Biology subjects

Kwon, C.

Publications and source records attributed to Kwon, C..

At least 19 recordsLinked to original sources

bioRxiv2026

CMAC: A deep learning framework for absolute cardiomyocyte transcriptional age estimation

Cardiomyocyte maturation spans embryogenesis through postnatal life and involves coordinated transcriptional transitions that are disrupted in disease and incompletely recapitulated by stem cell-derived cardiomyocytes. However, no unified framework exists for assigning absolute transcriptional maturation age across datasets, platforms, and experimental conditions. Here, we developed CMAC (Cardiomyocyte Maturation Age Clock), integrating 18 murine single-cell RNA-sequencing datasets spanning embryonic and postnatal timepoints, into a batch-invariant probabilistic atlas using single cell variational inference (scVI) with a 100-dimensional latent representation. Analysis of the developmental trajectory revealed highly non-uniform transcriptional change, with 63.9% of the total transcriptional journey completed by birth and the neonatal transition representing the largest single transcriptional step. An XGBoost regressor trained on the CMAC latent space predicted absolute chronological age with a leave-one-dataset-out mean absolute error (MAE) of 4.09 days (median 3.27 days), Pearson r = 0.903, and Spearman {rho} = 0.908 across 18 datasets and six sequencing platforms, outperforming linear regression (MAE = 6.18 days) and PCA-based representations (MAE = 6.35 days). In an independent P14 cohort comprising 98,163 cardiomyocytes from six biological replicates, CMAC achieved a mouse-level MAE of 0.33 days, with all six mice predicted within 1.1 days of chronological age. Application of CMAC to a previously published PGC1/{beta} double-knockout model with established delayed cardiomyocyte maturation independently recapitulated this phenotype, quantifying maturation deficits of 4.77 to 8.60 days across P7 to P28, with the largest deficit at P14. Together, these results establish CMAC as a quantitative, deep learning framework for assigning absolute transcriptional maturation age and quantifying deviations from normal cardiomyocyte development.

genomics↗

bioRxiv2026

SEME enables programmable transcript reconstruction through synthetic microexon incorporation

Precise correction of pathogenic mutations remains challenging for therapeutic applications because genome editing within protein-coding exons can generate unintended insertion-deletion byproducts that disrupt coding integrity and protein function. To address this limitation, we developed the Spliceable Editable Microexon Element (SEME), an intron-targeting platform that enables programmable microexon incorporation through endogenous splicing. In induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), SEME corrected aberrant splicing caused by the dilated cardiomyopathy-associated FLNC c.2003A>G variant by restoring the five nucleotides missing from exon 12, thereby recovering filamin C expression. Together, these findings establish programmable microexon incorporation as a proof-of-concept strategy for correcting diverse disease-causing transcript defects.

bioengineering↗

bioRxiv2026

Splicing Factor SF3B2 Regulates Cardiomyocyte Calcium Handling Through Alternative Splicing of Cardiac Ion Channel Genes

