Publications by authors named "Raymond C Wong"

62 Publications

Comparison of CRISPR/Cas Endonucleases for Retinal Gene Editing.

Front Cell Neurosci 2020 10;14:570917. Epub 2020 Sep 10.

Menzies Institute for Medical Research, University of Tasmania, Hobart, TAS, Australia.

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 ; however, with the expanding repertoire of CRISPR/Cas endonucleases, it is not clear which of these are most efficacious for retinal editing . 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 a sgRNA targeting , as well as a single vector system with SaCas9/YFP sgRNA were generated and validated in YFP-expressing HEK293A cell by flow cytometry and the 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 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 .
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http://dx.doi.org/10.3389/fncel.2020.570917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511709PMC
September 2020

Genetic Studies of Hypertrophic Cardiomyopathy in Singaporeans Identify Variants in and That Are Common in Chinese Patients.

Circ Genom Precis Med 2020 Oct 20;13(5):424-434. Epub 2020 Aug 20.

National Heart Centre Singapore (C.J.P., N.T., C.W.L.C., S.Q.L., S.C.K., P.M.L., A.A.H., C.X.Y., T.T.L., H.C.T., S.A.C.).

Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry.

Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies.

Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, <0.0001) but an excess of variants of uncertain significance (24%, <0.0001), as compared to Whites (pathogenic/likely pathogenic: 31%, excess of variants of uncertain significance: 7%). Two missense variants in thin filament encoding genes were commonly seen in Singaporean HCM (TNNI3:p.R79C, disease allele frequency [AF]=0.018; TNNT2:p.R286H, disease AF=0.022) and are enriched in Singaporean HCM when compared with Asian controls (TNNI3:p.R79C, Singaporean controls AF=0.0055, =0.0057, genome aggregation database-East Asian AF=0.0062, =0.0086; TNNT2:p.R286H, Singaporean controls AF=0.0017, <0.0001, genome aggregation database-East Asian AF=0.0009, <0.0001). Both these variants have conflicting annotations in ClinVar and are of low penetrance (TNNI3:p.R79C, 0.7%; TNNT2:p.R286H, 2.7%) but are predicted to be deleterious by computational tools. In population controls, TNNI3:p.R79C carriers had significantly thicker left ventricular walls compared with noncarriers while its etiological fraction is limited (0.70 [95% CI, 0.35-0.86]) and thus TNNI3:p.R79C is considered variant of uncertain significance. Mutant TNNT2:p.R286H iPSC-CMs (induced pluripotent stem cells derived cardiomyocytes) show hypercontractility, increased metabolic requirements, and cellular hypertrophy and the etiological fraction (0.93 [95% CI, 0.83-0.97]) support the likely pathogenicity of TNNT2:p.R286H.

Conclusions: As compared with Whites, Chinese HCM patients commonly have low penetrance risk alleles in or but exhibit few clinically actionable HCM variants overall. This highlights the need for greater study of HCM genetics in non-White populations.
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http://dx.doi.org/10.1161/CIRCGEN.119.002823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676617PMC
October 2020

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction.

Cardiovasc Res 2021 Feb;117(3):918-929

Departments of Medicine and Surgery, University of Melbourne, Melbourne, VIC, Australia.

Aims: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction.

Methods And Results: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs.

Conclusions: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.
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http://dx.doi.org/10.1093/cvr/cvaa088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898942PMC
February 2021

Apical hypertrophic cardiomyopathy complicated by apical aneurysm.

J Nucl Cardiol 2020 Feb 14. Epub 2020 Feb 14.

National University Heart Centre Singapore, National University Health System, Singapore, Singapore.

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http://dx.doi.org/10.1007/s12350-020-02066-8DOI Listing
February 2020

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

Front Cell Neurosci 2019 3;13:527. Epub 2019 Dec 3.

Save Sight Institute, Department of Clinical Ophthalmology and Eye Health, The University of Sydney, Sydney, NSW, Australia.

Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.
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http://dx.doi.org/10.3389/fncel.2019.00527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901827PMC
December 2019

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

Hum Gene Ther 2019 11 25;30(11):1349-1360. Epub 2019 Oct 25.

Menzies Institute for Medical Research, University of Tasmania, Hobart, Australia.

