sentiment seen with previous genetic biotechnologies.

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

Publisher: Proceedings of the National Academy of Sciences

Date: 06-09-2017

DOI: 10.1073/PNAS.1710430114

Abstract: Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, pharmacological treatments in these diseases are limited due to the lack of knowledge of underlying disease mechanisms, partly because appropriate human models to study AMD and related MDs are lacking. Furthermore, in the living human eye, the entire retina acts as a functional unit, making it difficult to investigate the specific contribution of a particular retinal cell type in the disease. Here, we established human models of multiple MDs, which demonstrated similar molecular and phenotypic manifestations within these diseases. Furthermore, we showed that dysfunction of an in idual cell type, retinal pigment epithelium cells in the retina, is sufficient for the development of key pathological features in these MDs.

Regenerative Biology of the Eye

Publisher: Springer New York

Date: 2014

DOI: 10.1007/978-1-4939-0787-8

Comparison of CRISPR/Cas Endonucleases for in vivo Retinal Gene Editing

Publisher: Frontiers Media SA

Date: 10-09-2020

DOI: 10.3389/FNCEL.2020.570917

Cell and Gene Therapy for Friedreich Ataxia: Progress to Date

Publisher: Mary Ann Liebert Inc

Date: 08-2014

DOI: 10.1089/HUM.2013.180

Abstract: Neurodegenerative disorders such as Friedreich ataxia (FRDA) present significant challenges in developing effective therapeutic intervention. Current treatments aim to manage symptoms and thus improve quality of life, but none can cure, nor are proven to slow, the neurodegeneration inherent to this disease. The primary clinical features of FRDA include progressive ataxia and shortened life span, with complications of cardiomyopathy being the major cause of death. FRDA is most commonly caused by an expanded GAA trinucleotide repeat in the first intron of FXN that leads to reduced levels of frataxin, a mitochondrial protein important for iron metabolism. The GAA expansion in FRDA does not alter the coding sequence of FXN. It results in reduced production of structurally normal frataxin, and hence any increase in protein level is expected to be therapeutically beneficial. Recently, there has been increased interest in developing novel therapeutic applications like cell and/or gene therapies, and these cutting-edge applications could provide effective treatment options for FRDA. Importantly, since in iduals with FRDA produce frataxin at low levels, increased expression should not elicit an immune response. Here we review the advances to date and highlight the future potential for cell and gene therapy to treat this debilitating disease.

Extending the photoelectrocatalytic detection range of KHP by eliminating self-inhibition at TiO2 nanoparticle electrodes

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.JELECHEM.2014.12.004

S1P inhibits gap junctions in astrocytes: involvement of G_i and Rho GTPase/ROCK

Publisher: Wiley

Date: 03-2006

DOI: 10.1111/J.1460-9568.2006.04671.X

Abstract: Sphingosine-1-phosphate (S1P) is a potent and pleiotropic bioactive lysophospholipid mostly released by activated platelets that acts on its target cells through its own G protein-coupled receptors. We have previously reported that mouse striatal astrocytes expressed mRNAs for S1P1 and S1P3 receptors and proliferate in response to S1P. Here, we investigated the effect of S1P on gap junctions. We show that a short-term exposure of astrocytes to S1P causes a robust inhibition of gap junctional communication, as demonstrated by dye coupling experiments and double voltage-cl recordings of junctional currents. The inhibitory effect of S1P on dye coupling involves the activation of both Gi and Rho GTPases. Rho-associated kinase (ROCK) also plays a critical role. The capacity of S1P to activate a Rho/ROCK axis in astrocytes is demonstrated by the typical remodeling of actin cytoskeleton. Connexin43, the protein forming gap junction channels, is a target of the Gi- and Rho/ROCK-mediated signaling cascades. Indeed, as shown by Western blots and confocal immunofluorescence, its nonphosphorylated form increases following S1P treatment and this change does not occur when both cascades are disrupted. This novel effect of S1P may have an important physiopathological significance when considering the proposed roles for astrocyte gap junctions on neuronal survival.

AAV capsid bioengineering in primary human retina

Publisher: Research Square Platform LLC

Date: 12-09-2023

DOI: 10.21203/RS.3.RS-3293367/V1

Sphingosine‐1‐phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Publisher: Wiley

Date: 06-2001

DOI: 10.1046/J.1460-9568.2001.0130122336.X

Pluripotent stem cell-based models to investigate retinal pigmented epithelium function and disease

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.DDMOD.2014.02.010

Single cell eQTL analysis identifies cell type-specific genetic control of gene expression in fibroblasts and reprogrammed induced pluripotent stem cells

Publisher: Springer Science and Business Media LLC

Date: 05-03-2021

DOI: 10.1186/S13059-021-02293-3

Abstract: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) has provided a foundation for in vitro human disease modelling, drug development and population genetics studies. Gene expression plays a critical role in complex disease risk and therapeutic response. However, while the genetic background of reprogrammed cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of in idual cells which would provide significant resolution. By integrating single cell RNA-sequencing (scRNA-seq) and population genetics, we apply a framework in which to evaluate cell type-specific effects of genetic variation on gene expression. Here, we perform scRNA-seq on 64,018 fibroblasts from 79 donors and map expression quantitative trait loci (eQTLs) at the level of in idual cell types. We demonstrate that the majority of eQTLs detected in fibroblasts are specific to an in idual cell subtype. To address if the allelic effects on gene expression are maintained following cell reprogramming, we generate scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type-specific eQTLs in iPSCs and show that the eQTLs in fibroblasts almost entirely disappear during reprogramming. This work provides an atlas of how genetic variation influences gene expression across cell subtypes and provides evidence for patterns of genetic architecture that lead to cell type-specific eQTL effects.

Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina

Publisher: Mary Ann Liebert Inc

Date: 11-2019

DOI: 10.1089/HUM.2019.021

Abstract: Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of

PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia

Publisher: Elsevier BV

Date: 10-2019

DOI: 10.1016/J.STEMCR.2019.08.004

Increased tau expression in theAPOE4blood-brain barrier model is associated with reduced anti-tau therapeutic antibody deliveryin vitro

Publisher: Cold Spring Harbor Laboratory

Date: 25-10-2023

DOI: 10.1101/2023.10.24.563706

Animal and Human Models of Retinal Diseases

Publisher: Elsevier

Date: 2020

DOI: 10.1016/B978-0-12-809324-5.24191-6

Study of Gap Junctions in Human Embryonic Stem Cells

Publisher: Springer New York

Date: 2014

DOI: 10.1007/7651_2014_83

Abstract: Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency.

Bone turnover markers: Defining a therapeutic target

Publisher: Elsevier BV

Date: 02-2017

DOI: 10.1016/J.CLINBIOCHEM.2016.09.010

Growth Factors and the Serum-free Culture of Human Pluripotent Stem Cells

Publisher: Elsevier

Date: 2004

DOI: 10.1016/B978-012436643-5/50060-2

Mitochondrial replacement in an iPSC model of Leber’s hereditary optic neuropathy

Publisher: Cold Spring Harbor Laboratory

Date: 26-03-2017

DOI: 10.1101/120659

Abstract: Cybrid technology was used to replace Leber hereditary optic neuropathy (LHON) causing mitochondrial DNA (mtDNA) mutations from patient-specific fibroblasts with wildtype mtDNA, and mutation-free induced pluripotent stem cells (iPSCs) were generated subsequently. Retinal ganglion cell (RGC) differentiation demonstrates increased cell death in LHON-RGCs and can be rescued in cybrid corrected RGCs.

Non-invasive in vivo hyperspectral imaging of the retina for potential biomarker use in Alzheimer’s disease

Publisher: Springer Science and Business Media LLC

Date: 17-09-2019

DOI: 10.1038/S41467-019-12242-1

Abstract: Studies of rodent models of Alzheimer’s disease (AD) and of human tissues suggest that the retinal changes that occur in AD, including the accumulation of amyloid beta (Aβ), may serve as surrogate markers of brain Aβ levels. As Aβ has a wavelength-dependent effect on light scatter, we investigate the potential for in vivo retinal hyperspectral imaging to serve as a biomarker of brain Aβ. Significant differences in the retinal reflectance spectra are found between in iduals with high Aβ burden on brain PET imaging and mild cognitive impairment ( n = 15), and age-matched PET-negative controls ( n = 20). Retinal imaging scores are correlated with brain Aβ loads. The findings are validated in an independent cohort, using a second hyperspectral camera. A similar spectral difference is found between control and 5xFAD transgenic mice that accumulate Aβ in the brain and retina. These findings indicate that retinal hyperspectral imaging may predict brain Aβ load.

