Lineage recording in human cerebral organoids

AbstractInduced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR–Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

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Lineage recording reveals dynamics of cerebral organoid regionalization

10.1101/2020.06.19.162032 ◽

2020 ◽

Author(s):

Zhisong He ◽

Tobias Gerber ◽

Ashley Maynard ◽

Akanksha Jain ◽

Rebecca Petri ◽

...

Keyword(s):

Time Course ◽

Light Sheet ◽

Brain Regions ◽

Patient Specific ◽

Light Sheet Microscopy ◽

Brain Organoid ◽

Induced Pluripotent ◽

Models Of Disease ◽

Fate Restriction ◽

Lineage Trees

Diverse regions develop within cerebral organoids generated from human induced pluripotent stem cells (iPSCs), however it has been a challenge to understand the lineage dynamics associated with brain regionalization. Here we establish an inducible lineage recording system that couples reporter barcodes, inducible CRISPR/Cas9 scarring, and single-cell transcriptomics to analyze lineage relationships during cerebral organoid development. We infer fate-mapped whole organoid phylogenies over a scarring time course, and reconstruct progenitor-neuron lineage trees within microdissected cerebral organoid regions. We observe increased fate restriction over time, and find that iPSC clones used to initiate organoids tend to accumulate in distinct brain regions. We use lineage-coupled spatial transcriptomics to resolve lineage locations as well as confirm clonal enrichment in distinctly patterned brain regions. Using long term 4-D light sheet microscopy to temporally track nuclei in developing cerebral organoids, we link brain region clone enrichment to positions in the neuroectoderm, followed by local proliferation with limited migration during neuroepithelial formation. Our data sheds light on how lineages are established during brain organoid regionalization, and our techniques can be adapted in any iPSC-derived cell culture system to dissect lineage alterations during perturbation or in patient-specific models of disease.

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Long-term effects of human induced pluripotent stem cell-derived retinal cell transplantation in Pde6b knockout rats

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00588-w ◽

2021 ◽

Author(s):

Jee Myung Yang ◽

Sunho Chung ◽

KyungA Yun ◽

Bora Kim ◽

Seongjun So ◽

...

Keyword(s):

Stem Cell ◽

Cell Transplantation ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Histological Evaluation ◽

Long Term Effects ◽

Retinal Cells ◽

Rpe Cells ◽

Induced Pluripotent

AbstractRetinal degenerative disorders, including age-related macular degeneration and retinitis pigmentosa (RP), are characterized by the irreversible loss of photoreceptor cells and retinal pigment epithelial (RPE) cells; however, the long-term effect of implanting both human induced pluripotent stem cell (hiPSC)-derived RPE and photoreceptor for retinal regeneration has not yet been investigated. In this study, we evaluated the long-term effects of hiPSC-derived RPE and photoreceptor cell transplantation in Pde6b knockout rats to study RP; cells were injected into the subretinal space of the right eyes of rats before the appearance of signs of retinal degeneration at 2–3 weeks of age. Ten months after transplantation, we evaluated the cells using fundus photography, optical coherence tomography, and histological evaluation, and no abnormal cell proliferation was observed. A relatively large number of transplanted cells persisted during the first 4 months; subsequently, the number of these cells decreased gradually. Notably, immunohistochemical analysis revealed that the hiPSC-derived retinal cells showed characteristics of both RPE cells and photoreceptors of human origin after transplantation. Functional analysis of vision by scotopic electroretinogram revealed significant preservation of vision after transplantation. Our study suggests that the transplantation of hiPSC-derived retinal cells, including RPE cells and photoreceptors, has a potential therapeutic effect against irreversible retinal degenerative diseases.

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Generation of 2.5D lung bud organoid from human induced pluripotent stem cell

Clinical Hemorheology and Microcirculation ◽

10.3233/ch-219111 ◽

2021 ◽

pp. 1-14

Author(s):

Xun Xu ◽

Yan Nie ◽

Weiwei Wang ◽

Imran Ullah ◽

Wing Tai Tung ◽

...

Keyword(s):

Single Cell ◽

Disease Modeling ◽

3D Culture ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Homogeneous Distribution ◽

Cell Seeding ◽

Differentiation Potential ◽

Induced Pluripotent ◽

Defined Culture

Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70%confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90%confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.

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Patient-Specific and Gene-Corrected Induced Pluripotent Stem Cell-Derived Cardiomyocytes Elucidate Single-Cell Phenotype of Short QT Syndrome

Circulation Research ◽

10.1161/circresaha.118.313518 ◽

2019 ◽

Vol 124 (1) ◽

pp. 66-78 ◽

Cited By ~ 10

Author(s):

Fengfeng Guo ◽

Yaxun Sun ◽

Xiaochen Wang ◽

Hao Wang ◽

Jue Wang ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Cell Phenotype ◽

Short Qt Syndrome ◽

Qt Syndrome ◽

Induced Pluripotent

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Batch effects and the effective design of single-cell gene expression studies

10.1101/062919 ◽

2016 ◽

Cited By ~ 11

Author(s):

Po-Yuan Tung ◽

John D. Blischak ◽

Chiaowen Joyce Hsiao ◽

David A. Knowles ◽

Jonathan E. Burnett ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Induced Pluripotent Stem Cell ◽

