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8 Publications visible to you, out of a total of 8

Abstract (Expand)

BACKGROUND: Human pluripotent stem cell-derived muscle models show great potential for translational research. Here, we describe developmentally inspired methods for the derivation of skeletal muscle cells and their utility in skeletal muscle tissue engineering with the aim to model skeletal muscle regeneration and dystrophy in vitro. METHODS: Key steps include the directed differentiation of human pluripotent stem cells to embryonic muscle progenitors followed by primary and secondary foetal myogenesis into three-dimensional muscle. To simulate Duchenne muscular dystrophy (DMD), a patient-specific induced pluripotent stem cell line was compared to a CRISPR/Cas9-edited isogenic control line. RESULTS: The established skeletal muscle differentiation protocol robustly and faithfully recapitulates critical steps of embryonic myogenesis in two-dimensional and three-dimensional cultures, resulting in functional human skeletal muscle organoids (SMOs) and engineered skeletal muscles (ESMs) with a regeneration-competent satellite-like cell pool. Tissue-engineered muscle exhibits organotypic maturation and function (up to 5.7 +/- 0.5 mN tetanic twitch tension at 100 Hz in ESM). Contractile performance could be further enhanced by timed thyroid hormone treatment, increasing the speed of contraction (time to peak contraction) as well as relaxation (time to 50% relaxation) of single twitches from 107 +/- 2 to 75 +/- 4 ms (P < 0.05) and from 146 +/- 6 to 100 +/- 6 ms (P < 0.05), respectively. Satellite-like cells could be documented as largely quiescent PAX7(+) cells (75 +/- 6% Ki67(-) ) located adjacent to muscle fibres confined under a laminin-containing basal membrane. Activation of the engineered satellite-like cell niche was documented in a cardiotoxin injury model with marked recovery of contractility to 57 +/- 8% of the pre-injury force 21 days post-injury (P < 0.05 compared to Day 2 post-injury), which was completely blocked by preceding irradiation. Absence of dystrophin in DMD ESM caused a marked reduction of contractile force (-35 +/- 7%, P < 0.05) and impaired expression of fast myosin isoforms resulting in prolonged contraction (175 +/- 14 ms, P < 0.05 vs. gene-edited control) and relaxation (238 +/- 22 ms, P < 0.05 vs. gene-edited control) times. Restoration of dystrophin levels by gene editing rescued the DMD phenotype in ESM. CONCLUSIONS: We introduce human muscle models with canonical properties of bona fide skeletal muscle in vivo to study muscle development, maturation, disease and repair.

Authors: M. Shahriyari, M. R. Islam, S. M. Sakib, M. Rinn, A. Rika, D. Kruger, L. Kaurani, V. Gisa, M. Winterhoff, H. Anandakumar, O. Shomroni, M. Schmidt, G. Salinas, A. Unger, W. A. Linke, J. Zschuntzsch, J. Schmidt, R. Bassel-Duby, E. N. Olson, A. Fischer, W. H. Zimmermann, M. Tiburcy

Date Published: 19th Oct 2022

Publication Type: Journal

Abstract (Expand)

Lung infections and smoking are risk factors for multiple sclerosis, a T-cell-mediated autoimmune disease of the central nervous system(1). In addition, the lung serves as a niche for the disease-inducing T cells for long-term survival and for maturation into migration-competent effector T cells(2). Why the lung tissue in particular has such an important role in an autoimmune disease of the brain is not yet known. Here we detected a tight interconnection between the lung microbiota and the immune reactivity of the brain. A dysregulation in the lung microbiome significantly influenced the susceptibility of rats to developing autoimmune disease of the central nervous system. Shifting the microbiota towards lipopolysaccharide-enriched phyla by local treatment with neomycin induced a type-I-interferon-primed state in brain-resident microglial cells. Their responsiveness towards autoimmune-dominated stimulation by type II interferons was impaired, which led to decreased proinflammatory response, immune cell recruitment and clinical signs. Suppressing lipopolysaccharide-producing lung phyla with polymyxin B led to disease aggravation, whereas addition of lipopolysaccharide-enriched phyla or lipopolysaccharide recapitulated the neomycin effect. Our data demonstrate the existence of a lung-brain axis in which the pulmonary microbiome regulates the immune reactivity of the central nervous tissue and thereby influences its susceptibility to autoimmune disease development.

Authors: L. Hosang, R. C. Canals, F. J. van der Flier, J. Hollensteiner, R. Daniel, A. Flugel, F. Odoardi

Date Published: 25th Feb 2022

Publication Type: Journal

Abstract (Expand)