Single-Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.ISCI.2018.08.016

Mitochondrial fission protein Drp1 inhibition promotes cardiac mesodermal differentiation of human pluripotent stem cells

Publisher: Springer Science and Business Media LLC

Date: 05-03-2018

DOI: 10.1038/S41420-018-0042-9

Abstract: Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or M i-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with M i-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Fgf2 improves functional recovery—decreasing gliosis and increasing radial glia and neural progenitor cells after spinal cord injury

Publisher: Wiley

Date: 13-01-2014

DOI: 10.1002/BRB3.172

Genetic variation affects morphological retinal phenotypes extracted from UK Biobank optical coherence tomography images

Publisher: Public Library of Science (PLoS)

Date: 12-05-2021

DOI: 10.1371/JOURNAL.PGEN.1009497

Abstract: Optical Coherence Tomography (OCT) enables non-invasive imaging of the retina and is used to diagnose and manage ophthalmic diseases including glaucoma. We present the first large-scale genome-wide association study of inner retinal morphology using phenotypes derived from OCT images of 31,434 UK Biobank participants. We identify 46 loci associated with thickness of the retinal nerve fibre layer or ganglion cell inner plexiform layer. Only one of these loci has been associated with glaucoma, and despite its clear role as a biomarker for the disease, Mendelian randomisation does not support inner retinal thickness being on the same genetic causal pathway as glaucoma. We extracted overall retinal thickness at the fovea, representative of foveal hypoplasia, with which three of the 46 SNPs were associated. We additionally associate these three loci with visual acuity. In contrast to the Mendelian causes of severe foveal hypoplasia, our results suggest a spectrum of foveal hypoplasia, in part genetically determined, with consequences on visual function.

Microglial amyloid beta clearance is driven by PIEZO1 channels

Publisher: Springer Science and Business Media LLC

Date: 15-06-2022

DOI: 10.1186/S12974-022-02486-Y

Abstract: Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer's disease (AD), toxic amyloid beta (Aβ) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aβ. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia had been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aβ pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. We show that PIEZO1 orchestrates Aβ clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aβ inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aβ clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single-cell datasets. These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aβ burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

Sphingosine‐1‐phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Publisher: Wiley

Date: 06-2001

DOI: 10.1046/J.0953-816X.2001.01585.X

Complexities of lysophospholipid signalling in glioblastoma

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/J.JOCN.2014.02.013

Abstract: Glioblastoma multiforme (GBM) is the most malignant brain tumour and continues to have a very poor median survival of 12-16 months despite current best therapies. These aggressive tumours always recur after treatment and are defined by their ability to diffusely infiltrate and invade normal brain parenchyma. Autotaxin is overexpressed in GBM, and is a potent chemotactic enzyme that produces lysophosphatidic acid. Lysophospholipid (LPL) signalling is known to increase invasion of solid tumours and is also dysregulated in GBM. The LPL pathway has been shown to interact with known cancer-related signalling pathways, including those for epidermal growth factor and yes-associated protein, which are also dysregulated in GBM. The interactions between these pathways provide insights into the complexities of cancer signalling and suggest potential novel targets for GBM.

Single cell RNA sequencing of stem cell-derived retinal ganglion cells

Publisher: Springer Science and Business Media LLC

Date: 13-02-2018

DOI: 10.1038/SDATA.2018.13

Abstract: We used single cell sequencing technology to characterize the transcriptomes of 1,174 human embryonic stem cell-derived retinal ganglion cells (RGCs) at the single cell level. The human embryonic stem cell line BRN3B-mCherry (A81-H7), was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells showed increased expression of genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature RGC phenotype.

A Global Social Media Survey of Attitudes to Human Genome Editing

Publisher: Elsevier BV

Date: 05-2016

DOI: 10.1016/J.STEM.2016.04.011

Abstract: Ongoing breakthroughs with CRISPR/Cas-based editing could potentially revolutionize modern medicine, but there are many questions to resolve about the ethical implications for its therapeutic application. We conducted a worldwide online survey of over 12,000 people recruited via social media to gauge attitudes toward this technology and discuss our findings here.

The Immortal Life of Ethics? The Alienation of Body Tissue, Ethics and the Informed Consent Procedure Within Induced Pluripotent Stem Cell Research

Publisher: Springer International Publishing

Date: 2017

DOI: 10.1007/978-3-319-55532-4_3

Data descriptor: Single cell RNA sequencing of stem cell-derived retinal ganglion cells

Publisher: Springer Science and Business Media LLC

Date: 10-07-2018

DOI: 10.1038/SDATA.2018.131

Abstract: Crab culture has gained prominence in the last decade due to the large global market demand for live crabs and crab products. Portunus sanguinolentus is one of the economically important crab species in the Indo-Pacific region, with distinct differences in growth and size between male and female crabs, thus, leading to huge difference in their market values. The culture of P. sanguinolentus is still in its infancy, with crab supplies heavily dependent on wild catch. In order to unravel the molecular differences between male and female crabs, we generated a comprehensive transcriptomic dataset for P. sanguinolentus by sequencing the gonads of both sexes using the Illumina Hiseq 2500 system. Transcriptomes were assembled using Trinity de novo assembly followed by annotation. This transcriptomic data set for P. sanguinolentus would serve as an important reference data for genomic and genetic studies in this crab and related species.

Differentiation of Retinal Glial Cells From Human Embryonic Stem Cells by Promoting the Notch Signaling Pathway

Publisher: Frontiers Media SA

Date: 03-12-2019

DOI: 10.3389/FNCEL.2019.00527

Induced pluripotent stem cell derived pericytes respond to endogenous mediators of proliferation and contractility

Publisher: Cold Spring Harbor Laboratory

Date: 10-2023

DOI: 10.1101/2023.09.29.560066

Culture Variabilities of Human iPSC-Derived Cerebral Organoids Are a Major Issue for the Modelling of Phenotypes Observed in Alzheimer’s Disease

Publisher: Springer Science and Business Media LLC

Date: 16-03-2021

DOI: 10.1007/S12015-021-10147-5

Abstract: Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer's disease (AD), with the presence of APOE-ε4 associated with increased risk of developing AD. Here, we reprogrammed human fibroblasts from in iduals with different APOE-ε genotypes into induced pluripotent stem cells (iPSCs), and generated isogenic lines with different APOE profiles. Following characterisation of the newly established iPSC lines, we used an unguided/unpatterning differentiation method to generate six-month-old cerebral organoids from all iPSC lines to assess the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels. We identified variabilities in the organoids' cell composition between cell lines, and between batches of differentiation for each cell line. We observed more homogenous cerebral organoids, and similar levels of APOE, β amyloid, and Tau when using the CRISPR-edited APOE isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the cell-line-to-cell-line and batch-to-batch variabilities need to be considered when using cerebral organoids.

Generation of a human induced pluripotent stem cell line CERAi001-A-6 using episomal vectors

Publisher: Elsevier BV

Date: 07-2017

DOI: 10.1016/J.SCR.2017.05.007

Abstract: We report the generation of the hiPSC line CERAi001-A-6 from primary human dermal fibroblasts. Reprogramming was performed using episomal vector delivery of OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA for p53.

Genetics of reticular pseudodrusen in age-related macular degeneration

Publisher: Elsevier BV

Date: 04-2022

DOI: 10.1016/J.TIG.2022.01.003

Abstract: Reticular pseudodrusen (RPD) are subretinal deposits that, when observed with age-related macular degeneration (AMD), form a distinct phenotype, often associated with late-stage disease. To date, RPD genetic risk associations overlap six well-established AMD-risk regions. Determining RPD-specific underlying genetic causes by using adequate imaging methods should improve our understanding of the pathophysiology of RPD.

Maintenance of Human Embryonic Stem Cells by Sphingosine-1-Phosphate and Platelet-Derived Growth Factor

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-800-9_13

Abstract: Embryonic stem cells are pluripotent and capable of indefinite self-renewal in vitro. Human embryonic stem cells (hESC) have generally been cultivated on feeder layers of primary mouse embryonic fibroblasts (MEF) in media supplemented with fetal calf serum (FCS). However, serum contains a wide variety of biologically active compounds that might adversely affect hESC growth and differentiation. Thus, cultivation of stem cells in FCS complicates experimental approaches to define the intracellular mechanisms required for hESC maintenance. This chapter describes the serum-free maintenance of hESC in culture by addition of sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). This complete protocol provides a chemically defined serum-free system that is advantageous for studying signaling pathways involved in hESC pluripotency.