BackgroundAlternative splicing is a critical determinant of protein diversity in the heart, where it drives the postnatal functional maturation of cardiomyocytes and specifies the ion-channel and calcium-handling isoforms required for mature contractile function; dysregulated splicing programs have in turn been implicated in cardiomyopathies and arrhythmias. However, the splicing regulators that control cardiomyocyte calcium handling remain largely unknown. ObjectiveWe systematically screened 276 splicing factor genes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to identify regulators of calcium handling, and we selected SF3B2, a core U2 snRNP spliceosome component, for detailed follow-up based on complex-level enrichment and prior identification by Murphy et al. 2021 [1]. MethodsA high-throughput siRNA screen targeting 276 splicing factor genes was performed in hiPSC-CMs using 384-well calcium transient imaging. SF3B2 knockdown was followed by deep bulk RNA-seq analysis (n = 3 per group) for differential gene expression with DESeq2 and alternative splicing quantification with rMATS. RNA immunoprecipitation sequencing (RIP-seq) using an epitope-tagged SF3B2 construct was performed to identify direct mRNA binding targets of SF3B2. ResultsThe screen identified multiple U2 snRNP components, including SF3A2, SF3B1, and SF3B4--as regulators of calcium transient duration. SF3B2 was previously identified as a regulator of contractility in this screen [1], and the enrichment of its complex partners above the significance threshold in the current CTD75 analysis supported its selection for follow-up characterization. SF3B2 knockdown resulted in 2,683 differentially expressed genes (adjusted p < 0.05), with downregulated genes enriched in cell cycle and DNA replication pathways. Alternative splicing analysis revealed significant changes across all five rMATS event types in 36 genes within the cardiac muscle cell action potential involved in contraction gene ontology term (GO:0086002), including calcium channel (CACNA1C, CACNA1D, CACNA2D1), potassium channel (KCNH2, KCNQ1), and sodium channel (SCN5A) genes, although the large number of affected genes is consistent with broad spliceosomal perturbation and a formal enrichment test would be needed to determine whether cardiac action potential genes are preferentially affected. Integration of RIP-seq data (597 SF3B2-enriched transcripts, FDR < 0.05) with splicing analysis identified CACNA2D1, which encodes an auxiliary subunit of L-type calcium channels, as both directly bound by SF3B2 and alternatively spliced upon knockdown. ConclusionThese findings identify SF3B2 as a regulator of cardiomyocyte calcium handling and suggest that SF3B2-dependent missplicing of CACNA2D1 may link core spliceosome function to the splicing programs underlying cardiomyocyte functional maturation. Significance StatementThis study provides the first systematic functional screen of splicing factors in cardiomyocyte calcium handling and identifies SF3B2, a U2 snRNP subunit, as a regulator of cardiac ion channel splicing. Transcriptomic, splicing, and RNA binding data converge on CACNA2D1, identifying a route by which a core splicing factor shapes cardiac contractility, extending cardiac splicing regulation beyond the accessory RBPs studied to date.

developmental biology↗

bioRxiv2026

SureQuant IS-PRM Enables Cross-Species Targeted Quantification of Retinoid Metabolism and Signaling Proteins in the Heart

Retinoic acid signaling is critical for cardiac development and homeostasis. Dysregulation of all-trans retinoic acid metabolism contributes to vascular atherogenesis, restenosis, calcification, and heart failure. Therefore, assessment of proteins involved in retinoid metabolism and signaling has gained interest for identifying potential biomarkers and therapeutic targets in cardiovascular disease. However, quantifying these proteins remains challenging due to limitations of antibody-based methods. We developed a targeted proteomics approach using SureQuant internal standard-triggered parallel reaction monitoring mass spectrometry to profile these proteins. We designed a panel of 80 stable isotope-labeled (heavy) peptides representing proteins involved in retinoid signaling and metabolism, with sequences applicable to human samples and conserved across multiple species. Survey experiments using directed data-dependent acquisition on the Orbitrap Fusion Lumos mass spectrometer determined precursor and product ion masses for each heavy peptide, which were programmed into the SureQuant method for continuous monitoring. Upon detection of these heavy internal standards, the instrument transitions to a targeted PRM acquisition mode in which repeated high-resolution MS/MS spectra of both endogenous (light) and heavy peptides are acquired. Using this method, retinoid pathway-associated proteins were quantified to as low as 10 attomoles for selected targets across multiple tissues and developmental stages. Distinct tissue-specific retinoid metabolic networks were identified across lung, liver, retinal cell lines and cardiac tissues. Developmental profiling of mouse and rat hearts revealed remodeling of retinoid pathway proteins from embryonic to postnatal and adult stages, suggesting a functional transition from retinoid-driven cardiac development toward maintenance of retinoid homeostasis in the mature heart.

biochemistry↗

bioRxiv2026

In Vivo Bioincubation Promotes Maturation of Human iPSC-Derived Cardiomyocytes in Neonatal Rat and Pig Hearts