Safe delivery of CRISPR/Cas endonucleases remains one of the major barriers to the widespread application of genome editing. 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 initially designed to target Cas9 (SpCas9) and after 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 vectors and intravitreally administered in C57BL/6 and transgenic mice. After 8 weeks, the expression of SpCas9 and the efficacy of gene disruption were quantified. A reduction of SpCas9 mRNA was found in retinas treated with AAV2-mediated YFP/SpCas9 targeting CRISPR/Cas compared with 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 with LacZ/SpCas9 targeting CRISPR/Cas) in the transfected retina of transgenic mice. In conclusion, our data suggest that a self-destructive "kamikaze" CRISPR/Cas system can be used as a robust tool for genome editing in the retina, without compromising on-target efficiency.
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http://dx.doi.org/10.1089/hum.2019.021DOI Listing
November 2019

Corrigendum: Potentials of Cellular Reprogramming as a Novel Strategy for Neuroregeneration.

Front Cell Neurosci 2019 3;13:147. Epub 2019 May 3.

Centre for Eye Research Australia, East Melbourne, VIC, Australia.

[This corrects the article DOI: 10.3389/fncel.2018.00460.].
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http://dx.doi.org/10.3389/fncel.2019.00147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510166PMC
May 2019

Screening of CRISPR/Cas base editors to target the AMD high-risk Y402H complement factor H variant.

Mol Vis 2019 16;25:174-182. Epub 2019 Mar 16.

Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia.

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H gene.

Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the locus or the gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy.

Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons).

Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the gene with minimal off-target effects.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441356PMC
August 2019

Rational design of silicon structures for optically controlled multiscale biointerfaces.

Nat Biomed Eng 2018 Jul 30;2(7):508-521. Epub 2018 Apr 30.

Department of Chemistry, University of Chicago, Chicago, IL, USA.

Silicon-based materials have been widely used. However, remotely controlled and interconnect-free silicon configurations have been rarely explored, because of limited fundamental understanding of the complex physicochemical processes that occur at interfaces between silicon and biological materials. Here, we describe rational design principles, guided by biology, for establishing intracellular, intercellular and extracellular silicon-based interfaces, where the silicon and the biological targets have matched properties. We focused on light-induced processes at these interfaces, and developed a set of matrices to quantify and differentiate the capacitive, Faradaic and thermal outputs from about 30 different silicon materials in saline. We show that these interfaces are useful for the light-controlled non-genetic modulation of intracellular calcium dynamics, of cytoskeletal structures and transport, of cellular excitability, of neurotransmitter release from brain slices, and of brain activity in vivo.
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http://dx.doi.org/10.1038/s41551-018-0230-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430241PMC
July 2018

A Simple Cloning-free Method to Efficiently Induce Gene Expression Using CRISPR/Cas9.

Mol Ther Nucleic Acids 2019 Mar 20;14:184-191. Epub 2018 Nov 20.

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia; Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, Australia; Shenzhen Eye Hospital, School of Medicine, Shenzhen University, Shenzhen, China. Electronic address:

Gain-of-function studies often require the tedious cloning of transgene cDNA into vectors for overexpression beyond the physiological expression levels. The rapid development of CRISPR/Cas technology presents promising opportunities to address these issues. Here, we report a simple, cloning-free method to induce gene expression at an endogenous locus using CRISPR/Cas9 activators. Our strategy utilizes synthesized sgRNA expression cassettes to direct a nuclease-null Cas9 complex fused with transcriptional activators (VP64, p65, and Rta) for site-specific induction of endogenous genes. This strategy allows rapid initiation of gain-of-function studies in the same day. Using this approach, we tested two CRISPR activation systems, dSpCas9VPR and dSaCas9VPR, for induction of multiple genes in human and rat cells. Our results showed that both CRISPR activators allow efficient induction of six different neural development genes (CRX, RORB, RAX, OTX2, ASCL1, and NEUROD1) in human cells, whereas the rat cells exhibit more variable and less-efficient levels of gene induction, as observed in three different genes (Ascl1, Neurod1, Nrl). Altogether, this study provides a simple method to efficiently activate endogenous gene expression using CRISPR/Cas9 activators, which can be applied as a rapid workflow to initiate gain-of-function studies for a range of molecular- and cell-biology disciplines.
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http://dx.doi.org/10.1016/j.omtn.2018.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307107PMC
March 2019

Potentials of Cellular Reprogramming as a Novel Strategy for Neuroregeneration.

Front Cell Neurosci 2018 30;12:460. Epub 2018 Nov 30.

Centre for Eye Research Australia, East Melbourne, VIC, Australia.