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Publisher: MyJove Corporation

Date: 20-08-2015

DOI: 10.3791/53174

Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1177/2472555217696797

Abstract: Patient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modeling, and drug discovery. However, the processes of reprogramming, maintenance, and differentiation are labor intensive and subject to intertechnician variability. To address these issues, we established and optimized protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60-positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase s le size, reduce variability during reprogramming or differentiation, and enable medium- to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

Retinal ganglion cell-specific genetic regulation in primary open angle glaucoma

Publisher: Cold Spring Harbor Laboratory

Date: 14-07-2021

DOI: 10.1101/2021.07.14.452417

Abstract: To assess the transcriptomic profile of disease-specific cell populations, fibroblasts from patients with primary open-angle glaucoma (POAG) were reprogrammed into induced pluripotent stem cells (iPSCs) before being differentiated into retinal organoids and compared to those from healthy in iduals. We performed single-cell RNA-sequencing of a total of 330,569 cells and identified cluster-specific molecular signatures. Comparing the gene expression profile between cases and controls, we identified novel genetic associations for this blinding disease. Expression quantitative trait mapping identified a total of 2,235 significant loci across all cell types, 58 of which are specific to the retinal ganglion cell subpopulations, which ultimately degenerate in POAG. Transcriptome-wide association analysis identified genes at loci previously associated with POAG, and analysis, conditional on disease status, implicated 54 statistically significant retinal ganglion cell-specific expression quantitative trait loci. This work highlights the power of large-scale iPSC studies to uncover context-specific profiles for a genetically complex disease.

Retinal ganglion cell-specific genetic regulation in primary open-angle glaucoma

Publisher: Elsevier BV

Date: 06-2022

DOI: 10.1016/J.XGEN.2022.100142

Gli1 Is an Inducing Factor in Generating Floor Plate Progenitor Cells from Human Embryonic Stem Cells  

Publisher: Oxford University Press (OUP)

Date: 10-2010

DOI: 10.1002/STEM.510

Abstract: Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer.

Peeking into the molecular trove of discarded surgical specimens

Publisher: Wiley

Date: 11-2016

DOI: 10.1111/CEO.12837

If Human Brain Organoids Are the Answer to Understanding Dementia, What Are the Questions?

Publisher: SAGE Publications

Date: 13-04-2020

DOI: 10.1177/1073858420912404

Abstract: Because our beliefs regarding our in iduality, autonomy, and personhood are intimately bound up with our brains, there is a public fascination with cerebral organoids, the “mini-brain,” the “brain in a dish”. At the same time, the ethical issues around organoids are only now being explored. What are the prospects of using human cerebral organoids to better understand, treat, or prevent dementia? Will human organoids represent an improvement on the current, less-than-satisfactory, animal models? When considering these questions, two major issues arise. One is the general challenge associated with using any stem cell–generated preparation for in vitro modelling (challenges lified when using organoids compared with simpler cell culture systems). The other relates to complexities associated with defining and understanding what we mean by the term “dementia.” We discuss 10 puzzles, issues, and stumbling blocks to watch for in the quest to model “dementia in a dish.”

Genome engineering in ophthalmology: Application of CRISPR/Cas to the treatment of eye disease

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.PRETEYERES.2016.05.001

Abstract: The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (Cas) system has enabled an accurate and efficient means to edit the human genome. Rapid advances in this technology could results in imminent clinical application, and with favourable anatomical and immunological profiles, ophthalmic disease will be at the forefront of such work. There have been a number of breakthroughs improving the specificity and efficacy of CRISPR/Cas-mediated genome editing. Similarly, better methods to identify off-target cleavage sites have also been developed. With the impending clinical utility of CRISPR/Cas technology, complex ethical issues related to the regulation and management of the precise applications of human gene editing must be considered. This review discusses the current progress and recent breakthroughs in CRISPR/Cas-based gene engineering, and outlines some of the technical issues that must be addressed before gene correction, be it in vivo or in vitro, is integrated into ophthalmic care. We outline a clinical pipeline for CRISPR-based treatments of inherited eye diseases and provide an overview of the important ethical implications of gene editing and how these may influence the future of this technology.

Engineering domain-inlaid SaCas9 adenine base editors with reduced RNA off-targets and increased on-target DNA editing

Publisher: Springer Science and Business Media LLC

Date: 25-09-2020

DOI: 10.1038/S41467-020-18715-Y

Abstract: Precision genome engineering has dramatically advanced with the development of CRISPR/Cas base editing systems that include cytosine base editors and adenine base editors (ABEs). Herein, we compare the editing profile of circularly permuted and domain-inlaid Cas9 base editors, and find that on-target editing is largely maintained following their intradomain insertion, but that structural permutation of the ABE can affect differing RNA off-target events. With this insight, structure-guided design was used to engineer an SaCas9 ABE variant (microABE I744) that has dramatically improved on-target editing efficiency and a reduced RNA-off target footprint compared to current N-terminal linked SaCas9 ABE variants. This represents one of the smallest AAV-deliverable Cas9-ABEs available, which has been optimized for robust on-target activity and RNA-fidelity based upon its stereochemistry.

Optimization of silk fibroin membranes for retinal implantation

Publisher: Elsevier BV

Date: 12-2019

DOI: 10.1016/J.MSEC.2019.110131

Abstract: Silk fibroin membrane displays potential for ocular tissue reconstruction as demonstrated by its ability to support a functioning retinal pigment epithelium (RPE) in vitro. Nevertheless, translation of these findings to the clinic will require the use of membranes that can be readily handled and implanted into diseased retinas, with minimal impact on the surrounding healthy tissue. To this end, we optimized the physical properties of fibroin membranes to enable surgical handling during implantation into the retina, without compromising biocompatibility or permeability. Our central hypothesis is that optimal strength and permeability can be achieved by combining the porogenic properties of poly(ethylene glycol) (PEG) with the crosslinking properties of horseradish peroxidase (HRP). Our study reveals that PEG used in conjunction with HRP enables the production of fibroin membranes with superior handling properties to conventional fibroin membranes. More specifically, the modified membranes could be more easily implanted into the retinas of rats and displayed good evidence of biocompatibility. Moreover, the modified membranes retained the ability to support construction of functional RPE derived from pluripotent stem cells. These findings pave the way for preclinical studies of RPE-implantation using the optimized fibroin membranes.

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells

Publisher: Springer Science and Business Media LLC

Date: 04-2011

DOI: 10.1186/SCRT57

Sphingosine-1-Phosphate

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-800-9

Presence of Functional Gap Junctions in Human Embryonic Stem Cells

Publisher: Oxford University Press (OUP)

Date: 11-2004

DOI: 10.1634/STEMCELLS.22-6-883

Abstract: Gap junctions are intercellular channels that allow both chemical and electrical signaling between two adjacent cells. Gap junction intercellular communication has been implicated in the regulation of various cellular processes, including cell migration, cell proliferation, cell differentiation, and cell apoptosis. This study aimed to determine the presence and functionality of gap junctions in human embryonic stem cells (hESCs). Using reverse transcription--polymerase chain reaction and immunocytochemistry, we demonstrate that human ES cells express two gap junction proteins, connexin 43 and connexin 45. Western blot analysis revealed the presence of three phosphorylated forms (nonphosphorylated [NP], P1, and P2) of connexin 43, NP being prominent. Moreover, scrape loading/dye transfer assay indicates that human ES cells are coupled through functional gap junctions that are inhibited by protein kinase C activation and extracellular signal-regulated kinase inhibition.

Generation of a gene-corrected human isogenic iPSC line from an Alzheimer’s disease iPSC line carrying the London mutation in APP (V717I)

Publisher: Elsevier BV

Date: 05-2021

DOI: 10.1016/J.SCR.2021.102373

Abstract: We report the genome-editing of an existing iPSC line carrying the London mutation in APP (V717I) into an iPSC line in which the pathogenic mutation was corrected. The resulting isogenic iPSC line maintained pluripotent stem cell morphology, a normal karyotype, expression of pluripotency markers and the ability to differentiate into the three germ-layers in vitro.

