Molecular cardiovascular developmental biology

The research within our LUMC departments is conducted within departmental research programmes. The research programme below is embedded within the department of Anatomy.

Aim and focus

The heart is the first organ to form during embryonic development and its proper connection to the vasculature and blood circulation are controlled by a complex interplay of multiple signalling pathways. Maladaption of these pathways not only leads to congenital heart and vascular defects but also to late onset cardiovascular disease. Conversely, recapitulating normal use can support directed differentiation of stem cells so that human derivatives of the cardiovascular lineages can be produced for a multiplicity of biomedical applications ranging from disease modelling and drug discovery to regenerative medicine. This research programme aims to define molecular and biomechanical mechanisms that : 1. are used by progenitors to form cellular components of the heart and vasculature, 2. support cardiovascular cell maturation to fully functional phenotypes and 3. cause deregulation in cardiac and vascular disease. We use (pluripotent) stem cells, embryos and foetuses of chicken, mouse and human origin to identify functional signalling pathways in combination with a range of classical (embryo culture, histology, gross morphology, electrophysiology) and modern molecular techniques (molecular imaging, functional genomics, proteomics and epigenomics, high content analysis, gene editing techniques) for this purpose.

Collaboration with clinical departments is an ongoing basis for deriving pluripotent stem cells bearing (cardiovascular and brain) disease genes for pathophysiology analysis in combination with drug discovery. A human fetal database and tissue bank has been established as a research resource for the community. A joint appointment in the department of Cardiology provides a clinical link to research on the developmental origin of congenital heart defects and long-term outcomes of childhood surgery. Development and pathomorphology of bicuspid aortic valves, cardiac conduction and innervation is explored in both genetic animal models and human tissues. The earliest stages of oocyte development and maturation in the context of infertility (as a result of cancer therapy in young females) and placental development in the context of repeated miscarriage provides underlying basic research that support clinical innovation in the department of Obstetrics and Gynaecology.

A new “organ on chip” research programme developing human tissue and disease modelling technology (hDMT) in microfluidic and biomechanical/electrical devices was initiated in 2015 as a collaboration between the three technical universities, seven general universities and university medical centres, the Hubrecht Institute and pharma/biotech (Galapagos, GenMab). Synthetic human tissues derived from stem cells are incorporated in devices to provide complex models for drug discovery and safety pharmacology. In the first instance cardiac and vascular diseases and cancer were modelled on chip formats with neural (in collaboration with Erasmus MC) and skin (in collaboration with the AMC) in 2016.

Research within programme 50201 is organized in two main themes

  1. (Ab)normal cardiovascular development and disease. The primary goal is to obtain new insights into heart disease and heart development with view to developing new treatment strategies that range from (repurposing) drugs and risk profiling to surgical intervention.
  2. Differentiation and Organ development. This embraces the primary competencies of the department and includes in depth knowledge of human embryo development through morphological, pathological, genetic and epigenetic analysis of an extensive collection of human fetal tissue. This forms the Centre for Human Development with links to a human fetal database (“Keygenes”) in which different organs at different developmental stages can be examined/compared (www.keygenes.nl).

These themes are lead by three senior (C.L. Mummery, S.M. Chuva de Sousa Lopes, M.C. DeRuiter) and three junior (M. Bellin, R.P. Davis, V.V. Orlova) Principal Investigators; PI M.R.M. Jongbloed has shared appointments in the depts. of Anatomy&Embryology and Cardiology, combining basic and clinical research with clinical activities. Senior PI R. Passier is also Professor of Applied Stem Cell Biology at the University of Twente since 2015 and C.L. Mummery also holds a part-time chair of Vascular Microfluidics at the same university, part of a strategic collaboration between the LUMC and UT. M.C. DeRuiter is responsible for the organization, execution and development of the anatomy and developmental biology teaching programme (0.7fte) in addition to research (0.3fte). Human (patient-derived) induced pluripotent stem cells used in multiple research lines are produced by the LUMC iPSC core facility (joint ANA/MCB).