Cellular reprogramming technology holds great potential for tissue repair and regeneration to replace cells that are lost due to diseases or injuries. In addition to the landmark discovery of induced pluripotent stem cells, advances in cellular reprogramming allow the direct lineage conversion of one somatic cell type to another using defined transcription factors. This direct reprogramming technology represents a rapid way to generate target cells in the laboratory, which can be used for transplantation and studies of biology and diseases. More importantly, recent work has demonstrated the exciting application of direct reprogramming to stimulate regeneration , providing an alternative approach to transplantation of donor cells. Here, we provide an overview of the underlying concept of using cellular reprogramming to convert cell fates and discuss the current advances in cellular reprogramming both and with particular focuses on the neural and retinal systems. We also discuss the potential of reprogramming in regenerative medicine, the challenges and potential solutions to translate this technology to the clinic.
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http://dx.doi.org/10.3389/fncel.2018.00460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284065PMC
November 2018

Effect of Cardiac Rehabilitation on Quality of Life, Depression and Anxiety in Asian Patients.

Int J Environ Res Public Health 2018 05 28;15(6). Epub 2018 May 28.

College of Healthcare Sciences, James Cook University, Singapore 387380, Singapore.

This study explored the effect of cardiac rehabilitation on quality of life, depression, and anxiety in Asian patients in Singapore. Out of the 194 patients who were recruited into the study, 139 patients (71.6%) completed both the pre- and post-cardiac rehabilitation questionnaires. Their ages ranged from 28 to 80 (M = 56.66, SD = 8.88), and 103 patients (74.1%) were males and 21 patients (15.1%) were females. As hypothesized, there was a statistically significant difference between the pre- and post-cardiac rehabilitation scores on the combined dependent variables, F (4, 135) = 34.84, < 0.001; Wilks' Lambda = 0.49; partial eta squared = 0.51. An inspection of the mean scores indicated that patients reported higher levels of physical and mental quality of life and lower levels of depression post-cardiac rehabilitation. The findings were discussed in regards to implications in cardiac rehabilitation in Singapore.
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http://dx.doi.org/10.3390/ijerph15061095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025099PMC
May 2018

Effect of multimorbidity on survival of patients diagnosed with heart failure: a retrospective cohort study in Singapore.

BMJ Open 2018 05 20;8(5):e021291. Epub 2018 May 20.

Health Services and Outcomes Research, National Healthcare Group, Singapore.

Objective: Multimorbidity in patients with heart failure (HF) results in poor prognosis and is an increasing public health concern. We aim to examine the effect of multimorbidity focusing on type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) on all-cause and cardiovascular disease (CVD)-specific mortality among patients diagnosed with HF in Singapore.

Design: Retrospective cohort study.

Setting: Primary and tertiary care in three (out of six) Regional Health Systems in Singapore.

Participants: Patients diagnosed with HF between 2003 and 2016 from three restructured hospitals and nine primary care polyclinics were included in this retrospective cohort study.

Primary Outcomes: All-cause mortality and CVD-specific mortality.

Results: A total of 34 460 patients diagnosed with HF from 2003 to 2016 were included in this study and were followed up until 31 December 2016. The median follow-up time was 2.1 years. Comorbidities prior to HF diagnosis were considered. Patients were categorised as (1) HF only, (2) T2DM+HF, (3) CKD+HF and (4) T2DM+CKD+HF. Cox regression model was used to determine the effect of multimorbidity on (1) all-cause mortality and (2) CVD-specific mortality. Adjusting for demographics, other comorbidities, baseline treatment and duration of T2DM prior to HF diagnosis, 'T2DM+CKD+HF' patients had a 56% higher risk of all-cause mortality (HR: 1.56, 95% CI 1.48 to 1.63) and a 44% higher risk of CVD-specific mortality (HR: 1.44, 95% CI 1.32 to 1.56) compared with patients diagnosed with HF only.

Conclusion: All-cause and CVD-specific mortality risks increased with increasing multimorbidity. This study highlights the need for a new model of care that focuses on holistic patient management rather than disease management alone to improve survival among patients with HF with multimorbidity.
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http://dx.doi.org/10.1136/bmjopen-2017-021291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961600PMC
May 2018

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

Cell Death Discov 2018 Dec 5;4:39. Epub 2018 Mar 5.

1St Vincent's Institute of Medical Research, Fitzroy, VIC 3065 Australia.

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 Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-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.
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http://dx.doi.org/10.1038/s41420-018-0042-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841367PMC
December 2018

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

Stem Cell Res 2017 07 19;22:13-15. Epub 2017 May 19.

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, Australia; Ophthalmology, Department of Surgery, The University of Melbourne, Australia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.scr.2017.05.007DOI Listing
July 2017

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

Proc Natl Acad Sci U S A 2017 09 6;114(39):E8214-E8223. Epub 2017 Sep 6.