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for cardiac regenerative medicine and disease modeling. However, hiPSC-CMs generated through conventional in vitro differentiation exhibit immature, fetal-like phenotypes. While in vivo bioincubation in neonatal rodent hearts promotes hiPSC-CM maturation toward adult-like phenotypes, studies in large animal models remain limited, particularly with detailed morphological characterization. In this study, we investigated bioincubation of fluorescently labeled hiPSC-CMs in both neonatal rat and pig hearts. Human iPSCs were differentiated into cardiomyocytes expressing GFP or RFP reporters and subsequently injected intramyocardially into neonatal rats (GFP-labeled) and pigs (RFP-labeled). After 4-8 weeks of bioincubation, fluorescent hiPSC-CMs were isolated using large-particle fluorescence-activated cell sorting (COPAS), which preserves cellular morphology of adult-like cardiomyocytes. Immunostaining for cardiac troponin T revealed well-organized sarcomeric structures in multinucleated hiPSC-CMs. Bioincubated hiPSC-CMs displayed rod-shaped morphology with binucleation, characteristic features of mature adult cardiomyocytes. Quantitative analysis demonstrated that bioincubated hiPSC-CMs from rat hearts exhibited sarcomere length and cell circularity comparable to native rat adult cardiomyocytes, though with higher intra-cellular variability in sarcomere organization. Histological examination confirmed successful engraftment of RFP-positive hiPSC-CMs within pig myocardium, with engrafted cells also displaying mature adult-like features. These findings provide critical proof-of-concept data for bioincubation in large animal models and support further investigation for disease modeling, drug screening, and regenerative cell therapies. SIGNIFICANCE STATEMENTHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer tremendous potential for cardiac disease modeling and regenerative therapies, but their clinical application is limited by their immature characteristics. Here we show that in vivo bioincubation in neonatal rat hearts enables hiPSC-CMs to achieve structural maturity, exhibiting features of adult cardiomyocytes, including organized sarcomeres, rod-shaped morphology, and multinucleation. We further provided proof-of-concept evidence for engraftment in neonatal pig hearts for maturation, supporting feasibility in large animal models. The use of large-particle cell sorting enables recovery of intact, adult-sized cardiomyocytes for subsequent analysis. These findings establish a practical and scalable platform for generating structurally mature human cardiomyocytes through in vivo bioincubation.

developmental biology↗

bioRxiv2026

Muscle Loss as a Foundational Step in the Development and Evolution of the Turtle Shell

Modern biodiversity is built on a series of disparate body plans whose origins are obscured by deep time. Our most direct source of clarifying data is the fossilized skeleton, where morphology reflects an evolving functionality realized through the development of associated tissues. We apply this dualistic perspective to the shelled body plan of turtles whose Paleozoic initiation is marked by a derived relationship between ribs and dermis (Lyson & Bever, 2020). Current developmental models remain in conflict with an increasingly informative fossil record, suggesting critical steps remain unrecognized. Here we explore the hypothesis that the breakdown of rib-spanning muscles--an evolutionary transformation mirrored in embryogenesis--is one such step. Multi-modal imaging of turtle embryos, including a novel application of histology-based deep learning (Kiemen et al., 2022; Matos-Romero et al., 2025; Forjaz et al., 2026), establishes intercostal muscle degradation as preceding turtle-specific rib development and highlights the heuristic power of 3D, whole-embryo analysis (Forjaz et al., 2026). Quantified divergence from mouse pinpoints the timing and tempo of this organized, apoptotic breakdown. Initial evidence suggests an associated non-pathological inflammatory response, which has been shown capable of driving evolutionarily stable hyperossification (Rashid et al., 2023). These patterns support trunk muscles as a critical signalling centre whose ontogenetic loss set the phylogenetic stage for a morphogenetic transformation remarkable in a non-metamorphic species.

evolutionary biology↗

bioRxiv2025

In vivo bioreactor matures iPSC-CMs for MLP disease modeling

Muscle LIM protein (MLP) is a critical regulator of cardiomyocytes (CMs) cytoarchitecture, and its deficiency results in late-onset dilated cardiomyopathy (DCM) in both mice and human. However, recapitulating this phenotype in vitro using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) has been challenging largely due to their immature state. Here, we generated MLP knockout (MLP-KO) mouse iPSCs and differentiated them into CMs. We then employed both conventional in vitro and in vivo transplantation approach using immunocompromised rat hearts to promote cardiomyocytes maturation. Our results showed that while in vitro-matured MLP-KO iPSC-CMs failed to exhibit disease phenotypes, in vivo-matured MLP-KO iPSC-CMs successfully recapitulated the hallmarks of DCM, including disrupted sarcomeric architecture and upregulation of atrial natriuretic peptide (ANP), closely mirroring disease progression observed in MLP-deficient mice. These findings demonstrate that the in vivo maturation environment is essential for the maturation of iPSC-derived cardiomyocytes to better model genetic cardiac diseases like DCM and provide valuable insights for future therapeutic strategies.