Position in international context

The department is internationally leading in the area of cardiovascular disease modelling using human pluripotent stem cells. This is reflected in the large number of invitations to speak (including plenary) and be part of the programme committee of premier international conferences on stem cells (ESHRE, ISSCR, EMBO, New York Stem Cell Foundation), cardiology (eg ESC, AEPC, EuroEcho, ESHG, Dutch/German meeting, Keystone), pharmaceuticals/ safety pharmacology (eg SelectbioSciences, British Society Pharmacology, as well as more general international conferences (FEBS, Gordon conference).

Members of the department hold several individual prestigious international and national grants including three from  the ERC (ERC-AdG, ERC-CoG, ERC-StG), VIDI, Marie Curie and Gisela Their fellowships. 

Most PIs have multiple international collaborations that include joint publications (with eg Germany, US, Australia, UK, Portugal, Brazil); in some cases, these are funded by competitive international grants (eg EU FP7 and H2020, Marie Curie, NC3R/GSK, HFSP). Editorial board or editor-in-chief functions in multiple high profile journals (Stem Cell Reports, Scientific Reports, Cell Stem Cell, Cardiovascular Research) are testimony of the strong international position of several staff members in their respective fields.

Invited reviews to major journals include:

  • Bellin M, Marchetto MC, Gage FH, Mummery CL. Induced pluripotent stem cells: the new patient? Nature Rev Mol Cell Biol., 2012. 13(11):713-26
  • Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview. Circulation Research 2012 111(3):344-58
  • Passier R, Orlova V, Mummery C. Complex Tissue and Disease Modeling using hiPSCs  Cell Stem Cell. 2016 Mar 3;18(3):309-21. doi: 10.1016/j.stem.2016.02.011
  • Van der Jeught M, O'Leary T, Duggal G, De Sutter P, Chuva de Sousa Lopes S*, Heindryckx B* (2015). The post-inner cell mass intermediate: implications for stem cell biology and assisted reproductive technology. Hum Reprod Update 21:616-626

International positions of trust include: 

  • Departments staff are members of the Scientific Advisory or Non-Executive Boards of the New York Stem Cell Foundation, EU ESTOOLS (Chair); the International Society for Hereditary Hemorrhagic Telangiectasia.(HHT), CellCure (biotech company developing stem cell therapy for macular degeneration), EU IMI StemBANCC, ESHRE, Dutch-German Heart consortium.
  • Member of the Wellcome Trust Committee, UK 2002-2006 “Molecules, Genes, Cells” and ad hoc specialist committee member 2012.
  • Netherlands scientific representative of the International Stem Cell Initiative (ISCI) 2007.
  • Member of the NWO Roadmap selection committee 2012, 2014.
  • Member of the ERC Starters grant selection committee 2012, 2015, 2018.
  • Chair of the European WG cardiovascular morphology of the AEPC. Member of the European WG of Development, Anatomy and Pathology of the ESC.
  • Chair of the Netherlands/Belgian Anatomical Society 2009-2015.
  • Programme committee/organizer of the following (selected) conferences: ISSCR 2013, 2015; DSSCR 2011- ; ISD 2012; De Snoo 2013, 2016; Keystone 2016, 2018, ESHRE 2016; AEPC 2014, 2015, 2016, 2017.

Content / highlights / achievements

The department has established a strong international profile in (i) creating human stem cell-based models for (genetic forms of cardiac and vascular disease and (ii) describing human development and morphogenesis with associated genetic and epigenetic profiles. This is highlighted by the award of three ERC grants (Advanced, Consolidator, Starter), VIDI, Marie Curie, NHS Dekker, ZonMW and invitations to participate in consortia funded by the EU (FP7, H2020, ERA-CVD) and NHS (CVON). In addition, an NWO Gravitiy grant (18.8ME) was awarded in 2017.

Major scientific achievements include:

