scholarly journals Islet1 and Brn3 Expression Pattern Study in Human Retina and hiPSC-Derived Retinal Organoid

2019 ◽  
Vol 2019 ◽  
pp. 1-14
Author(s):  
Ziming Luo ◽  
Chaochao Xu ◽  
Kaijing Li ◽  
Bikun Xian ◽  
Yuchun Liu ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Ganglion Cells ◽  
Induced Pluripotent Stem Cell ◽  
Positive Cone ◽  
Adult Retina ◽  
Retina Development ◽  
Human Adult ◽  
A Cell ◽  
Induced Pluripotent ◽  
Fetal Retina

This study was conducted to determine the dynamic Islet1 and Brn3 (POU4F) expression pattern in the human fetal retina and human-induced pluripotent stem cell- (hiPSC-) derived retinal organoid. Human fetal eyes from 8 to 27 fetal weeks (Fwks), human adult retina, hiPSC-derived retinal organoid from 7 to 31 differentiation weeks (Dwks), and rhesus adult retina were collected for cyrosectioning. Immunofluorescence analysis showed that Islet1 was expressed in retinal ganglion cells in the fetal retina, human adult retina, and retinal organoids. Unexpectedly, after Fwk 20, Brn3 expression gradually decreased in the fetal retina. In the midstage of development, Islet1 was detected in bipolar and developing horizontal cells. As the photoreceptor developed, the Islet1-positive cone precursors gradually became Islet1-negative/S-opsin-positive cones. This study highlights the distinguishing characteristics of Islet1 dynamic expression in human fetal retina development and proposes more concerns which should be taken regarding Brn3 as a cell-identifying marker in mature primate retina.

scholarly journals Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches

Genes ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 112
Author(s):  
Marta García-López ◽  
Joaquín Arenas ◽  
M. Esther Gallardo
Keyword(s):  
Stem Cell ◽  
Retinal Ganglion Cells ◽  
Pluripotent Stem Cell ◽  
Ganglion Cells ◽  
Genetic Disorders ◽  
Three Dimensional ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Mechanism ◽  
Retinal Ganglion ◽  
Induced Pluripotent

Inherited optic neuropathies share visual impairment due to the degeneration of retinal ganglion cells (RGCs) as the hallmark of the disease. This group of genetic disorders are caused by mutations in nuclear genes or in the mitochondrial DNA (mtDNA). An impaired mitochondrial function is the underlying mechanism of these diseases. Currently, optic neuropathies lack an effective treatment, and the implementation of induced pluripotent stem cell (iPSC) technology would entail a huge step forward. The generation of iPSC-derived RGCs would allow faithfully modeling these disorders, and these RGCs would represent an appealing platform for drug screening as well, paving the way for a proper therapy. Here, we review the ongoing two-dimensional (2D) and three-dimensional (3D) approaches based on iPSCs and their applications, taking into account the more innovative technologies, which include tissue engineering or microfluidics.

Induced Pluripotent Stem Cell Elimination in a Cell Sheet by Methionine-Free and 42°C Condition for Tumor Prevention

2018 ◽  
Vol 24 (10) ◽  
pp. 605-615 ◽  
Author(s):  
Katsuhisa Matsuura ◽  
Kyoji Ito ◽  
Nobuaki Shiraki ◽  
Shoen Kume ◽  
Nobuhisa Hagiwara ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cell Sheet ◽  
Induced Pluripotent Stem Cell ◽  
Tumor Prevention ◽  
A Cell ◽  
Induced Pluripotent

scholarly journals Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types

2019 ◽  
Author(s):  
Phuong T. Lam ◽  
Christian Gutierrez ◽  
Katia Del Rio-Tsonis ◽  
Michael L. Robinson
Keyword(s):  
Fluorescent Protein ◽  
Ganglion Cells ◽  
Fluorescent Proteins ◽  
Retinal Development ◽  
Retinal Disease ◽  
Cell Types ◽  
Retinal Cell ◽  
Therapeutic Drug ◽  
Retina Development ◽  
Induced Pluripotent

ABSTRACTEarly in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retina progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist. Here, we describe a CRISPR/Cas9 gene editing strategy to generate a triple transgenic reporter hiPSC line (PGP1) that utilizes the endogenous VSX2, BRN3b, and RCVRN promoters to specifically express fluorescent proteins (Cerulean in NRPs, eGFP in RGCs and mCherry in PRs) without disrupting the function of the endogenous alleles. Retinal organoid formation from the PGP1 line demonstrated the ability of the edited cells to undergo normal retina development while exhibiting appropriate fluorescent protein expression consistent with the onset of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types. The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.