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells

Publisher: Association for Research in Vision and Ophthalmology (ARVO)

Date: 05-2014

DOI: 10.1167/TVST.3.3.7

Single Cell RNA Sequencing of stem cell-derived retinal ganglion cells

Publisher: Cold Spring Harbor Laboratory

Date: 22-09-2017

DOI: 10.1101/191395

Abstract: We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and subsequently prepared for single cell RNA sequencing. Our data indicates the presence of three distinct subpopulations of cells, with various degrees of maturity. One cluster of 288 cells upregulated genes involved in axon guidance together with semaphorin interactions, cell-extracellular matrix interactions and ECM proteoglycans, suggestive of a more mature phenotype.

Development and validation of the Scleroderma Clinical Trials Consortium Damage Index (SCTC-DI): a novel instrument to quantify organ damage in systemic sclerosis

Publisher: BMJ

Date: 30-03-2019

DOI: 10.1136/ANNRHEUMDIS-2018-214764

Abstract: We sought to develop the first Damage Index (DI) in systemic sclerosis (SSc). The conceptual definition of ‘damage’ in SSc was determined through consensus by a working group of the Scleroderma Clinical Trials Consortium (SCTC). Systematic literature review and consultation with patient partners and non-rheumatologist experts produced a list of potential items for inclusion in the DI. These steps were used to reduce the items: (1) Expert members of the SCTC (n=331) were invited to rate the appropriateness of each item for inclusion, using a web-based survey. Items with % consensus were retained (2) Using a prospectively acquired Australian cohort data set of 1568 patients, the univariable relationships between the remaining items and the endpoints of mortality and morbidity (Physical Component Summary score of the Short Form 36) were analysed, and items with p .10 were retained (3) using multivariable regression analysis, coefficients were used to determine a weighted score for each item. The DI was externally validated in a Canadian cohort. Ninety-three (28.1%) complete survey responses were analysed 58 of 83 items were retained. The univariable relationships with death and/or morbidity endpoints were statistically significant for 22 items, with one additional item forced into the multivariable model by experts due to clinical importance, to create a 23-item weighted SCTC DI (SCTC-DI). The SCTC-DI was predictive of morbidity and mortality in the external cohort. Through the combined use of consensus and data-driven methods, a 23-item SCTC-DI was developed and retrospectively validated.

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration

Publisher: Cold Spring Harbor Laboratory

Date: 20-08-2021

DOI: 10.1101/2021.08.19.457044

Abstract: Induced pluripotent stem cells generated from patients with geographic atrophy as well as healthy in iduals were differentiated to retinal pigment epithelium (RPE) cells. By integrating transcriptional profiles of 127,659 RPE cells generated from 43 in iduals with geographic atrophy and 36 controls with genotype data, we identified 439 expression Quantitative Trait (eQTL) loci in cis that were associated with disease status and specific to subpopulations of RPE cells. We identified loci linked to two genes with known associations with geographic atrophy - PILRB and PRPH2, in addition to 43 genes with significant genotype x disease interactions that are candidates for novel genetic associations for geographic atrophy. On a transcriptome-only level, we identified molecular pathways significantly upregulated in geographic atrophy-RPE including in extracellular cellular matrix reorganisation, neurodegeneration, and mitochondrial functions. We subsequently implemented a large-scale proteomics analysis, confirming modification in proteins associated with these pathways. We also identified six significant protein (p) QTL that regulate protein expression in the RPE cells and in geographic atrophy - two of which share variants with cis-eQTL. Transcriptome-wide association analysis identified genes at loci previously associated with age-related macular degeneration. Further analysis conditional on disease status, implicated statistically significant RPE-specific eQTL. This study uncovers important differences in RPE homeostasis associated with geographic atrophy.

Neural Differentiation of Human Embryonic Stem Cells

Publisher: Humana Press

Date: 2009

DOI: 10.1007/978-1-60761-292-6_4

Stem cell regulation by lysophospholipids

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.PROSTAGLANDINS.2007.08.004

Abstract: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) regulate a erse range of mammalian cell processes, largely through engaging multiple G protein-coupled receptors specific for these lysophospholipids. LPA and S1P have been clearly identified to have widespread physiological and pathophysiological actions, controlling events within the reproductive, gastrointestinal, vascular, nervous and immune systems, and also having a prominent role in cancer. Here we review the recent literature showing the additional emerging role for LPA and S1P in the regulation of stem cells and their progenitors. We discuss the role of these lysophospholipids in regulating the proliferation, survival, differentiation and migration of a range of adult and embryonic stem cells and progenitors, and thus are likely to play a substantial role in the maintenance, generation, mobilisation and homing of stem cell and progenitor populations in the body.

Human fibroblast and stem cell resource from the Dominantly Inherited Alzheimer Network

Publisher: Springer Science and Business Media LLC

Date: 25-07-2018

DOI: 10.1186/S13195-018-0400-0

Study of Gap Junctions in Human Embryonic Stem Cells

Publisher: Humana Press

Date: 2009

DOI: 10.1007/978-1-60761-369-5_12

Abstract: Gap junctional intercellular communication (GJIC) has been described in different cell types including stem cells and has been involved in different biological events. GJIC is required for mouse embryonic stem cell maintenance and proliferation, and various studies suggest that functional GJIC is a common characteristic of human embryonic stem cells (hESC) maintained in different culture conditions. This chapter introduces methods to study gap junctions in hESC, from expression of gap junction proteins to functional study of GJIC in hESC proliferation, apoptosis, colony growth, and pluripotency.

Genotype-free demultiplexing of pooled single-cell RNA-seq

Publisher: Springer Science and Business Media LLC

Date: 12-2019

DOI: 10.1186/S13059-019-1852-7

Abstract: A variety of methods have been developed to demultiplex pooled s les in a single cell RNA sequencing (scRNA-seq) experiment which either require hashtag barcodes or s le genotypes prior to pooling. We introduce scSplit which utilizes genetic differences inferred from scRNA-seq data alone to demultiplex pooled s les. scSplit also enables mapping clusters to original s les. Using simulated, merged, and pooled multi-in idual datasets, we show that scSplit prediction is highly concordant with demuxlet predictions and is highly consistent with the known truth in cell-hashing dataset. scSplit is ideally suited to s les without external genotype information and is available at: on-xu/scSplit

Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium

Publisher: Springer Science and Business Media LLC

Date: 20-11-2015

DOI: 10.1007/S12015-015-9636-2

Abstract: We demonstrate that a combination of Noggin, Dickkopf-1, Insulin Growth Factor 1 and basic Fibroblast Growth Factor, promotes the differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) cells. We describe an efficient one-step approach that allows the generation of RPE cells from both human embryonic stem cells and human induced pluripotent stem cells within 40-60 days without the need for manual excision, floating aggregates or imbedded cysts. Compared to methods that rely on spontaneous differentiation, our protocol results in faster differentiation into RPE cells. This pro-retinal culture medium promotes the growth of functional RPE cells that exhibit key characteristics of the RPE including pigmentation, polygonal morphology, expression of mature RPE markers, electrophysiological membrane potential and the ability to phagocytose photoreceptor outer segments. This protocol can be adapted for feeder, feeder-free and serum-free conditions. This method thereby provides a rapid and simplified production of RPE cells for downstream applications such as disease modelling and drug screening.

Decreased anti-regenerative effects after spinal cord injury in spry4−/− mice

Publisher: Elsevier BV

Date: 02-2015

DOI: 10.1016/J.NEUROSCIENCE.2014.12.020

Abstract: Previously, we have demonstrated a role for fibroblast growth factor (Fgf) in spinal cord regeneration in both zebrafish and mouse. We have shown that exogenous Fgf2 treatment attenuates astrocytic gliosis and induces glia cells to become progenitors that undergo neurogenesis as well as differentiating into bipolar astrocytes that support axonal regeneration (Goldshmit et al., 2012, 2014). One of the downstream signaling target genes of Fgf is spry4, which acts as a feedback inhibitor for Fgf signaling. In this study we examined the effects of increased endogenous Fgf signaling, in spry4-/- mice, on the early events that occur after spinal cord injury (SCI). We demonstrate that in spry4-/- mice inflammatory responses, such as tumor necrosis factor α (TNFα) secretion and macrophage/neutrophil invasion into the lesion site are reduced. In addition, astrocytic gliosis is attenuated and neuronal survival is increased. These results further support a pro-regenerative role of Fgf after SCI, and suggest that increased endogenous Fgf signaling after SCI may contribute to functional recovery and therefore presents this pathway as a target for new therapy development.