Department of Ophthalmology, University of Rochester, Rochester, NY 14642;

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby's fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich "drusen-like" composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE-ECM interface.
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http://dx.doi.org/10.1073/pnas.1710430114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625924PMC
September 2017

Thymosin Beta-4 Is Elevated in Women With Heart Failure With Preserved Ejection Fraction.

J Am Heart Assoc 2017 Jun 13;6(6). Epub 2017 Jun 13.

Cardiovascular Research Institute, National University Health System, Singapore

Background: Thymosin beta-4 (TB4) is an X-linked gene product with cardioprotective properties. Little is known about plasma concentration of TB4 in heart failure (HF), and its relationship with other cardiovascular biomarkers. We sought to evaluate circulating TB4 in HF patients with preserved (HFpEF) or reduced (HFrEF) ejection fraction compared to non-HF controls.

Methods And Results: TB4 was measured using a liquid chromatography and mass spectrometry assay in age- and sex-matched HFpEF (n=219), HFrEF (n=219) patients, and controls (n=219) from a prospective nationwide study. Additionally, a 92-marker multiplex proximity extension assay was measured to identify biomarker covariates. Compared with controls, plasma TB4 was elevated in HFpEF (985 [421-1723] ng/mL versus 1401 [720-2379] ng/mL, <0.001), but not in HFrEF (1106 [556-1955] ng/mL, =0.642). Stratifying by sex, only women (1623 [1040-2625] ng/mL versus 942 [386-1891] ng/mL, <0.001), but not men (1238.5 [586-1967] ng/mL versus 1004 [451-1538] ng/mL, =1.0), had significantly elevated TB4 in the setting of HFpEF. Adjusted for New York Heart Association class, N-terminal pro B-type natriuretic peptide, age, and myocardial infarction, hazard ratio to all-cause mortality is significantly higher in women with elevated TB4 (1.668, =0.036), but not in men (0.791, =0.456) with HF. TB4 is strongly correlated with a cluster of 7 markers from the proximity extension assay panel, which are either X-linked, regulated by sex hormones, or involved with NF-κB signaling.

Conclusions: We show that plasma TB4 is elevated in women with HFpEF and has prognostic information. Because TB4 can preserve EF in animal studies of cardiac injury, the relation of endogenous, circulating TB4 to X chromosome biology and differential outcomes in female heart disease warrants further study.
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http://dx.doi.org/10.1161/JAHA.117.005586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669175PMC
June 2017

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

SLAS Technol 2018 08 2;23(4):315-325. Epub 2017 Jun 2.

1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria, Australia.

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.
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http://dx.doi.org/10.1177/2472630317712220DOI Listing
August 2018

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

Aging (Albany NY) 2017 04;9(4):1341-1350

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, Australia.

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.
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http://dx.doi.org/10.18632/aging.101231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425131PMC
April 2017

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

Methods Mol Biol 2018 ;1697:133-140

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, The University of Melbourne, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.

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.
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http://dx.doi.org/10.1007/7651_2017_4DOI Listing
July 2018

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

SLAS Discov 2017 09 13;22(8):1016-1025. Epub 2017 Mar 13.

1 Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital & Department of Ophthalmology, the University of Melbourne, East Melbourne, Victoria, Australia.

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 sample 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.
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http://dx.doi.org/10.1177/2472555217696797DOI Listing
September 2017

Drug discovery using induced pluripotent stem cell models of neurodegenerative and ocular diseases.

Pharmacol Ther 2017 Sep 14;177:32-43. Epub 2017 Feb 14.

Centre for Eye Research Australia & Ophthalmology, Department of Surgery, University of Melbourne, Australia. Electronic address:

The revolution of induced pluripotent stem cell (iPSC) technology provides a platform for development of cell therapy, disease modeling and drug discovery. Recent technological advances now allow us to reprogram a patient's somatic cells into induced pluripotent stem cells (iPSCs). Together with methods to differentiate these iPSCs into disease-relevant cell types, we are now able to model disease in vitro using iPSCs. Importantly, this represents a robust in vitro platform using patient-specific cells, providing opportunity for personalized precision medicine. Here we provide a review of advances using iPSC for drug development, and discuss the potential and limitations of iPSCs for drug discovery in neurodegenerative and ocular diseases. Emerging technologies that can facilitate the search for new drugs by assessment using in vitro disease models will also be discussed, including organoid differentiation, organ-on-chip, direct reprogramming and humanized animal models.
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http://dx.doi.org/10.1016/j.pharmthera.2017.02.026DOI Listing
September 2017

Enriched retinal ganglion cells derived from human embryonic stem cells.