scholarly journals Cardiac Progenitor Cells from Stem Cells: Learning from Genetics and Biomaterials

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1536 ◽  
Author(s):  
Sara Barreto ◽  
Leonie Hamel ◽  
Teresa Schiatti ◽  
Ying Yang ◽  
Vinoj George
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Genetic Factors ◽  
Induced Pluripotent Stem Cell ◽  
Significant Shift ◽  
Cardiac Progenitor Cells ◽  
Recent Developments ◽  
A Cell ◽  
Induced Pluripotent

Cardiac Progenitor Cells (CPCs) show great potential as a cell resource for restoring cardiac function in patients affected by heart disease or heart failure. CPCs are proliferative and committed to cardiac fate, capable of generating cells of all the cardiac lineages. These cells offer a significant shift in paradigm over the use of human induced pluripotent stem cell (iPSC)-derived cardiomyocytes owing to the latter’s inability to recapitulate mature features of a native myocardium, limiting their translational applications. The iPSCs and direct reprogramming of somatic cells have been attempted to produce CPCs and, in this process, a variety of chemical and/or genetic factors have been evaluated for their ability to generate, expand, and maintain CPCs in vitro. However, the precise stoichiometry and spatiotemporal activity of these factors and the genetic interplay during embryonic CPC development remain challenging to reproduce in culture, in terms of efficiency, numbers, and translational potential. Recent advances in biomaterials to mimic the native cardiac microenvironment have shown promise to influence CPC regenerative functions, while being capable of integrating with host tissue. This review highlights recent developments and limitations in the generation and use of CPCs from stem cells, and the trends that influence the direction of research to promote better application of CPCs.

scholarly journals Embryoid Body-Explant Outgrowth Cultivation from Induced Pluripotent Stem Cells in an Automated Closed Platform

2016 ◽  
Vol 2016 ◽  
pp. 1-7
Author(s):  
Hiroshi Tone ◽  
Saeko Yoshioka ◽  
Hirokazu Akiyama ◽  
Akira Nishimura ◽  
Masaki Ichimura ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Expansion ◽  
Embryoid Body ◽  
Induced Pluripotent Stem Cell ◽  
Explant Culture ◽  
Model Case ◽  
System P ◽  
Closed Cell ◽  
A Cell ◽  
Induced Pluripotent

Automation of cell culture would facilitate stable cell expansion with consistent quality. In the present study, feasibility of an automated closed-cell culture system “P 4C S” for an embryoid body- (EB-) explant outgrowth culture was investigated as a model case for explant culture. After placing the induced pluripotent stem cell- (iPSC-) derived EBs into the system, the EBs successfully adhered to the culture surface and the cell outgrowth was clearly observed surrounding the adherent EBs. After confirming the outgrowth, we carried out subculture manipulation, in which the detached cells were simply dispersed by shaking the culture flask, leading to uniform cell distribution. This enabled continuous stable cell expansion, resulting in a cell yield of 3.1 × 107. There was no evidence of bacterial contamination throughout the cell culture experiments. We herewith developed the automated cultivation platform for EB-explant outgrowth cells.

scholarly journals Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy

2021 ◽  
Vol 15 ◽  
Author(s):  
Marcela Garita-Hernandez ◽  
Antoine Chaffiol ◽  
Laure Guibbal ◽  
Fiona Routet ◽  
Hanen Khabou ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Therapy ◽  
Transgene Expression ◽  
Induced Pluripotent Stem Cell ◽  
Fluorescent Reporter ◽  
Human Organ ◽  
Gene Therapies ◽  
Transcriptional Changes ◽  
A Cell ◽  
Induced Pluripotent

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.

scholarly journals Single Cell Sequencing of Induced Pluripotent Stem Cell Derived Retinal Ganglion Cells (iPSC-RGC) Reveals Distinct Molecular Signatures and RGC Subtypes

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 2015
Author(s):  
Harini V. Gudiseva ◽  
Vrathasha Vrathasha ◽  
Jie He ◽  
Devesh Bungatavula ◽  
Joan M. O’Brien ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Single Cell ◽  
Retinal Ganglion Cells ◽  
Pluripotent Stem Cell ◽  
Ganglion Cells ◽  
Induced Pluripotent Stem Cell ◽  
Retinal Ganglion ◽  
Single Cell Sequencing ◽  
Induced Pluripotent

We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3’ V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.

scholarly journals Human iPSC-derived astrocytes transplanted into the mouse brain display three morphological responses to amyloid-β plaques

