Developmental dynamics of cardiac progenitors and their role in congenital heart defects

Cardiac progenitors are the fundamental building blocks of the heart and play a central role in the pathogenesis of congenital heart defects. Recent single-cell RNA sequencing studies have revealed that the cell populations in both developing and mature heart are more diverse than previously recognized. In particular, there has been a great interest in characterizing the transcriptional signatures of human cardiac progenitor populations and building a roadmap of the early heart lineages. This thesis work is a part of an ongoing effort to map the early human heart progenitors, their developmental dynamics and their role in the pathogenesis of congenital heart defects at a single-cell resolution. In paper I, we present a single-cell characterization of in vitro cardiac differentiation of human embryonic stem cells to beating cardiomyocytes and other populations. We used single-cell RNA sequencing data integration to compare the in vitro-derived cardiac cells to human embryonic heart, studied the developmental dynamics of cardiac progenitors by building a differentiation roadmap, and investigated the effect of loss of ISL1 transcription factor on the differentiation process. In paper II, we mapped the genetic landscape of non-syndromic Tetralogy of Fallot, a form of complex congenital heart defect, in a cohort of 146 patient-parent trios. We intersected the identified disease-associated genes with single-cell RNA sequencing analysis of in vivo and in vitro human cardiac development generated by us and other laboratories. Interestingly, we could pinpoint cardiac progenitors as a cellular hotspot in cardiac disease pathogenesis. Paper III introduces a novel cardiac progenitor marked by LGR5, which exists both in the human embryonic heart in vivo and the human embryonic stem cell cardiac differentiation system in vitro. The LGR5+ cono-ventricular progenitor population originates from the ISL1+ progenitor pool and populates the cardiac outflow tract, a cardiac region often affected in congenital heart defects. Taken together, these studies support the importance of cardiac progenitors in the pathogenesis of cardiac abnormalities and explore their developmental dynamics at a singlecell level

Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.The Knut and Alice Wallenberg Foundation (KAW Dnr 2013.0028)Croucher Foundation, Hong KongSwedish Research Council Grant no 541-2013-8351 and 539‐2013‐7002European Research Council Advanced Research Grant Award (AdG743225)Publishe

Simulation of Subject-Specific Progression of Knee Osteoarthritis and Comparison to Experimental Follow-up Data : Data from the Osteoarthritis Initiative

Economic costs of osteoarthritis (OA) are considerable. However, there are no clinical tools to predict the progression of OA or guide patients to a correct treatment for preventing OA. We tested the ability of our cartilage degeneration algorithm to predict the subject-specific development of OA and separate groups with different OA levels. The algorithm was able to predict OA progression similarly with the experimental follow-up data and separate subjects with radiographical OA (Kellgren-Lawrence (KL) grade 2 and 3) from healthy subjects (KL0). Maximum degeneration and degenerated volumes within cartilage were significantly higher (p <0.05) in OA compared to healthy subjects, KL3 group showing the highest degeneration values. Presented algorithm shows a great potential to predict subjectspecific progression of knee OA and has a clinical potential by simulating the effect of interventions on the progression of OA, thus helping decision making in an attempt to delay or prevent further OA symptoms.Peer reviewe

Population and single-cell analysis of human cardiogenesis reveals unique LGR5 ventricular progenitors in embryonic outflow tract.

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.The Knut and Alice Wallenberg Foundation (KAW Dnr 2013.0028)Swedish Research Council Distinguished Professor Grant Dnr 541-2013-8351AstraZeneca PharmaceuticalsKarolinska InstitutetSwedish Heart Lung Foundation No. 20150421EMBO long-term fellowship (ALTF 620-2014)European Research Council Advanced Research Grant Award (AdG743225)Publishe

Sequencing of a Chinese tetralogy of Fallot cohort reveals clustering mutations in myogenic heart progenitors