Sci Rep 2016 08 10;6:30552. Epub 2016 Aug 10.

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital &Ophthalmology, Department of Surgery, the University of Melbourne, Australia.

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.
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http://dx.doi.org/10.1038/srep30552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978994PMC
August 2016

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

Invest Ophthalmol Vis Sci 2016 Jun;57(7):3470-6

Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia 2Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, Tasmania, Australia.

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1167/iovs.16-19316DOI Listing
June 2016

Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces.

Nat Mater 2016 09 27;15(9):1023-30. Epub 2016 Jun 27.

Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.

Silicon-based materials have widespread application as biophysical tools and biomedical devices. Here we introduce a biocompatible and degradable mesostructured form of silicon with multi-scale structural and chemical heterogeneities. The material was synthesized using mesoporous silica as a template through a chemical vapour deposition process. It has an amorphous atomic structure, an ordered nanowire-based framework and random submicrometre voids, and shows an average Young's modulus that is 2-3 orders of magnitude smaller than that of single-crystalline silicon. In addition, we used the heterogeneous silicon mesostructures to design a lipid-bilayer-supported bioelectric interface that is remotely controlled and temporally transient, and that permits non-genetic and subcellular optical modulation of the electrophysiology dynamics in single dorsal root ganglia neurons. Our findings suggest that the biomimetic expansion of silicon into heterogeneous and deformable forms can open up opportunities in extracellular biomaterial or bioelectric systems.
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http://dx.doi.org/10.1038/nmat4673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5388139PMC
September 2016

Study of mitochondrial respiratory defects on reprogramming to human induced pluripotent stem cells.

Aging (Albany NY) 2016 05;8(5):945-57

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital and Ophthalmology, Department of Surgery, The University of Melbourne, Melbourne, Australia.

Reprogramming of somatic cells into a pluripotent state is known to be accompanied by extensive restructuring of mitochondria and switch in metabolic requirements. Here we utilized Leber's hereditary optic neuropathy (LHON) as a mitochondrial disease model to study the effects of homoplasmic mtDNA mutations and subsequent oxidative phosphorylation (OXPHOS) defects in reprogramming. We obtained fibroblasts from a total of 6 LHON patients and control subjects, and showed a significant defect in complex I respiration in LHON fibroblasts by high-resolution respiratory analysis. Using episomal vector reprogramming, our results indicated that human induced pluripotent stem cell (hiPSC) generation is feasible in LHON fibroblasts. In particular, LHON-specific OXPHOS defects in fibroblasts only caused a mild reduction and did not significantly affect reprogramming efficiency, suggesting that hiPSC reprogramming can tolerate a certain degree of OXPHOS defects. Our results highlighted the induction of genes involved in mitochondrial biogenesis (TFAM, NRF1), mitochondrial fusion (MFN1, MFN2) and glycine production (GCAT) during reprogramming. However, LHON-associated OXPHOS defects did not alter the kinetics or expression levels of these genes during reprogramming. Together, our study provides new insights into the effects of mtDNA mutation and OXPHOS defects in reprogramming and genes associated with various aspects of mitochondrial biology.
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http://dx.doi.org/10.18632/aging.100950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931846PMC
May 2016

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.

Eur Heart J 2016 Sep 22;37(33):2586-90. Epub 2016 Apr 22.

Bruce Lefroy Centre for Genetic Health Research, Murdoch Childrens Research Institute, Flemington Road, Parkville 3052, Victoria, Australia Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville 3052, Victoria, Australia

Aims: 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.

Methods And Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1093/eurheartj/ehw160DOI Listing
September 2016

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

Cell Stem Cell 2016 Mar;18(3):307-8

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; University of Melbourne, Department of Surgery, 3002, VIC, Australia; Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Hobart, 7000, TAS, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.stem.2016.02.006DOI Listing
March 2016

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells.

Stem Cells Int 2016 14;2016:1718041. Epub 2015 Dec 14.

O'Brien Institute Department, St Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Surgery, University of Melbourne, East Melbourne, VIC 3002, Australia.

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used.
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http://dx.doi.org/10.1155/2016/1718041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691644PMC
January 2016

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

Stem Cell Rev Rep 2016 Apr;12(2):179-88

Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital; Ophthalmology, University of Melbourne, Department of Surgery, 32 Gisborne Street, East Melbourne, VIC, 3002, Australia.

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.
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http://dx.doi.org/10.1007/s12015-015-9636-2DOI Listing
April 2016