bioengineering↗

bioRxiv2024

Mesendodermal cells fail to contribute to heart formation following blastocyst injection

Blastocyst complementation offers an opportunity for generating transplantable whole organs from donor sources. Pluripotent stem cells (PSCs) have traditionally served as the primary donor cells due to their ability to differentiate into any type of body cell. However, the use of PSCs raises ethical concerns, particularly regarding their uncontrollable differentiation potential to undesired cell lineages such as brain and germline cells. To address this issue, various strategies have been explored, including the use of genetically modified PSCs with restricted lineage potential or lineage-specified progenitor cells as donors. In this study, we tested whether nascent mesendodermal cells (MECs), which appear during early gastrulation, can be used as donor cells. To do this, we induced Bry-GFP+ MECs from mouse embryonic stem cells (ESCs) and introduced them into the blastocyst. While donor ESCs gave rise to various regions of embryos, including the heart, Bry-GFP+ MECs failed to contribute to the host embryos. This finding suggests that MECs, despite being specified from PSCs within a few days, lack the capacity to assimilate into the developing embryo.

developmental biology↗

bioRxiv2023

False positive glycopeptide identification via in-FAIMS fragmentation

High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates glycopeptides in the gas phase prior to mass spectrometry (MS) analysis, thus offering the potential to analyze glycopeptides without prior enrichment. Several studies have demonstrated the ability of FAIMS to enhance glycopeptide detection but have primarily focused on N-glycosylation. Here, we evaluated FAIMS for O-glycoprotein and mucin-domain glycoprotein analysis using samples of varying complexity. We demonstrated that FAIMS was useful in increasingly complex samples, as it allowed for the identification of more glycosylated species. However, during our analyses, we observed a phenomenon called "in FAIMS fragmentation" (IFF) akin to in source fragmentation but occurring during FAIMS separation. FAIMS experiments showed a 2-5-fold increase in spectral matches from IFF compared to control experiments. These results were also replicated in previously published data, indicating that this is likely a systemic occurrence when using FAIMS. Our study highlights that although there are potential benefits to using FAIMS separation, caution must be exercised in data analysis because of prevalent IFF, which may limit its applicability in the broader field of O-glycoproteomics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542648v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@d94300org.highwire.dtl.DTLVardef@1bdc6dforg.highwire.dtl.DTLVardef@4b8ea8org.highwire.dtl.DTLVardef@82beed_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of biological functions. In particular, the T cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, -3, -4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in terms of glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamics (MD) simulations of TIM-3 and -4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

cancer biology↗

bioRxiv2022

Platform-Agnostic CellNet (PACNet) enables cross-study meta-analysis of cell fate engineering protocols