2020 ◽  
Author(s):  
Pranav Preman ◽  
Julia TCW ◽  
Sara Calafate ◽  
An Snellinx ◽  
Maria Alfonso-Triguero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Brain ◽  
Morphological Changes ◽  
Amyloid Β ◽  
Induced Pluripotent Stem Cell ◽  
Immunodeficient Mice ◽  
A Cell ◽  
Induced Pluripotent ◽  
Chimeric Model

ABSTRACTBackgroundIncreasing evidence for a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer’s disease comes from molecular studies in rodent models. However, these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression.MethodsTo address these challenges, we established an approach to study human astroglia within the context of the mouse brain by transplanting human induced pluripotent stem cell (hiPSC)-derived glia progenitors into neonatal brains of immunodeficient mice.ResultsXenografted (hiPSC)-derived glia progenitors differentiate into astrocytes that integrate functionally within the mouse host brain and mature in a cell-autonomous way retaining human-specific morphologies, unique features and physiological properties. In Alzheimer’s chimeric brains, transplanted hiPSC-derived astrocytes respond to the presence of amyloid plaques with various morphological changes that seem independent of the APOE allelic background.ConclusionIn sum, this chimeric model has great potential to analyze the role of patient-derived and genetically modified astroglia in Alzheimer’s disease.

scholarly journals Correction of a pathogenic mutation in iPSCs derived from a patient with Christianson syndrome using CRISPR/Cas9 genome editing

2021 ◽  
Author(s):  
Li Ma ◽  
Qing Wu ◽  
Michael Schmidt ◽  
Eric M Morrow
Keyword(s):  
Reference Genome ◽  
Cell Model ◽  
Control Cell ◽  
Induced Pluripotent Stem Cell ◽  
Pathogenic Mutation ◽  
Loss Of Function ◽  
Homology Directed Repair ◽  
A Cell ◽  
Translational Studies ◽  
Induced Pluripotent

SLC9A6 (also termed NHE6) encodes the endosomal Na+/H+ exchanger 6 (NHE6). Pathogenic, loss-of-function mutations in NHE6 cause the X-linked neurogenetic disorder Christianson syndrome (CS). We developed induced pluripotent stem cell (iPSC) lines derived from a patient with CS and from a biologically related control. The patient with CS contained the nonsense mutation c.1569G>A (p.(W523X)), which caused a significant reduction in NHE6 mRNA and a lack of detectable NHE6 protein in CS iPSCs in comparison to control iPSCs. To establish a cell model for study of CS with an isogenic control, we corrected the c.1569G>A mutation to the NHE6 reference genome sequence using CRISPR/Cas9-mediated homology directed repair knock-in methodology. Multiple subclonal lines were generated, and notably, NHE6 protein was expressed in all analyzed c.1569G>A (p.(W523X)) genome-corrected iPSC lines. This CS iPSC model together with the associated biologically related and isogenic control cell lines will serve as a valuable resource for both basic and translational studies in CS.

scholarly journals Human iPSC-derived astrocytes transplanted into the mouse brain undergo morphological changes in response to amyloid-β plaques

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Pranav Preman ◽  
Julia TCW ◽  
Sara Calafate ◽  
An Snellinx ◽  
Maria Alfonso-Triguero ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Morphological Changes ◽  
Amyloid Β ◽  
Induced Pluripotent Stem Cell ◽  
Functional Studies ◽  
Physiological Properties ◽  
A Cell ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
Human Specific

Abstract Background Increasing evidence for a direct contribution of astrocytes to neuroinflammatory and neurodegenerative processes causing Alzheimer’s disease comes from molecular and functional studies in rodent models. However, these models may not fully recapitulate human disease as human and rodent astrocytes differ considerably in morphology, functionality, and gene expression. Results To address these challenges, we established an approach to study human astrocytes within the mouse brain by transplanting human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors into neonatal brains. Xenografted hiPSC-derived astrocyte progenitors differentiated into astrocytes that integrated functionally within the mouse host brain and matured in a cell-autonomous way retaining human-specific morphologies, unique features, and physiological properties. In Alzheimer´s chimeric brains, transplanted hiPSC-derived astrocytes responded to the presence of amyloid plaques undergoing morphological changes that seemed independent of the APOE allelic background. Conclusions In sum, we describe here a promising approach that consist of transplanting patient-derived and genetically modified astrocytes into the mouse brain to study human astrocyte pathophysiology in the context of Alzheimer´s disease.

Sign in / Sign up

Export Citation Format

Share Document