The past few years have seen unexpected new developments in direct cardiomyocyte reprogramming. Direct cardiomyocyte reprogramming potentially offers an entirely novel approach to cardiovascular ...regenerative medicine by converting cardiac fibroblasts into functional cardiomyocytes in situ. There is much to be learned, however, about the mechanisms of direct reprogramming in order that the process can be made more efficient. Early efforts have suggested that this new technology can be technically challenging. Moreover, new methods of inducing heart reprogramming will need to be developed before this approach can be translated to the bedside. Despite this, direct cardiomyocyte reprogramming may lead to new therapeutic options for sufferers of heart disease.
BACKGROUND:Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, ...the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown.
METHODS:We took advantage of Acvrl1 mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1 mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans.
RESULTS:Acvrl1 retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial “tip cells” at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1 mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1 embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1 (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1 embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1 endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1 mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1 tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies.
CONCLUSIONS:Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.
Investigating the signalling pathways that regulate heart development is essential if stem cells are to become an effective source of cardiomyocytes that can be used for studying cardiac physiology ...and pharmacology and eventually developing cell-based therapies for heart repair. Here, we briefly describe current understanding of heart development in vertebrates and review the signalling pathways thought to be involved in cardiomyogenesis in multiple species. We discuss how this might be applied to stem cells currently thought to have cardiomyogenic potential by considering the factors relevant for each differentiation step from the undifferentiated cell to nascent mesoderm, cardiac progenitors and finally a fully determined cardiomyocyte. We focus particularly on how this is being applied to human embryonic stem cells and provide recent examples from both our own work and that of others.
Full text
Available for:
EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
The human adult cardiomyocyte phenotype BIRD, S. D; DOEVENDANS, P. A; VAN ROOIJEN, M. A ...
Cardiovascular research,
05/2003, Volume:
58, Issue:
2
Journal Article
Peer reviewed
Open access
Determination of the phenotype of adult human atrial and ventricular myocytes based on gene expression and morphology.
Atrial and ventricular cardiomyocytes were obtained from patients undergoing ...cardiac surgery using a modified isolation procedure. Myocytes were isolated and cultured with or without serum. The relative cell attachment promoting efficiency of several reagents was evaluated and compared. Morphological changes during long-term culture were assessed with phase contrast microscopy, morphometric analysis and immunocytochemistry or RT-PCR of sarcomeric markers including alpha-actinin, myosin light chain-2 (MLC-2) and the adhesion molecule, cadherin.
The isolation method produced viable rod-shaped atrial (16.6+/-6.0%, mean+/-S.E.; n=5) and ventricular cells (22.4+/-8.0%, mean+/-S.E.; n=5) in addition to significant numbers of apoptotic and necrotic cells. Cell dedifferentiation was characterized by the loss of sarcomeric structure, condensation and extrusion of sarcomeric proteins. Cells cultured with low serum recovered and assumed a flattened, spread form with two distinct morphologies apparent. Type I cells were large, had extensive sarcolemmal spreading, with stress fibers and nascent myofibrils, whilst type II cells appeared smaller, with more mature myofibril organisation and focal adhesions.
Characterization of the redifferentiation capabilities of cultured adult cardiac myocytes in culture, provides an important system for comparing cardiomyocytes differentiating from human stem cells and provides the basis for an in vitro transplantation model to study interaction and communication between primary adult and stem cell-derived cardiomyocytes.
Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Organisation for Health Research and Development ZonMW ...Background Human induced pluripotent stem cells (hiPSCs) have demonstrated enormous potential for advancing in vitro disease modelling and genetically modified hiPSCs carrying disease-associated variants are particularly useful tools for this purpose. Until now, strategies for efficient and rapid integration of large DNA payloads (>10 kb) into specific genomic regions are still limited, thus precluding efficient and rapid targeting of hiPSC lines. Purpose and Methods To overcome this, the STRAIGHT-IN platform (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation) was developed that combined different classes of site-specific recombinases with CRISPR-Cas9 mediated homologous recombination and allowed stringent site-specific replacement of large genomic fragments in hiPSCs. Here, we use STRAIGHT-IN to simultaneously generate a library of genetically matched hiPSC lines carrying multiple heterozygous mutations in LMNA gene. Mutations in LMNA, encoding Lamin A/C protein, have been associated with 5-10% cases of dilated cardiomyopathy (DCM) with conduction defects. Results Through detailed online searches of the LMNA database and literature, we identified and selected 11 LMNA mutations to insert into a wild-type hiPSC line based on the following criteria: (i) mutations associated with cardiac abnormalities, in terms of structural (DCM/heart failure) and arrhythmic phenotypes; (ii) mutations with known familiar/pedigree-relationship; (iii) different types of mutations (missense and nonsense). We then applied the workflow of STRAIGHT-IN consisting of: (1) replacing the entire LMNA genomic locus (33 kb) on one allele with a Landing Pad (LP) cassette containing Serine Recombinase (Bxb1) recognition sites; (2) reintroducing LMNA gene into the LP cassette through integration of Bxb1-Donor plasmids carrying the LMNA variants of interest via Bxb1 expression; (3) expressing a tyrosine recombinase (Cre) to excise the majority of the auxiliary exogenous DNA sequences. Conclusions STRAIGHT-IN allows simultaneous generation of a panel of 11 isogenic hiPSC lines carrying selected LMNA mutations rapidly (5-6 weeks), efficiently and cost-effectively, thus facilitating the production of specific mutated hiPSC lines for disease modelling and personalized medicine applications.
Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ECR (European research council) and ReNew (the Novo Nordisk Foundation Center for Stem ...Cell Medicine supported by Novo Nordisk Foundation grants) Introduction Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are functionally immature and this limits the application of these cells to cardiovascular research. Maturation can be improved by aggregating hiPSC-CMs into three-dimensional (3D) microtissues together with hiPSC-cardiac fibroblasts (hiPSC-CFs) and hiPSC-endothelial cells (hiPSC-ECs). These cardiac microtissues showed improved structural (increase of sarcomere length and organisation) and functional (more mature action potential profile) properties. This effect was only observed when the cardiac microtissues contained hiPSC-CFs. Since connexin 43 (CX43), the most ubiquitous connexin isoform in the heart, was significantly upregulated in hiPSC-CMs from cardiac microtissues, we hypothesise that mechanisms underlying hiPSC-CMs maturation involves the coupling of hiPSC-CMs with hiPSC-CFs through this protein. Purpose The aim of this study is to unravel the role of CX43 in the maturation of hiPSC-CMs in the context of 3D multicellular cardiac tissues. Methods We used the CRISPR/Cas9 technology to generate a CX43 knock-out (KO) hiPSC line. We designed two sgRNAs annealing to exon 2 of GJA1 gene, encoding CX43; indels generated by the non-homologous end joining after the Cas9 double strand break will result in a non-functional protein. Cells were differentiated in vitro into CMs, epicardial cells (EPI) and then CFs and characterized for the expression of cell-specific markers and compared with the wild-type parental control cells. Results and conclusions We screened 31 hiPSC clones and selected those in which both GJA1 alleles contained a deletion. Copy number variation analysis by ddPCR of the flanking regions confirmed that the deletion was specific and confined. The lack of GJA1 expression in the KO clones was confirmed by qPCR, while the lack of the CX43 protein was confirmed by immunofluorescence and Western Blot. This did not affect CX43-KO hiPSCs pluripotency state shown by the presence of pluripotent markers such as NANOG, OCT3/4, SSEA4. Additionally, CX43-KO hiPSCs were able to differentiate into the three germ layers. CX43-KO hiPSCs were successfully differentiated into hiPSC-CMs, -EPI, and -CFs. CX43-KO hiPSC-CMs and controls showed comparable gene expression of cardiac troponin T. Since immunofluorescent staining showed that CX43-KO hiPSC-EPI expressed the epicardial markers WT1 and ZO1, we continued to differentiate them into hiPSC-CFs, which expressed markers such as VIM and COL1A1, similar to the control. Differentiated CX43-KO hiPSC-CMs and CFs will be assembled into cardiac microtissues. Functional and structural assays that include patch-clamp and immunofluorescence will be performed on these microtissue to assess the effect of the CX43 KO on the maturation of hiPSC-CMs. Our results show that the lack of CX43 expression does not impede hiPSCs to differentiate into various cell types originating from the mesoderm germ layer.
The hematopoietic stem cell (HSC) is the prototype organ-regenerating stem cell (SC), and by far the most studied type of SC in the body. Currently, HSC-based therapy is the only routinely used SC ...therapy; however, advances in the field of embryonic SCs and induced pluripotent SCs may change this situation. Interest into in vitro generation of HSCs, including signals for HSC expansion and differentiation from these more primitive SCs, as well as advances in other organ-specific SCs, in particular the intestine, provide promising new applications for SC therapies. Here, we review the basic principles of different SC systems, and on the basis of the experience with HSC-based SC therapy, provide recommendations for clinical application of emerging SC technologies.