Single cell eQTL analysis identifies cell type-specific genetic control of gene expression in fibroblasts and reprogrammed induced pluripotent stem cells

Publisher: Cold Spring Harbor Laboratory

Date: 21-06-2020

DOI: 10.1101/2020.06.21.163766

Abstract: The discovery that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) - cells that can be differentiated into any cell type of the three germ layers - has provided a foundation for in vitro human disease modelling 1,2 , drug development 1,2 , and population genetics studies 3,4 . In the majority of instances, the expression levels of genes, plays a critical role in contributing to disease risk, or the ability to identify therapeutic targets. However, while the effect of the genetic background of cell lines has been shown to strongly influence gene expression, the effect has not been evaluated at the level of in idual cells. Differences in the effect of genetic variation on the gene expression of different cell-types, would provide significant resolution for in vitro research using preprogramed cells. By bringing together single cell RNA sequencing 15–21 and population genetics, we now have a framework in which to evaluate the cell-types specific effects of genetic variation on gene expression. Here, we performed single cell RNA-sequencing on 64,018 fibroblasts from 79 donors and we mapped expression quantitative trait loci (eQTL) at the level of in idual cell types. We demonstrate that the large majority of eQTL detected in fibroblasts are specific to an in idual sub-type of cells. To address if the allelic effects on gene expression are dynamic across cell reprogramming, we generated scRNA-seq data in 19,967 iPSCs from 31 reprogramed donor lines. We again identify highly cell type specific eQTL in iPSCs, and show that that the eQTL in fibroblasts are almost entirely disappear during reprogramming. This work provides an atlas of how genetic variation influences gene expression across cell subtypes, and provided evidence for patterns of genetic architecture that lead to cell-types specific eQTL effects.

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors

Publisher: Elsevier BV

Date: 07-2018

DOI: 10.1016/J.BBALIP.2018.04.007

Abstract: The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent giant cell arteritis

Publisher: Springer Science and Business Media LLC

Date: 23-07-2018

DOI: 10.1186/S12920-018-0376-4

Biological Effects of Lysophosphatidic Acid in the Nervous System

Publisher: Elsevier

Date: 2012

DOI: 10.1016/B978-0-12-394307-1.00005-9

Gap junctions modulate apoptosis and colony growth of human embryonic stem cells maintained in a serum-free system

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.BBRC.2006.03.127

Abstract: We investigated the gap junctional properties of human embryonic stem cells (hESC) cultivated in a serum-free system using sphingosine-1-phosphate and platelet-derived growth factor (S1P/PDGF). We compared this condition to hESC grown on Matrigel in mouse embryonic fibroblast conditioned medium (MEF-CM) or unconditioned medium (UM). We show that in all culture systems, hESC express connexins 43 and 45. hESC maintained in S1P/PDGF conditions and hESC grown in presence of MEF-CM are coupled through gap junctions while hESC maintained on Matrigel in UM do not exhibit gap junctional intercellular communication. In this latter condition, coupling was retrieved by addition of noggin, suggesting that BMP-like activity in UM inhibits gap junctional communication. Last, our data indicate that the closure of gap junctions by the decoupling agent alpha-glycyrrhetinic acid increases cell apoptosis and inhibits hESC colony growth. Altogether, these results suggest that gap junctions play an important role in hESC maintenance.

An Interactive Multimedia Approach to Improving Informed Consent for Induced Pluripotent Stem Cell Research

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.STEM.2016.02.006

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

Publisher: Oxford University Press (OUP)

Date: 13-05-2020

DOI: 10.1093/NARGAB/LQAA034

Abstract: The libraries generated by high-throughput single cell RNA-sequencing (scRNA-seq) platforms such as the Chromium from 10× Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use these data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3′ libraries consisting of over 70 000 cells generated on the 10× Genomics Chromium platform. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and the detection of genes, cell barcodes, Unique Molecular Identifiers. The performance of the NextSeq 500 was also similarly comparable to the MGISEQ-2000 based on the same metrics. Data generated by both sequencing platforms yielded similar analytical outcomes for general single-cell analysis. The performance of the NextSeq 500 and MGISEQ-2000 were also comparable for the deconvolution of multiplexed cell pools via variant calling, and detection of guide RNA (gRNA) from a pooled CRISPR single-cell screen. Our study provides a benchmark for high-capacity sequencing platforms applied to high-throughput scRNA-seq libraries.

A semi-automated pipeline for quantifying drusen-like deposits in human induced pluripotent stem cell-derived retinal pigment epithelium cells

Publisher: Elsevier BV

Date: 08-2023

DOI: 10.1016/J.SLAST.2023.08.006

A role for neurotrophins in embryonic stem cell growth

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1016/J.DEVCEL.2006.01.007

Abstract: Neurotrophins act on embryonic cells through TRK receptors to inhibit apoptosis by phosphorylation of AKT. In a recent paper in Nature Biotechnology, show that the presence of selected neurotrophins enables cloning of trypsinized single embryonic stem cells and potentially increases the availability and usefulness of these stem cells.

Role of ectonucleotide pyrophosphatase/phosphodiesterase 2 in the midline axis formation of zebrafish

Publisher: Springer Science and Business Media LLC

Date: 24-11-2016

DOI: 10.1038/SREP37678

Abstract: Lysophosphatidic acid (LPA) is a unique bioactive lysophospholipid that induces pleiotropic effects in various cell types and organisms by acting on its specific receptors. LPA is mainly synthetised extracellularly by the ectonucleotide pyrophosphatase hosphodiesterase 2/autotaxin (enpp2). Altered LPA signalling is associated with embryonic abnormalities, suggesting critical roles for LPA during development. However, the role of LPA signalling during early embryogenesis is not well established. We demonstrate that enpp2/LPA signalling in the early zebrafish embryo results in altered axis and midline formation, defects in left right (L-R) patterning, ciliogenesis of the Kupffer’s vesicle (KV), through the modulation of cell migration during gastrulation in a lpar 1–3 Rho/ROCK-dependant manner. Overall, this study demonstrates an essential role of enpp2/LPA signalling during early embryogenesis.

Automated Cell Culture Systems and Their Applications to Human Pluripotent Stem Cell Studies

Publisher: Elsevier BV

Date: 08-2018

DOI: 10.1177/2472630317712220

Abstract: Pluripotent stem cells are an extremely powerful tool in modeling human diseases and hold much promise for personalized regenerative or cell replacement therapies. There is an increasing need for reproducible large-scale stem cell and differentiated progeny production, with minimal variation, rendering manual approaches impracticable. Here, we provide an overview of systems currently available for automated stem cell culture, and undertake a review of their capacities, capabilities, and relative limitations. With the merging of modern technology and stem cell biology, an increased demand and implementation of automated platforms for stem cell studies is anticipated.

Human Pluripotent Stem Cell Strategies for Age-Related Macular Degeneration

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 08-2014

DOI: 10.1097/OPX.0000000000000282

Friedreich’s ataxia induced pluripotent stem cell-derived cardiomyocytes display electrophysiological abnormalities and calcium handling deficiency

Publisher: Impact Journals, LLC

Date: 30-05-2017

DOI: 10.18632/AGING.101247

Anti-Apoptotic Effect of Sphingosine-1-Phosphate and Platelet-Derived Growth Factor in Human Embryonic Stem Cells

Publisher: Mary Ann Liebert Inc

Date: 12-2007

DOI: 10.1089/SCD.2007.0057

Abstract: Human embryonic stem (hES) cells hold great promise for use in regenerative medicine. However, technologies first need to be established to maintain hES cells efficiently in vitro. Understanding the signaling networks involved in hES cell maintenance will prove to be essential to the development of such culture systems. Previously, we described a serum-free medium capable of supporting prolonged hES cell maintenance using sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF). Here, we describe an anti-apoptotic effect of S1P and PDGF in hES cells and demonstrate a direct effect of S1P in preventing hES cell apoptosis. Western blot analysis shows that S1P stimulates the phosphorylation of the mitogen-activated protein (MAP) kinases Erk1/2 but not of Akt, whereas PDGF stimulates both Erk1/2 and Akt phosphorylation. Moreover, our study suggests that the Erk1/2 and PI3K/Akt signaling pathways act independently of each other. Furthermore, neither S1P nor PDGF modify intracellular calcium concentration ([Ca(2+)]( i )) and Smad2 phosphorylation. Using pharmacological inhibitors of Erk1/2 and PI3K, our results demonstrate a critical role of the Erk1/2 and PI3K/Akt signaling pathways in mediating the anti-apoptotic effect of S1P and PDGF on hES cells. However, inhibition of the mammalian target of rapamycin (mTOR), a common downstream effector of Erk1/2 and PI3K/Akt, has no effect on hES cell apoptosis.