The optimization of cell fate engineering protocols requires evaluating their fidelity, efficiency, or both. We previously adopted CellNet, a computational tool to quantitatively assess the transcriptional fidelity of engineered cells and tissues as compared to their in vivo counterparts based on bulk RNA-Seq. However, this platform and other similar approaches are sensitive to experimental and analytical aspects of transcriptomics methodologies. This makes it challenging to capitalizing on the expansive, publicly available sets of transcriptomic data that reflect the diversity of cell fate engineering protocols. Here, we present Platform-Agnostic CellNet (PACNet), which extends the functionality of CellNet by enabling the assessment of transcriptional profiles in a platform-agnostic manner, and by enabling the comparison of user-supplied data to panels of engineered cell types from state-of-the-art protocols. To demonstrate the utility of PACNet, we evaluated a range of cell fate engineering protocols for cardiomyocytes and hepatocytes. Through this analysis, we identified the best-performing methods, characterized the extent of intra-protocol and inter-lab variation, and identified common off-target signatures, including a surprising neural and neuroendocrine signature in primary liver-derived organoids. Finally, we made our tool accessible as a user-friendly web application that allows users to upload their own transcriptional profiles and assess their protocols relative to our database of reference engineered samples. Highlights* The development of Platform-Agnostic CellNet (PACNet) that classifies engineered cell populations from transcriptome data regardless of profiling method or transcript abundance estimation method * PACNet enables cross-study comparisons of cell fate engineering protocols * Comparison of cardiomyocyte engineering protocols emphasizes metabolic selection as a key step in achieving a strong cardiomyocyte fate. * PACNet identifies an unexpected off-target neural and neuroendocrine signature in primary liver-derived organoids. eTOC BlurbCahan and colleagues created a computational resource, PACNet, which evaluates the fidelity of cell engineering expression profiles in a platform-agnostic manner to facilitate cross-protocol benchmarking. Examining state-of-the-field cardiomyocyte and hepatocyte derivation protocols, they identified that two techniques in cardiomyocyte engineering best increase cardiac identity and that an off-target neural/neuroendocrine signature in primary liver-derived organoids may reflect a cholangiopathic signature. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/506886v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@e4e985org.highwire.dtl.DTLVardef@9f485borg.highwire.dtl.DTLVardef@1475d0borg.highwire.dtl.DTLVardef@2d8c31_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

A Mouse Model with Complete Penetrance for Atrioventricular Septal Defect/Complete AV Canal

Complete Atrioventricular Septal Defect/complete AV canal (AVSD/ cAVC) occurs via defective formation of the Dorsal Mesenchymal Protrusion (DMP) which grows from the interior dorsal wall of the fetal heart. The DMP is derived from cells of the second heart field (SHF). AVSD occurs in about 1/10,000 births in the general population, but is 2000 times more prevalent in individuals with Down syndrome (DS). The low penetrance of AVSD in the general population remains a fundamental challenge to investigation of the genetic and developmental basis for this condition. Analysis of the etiology of this condition in mouse models is limited by the fact that the developmental events producing the DMP begin around embryonic day 9 (E9) but AVSD cannot be diagnosed until E14, and no model has been described previously in which AVSD occurs in 100% of embryos. We describe a trisomic mouse DS model with conditional mutations in the SHH pathway that produces AVSD with 100% penetrance. This new mouse model can serve as an important tool to understand the mechanisms underlying AVSD pathogenesis.

developmental biology↗

bioRxiv2021

Noncanonical Notch signals have opposing roles during cardiac development

The Notch pathway is an ancient intercellular signaling system with crucial roles in numerous cell-fate decision processes across species. While the canonical pathway is activated by ligand-induced cleavage and nuclear localization of membrane-bound Notch, Notch can also exert its activity in a ligand/transcription-independent fashion, which is conserved in Drosophila, Xenopus, and mammals. However, the noncanonical role remains poorly understood in in vivo processes. Here we show that increased levels of the Notch intracellular domain (NICD) in the early mesoderm inhibit heart development, potentially through impaired induction of the second heart field (SHF), independently of the transcriptional effector RBP-J. Similarly, inhibiting Notch cleavage, shown to increase noncanonical Notch activity, suppressed SHF induction in embryonic stem cell (ESC)-derived mesodermal cells. In contrast, NICD overexpression in late cardiac progenitor cells lacking RBP-J resulted in an increase in heart size. Our study suggests that noncanonical Notch signaling has stagespecific roles during cardiac development.

developmental biology↗

bioRxiv2021

Cross-organ transcriptomic comparison reveals universal factors during maturation

Various cell types can be derived from stem cells. However, these cells are immature and do not match their adult counterparts in functional capabilities, limiting their use in disease modeling and cell therapies. Thus, it is crucial to understand the mechanisms of maturation in vivo. However, it is unknown if there are genes and pathways conserved across organs during maturation. To address this, we performed a time-series analysis of the transcriptome of the mouse heart, brain, liver, and kidney and analyzed their trajectories over time. In addition, gene regulatory networks were reconstructed to determine overlapping expression patterns. Based on these, we identified commonly upregulated and downregulated pathways across all four organs. Key upstream regulators were also predicted based on the temporal expression of downstream genes. These findings suggest the presence of universal regulators during organ maturation, which may help us develop a general strategy to mature stem cell-derived cells in vitro.