The highly conserved YrdC domain-containing protein (YRDC) interacts with the well-described KEOPS complex, regulating specific tRNA modifications to ensure accurate protein synthesis. Previous studies have linked the KEOPS complex to a role in promoting telomere maintenance and controlling genome integrity. Here, we report on a newborn with a severe neonatal progeroid phenotype including generalized loss of subcutaneous fat, microcephaly, growth retardation, wrinkled skin, renal failure, and premature death at the age of 12 days. By trio whole-exome sequencing, we identified a novel homozygous missense mutation, c.662T > C, in YRDC affecting an evolutionary highly conserved amino acid (p.Ile221Thr). Functional characterization of patient-derived dermal fibroblasts revealed that this mutation impairs YRDC function and consequently results in reduced t(6)A modifications of tRNAs. Furthermore, we established and performed a novel and highly sensitive 3-D Q-FISH analysis based on single-telomere detection to investigate the impact of YRDC on telomere maintenance. This analysis revealed significant telomere shortening in YRDC-mutant cells. Moreover, single-cell RNA sequencing analysis of YRDC-mutant fibroblasts revealed significant transcriptome-wide changes in gene expression, specifically enriched for genes associated with processes involved in DNA repair. We next examined the DNA damage response of patient's dermal fibroblasts and detected an increased susceptibility to genotoxic agents and a global DNA double-strand break repair defect. Thus, our data suggest that YRDC may affect the maintenance of genomic stability. Together, our findings indicate that biallelic variants in YRDC result in a developmental disorder with progeroid features and might be linked to increased genomic instability and telomere shortening.

Authors: J. Schmidt, J. Goergens, T. Pochechueva, A. Kotter, N. Schwenzer, M. Sitte, G. Werner, J. Altmuller, H. Thiele, P. Nurnberg, J. Isensee, Y. Li, C. Muller, B. Leube, H. C. Reinhardt, T. Hucho, G. Salinas, M. Helm, R. D. Jachimowicz, D. Wieczorek, T. Kohl, S. E. Lehnart, G. Yigit, B. Wollnik

Date Published: 22nd Sep 2021

Publication Type: Journal

Abstract

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Authors: J. Peper, D. Kownatzki-Danger, G. Weninger, F. Seibertz, J. R. D. Pronto, H. Sutanto, D. Pacheu-Grau, R. Hindmarsh, S. Brandenburg, T. Kohl, G. Hasenfuss, M. Gotthardt, E. A. Rog-Zielinska, B. Wollnik, P. Rehling, H. Urlaub, J. Wegener, J. Heijman, N. Voigt, L. Cyganek, C. Lenz, S. E. Lehnart

Date Published: 19th Mar 2021

Publication Type: Journal

Abstract (Expand)

BACKGROUND: Noonan syndrome (NS) is a multisystemic developmental disorder characterized by common, clinically variable symptoms, such as typical facial dysmorphisms, short stature, developmental delay, intellectual disability as well as cardiac hypertrophy. The underlying mechanism is a gain-of-function of the RAS-mitogen-activated protein kinase signaling pathway. However, our understanding of the pathophysiological alterations and mechanisms, especially of the associated cardiomyopathy, remains limited and effective therapeutic options are lacking. METHODS: Here, we present a family with two siblings displaying an autosomal recessive form of NS with massive hypertrophic cardiomyopathy as clinically the most prevalent symptom caused by biallelic mutations within the leucine zipper-like transcription regulator 1 (LZTR1). We generated induced pluripotent stem cell-derived cardiomyocytes of the affected siblings and investigated the patient-specific cardiomyocytes on the molecular and functional level. RESULTS: Patients' induced pluripotent stem cell-derived cardiomyocytes recapitulated the hypertrophic phenotype and uncovered a so-far-not-described causal link between LZTR1 dysfunction, RAS-mitogen-activated protein kinase signaling hyperactivity, hypertrophic gene response and cellular hypertrophy. Calcium channel blockade and MEK inhibition could prevent some of the disease characteristics, providing a molecular underpinning for the clinical use of these drugs in patients with NS, but might not be a sustainable therapeutic option. In a proof-of-concept approach, we explored a clinically translatable intronic CRISPR (clustered regularly interspaced short palindromic repeats) repair and demonstrated a rescue of the hypertrophic phenotype. CONCLUSIONS: Our study revealed the human cardiac pathogenesis in patient-specific induced pluripotent stem cell-derived cardiomyocytes from NS patients carrying biallelic variants in LZTR1 and identified a unique disease-specific proteome signature. In addition, we identified the intronic CRISPR repair as a personalized and in our view clinically translatable therapeutic strategy to treat NS-associated hypertrophic cardiomyopathy.

Authors: U. Hanses, M. Kleinsorge, L. Roos, G. Yigit, Y. Li, B. Barbarics, I. El-Battrawy, H. Lan, M. Tiburcy, R. Hindmarsh, C. Lenz, G. Salinas, S. Diecke, C. Muller, I. Adham, J. Altmuller, P. Nurnberg, T. Paul, W. H. Zimmermann, G. Hasenfuss, B. Wollnik, L. Cyganek

Date Published: 15th Sep 2020

Publication Type: Journal

Abstract (Expand)

Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell-induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.

Authors: X. Zheng, L. Zhang, Y. Kuang, V. Venkataramani, F. Jin, K. Hein, M. P. Zafeiriou, C. Lenz, W. Moebius, E. Kilic, D. M. Hermann, M. S. Weber, H. Urlaub, W. H. Zimmermann, M. Bahr, T. R. Doeppner

Date Published: 4th May 2020

Publication Type: Journal

Abstract (Expand)

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca(2+) cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca(2+) handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca(2+)-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca(2+) regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions.

Authors: S. Khadjeh, V. Hindmarsh, F. Weber, L. Cyganek, R. O. Vidal, S. Torkieh, K. Streckfuss-Bomeke, D. Lbik, M. Tiburcy, B. A. Mohamed, S. Bonn, K. Toischer, G. Hasenfuss

Date Published: 7th Mar 2020

Publication Type: Journal

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