Enriched retinal ganglion cells derived from human embryonic stem cells

Publisher: Springer Science and Business Media LLC

Date: 10-08-2016

DOI: 10.1038/SREP30552

Abstract: Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro . In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies.

Cellular pathophysiology of Friedreich's ataxia cardiomyopathy

Publisher: Elsevier BV

Date: 2022

DOI: 10.1016/J.IJCARD.2021.11.033

Human pluripotent stem cells for the modelling of retinal pigment epithelium homeostasis and disease: A review.

Publisher: Wiley

Date: 11-07-2022

DOI: 10.1111/CEO.14128

Abstract: Human pluripotent stem cells (hPSCs), which include induced pluripotent stem cells and embryonic stem cells, are powerful tools for studying human development, physiology and disease, including those affecting the retina. Cells from selected in iduals, or specific genetic backgrounds, can be differentiated into distinct cell types allowing the modelling of diseases in a dish for therapeutic development. hPSC‐derived retinal cultures have already been used to successfully model retinal pigment epithelium (RPE) degeneration for various retinal diseases including monogenic conditions and complex disease such as age‐related macular degeneration. Here, we will review the current knowledge gained in understanding the molecular events involved in retinal disease using hPSC‐derived retinal models, in particular RPE models. We will provide ex les of various conditions to illustrate the scope of applications associated with the use of hPSC‐derived RPE models.

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

Publisher: Association for Research in Vision and Ophthalmology (ARVO)

Date: 29-06-2016

DOI: 10.1167/IOVS.16-19316

Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

Transcriptomic Profiling of Human Pluripotent Stem Cell-derived Retinal Pigment Epithelium over Time

Publisher: Elsevier BV

Date: 04-2021

DOI: 10.1016/J.GPB.2020.08.002

Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

Publisher: Cold Spring Harbor Laboratory

Date: 14-11-2019

DOI: 10.1101/842328

Abstract: Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of disease associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate points in time for RPE cultures to reflect native counterparts, we characterised the transcriptomic profiles of hPSC-derived retinal pigment epithelium (RPE) cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single cell RNA-sequencing of a total of 16,576 cells, and analysed the resulting data to assess the molecular changes of RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial – mesenchymal transition, and with maturing populations of RPE observed with time in culture. Assessment of gene ontology pathways revealed that as cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirmed a maturing transcriptional profile of RPE cells in culture. These results suggest that in vitro long-term culture of RPE cells allow the modelling of specific phenotypes observed in native mature tissue. Our work highlights the transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient-s les to be benchmarked against.

Roles of lysophosphatidic acid and sphingosine-1-phosphate in stem cell biology.

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.PLIPRES.2018.09.001

Abstract: Stem cells are unique in their ability to self-renew and differentiate into various cell types. Because of these features, stem cells are key to the formation of organisms and play fundamental roles in tissue regeneration and repair. Mechanisms controlling their fate are thus fundamental to the development and homeostasis of tissues and organs. Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive phospholipids that play a wide range of roles in multiple cell types, during developmental and pathophysiological events. Considerable evidence now demonstrates the potent roles of LPA and S1P in the biology of pluripotent and adult stem cells, from maintenance to repair. Here we review their roles for each main category of stem cells and explore how those effects impact development and physiopathology.

Biologically active constituents of the secretome of human W8B2(+) cardiac stem cells

Publisher: Springer Science and Business Media LLC

Date: 25-01-2018

DOI: 10.1038/S41598-018-19855-4

Abstract: The benefits of adult stem cells for repair of the heart have been attributed to the repertoire of salutary paracrine activities they appear to exert. We previously isolated human W8B2 + cardiac stem cells (CSCs) and found they powerfully influence cardiomyocytes and endothelial cells to collectively promote cardiac repair and regeneration. Here, the complexity of the W8B2 + CSC secretomes was characterised and examined in more detail. Using ion exchange chromatography to separate soluble proteins based on their net surface charge, the secreted factors responsible for the pro-survival activity of W8B2 + CSCs were found within the low and medium cation fractions. In addition to the soluble proteins, extracellular vesicles generated from W8B2 + CSCs not only exhibited pro-survival and pro-angiogenic activities, but also promoted proliferation of neonatal cardiomyocytes. These extracellular vesicles contain a cargo of proteins, mRNA and primary microRNA precursors that are enriched in exosomes and are capable of modulating collectively many of the cellular pathways involved in protein metabolism, cell growth, as well as cellular responses to stress and organisation of the extracellular matrix. Thus the W8B2 + CSC secretome contains a multitude of bioactive paracrine factors we have now characterised, that might well be harnessed for therapeutic application for cardiac repair and regeneration.

Longitudinal expression profiling of CD4+ and CD8+ cells in patients with active to quiescent Giant Cell Arteritis

Publisher: Cold Spring Harbor Laboratory

Date: 05-01-2018

DOI: 10.1101/243493

Abstract: Giant cell arteritis (GCA) is the most common form of vasculitis affecting elderly people. It is one of the few true ophthalmic emergencies. GCA is a heterogenous disease, symptoms and signs are variable thereby making it challenging to diagnose and often delaying diagnosis. A temporal artery biopsy is the gold standard to test for GCA, and there are currently no specific biochemical markers to categorize or aid diagnosis of the disease. We aimed to identify a less invasive method to confirm the diagnosis of GCA, as well as to ascertain clinically relevant predictive biomarkers by studying the transcriptome of purified peripheral CD4+ and CD8+ T lymphocytes in patients with GCA. We recruited 16 patients with histological evidence of GCA at the Royal Victorian Eye and Ear Hospital (RVEEH), Melbourne, Australia, and aimed to collect blood s les at six time points: acute phase, 2–3 weeks, 6–8 weeks, 3 months, 6 months and 12 months after clinical diagnosis. CD4+ and CD8+ T-cells were positively selected at each time point through magnetic-assisted cell sorting (MACS). RNA was extracted from all 195 collected s les for subsequent RNA sequencing. The expression profiles of patients were compared to those of 16 age-matched controls. Over the 12-month study period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. In CD8+ cells, we identified two transcripts that remained differentially expressed after 12 months, namely SGTB , associated with neuronal apoptosis, and FCGR3A , which has been found in association with Takayasu arteritis (TA), another large vessel vasculitis. We detected genes that correlate with both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, both visual disturbance and raised neutrophils at the time of presentation, bilateral blindness and death within 12 months. Altered IL32 gene expression could provide risk evaluation of GCA diagnosis at the time of presentation and give an indication of prognosis, which may influence management. This is the first longitudinal gene expression study undertaken to identify robust transcriptomic biomarkers of GCA. Our results show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways for this disease. These data significantly enhance the current knowledge of relevant biomarkers, their association with clinical prognostic markers, as well as potential candidates for detecting disease activity in whole blood s les. In the acute phase, the gene-phenotype relationships we have identified could provide insight to potential disease severity and as such guide us in initiating appropriate patient management.

Regulation of Stem Cell Pluripotency and Neural Differentiation by Lysophospholipids

Publisher: S. Karger AG

Date: 2009

DOI: 10.1159/000231891

Abstract: Lysophospholipids are bioactive signalling molecules able to act through the binding of their specific G-protein-coupled receptors to exert pleiotropic effects on a wide range of cells. The most widely studied signalling lysophospholipids are lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). LPA and S1P have been identified to have widespread developmental, physiological and pathological actions in the central nervous system and more recently have been shown to induce biological effects on various stem cell types. This review aims to summarise the current knowledge on LPA and S1P regulation of embryonic and neural stem cell biology.

Targeted therapeutic mild hypercapnia after cardiac arrest: A phase II multi-centre randomised controlled trial (the CCC trial)

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.RESUSCITATION.2016.03.023

Abstract: In intensive care observational studies, hypercapnia after cardiac arrest (CA) is independently associated with improved neurological outcome. However, the safety and feasibility of delivering targeted therapeutic mild hypercapnia (TTMH) for such patients is untested. In a phase II safety and feasibility multi-centre, randomised controlled trial, we allocated ICU patients after CA to 24h of targeted normocapnia (TN) (PaCO2 35-45mmHg) or TTMH (PaCO2 50-55mmHg). The primary outcome was serum neuron specific enolase (NSE) and S100b protein concentrations over the first 72h assessed in the first 50 patients surviving to day three. Secondary end-points included global measure of function assessment at six months and mortality for all patients. We enrolled 86 patients. Their median age was 61 years (58, 64 years) and 66 (79%) were male. Of these, 50 patients (58%) survived to day three for full biomarker assessment. NSE concentrations increased in the TTMH group (p=0.02) and TN group (p=0.005) over time, with the increase being significantly more pronounced in the TN group (p(interaction)=0.04). S100b concentrations decreased over time in the TTMH group (p<0.001) but not in the TN group (p=0.68). However, the S100b change over time did not differ between the groups (p(interaction)=0.23). At six months, 23 (59%) TTMH patients had good functional recovery compared with 18 (46%) TN patients. Hospital mortality occurred in 11 (26%) TTMH patients and 15 (37%) TN patients (p=0.31). In CA patients admitted to the ICU, TTMH was feasible, appeared safe and attenuated the release of NSE compared with TN. These findings justify further investigation of this novel treatment.