Full text
Available for:
DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Clinical trial satisfaction is increasingly important for future trial designs and is associated with treatment adherence and willingness to enroll in future research studies or to recommend trial ...participation. In this post-trial survey, we examined participant satisfaction and attitudes toward future clinical trials in the Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU).
We developed an anonymous, participant satisfaction survey tailored to participants enrolled in the DIAN-TU-001 double-blind clinical trial of solanezumab or gantenerumab and requested that all study sites share the survey with their trial participants. A total of 194 participants enrolled in the trial at 24 study sites. We utilized regression analysis to explore the link between participants' clinical trial experiences, their satisfaction, and their willingness to participate in upcoming trials.
Survey responses were received over a sixteen-month window during 2020-2021 from 58 participants representing 15 study sites. Notably, 96.5% of the survey respondents expressed high levels of satisfaction with the trial, 91.4% would recommend trial participation, and 96.5% were willing to enroll again. Age, gender, and education did not influence satisfaction levels. Participants reported enhanced medical care (70.7%) and pride in contributing to the DIAN-TU trial (84.5%). Satisfaction with personnel and procedures was high (98.3%). Respondents had a mean age of 48.7 years, with most being from North America and Western Europe, matching the trial's demographic distribution. Participants' decisions to learn their genetic status increased during the trial, and most participants endorsed considering future trial participation regardless of the DIAN-TU-001 trial outcome.
Results suggest that DIAN-TU-001 participants who responded to the survey exhibited high motivation to participate in research, overall satisfaction with the clinical trial, and willingness to participate in research in the future, despite a long trial duration of 4-7 years with detailed annual clinical, cognitive, PET, MRI, and lumbar puncture assessments. Implementation of features that alleviate barriers and challenges to trial participation is like to have a high impact on trial satisfaction and reduce participant burden.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Background: Patients with a hereditary vascular disorder called Rendu-Osler-Weber syndrome (Hereditary Haemorrhagic Telangiectasia, HHT) haemorrhage easily due to weak-walled vessels. Haemorrhage in ...lungs or brain can be fatal but patients suffer most from chronic and prolonged nosebleeds (epistaxis), the frequency and intensity of which increases with age. Several years ago, it was discovered serendipitously that the drug Thalidomide had beneficial effects on the disease symptoms in several of a small group of HHT patients: epistaxis and the incidence of anaemia were reduced and patients required fewer blood transfusions. In addition, they reported a better quality of life. However, Thalidomide has significant negative side effects, including neuropathy and fatigue. Methods: We followed up all HHT patients in the Netherlands who had been taking Thalidomide at the time the original study was completed to find out (i) how many had continued taking Thalidomide and for how long (ii) the nature and severity of any side-effects and (iii) whether side-effects had influenced their decision to continue taking Thalidomide. Results: Only a minority of patients had continued taking the drug despite its beneficial effects on their symptoms and that the side effects were the primary reason to stop. Conclusion: Despite symptom reduction, alternative treatments are still necessary for epistaxis in HHT patients and a large-scale clinical trial is not justified although incidental use in the most severely affected patients can be considered.
Patients with a hereditary vascular disorder called Rendu-Osler-Weber syndrome (Hereditary Haemorrhagic Telangiectasia, HHT) haemorrhage easily due to weak-walled vessels. Haemorrhage in lungs or ...brain can be fatal but patients suffer most from chronic and prolonged nosebleeds (epistaxis), the frequency and intensity of which increases with age. Several years ago, it was discovered serendipitously that the drug Thalidomide had beneficial effects on the disease symptoms in several of a small group of HHT patients: epistaxis and the incidence of anaemia were reduced and patients required fewer blood transfusions. In addition, they reported a better quality of life. However, Thalidomide has significant negative side effects, including neuropathy and fatigue.
We followed up all HHT patients in the Netherlands who had been taking Thalidomide at the time the original study was completed to find out (i) how many had continued taking Thalidomide and for how long (ii) the nature and severity of any side-effects and (iii) whether side-effects had influenced their decision to continue taking Thalidomide.
Only a minority of patients had continued taking the drug despite its beneficial effects on their symptoms and that the side effects were the primary reason to stop.
Despite symptom reduction, alternative treatments are still necessary for epistaxis in HHT patients and a large-scale clinical trial is not justified although incidental use in the most severely affected patients can be considered.
PDF