Tetralogy of Fallot (TOF) is the most common cyanotic heart defect, yet the underlying genetic mechanisms remain poorly understood. Here, we performed whole-genome sequencing analysis on 146 nonsyndromic TOF parent-offspring trios of Chinese ethnicity. Comparison of de novo variants and recessive genotypes of this data set with data from a European cohort identified both overlapping and potentially novel gene loci and revealed differential functional enrichment between cohorts. To assess the impact of these mutations on early cardiac development, we integrated single-cell and spatial transcriptomics of early human heart development with our genetic findings. We discovered that the candidate gene expression was enriched in the myogenic progenitors of the cardiac outflow tract. Moreover, subsets of the candidate genes were found in specific gene coexpression modules along the cardiomyocyte differentiation trajectory. These integrative functional analyses help dissect the pathogenesis of TOF, revealing cellular hotspots in early heart development resulting in cardiac malformations

Application of a semi-automatic cartilage segmentation method for biomechanical modeling of the knee joint

Abstract Manual segmentation of articular cartilage from knee joint 3D magnetic resonance images (MRI) is a time consuming and laborious task. Thus, automatic methods are needed for faster and reproducible segmentations. In the present study, we developed a semi-automatic segmentation method based on radial intensity profiles to generate 3D geometries of knee joint cartilage which were then used in computational biomechanical models of the knee joint. Six healthy volunteers were imaged with a 3T MRI device and their knee cartilages were segmented both manually and semi-automatically. The values of cartilage thicknesses and volumes produced by these two methods were compared. Furthermore, the influences of possible geometrical differences on cartilage stresses and strains in the knee were evaluated with finite element modeling. The semi-automatic segmentation and 3D geometry construction of one knee joint (menisci, femoral and tibial cartilages) was approximately two times faster than with manual segmentation. Differences in cartilage thicknesses, volumes, contact pressures, stresses, and strains between segmentation methods in femoral and tibial cartilage were mostly insignificant (p > 0.05) and random, i.e. there were no systematic differences between the methods. In conclusion, the devised semi-automatic segmentation method is a quick and accurate way to determine cartilage geometries; it may become a valuable tool for biomechanical modeling applications with large patient groups

Application of a semi-automatic cartilage segmentation method for biomechanical modeling of the knee joint

<p>Manual segmentation of articular cartilage from knee joint 3D magnetic resonance images (MRI) is a time consuming and laborious task. Thus, automatic methods are needed for faster and reproducible segmentations. In the present study, we developed a semi-automatic segmentation method based on radial intensity profiles to generate 3D geometries of knee joint cartilage which were then used in computational biomechanical models of the knee joint. Six healthy volunteers were imaged with a 3T MRI device and their knee cartilages were segmented both manually and semi-automatically. The values of cartilage thicknesses and volumes produced by these two methods were compared. Furthermore, the influences of possible geometrical differences on cartilage stresses and strains in the knee were evaluated with finite element modeling. The semi-automatic segmentation and 3D geometry construction of one knee joint (menisci, femoral and tibial cartilages) was approximately two times faster than with manual segmentation. Differences in cartilage thicknesses, volumes, contact pressures, stresses, and strains between segmentation methods in femoral and tibial cartilage were mostly insignificant (<i>p</i> > 0.05) and random, i.e. there were no systematic differences between the methods. In conclusion, the devised semi-automatic segmentation method is a quick and accurate way to determine cartilage geometries; it may become a valuable tool for biomechanical modeling applications with large patient groups.</p

Estimation of the effect of body weight on the development of osteoarthritis based on cumulative stresses in cartilage:data from the osteoarthritis initiative

Abstract Evaluation of the subject-specific biomechanical effects of obesity on the progression of OA is challenging. The aim of this study was to create 3D MRI-based finite element models of the knee joints of seven obese subjects, who had developed OA at 4-year follow-up, and of seven normal weight subjects, who had not developed OA at 4-year follow-up, to test the sensitivity of cumulative maximum principal stresses in cartilage in quantitative risk evaluation of the initiation and progression of knee OA. Volumes of elements with cumulative stresses over 5 MPa in tibial cartilage were significantly (p &lt; 0.05) larger in obese subjects as compared to normal weight subjects. Locations of high peak cumulative stresses at the baseline in most of the obese subjects showed a good agreement with the locations of the cartilage loss and MRI scoring at follow-up. Simulated weight loss (to body mass index 24 kg/m²) in obese subjects led to significant reduction of the highest cumulative stresses in tibial and femoral cartilages. The modeling results suggest that an analysis of cumulative stresses could be used to evaluate subject-specific effects of obesity and weight loss on cartilage responses and potential risks for the progression of knee OA