Characterization of the retinal pigment epithelium in Friedreich ataxia

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.BBREP.2015.09.003

Comparison of CRISPR/Cas endonucleases for in vivo retinal gene editing

Publisher: Cold Spring Harbor Laboratory

Date: 09-06-2020

DOI: 10.1101/2020.06.09.141705

Abstract: CRISPR/Cas has opened the prospect of direct gene correction therapy for some inherited retinal diseases. Previous work has demonstrated the utility of adeno-associated virus (AAV) mediated delivery to retinal cells in vivo however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing in vivo . We sought to compare CRISPR/Cas endonuclease activity using both single and dual AAV delivery strategies for gene editing in retinal cells. Plasmids of a dual vector system with SpCas9, SaCas9, Cas12a, CjCas9 and sgRNA targeting YFP and a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and T7E1 assay. Paired CRISPR/Cas endonuclease and its best performing sgRNA was then packaged into an AAV2 capsid derivative, AAV7m8, and injected intravitreally into CMV-Cre::Rosa26-YFP mice. SpCas9 and Cas12a achieved better knockout efficiency than SaCas9 and CjCas9. Moreover, no significant difference in YFP gene editing was found between single and dual CRISPR/SaCas9 vector systems. With a marked reduction of YFP-positive retinal cells, AAV7m8 delivered SpCas9 was found to have the highest knockout efficacy among all investigated endonucleases. We demonstrate that the AAV7m8-mediated delivery of CRISPR/SpCas9 construct achieves the most efficient gene modification in neurosensory retinal cells in vitro and in vivo .

Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA-sequencing

Publisher: Cold Spring Harbor Laboratory

Date: 17-02-2019

DOI: 10.1101/552588

Abstract: The libraries generated by high-throughput single cell RNA-sequencing platforms such as the Chromium from 10X Genomics require considerable amounts of sequencing, typically due to the large number of cells. The ability to use this data to address biological questions is directly impacted by the quality of the sequence data. Here we have compared the performance of the Illumina NextSeq 500 and NovaSeq 6000 against the BGI MGISEQ-2000 platform using identical Single Cell 3’ libraries consisting of over 70,000 cells. Our results demonstrate a highly comparable performance between the NovaSeq 6000 and MGISEQ-2000 in sequencing quality, and cell, UMI, and gene detection. However, compared with the NextSeq 500, the MGISEQ-2000 platform performs consistently better, identifying more cells, genes, and UMIs at equalised read depth. We were able to call an additional 1,065,659 SNPs from sequence data generated by the BGI platform, enabling an additional 14% of cells to be assigned to the correct donor from a multiplexed library. However, both the NextSeq 500 and MGISEQ-2000 detected similar frequencies of gRNAs from a pooled CRISPR single cell screen. Our study provides a benchmark for high capacity sequencing platforms applied to high-throughput single cell RNA-seq libraries.

Growth Factors and the Serum-free Culture of Human Pluripotent Stem Cells

Publisher: Elsevier

Date: 2009

DOI: 10.1016/B978-0-12-374729-7.00043-3

Mitochondrial replacement in an iPSC model of Leber's hereditary optic neuropathy

Publisher: Impact Journals, LLC

Date: 29-04-2017

DOI: 10.18632/AGING.101231

A sporadic Alzheimer’s blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies

Publisher: Cold Spring Harbor Laboratory

Date: 07-03-2022

DOI: 10.1101/2022.03.06.483200

Abstract: The blood-brain barrier (BBB) is a major impediment to therapeutic intracranial drug delivery for the treatment of neurodegenerative diseases, including Alzheimer’s disease (AD). Focused ultrasound applied together with microbubbles (FUS +MB ) is a novel technique to transiently open the BBB and increase drug delivery. Evidence suggests that FUS +MB is safe, however the effects of FUS +MB on human BBB cells, especially in the context of AD, remain sparsely investigated. Here we generated BBB cells (induced brain endothelial cells (iBECs) and astrocytes (iAstrocytes)) from apolipoprotein E gene allele E4 ( APOE4 , high AD risk) and allele E3 ( APOE3 , lower AD risk) carrying patient-derived induced pluripotent stem cells (iPSCs). We then developed a human sporadic AD BBB cell platform to investigate the effects of FUS +MB on BBB cells and screen for the delivery of two potentially therapeutic AD antibodies. We utilized this robust and reproducible human BBB model to demonstrate increased delivery of therapeutic AD antibodies across the BBB following FUS +MB treatment, including an analogue of Aducanumab (Aduhelm TM anti-amyloid-β) and a novel anti-Tau antibody RNF5. Our results also demonstrate the safety of FUS +MB indicated by minimal changes in the cell transcriptome as well as little or no changes in cell viability and inflammatory responses within the first 24 h post FUS +MB . Finally, we report a more physiologically relevant hydrogel-based 2.5D BBB model as a key development for FUS +MB -mediated drug delivery screening, with potentially higher translational utility. Our results demonstrate an important translatable patient BBB cell model for identifying FUS +MB -deliverable drugs and screening for cell- and patient-specific effects of FUS +MB , accelerating the use of FUS +MB as a therapeutic modality in AD. Focused ultrasound increases the in vitro delivery of therapeutic antibodies Aducanumab and anti-Tau in a sporadic Alzheimer’s disease patient-derived blood-brain barrier cell model.

Microglia orchestrate neuronal activity in brain organoids

Publisher: Cold Spring Harbor Laboratory

Date: 09-12-2020

DOI: 10.1101/2020.12.08.416388

Abstract: Human stem cell-derived brain organoids provide a physiologically relevant in vitro 3D brain model for studies of neurological development that are unique to the human nervous system. Prior studies have reported protocols that support the maturation of microglia from mesodermal progenitors leading to innately developing microglia within the organoids. However, although microglia are known to support neuronal development in rodents, none of the previous studies have reported what is the impact of microglia on neuronal growth and maturation in human brain organoids. Here we show that incorporating microglial progenitors into the developing organoid supports neuronal maturation, the emergence of neurons capable of firing repetitive action potentials and the appearance of synaptic and neuronal bursting activity. Immunocompetent organoids enable experimental strategies for interrogating fundamental questions on microglial and neuronal ersity and function during human brain development.

A New Feeder-Free Technique to Expand Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Publisher: Bentham Science Publishers Ltd.

Date: 16-11-2009

DOI: 10.2174/1876893800901010076

Investigating the role of APOE: limitations of human pluripotent stem cell-derived cerebral organoids

Publisher: Cold Spring Harbor Laboratory

Date: 12-2020

DOI: 10.1101/2020.11.30.405654

Abstract: Apolipoprotein E (APOE) is the most important susceptibility gene for late onset of Alzheimer’s disease, with the presence of APOE-ε4 associated with increased risk of developing Alzheimer’s disease. Here, we reprogrammed human fibroblasts from in iduals with different APOE-ε genotypes into induced pluripotent stem cells, and generated isogenic lines with different APOE profiles. We then differentiated these into cerebral organoids for six months and assessed the suitability of this in vitro system to measure APOE, β amyloid, and Tau phosphorylation levels. We identified intra- and inter-variabilities in the organoids’ cell composition. Using the CRISPR-edited APOE isogenic lines, we observed more homogenous cerebral organoids, and similar levels of APOE, β amyloid, and Tau between the isogenic lines, with the exception of one site of Tau phosphorylation which was higher in the APOE-ε4/ε4 organoids. These data describe that pathological hallmarks of AD are observed in cerebral organoids, and that their variation is mainly independent of the APOE-ε status of the cells, but associated with the high variability of cerebral organoid differentiation. It demonstrates that the batch-to-batch and cell-line-to-cell-line variabilities need to be considered when using cerebral organoids.