  • Generic methods for efficient targeted gene insertion, mutation repair and cardiomyocyte differentiation of human pluripotent stem cells have been developed (EMBO Mol Med 2015a; PNAS; Methods Mol Biol 2016).
  • A prototype multiplexed drug screening platform based on human (stem cell derived) cardiomyocytes was developed.
  • A method for measuring cardiomyocyte contractility was established (Cell Reports 2015).
  • A novel sarcomeric protein important in cardiac development and hypertrophy was discovered (EMBO Mol Med 2016).
  • The stem cell groups published invited reviews in high impact journals (Nature, Cell, Cell Stem Cell) on cardiac repair.
  • Cardiac and vascular disease models were established by reprogramming somatic cells from patients (PNAS 2015, EMBO J 2013).
  • Methods for deriving functional vascular cells from stem cells were established (ATVB 2014; Nature Protocols 2014).
  • A prediction algorithm KeyGenes was generated and (epi)genomic signatures of (isogenic) human fetal organs were obtained as a resource (Stem Cell Reports 2015; Nature 2015; Plos Genetics 2015).
  • Novel protocols for deriving naïve human embryonic stem cells and epiblast stem cells were established (Stem Cell Reports 2014; Nature Communications 2017)
  • A mesodermal reporter line was generated in human pluripotent stem cells and molecular pathways in early cardiac differentiations were studied (Stem Cells 2014).
  • A protocol for generating atrial cardiomyocytes from human pluripotent stem cells and its use as a robust preclinical model for assessing atrial-selective pharmacology was established (EMBO Mol Med 2015b).
  • Developmental origins of cardiac arrhythmias in neonates and adults were identified (Int J Cardiol. 2015; J Cell Mol Med. 2015; J Mol Cell Cardiol. 2015).
  • (Animal) models for congenital heart disease enabled identification of molecular pathways underlying congenital heart disease (Dis Model Mech. 2016).
  • Adult human epicardium derived cells were shown to contribute to cardiac repair (reviewed in Differentiation 2012)
  • Clinical anatomy publications were cited in the European clinical guidelines for treatment of cardiovascular diseases (J Cell Mol Med 2015 in Europace 2016;  J Thorac Cardiovascular Surg. in Circulation 2015).

Outreach

  • The department has an active “outreach” programme in providing accurate and timely information to patient groups, the general public/media, schools and governmental agencies on stem cells and their relevance to medicine and biomedical science. The department also hosts and supervises high school students in their final year of study to undertake their own small research project. A lay guide to stem cells “Stem Cell: Scientific Fact and Fiction was published by Elsevier (2nd edition 2014: Authors C. Mummery, A. van de Stolpe, B. Roelen, H. Clevers) which is widely used as a text book in university medical, ethics and biology courses, by high school students in final year projects, patients and organizations, among others. We have contributed two chapters to the seminal and most influential stem cell book to date “Handbook of Embryonic Stem Cells” (2nd edition 2012: publisher Elsevier. Authors S. Chuva de Sousa Lopes, C. Mummery).
  • Clinical anatomy contributed chapters to several general textbooks: the European Society of Cardiology “Textbook of Cardiovascular Medicine”  (Barge-Schaapsveld, DeRuiter, Jongbloed), “Cardiac Electrophysiology: From Cell to Bedside” (Jongbloed, Schalij/Zeppenfeld (HTZ) and “Fetal Cardiology and Neonatal Cardiology”  (Jongbloed, Bartelings, DeRuiter).  Further, DeRuiter contributed  “De strijd tegen de vergankelijkheid” to the cahier “The human body” published in 2015 by Biowetenschappen en Maatschappij. He is interviewed on anatomy issues by national and local newspapers.
  • Human stem cell research is increasingly regarded as an alternative for laboratory animal experiments. The department is engaged in a number of these activities: R. Passier, C. Mummery, D. Salvatori and C. Freund have grants awarded to investigate 3R approaches (total value (1.25M€); R. Passier and C. Mummery have received several awards for 3R based research (“Lef in het lab award” and Hugo van de Poelgeest Prize: value ~15K€). This involved using heart cells derived from human stem cells as models of human cardiac physiology and disease and for safety pharmacology, or validating alternatives for pluripotency assessment based on teratoma formation in mice.  The department provides information to the media on stem cell issues, either medical breakthroughs or discussion of ethical problems. The basis of the expertise is in the stem cell research programme and is highly relevant for societal questions on the impact and importance of this area of biomedical science.
  • S.M Chuva de Sousa Lopes was Interviewed in “Kennis café: Baby’s maken”, De Balie, a cultural and political center in Amsterdam in 2015. C.L.Mummery and S.M Chuva de Sousa Lopes wrote Chapters “Baby op bestelling” and Organs-on-Chip  in  the cahier “Stamcellen”, published in 2016 by Biowetenschappen en Maatschappij; the cahiers are lay guides to scientific developments. Following the ERC consolidator award, she gave several radio and newspaper interviews (Trouw, De Correspondent) on artificial gametes and wrote commentaries for Oncologica for Dutch nurses, and Focus on Reproduction, the society magazine of ESHRE. Mummery took part in a TV documentary of cloning of animals in 2016,
  • Members of the department are regularly consulted by the media on issues of content regarding stem cells, development and human embryo modification, which includes providing ethical perspectives.
  • The department also provides a (fetal) pathology service with special focus on congenital heart disease to patients/clinical specialists requesting this after a spontaneous or induced abortion. M. Bartelings is one of the few experts in this area in the Netherlands and for potential parents it is of upmost importance to know why this occurred and what the likelihood is that another pregnancy will end the same way. This service is not charged for. The department also has a major role in developing 3D print models of cardiovascular/congenital heart disease, that are used in the clinic both as a guide for percutaneous and surgical interventions, and for education of patients and medical specialists (funded by LUF and AEPC grants).