bioinformatics↗

bioRxiv2021

Conserved Transcription Factors Control Chromatin Accessibility and Gene Expression to Maintain Cell Fate Stability and Restrict Reprogramming of Differentiated Cells

The comprehensive characterization of mechanisms safeguarding cell fate identity in differentiated cells is crucial for 1) our understanding of how differentiation is maintained in healthy tissues or misregulated in disease states and 2) to improve our ability to use direct reprogramming for regenerative purposes. To uncover novel fate-stabilizing regulators, we employed a genome-wide TF siRNA screen followed by a high-complexity combinatorial evaluation of top performing hits, in a cardiac reprogramming assay in mouse embryonic fibroblasts, and subsequently validated our findings in cardiac, neuronal and iPSCs reprogramming assays in primary human fibroblasts and adult endothelial cells. This approach identified a conserved set of 4 TFs (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming, as demonstrated by up to 6-fold increases in efficiency upon AJSZ knockdown in both lineage- and cell type-independent manners. Mechanistically, ChIP-seq and single-cell ATAC-seq analyses, revealed that AJSZ bind to both open and closed chromatin in a genome-wide and regionalized fashion, thereby limiting reprogramming TFs access to target DNA and ability to remodel the chromatin. In parallel, integration of ChIP-seq and RNA-seq data followed by systematic functional gene testing, identified that AJSZ also promote cell fate stability by proximally down-regulating a conserved set of genes involved in the regulation of cell fate specification (MEF2C), proteome remodeling (TPP1, PPIC), ATP homeostasis (EFHD1), and inflammation signaling (IL7R), thereby limiting cells ability to undergo large-scale phenotypic changes. Finally, simultaneous knock-down of AJSZ in combination with cardiac reprogramming TFs overexpression improved heart function by 250% as compared to no treatment and 50% as compared to MGT, 1 month after myocardial infarction. In sum, this study uncovers a novel evolutionarily conserved mechanism mediating cell fate stability in differentiated cells and also identifies AJSZ as promising therapeutic targets for regenerative purposes in adult organs. Significance StatementDifferentiated cells can be converted from one cell type into another by overexpressing lineage-determining transcription factors. Direct lineage reprogramming represents a promising strategy for regenerative medicine, but current clinical applications remain limited by the low yield of the reprogramming process. Here, we present the identification and detailed mechanistic study of a novel mechanisms opposing reprogramming process and promoting cell fate stability. Using a highthroughput screening technique, we identified four transcription factors that act as blockades to cell type change. By tracking chromatin and gene expression changes, we reconstructed a conserved pathway mediating cell fate stability in differentiated cells.

cell biology↗

bioRxiv2021

Cardiac progenitors auto-regulate second heart field cell fate via Wnt secretion

Proper heart formation requires coordinated development of two anatomically distinct groups of cells - the first and second heart fields (FHF and SHF). Given that congenital heart defects are often restricted to derivatives of the FHF or SHF, it is crucial to understand the mechanisms controlling their development. Wnt signaling has previously been implicated in SHF proliferation; however, the source of Wnts remains unknown. Through comparative gene analysis, we found upregulation of Wnts and Wnt receptor/target genes in the FHF and SHF, respectively, raising the possibility that early cardiac progenitors may secrete Wnts to influence SHF cell fate. To probe this further, we deleted Wntless (Wls), a gene required for Wnt ligand secretion, in various populations of precardiac cells. Deletion of Wls in Mesp1+ cells resulted in formation of a single chamber heart with left ventricle identity, implying compromised SHF development. This phenotype was recapitulated by deleting Wls in cells expressing Islet1, a pan-cardiac marker. Similarly, Wls deletion in cells expressing Nkx2.5, a later-expressed pan-cardiac marker, resulted in hypoplastic right ventricle, a structure derived from the SHF. However, no developmental defects were observed when deleting Wls in SHF progenitors. To gain mechanistic insights, we isolated Mesp1-lineage cells from developing embryos and performed single-cell RNA-sequencing. Our comprehensive single cell transcriptome analysis revealed that Wls deletion dysregulates developmental trajectories of both anterior and posterior SHF cells, marked by impaired proliferation and premature differentiation. Together, these results demonstrate a critical role of local precardiac mesodermal Wnts in SHF fate decision, providing fundamental insights into understanding heart field development and chamber formation. Significance StatementThere is significant interest in understanding the mechanisms underlying heart formation to develop treatments and cures for patients suffering from congenital heart disease. In particular, we were interested in the intricacies of first (FHF) and second heart field (SHF) development, as many congenital heart defects present with heart field-specific etiologies. Here, we uncovered a novel relationship between specified cardiac progenitor cells and second heart field progenitors. Through genetic manipulation of Wnt secretion in developing mouse embryos, we identified a population of cardiac progenitor cells that acts as a local source of Wnts which are necessary for proper SHF development. Our single cell transcriptomic analysis of developing anterior mesoderm showed cardiac progenitor-secreted Wnts function through regulation of differentiation and proliferation among SHF progenitors. Thus, this study provides insight into the source and timing of Wnts required for SHF development, and points to the crucial role of co-developing cell populations in heart development.