Cardiac Repair with a Novel Population of Mesenchymal Stem Cells Resident in the Human Heart

Publisher: Oxford University Press (OUP)

Date: 29-07-2015

DOI: 10.1002/STEM.2101

Abstract: Cardiac resident stem cells (CRSCs) hold much promise to treat heart disease but this remains a controversial field. Here, we describe a novel population of CRSCs, which are positive for W8B2 antigen and were obtained from adult human atrial appendages. W8B2+ CRSCs exhibit a spindle-shaped morphology, are clonogenic and capable of self-renewal. W8B2+ CRSCs show high expression of mesenchymal but not hematopoietic nor endothelial markers. W8B2+ CRSCs expressed GATA4, HAND2, and TBX5, but not C-KIT, SCA-1, NKX2.5, PDGFRα, ISL1, or WT1. W8B2+ CRSCs can differentiate into cardiovascular lineages and secrete a range of cytokines implicated in angiogenesis, chemotaxis, inflammation, extracellular matrix remodeling, cell growth, and survival. In vitro, conditioned medium collected from W8B2+ CRSCs displayed prosurvival, proangiogenic, and promigratory effects on endothelial cells, superior to that of other adult stem cells tested, and additionally promoted survival and proliferation of neonatal rat cardiomyocytes. Intramyocardial transplantation of human W8B2+ CRSCs into immunocompromised rats 1 week after myocardial infarction markedly improved cardiac function (∼40% improvement in ejection fraction) and reduced fibrotic scar tissue 4 weeks after infarction. Hearts treated with W8B2+ CRSCs showed less adverse remodeling of the left ventricle, a greater number of proliferating cardiomyocytes (Ki67+cTnT+ cells) in the remote region, higher myocardial vascular density, and greater infiltration of CD163+ cells (a marker for M2 macrophages) into the border zone and scar regions. In summary, W8B2+ CRSCs are distinct from currently known CRSCs found in human hearts, and as such may be an ideal cell source to repair myocardial damage after infarction. Stem Cells 2015 :3100–3113

Bio-engineering a tissue flap utilizing a porous scaffold incorporating a human induced pluripotent stem cell-derived endothelial cell capillary network connected to a vascular pedicle

Publisher: Elsevier BV

Date: 08-2019

DOI: 10.1016/J.ACTBIO.2019.05.067

Abstract: Tissue flaps are used to cover large oorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ± 100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31

The current state of stem cell therapy for ocular disease

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1016/J.EXER.2018.07.019

Abstract: Herein, we review the safety, efficacy, regulatory standards and ethical implications of the use of stem cells in ocular disease. A literature review was conducted, registered clinical trials reviewed, and expert opinions sought. Guidelines and codes of conduct from international societies and professional bodies were also reviewed. Collated data is presented on current progress in the field of ocular regenerative medicine, future challenges, the clinical trial process and ethical considerations in stem cell therapy. A greater understanding of the function and location of ocular stem cells has led to rapid advances in possible therapeutic applications. However, in the context of significant technical challenges and potential long-term complications, it is imperative that stem cell practices operate within formal clinical trial frameworks. While there remains broad scope for innovation, ongoing evidence-based review of potential interventions and the development of standardized protocols are necessary to ensure patient safety and best practice in ophthalmic care.

Role of Gap Junctions in Embryonic and Somatic Stem Cells

Publisher: Springer Science and Business Media LLC

Date: 14-08-2008

DOI: 10.1007/S12015-008-9038-9

Abstract: Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.

Village in a dish: a model system for population-scale hiPSC studies

Publisher: Cold Spring Harbor Laboratory

Date: 19-08-2021

DOI: 10.1101/2021.08.19.457030

Abstract: The mechanisms by which DNA alleles contribute to disease risk, drug response, and other human phenotypes are highly context-specific, varying across cell types and under different conditions. Human induced pluripotent stem cells (hiPSCs) are uniquely suited to study these context-dependent effects, but to do so requires cell lines from hundreds or potentially thousands of in iduals. Village cultures, where multiple hiPSC lines are cultured and differentiated together in a single dish, provide an elegant solution for scaling hiPSC experiments to the necessary s le sizes required for population-scale studies. Here, we show the utility of village models, demonstrating how cells can be assigned back to a donor line using single cell sequencing, and addressing whether line-specific signaling alters the transcriptional profiles of companion lines in a village culture. We generated single cell RNA sequence data from hiPSC lines cultured independently (uni-culture) and in villages at three independent sites. We show that the transcriptional profiles of hiPSC lines are highly consistent between uni- and village cultures for both fresh (0.46 R 0.88) and cryopreserved s les (0.46 R 0.62). Using a mixed linear model framework, we estimate that the proportion of transcriptional variation across cells is predominantly due to donor effects, with minimal evidence of variation due to culturing in a village system. We demonstrate that the genetic, epigenetic or hiPSC line-specific effects on gene expression are consistent whether the lines are uni- or village-cultured (0.82 R 0.94). Finally, we identify the consistency in the landscape of cell states between uni- and village-culture systems. Collectively, we demonstrate that village methods can be effectively used to detect hiPSC line-specific effects including sensitive dynamics of cell states.

Organelle mapping in dendrites of human iPSC-derived neurons reveals dynamic functional dendritic Golgi structures

Publisher: Elsevier BV

Date: 07-2023

DOI: 10.1016/J.CELREP.2023.112709

Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration

Publisher: Springer Science and Business Media LLC

Date: 26-07-2022

DOI: 10.1038/S41467-022-31707-4

Abstract: There are currently no treatments for geographic atrophy, the advanced form of age-related macular degeneration. Hence, innovative studies are needed to model this condition and prevent or delay its progression. Induced pluripotent stem cells generated from patients with geographic atrophy and healthy in iduals were differentiated to retinal pigment epithelium. Integrating transcriptional profiles of 127,659 retinal pigment epithelium cells generated from 43 in iduals with geographic atrophy and 36 controls with genotype data, we identify 445 expression quantitative trait loci in cis that are asssociated with disease status and specific to retinal pigment epithelium subpopulations. Transcriptomics and proteomics approaches identify molecular pathways significantly upregulated in geographic atrophy, including in mitochondrial functions, metabolic pathways and extracellular cellular matrix reorganization. Five significant protein quantitative trait loci that regulate protein expression in the retinal pigment epithelium and in geographic atrophy are identified - two of which share variants with cis- expression quantitative trait loci, including proteins involved in mitochondrial biology and neurodegeneration. Investigation of mitochondrial metabolism confirms mitochondrial dysfunction as a core constitutive difference of the retinal pigment epithelium from patients with geographic atrophy. This study uncovers important differences in retinal pigment epithelium homeostasis associated with geographic atrophy.

Anti-lysophosphatidic acid antibodies improve traumatic brain injury outcomes

Publisher: Springer Science and Business Media LLC

Date: 2014

DOI: 10.1186/1742-2094-11-37

Automation of Organoid Cultures: Current Protocols and Applications

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1177/24725552211024547

Abstract: Organoids are three-dimensional, functional structures that mimic in vivo organs. They offer new opportunities for the modeling of cancer and infectious and rare hereditary diseases. Furthermore, the advent of organoid biobanks opens new avenues for drug screening in a personalized fashion and holds much promise for personalized regenerative medicine. Thus, there is a need for reproducible, large-scale organoid generation with minimal variability, making manual approaches impracticable. Here, we review the current use of automation in organoid culture and analysis, using cerebral and retinal organoids as illustrations of current applications. An increased demand for automated organoid platforms is anticipated.

Single Cell Profiling Identifies Key Pathways Expressed by iPSCs Cultured in Different Commercial Media

Publisher: Elsevier BV

Date: 2018

DOI: 10.2139/SSRN.3188388

Université Paris 8 UFR De Psychologie

Location: France

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Université De Strasbourg

Location: France

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University Of Melbourne

Location: Australia

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Université Pierre Et Marie Curie

Location: France

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Monash University

Location: Australia

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University Of Western Australia

Location: Australia

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Collège De France

Location: France

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Centre For Eye Research Australia Ltd

Location: Australia

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Bernard O'Brien Institute Of Microsurgery

Location: Australia

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ARC Future Fellowships - Grant ID: FT140100047

Start Date: 2015

End Date: 12-2018

Amount: $890,552.00

Funder: Australian Research Council

Industrial Transformation Training Centres - Grant ID: IC170100016

Start Date: 05-2018

End Date: 12-2024

Amount: $3,123,492.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR1101002

Start Date: 07-2011

End Date: 12-2019

Amount: $21,000,000.00

Funder: Australian Research Council