Future themes

The department has a strong cardiovascular research profile and has an advanced blended learning educational programme in Anatomy and Embryology for (bio)medical,clinical technology and paramedical students. Future themes are based on strengthening these areas further through creation of opportunities for talented junior staff to build independent research groups and through implementing research in teaching methods, including e-learning.

Specific research objectives for the next five years include the following ambitions:

Scientific:

  • Development of a repertoire of clinically relevant cardiovascular disease models based on pluripotent stem cells, including methods for measuring phenotype in vitro.
  • Benchmark different human organs and cell/tissue types during development into adulthood to guide stem cell differentiation and identify congenital defects.
  • via the hiPSC core facility: create a biobank of diseased stem cells with genetically corrected controls for the LUMC and collaborators, incorporate in complex multicellular models with appropriate bioassay readouts.
  • By understanding the embryonic origin of conduction/innervation disorders in congenital heart malformations, develop new treatment or prevention strategies.
  • Creating clinical grade human pluripotent stem cells for Regnerative Medicine within the V&RM profile area.

Social:

  • Validate disease models that promote implementation of the 3R’s in research to reduce, refine and replace animal experiments.
  • Improve paediatric treatment strategies of heart patients based on developmental principles and provide present and future (young) heart patients with better treatment for and education on their heart conditions.

Strategic:

  • To make the LUMC national leader in pluripotent stem cell technology for modelling disease and for clinical translation of hPSC-based therapies (in collaboration with GMP/IHB). The ambition includes establishing an iPSC “hotel” in the LUMC as a national expertise centre, with a business case designed to support through hosting external users.
  • To use web-based platforms to aid/guide stem cell differentiation on the basis of human (fetal) tissue profiles.
  • To establish the LUMC as a major expertise centre for congenital heart defects in humans and underlying developmental mechanisms, using unique access to human fetal tissue, including historic samples with congenital (heart) defects, and tissue from heart patients.
  • Promote and develop Organ- on-Chip human tissue and disease modelling technologies with national partners. Synthetic human tissues derived from stem cells is incorporated into devices to provide complex models for drug discovery and safety pharmacology. Cardiac and vascular diseases and cancer are being modelled on chip formats with neural (in collaboration with Erasmus MC), intestine (with WU) and skin (with the VUMC). This reflects international interest in this area (from the Wyss Institute/EMULATE in Boston, ETH in Zurich, TU Berlin) and has strong affiliation with the aims of the 3Rs in animal experiments (reduce, refine, replace) programmes of ZonMW and the EU.  Opportunities for drug repurposing and target discovery in human disease present translational applications that are requirements of the Top Sector and EU Horizon 2020 and the Netherlands WetenschapsAgenda (end 2015).

The department has several new educational objectives initiated in response to societal, research, healthcare and technology demands to alter the content and form of our educational programs. Internationally, open and online education has increased enormously and the department has embraced this by producing “in house” online education like MOOCs, which are not just a collection of digitalized existing learning materials, but an educational environment with well-tuned interactive content, short knowledge clips, assignments, (student and teacher) feedback systems and various formative and summative tests.

Our teaching aim is to further implement the didactic blended learning concept, i.e. an integrated combination of face-to-face and online education, in all the anatomy and developmental biology lessons. A new Frontiers of Sciences course for MSc biomedical students  on “Research Applications of Developmental Biology” is being developed that includes new stem cell technologies and embryonic models. The course design is based on the TeamBased Learning concept.

