A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles

Zhang, Joe Z.; Termglinchan, Vittavat; Shao, Ning; Itzhaki, Ilanit; Li, Chun; Ma, Ning; Tian, Lei; Wang, Vicky Y.; Chang, Alex Chia Yu; Guo, Hongchao; Kitani, Tomoya; Wu, Haodi; Lam, Chi Keung; Kodo, Kazuki; Sayed, Nazish; Blau, Helen M.; Wu, Joseph C.

doi:10.1016/j.stem.2019.02.015

Cited by 107 publications

(89 citation statements)

References 39 publications

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“…Wildtype (WT) , GATA6 +/- , GATA6 -/- , and GATA6 R456G/R456G hiPSCs were processed for differentiation into cardiomyocytes (hiPSC-CMs) by modulation of the Wnt signaling pathway and subsequent metabolic selection via glucose deprivation ( Sharma et al, 2018b ). This protocol yields hiPSC-CMs that express first and second heart field genes ( Zhang et al, 2019 ). As previous single-cell RNA- sequencing (RNA-Seq) of cardiomyocytes isolated from developing mouse hearts ( DeLaughter et al, 2016 ) identified peak Gata6 expression in cardiac progenitors and early cardiomyocytes, we studied hiPSC-CMs at differentiation days 4 and 8, which approximate these in vivo developmental stages.…”

Section: Resultsmentioning

confidence: 99%

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

Sharma

Wasson

Willcox

et al. 2020

eLife

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Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. Loss of function variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2 and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

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Section: Resultsmentioning

confidence: 99%

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

Sharma

Wasson

Willcox

et al. 2020

eLife

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“…Generation of SAN-like pacemaker cells from hPSCs has been achieved by mimicking the developmental steps known to be involved in SAN specification in vivo. As already described above, by using a standard CM differentiation protocol, Zhang and colleagues identified a subpopulation of NKX2.5 − /TBX5 + cells that further differentiated into functional SAN-like CMs, expressing SHOX2 , TBX3 and TBX18 [ 29 ], reinforcing the idea that SAN-like CMs arise from an NKX2.5 − population. Recently, Protze and colleagues [ 45 ] reported a directed differentiation protocol for generation of SAN-like CMs from hPSCs ( Figure 3 B).…”

Section: Cardiovascular Lineages Specification From Hpscs—lessons mentioning

confidence: 77%

“…Moving further along in the identification of the origin of the different subpopulations of CMs, a more recent work from Zhang and colleagues, using in vitro differentiation of hPSCs into cardiac progenitor cells [ 29 ], showed that NKX2.5 + /TBX5 + cells represent an FHF-like derived population, which predominantly differentiates into ventricular-like CMs that are genetically and functionally similar to left ventricular CMs, expressing HAND1 and KCNJ2 markers [ 29 ]. They also identified CORIN as a specific cell surface marker for the NKX2.5 + /TBX5 + subpopulation, enabling in this way the isolation of left ventricular CMs from a mixed population of hPSC-derived CMs.…”

Section: Cardiovascular Lineages Specification From Hpscs—lessons mentioning

confidence: 99%

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

Branco

Diogo

2020

Bioengineering

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The knowledge acquired throughout the years concerning the in vivo regulation of cardiac development has promoted the establishment of directed differentiation protocols to obtain cardiomyocytes (CMs) and other cardiac cells from human pluripotent stem cells (hPSCs), which play a crucial role in the function and homeostasis of the heart. Among other developments in the field, the transition from homogeneous cultures of CMs to more complex multicellular cardiac microtissues (MTs) has increased the potential of these models for studying cardiac disorders in vitro and for clinically relevant applications such as drug screening and cardiotoxicity tests. This review addresses the state of the art of the generation of different cardiac cells from hPSCs and the impact of transitioning CM differentiation from 2D culture to a 3D environment. Additionally, current methods that may be employed to generate 3D cardiac MTs are reviewed and, finally, the adoption of these models for in vitro applications and their adaptation to medium- to high-throughput screening settings are also highlighted.

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“…This approach would be applicable to other human cell models, such as iPSC and ES-derived organoids. Although in these systems successful knock-in via conventional HDR approaches has already repeatedly been achieved [47][48][49] , use of an homology-independent-based strategy may increase the efficiency of knock-in generation.…”

Section: Discussionmentioning

confidence: 99%

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

Artegiani

Hendriks

Beumer

et al. 2020

Preprint

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CRISPR/Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences in human organoids awaits robust knock-in approaches. Here, we describe CRISPR/Cas9-mediated Homologyindependent Organoid Transgenesis (CRISPR-HOT), which allows efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences at desired loci, without the necessity to inactivate TP53 in untransformed cells, previously used to increase HDR-mediated knock-in. CRISPR-HOT was employed to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter labelling the mitotic spindle by tagged tubulin and the cell membrane by tagged E-cadherin uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed TP53 involvement in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.

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A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles

Cited by 107 publications

References 39 publications

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

From Human Pluripotent Stem Cells to 3D Cardiac Microtissues: Progress, Applications and Challenges

Fast and efficient generation of knock-in human organoids using homology-independent CRISPR/Cas9 precision genome editing

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