developmental biology↗

bioRxiv2021

Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

A primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs. Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

cell biology↗

bioRxiv2020

Haplotracker: a web application for simple and accurate mitochondrial haplogrouping using short DNA fragments

Mitochondrial DNA (mtDNA) haplogrouping is widely used in population genetics, forensics, and medical research to study evolutionary questions in human populations, examine degraded remains, and search for mtDNA-associated diseases. Next-generation sequencing methods have become a revolutionary tool for mtDNA analysis, particularly for degraded DNA, but they remain costly, laborious, and time-consuming. If an accurate and simple haplogroup-tracking tool were available, haplogrouping could be performed easily and rapidly. Here, we present Haplotracker, a web application for highly accurate and simple haplogroup tracking using a few sequence fragments. Haplotracker offers a unique user-friendly interface and estimates highly probable haplogroups from control-region sequences using a novel algorithm based on Phylotree Build 17 and our haplotype database (n=118,869). Haplotracker provides a simple, novel HG tracking solution, which was established through repeated blind simulation tests. It narrows down potential haplogroups and identifies their differential coding-region variants to confirm the haplogroups or to track sub-haplogroups. Haplotracker gives detailed information on sample variants, including their frequency in a large mtGenome database, which may give researchers an insight into common, rare, and potentially pathogenic mutations. It also offers a conserved region mapping tool for PCR primer design for successful tracking. Haplotracker produced high haplogroup prediction accuracy using 8,216 control-region Phylotree-provided sequences. It estimated top-ranking haplogroups with a higher concordance rate (56.6%, p<0.0001) than the similar tools MitoTool (29.4%) and HaploGrep 2 (33.9%). The significance persisted up to Rank 30. Haplotracker accurately estimated super-HGs from 94% of the control-region sequences at Rank 1. Further evaluation of the accuracy with 46,322 control-region sequences was significant. Laboratory application of Haplotracker to an ancient DNA extract demonstrated its practical usefulness. These results highlight the potential for the use of our web application as an alternative to full genome sequencing for easy haplogrouping, which may be useful in related fields. Free access: https://haplotracker.cau.ac.kr. Author summaryMitochondrial haplogroup (HG) classification is required in the search for answers to evolutionary questions about human populations, in the investigation of forensic samples, and in the search for disease-associated mutations. The sequencing of mitochondrial DNA (mtDNA) at the genome level is now frequently used due to the falling cost of next-generation sequencing. It offers accurate and detailed analysis of mtDNA variation, but it is still costly, laborious, and time-consuming. If an accurate and simple HG tracking tool were available, haplogrouping could be performed easily and rapidly. Here, we developed Haplotracker, a simple and accurate HG tracking web application with a novel algorithm and a novel tracking tool. The highly accurate prediction performance of Haplotracker was demonstrated in a series of tests. Using only control-region sequences, it accurately predicted HGs in more than half of the total Phylotree-provided mtGenome sequences and approximately 80% up to Rank 5; for super-HGs, it accurately predicted 94% at Rank 1 and more than 98% up to Rank 3. We demonstrated simple tracking for HG confirmation using Haplotracker. These results show that mtDNA haplogrouping with our web application may be useful in related fields.

bioinformatics↗