Expression Levels ◽

Amplification Bias ◽

Expression Studies ◽

Cell Gene Expression ◽

Gene Expression Studies ◽

Induced Pluripotent ◽

Gene Expression Levels

AbstractSingle cell RNA sequencing (scRNA-seq) can be used to characterize variation in gene expression levels at high resolution. However, the sources of experimental noise in scRNA-seq are not yet well understood. We investigated the technical variation associated with sample processing using the single cell Fluidigm C1 platform. To do so, we processed three C1 replicates from three human induced pluripotent stem cell (iPSC) lines. We added unique molecular identifiers (UMIs) to all samples, to account for amplification bias. We found that the major source of variation in the gene expression data was driven by genotype, but we also observed substantial variation between the technical replicates. We observed that the conversion of reads to molecules using the UMIs was impacted by both biological and technical variation, indicating that UMI counts are not an unbiased estimator of gene expression levels. Based on our results, we suggest a framework for effective scRNA-seq studies.

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Using High-Content Screening Technology as a Tool to Generate Single-Cell Patient-Derived Gene-Corrected Isogenic iPS Clones for Parkinson’s Disease Research

10.20944/preprints202005.0001.v1 ◽

2020 ◽

Author(s):

Peter A. Barbuti ◽

Paul M. Antony ◽

Bruno F.R. Santos ◽

François Massart ◽

Gérald Cruciani ◽

...

Keyword(s):

Single Cell ◽

Clonal Selection ◽

Induced Pluripotent Stem Cell ◽

Alpha Synuclein ◽

Screening Strategy ◽

High Content Screening ◽

Selection Strategy ◽

Microtiter Plates ◽

Automated Platform ◽

Induced Pluripotent

The generation of isogenic induced pluripotent stem cell (iPSC) lines using CRISPR-Cas9 technology is a technically challenging, time-consuming process with variable efficiency. Here we use fluorescence-activated cell sorting (FACS) to sort biallelic CRISPR-Cas9 edited single-cell iPS clones into high-throughput 96-well microtiter plates. We used high-content screening (HCS) technology and generated an in-house developed algorithm to select the correctly edited isogenic clones for continued expansion and validation. In our model we have gene-corrected the iPSCs of a Parkinson’s disease (PD) patient carrying the autosomal dominantly inherited heterozygous c.88G>C mutation in the SNCA gene, which leads to the pathogenic p.A30P form of the alpha-synuclein protein. Undertaking a PCR restriction-digest mediated clonal selection strategy prior to sequencing, we were able to post-sort validate each isogenic clone using a quadruple screening strategy. Subsequent transfection with mRNA encoding excision-only transposase allows for the generation of footprint-free isogenic iPSC lines. These monoclonal isogenic iPSC lines retain a normal molecular genotype, express pluripotency markers and have the ability to differentiate into the three germ layers. This combinatory approach of FACS, HCS and post-sorted restriction digestion facilitates the generation of isogenic cell lines for disease modelling to be scaled-up on an automated platform.

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Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Cells ◽

10.3390/cells10123370 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3370

Author(s):

Christina Schmid ◽

Najah Abi-Gerges ◽

Michael Georg Leitner ◽

Dietmar Zellner ◽

Georg Rast

Keyword(s):

Stem Cell ◽

Single Cell ◽

Pluripotent Stem Cell ◽

Quantitative Polymerase Chain Reaction ◽

Drug Effects ◽

Induced Pluripotent Stem Cell ◽

Ionic Currents ◽

Human Cardiomyocytes ◽

Ventricular Cells ◽

Induced Pluripotent

Subtype-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising tools, e.g., to assess the potential of drugs to cause chronotropic effects (nodal hiPSC-CMs), atrial fibrillation (atrial hiPSC-CMs), or ventricular arrhythmias (ventricular hiPSC-CMs). We used single-cell patch-clamp reverse transcriptase-quantitative polymerase chain reaction to clarify the composition of the iCell cardiomyocyte population (Fujifilm Cellular Dynamics, Madison, WI, USA) and to compare it with atrial and ventricular Pluricytes (Ncardia, Charleroi, Belgium) and primary human atrial and ventricular cardiomyocytes. The comparison of beating and non-beating iCell cardiomyocytes did not support the presence of true nodal, atrial, and ventricular cells in this hiPSC-CM population. The comparison of atrial and ventricular Pluricytes with primary human cardiomyocytes showed trends, indicating the potential to derive more subtype-specific hiPSC-CM models using appropriate differentiation protocols. Nevertheless, the single-cell phenotypes of the majority of the hiPSC-CMs showed a combination of attributes which may be interpreted as a mixture of traits of adult cardiomyocyte subtypes: (i) nodal: spontaneous action potentials and high HCN4 expression and (ii) non-nodal: prominent INa-driven fast inward current and high expression of SCN5A. This may hamper the interpretation of the drug effects on parameters depending on a combination of ionic currents, such as beat rate. However, the proven expression of specific ion channels supports the evaluation of the drug effects on ionic currents in a more realistic cardiomyocyte environment than in recombinant non-cardiomyocyte systems.

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CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

10.1101/2021.05.24.445360 ◽

2021 ◽

Author(s):

Zhengyu Ouyang ◽

Nathanael Bourgeois ◽

Eugenia Lyashenko ◽

Paige Cundiff ◽

Patrick F Cullen ◽

...

Keyword(s):

Single Cell ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Model Systems ◽

Rna Seq ◽

Cell Type ◽

Fine Grained ◽

Single Nucleus ◽

Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

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