Despite centuries of textbook based educational experience, the basic principles of anatomy and embryology are difficult to comprehend by many students because conceptualizing 3D relationships between structures remains challenging. However, using new 3D imaging technologies (stereographics, virtual reality and mixed reality) we are for the first time able enhance lessons with new learning strategies that increase knowledge acquisition, comprehension and its retention by students. In the coming years we plan to evaluate these new methods through learning analytics (new research line B.P. Hierck).

Cohesion within LUMC

The department is part of the LUMC profile area “Vascular and Regenerative Medicine” but has strong collaborative links with “Biomedical Imaging” and “Translational Neuroscience”.

There are multiple collaborations with clinical and preclinical research groups in the LUMC, both within and outside profile areas. These principally involve the depts of Cardiology (Atsma, Schalij), Molecular Cell Biology (ten Dijke, Goumans, Mikkers, Hoeben), Nephrology (de Koning, Rabelink) and Gynaecology (Lith, Trimbos) through shared PhD students and postdocs and common research interests in cardiovascular biology, growth factor signaling and early development. Funding for the iPSC core facility, jointly run by ANA and MCB and funded and funded by the LUMC have generated >100 cell lines from patients with a multiplicity of genetic diseases (neural, cardiac, bone, lung, vascular) with many more in the pipeline. This has provided opportunities to integrate unique human disease models into translational research on diseases within the dept and through the profile areas. Discoveries using hPSC already include (i) identification of the mechanisms underlying specific drug sensitivities in patients with a cardiac syndrome known as LQT2 or taking secondary drugs that partially inhibit a specific potassium channel (Ikr) in human cardiomyocytes (ii) establishment of a human disease model for hereditary hemorrhagic telengiectasia (HHT) which manifests as weak walled vessels and is caused by mutations in the transforming growth factor β receptors ALK1 and endoglin (iii) the establishment of cell models for migraine and “ Katwijk Disease”. We have successfully targeted genes in hESC by homologous recombination to generate lineage reporters (Orlova, Davis) for dissecting developmental signaling pathways and in hESC and hiPSC, to create and rescue cardiac disease phenotypes (Bellin; Passier; Mummery). Pluriomics was founded in 2010 after being awarded NGI venture challenge and STW grants, to license the hPSC technology and create platforms for high throughput drug screening and drug discovery. This is again in line with PPP policies to valorize basic science discoveries and has led to new co-funded pharma GSK and NC3R grants that involve both the LUMC and Pluriomics.

The research on heart development and congenital heart defects benefits from the joint appointment of PI Jongbloed in the depts of Anatomy and Cardiology. This research is internationally renowned for its impeccable descriptions of normal and abnormal heart and vascular development in mouse, chick and human (Jongbloed, Bartelings, DeRuiter,) which continues to include a fetal pathology service for the diagnosis of congenital heart defects in terminated pregnancies. This is in part funded by ZonMW (clinical fellowship to Jongbloed) and Dutch Heart Foundation (DeRuiter) and has recently been extended to investigate mechanisms underlying congenital aortic aneurysms.

In line with policy to link teaching and ongoing clinical education with contemporary research, e-learning modalities are being coupled to basic anatomical research. Funding has been obtained from Horizon 2020 and STW (DeRuiter), for example, for in depth mapping of the abdomen which should lead to more insight in inter-individual variability and, when coupled to appropriate training modules, better surgical intervention with fewer unintentional mishaps. Clinical anatomy as a minor research programme is ongoing to create opportunities for clinical fellows (from surgery, orthopedics, cardiology, urology, neurosurgery) to carry out basic research projects within the clinic towards a PhD. In the past years, 3D modeling and printing has become pivotal in creating educational modalities for patients and medical specialists (funded by Leiden University Fund). These modules are now increasingly used in MSc programs of Medicine, Biomedical Sciences and Clinical Technology.

Chuva de Sousa Lopes carries out single cell (transcriptome and methylome) profiling of human organs during development to understand the human embryo and benchmark stem cells following differentiation. In parallel, she carries out classical histology of developing human organs. In this context, she collaborates with the LUMC sequencing facility (LGTC) and the bioinformatics service (SASC).