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Spuler Lab

Myology

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We take our muscles for granted. They permit us to go about our daily lives and with perhaps the exception of a few aches and pains they serve us well until our lives have ended.

However, for persons with genetic muscle diseases, this happy state-of-affairs is not the case.

These persons are wheel chair bound from child- or young adulthood until they die. There are many such patients. Genetic muscle diseases are not rare. But acquired muscle diseases such as critical care myopathy, muscle cachexia following cancer, heart failure or renal failure, traumatic muscle ischemia, or myopathy following drug reactions are definitely even more common.

We still have no treatment for any genetic muscle disease. However, we have made marked progress on an understanding of their pathologies. For acquired muscle disease, we are in the process of developing treatment strategies, although as always, “vigilance equals avoidance”.

Team

Group Leader

Prof. Dr. Simone Spuler

simone.spuler@charite.de
simone.spuler@mdc-berlin.de

Phone +49 30 450 540501
  
Phone +49 30 450 540504
 

Project Referee

Monique Wysterski

monique.wysterski@charite.de
monique.wysterski@mdc-berlin.de
Phone +49 30 450 540504

 

Senior Scientist

Dr. rer. nat. Andreas Marg

Member of the Spuler Lab since 2010

Area of expertise: Muscle stem cells   
Current Project: Satellite cells and heterogeneity

andreas.marg@charite.de
Phone +49 30 450 540524

 

Scientists, Physicians

Biniam Bekele, M.D., M.Sc.

Member of the Spuler Lab since 2019

Area of Expertise: Clinical development, regulatory affairs, Trial design 
Current Project: MUST-Trial (Clinical Trial)

biniam.bekele@charite.de
Phone +49 30 450 540518
 

Dr. Helena Escobar Fernandez

Member of the Spuler Lab since 2016

Current project: Precise gene editing of muscular dystrophy-causing mutations in patient-derived induced pluripotent stem cells

helena.escobar@charite.de
helena.escobar@mdc-berlin.de
Phone +49 30 450 540523

 

Dr. med. Elisabetta Gazzerro
Senior Physician of the out-patient clinic

Member of the Spuler Lab since 2017

Area of expertise: Neuromuscular Diseases, Metabolic Diseases
Current project: Immunological impact of muscular dystrophies

elisabetta.gazzerro@charite.de
Phone +49 30 450 540514

 

Janine Kieshauer, M.Sc.

Member of the Spuler Lab since 2017

Area of expertise: Muscle Cell biology, regulatory affairs, Advanced Therapy Medicinal Product (ATMP) development
Current project: Validation of processing and manufacturing human muscle stem cells as an ATMP

janine.kieshauer@charite.de
Phone +49 30 450 540518

 

Anne Krause, M.Sc.

Member of the Spuler Lab since 2018

Area of expertise: Molecular biology, induced pluripotent stem cells (iPSCs)

anne.krause@charite.de
anne.krause@mdc-berlin.de

Phone +49 30 450 540506
 

Dr. Eric Metzler

Member of the Spuler Lab since 2015

Area of expertise: human induced Pluripotent Stem Cells (hiPSCs), Reprogramming, Myogenic Differentiation, Primary human muscle cell
Current project: Primary and induced pluripotent stem cell-derived human satellite cells for cell-based therapies

eric.metzler@charite.de
eric.metzler@mdc-berlin.de
Phone +49 30 450 540523

 

Dr. Stefanie Müthel

Member of the Spuler Lab since 2018

Area of expertise: Calpain, LGMD2A, Gene editing, Epigenetics 
Current project: Precise gene editing of LGMD2A causing mutations

stefanie.muethel@charite.de
Phone +49 30 450 540518

 

Dr. Hans-Jürgen Peter

Member of the Spuler Lab since 2018

Area of expertise: Biotechnology, immunology, plant biochemistry, immunoassays, protein purification, HPLC,
GC-MS, blotting techniques, fluorescence microscopy, analysis & isolation of secondary plant compounds, bacteria & plant cell cultures, human mast cells, 2D/3D-CAD
Current project: Production of bacterial nanocellulose

hans-juergen.peter@charite.de
hans-juergen.peter@mdc-berlin.de


Phone: +49 30 450 540584

Dr. Joanna Schneider 

Member of the Spuler Lab since 2016

Current project: Epigenetic changes in critical illness myopathy

joanna.schneider@charite.de
joanna.schneider@mdc-berlin.de
Phone +49 30 450 540
514
 

Dr. med. Verena Schöwel-Wolf, MBA Clinical Scientist

Member of the Spuler Lab since 2008

Area of expertise: Stem cell therapy development: Advanced Therapy Medicinal Product (ATMP) product development, planning first-in-human trial, strategy debelopment according to ATMP market, orphan drug specifications, fundraising (public/NGO)
Current project: Development of a muscle stem cell therapy to fight muscle wasting

Research topic: Metabolism in dysferlinopathy

verena.schoewel@charite.de
verena.schoewel@mdc-berlin.de
Phone +49 30 450 540518

Dr. rer. nat. Haicui Wang

Member of the Spuler Lab since 2020

Area of expertise: Human genetics, Cell biology, Biochemistry
Current Project: Gene editing in LMNA related muscular dystrophy patient-derived cells

haicui.wang@charite.de
haicui.wang@mdc-berlin.de

Phone +49 30 450 540518

 

PhD Candidates, Students

Silvia Di Francescantonio, M.Sc. Medical Biology

Member of the Spuler Lab since 2017

Area of expertise: Stem cell biology, Muscle biology, Quiescence, Gene Editing (CRISPR-Cas), Dysferlin
Current project: Bacterial nano-cellulose: human muscle stem cells culture strategy for improving gene-editing approaches

silvia.di-francescantonio@charite.de
Phone +49 30 450 540506  

 

Christian Stadelmann, M.Sc. Translational Medicine

Member of the Spuler Lab since 2020

Area of expertise: Molecular Biology, Genetics, Gene Editing using CRISPR-based methods, Stem Cell Biology
Current project: GMP-compliant Gene Editing in Primary Muscle Stem Cells for Autologous Transplantation 

christian.stadelmann@charite.de
christian.stadelmann@mdc-berlin.de
Phone +49 30 450 540523
 

Alexej Zhogov

Member of the Spuler Lab since 2019

Current project: Characterization of humanized mouse models for muscular dystrophy

alexej.zhogov@charite.de
Phone +49 30 450 540518


 

Technical Assistents

Stephanie Meyer-Liesener
Head technician

Member of the Spuler Lab since 2008

Area of expertise: cell culture techniques, lab management 
Current project: Regulation und Fehlregulation von Muskelwachstum

stephanie.meyer@charite.de
Phone +49 30 450 540506

 

Stefanie Haafke, Biology laboratory technician 

Member of the Spuler Lab since 2012

Area of expertise: Molecular biology, immunofluorescence staining, cell culture

stefanie.haafke@charite.de
Phone +49 30 450 540518

 

Adrienne Rothe, Biological- technical assistant

Member of the Spuler Lab since 2013

Area of expertise: histology, mouse preparation
Current project: Human diagnostics, Sgca- mice

adrienne.rothe@charite.de
Phone +49 30 450 540518

 

Interns

Magdalena Bolsinger
Lucia Link Dopazo
 

Alumni

Dr. rer. nat. Jakub Malcher

Postdoctoral fellow 2018 - 2020
MyoGrad PhD fellowship from 2013 - 2018

Former project: Exon skipping and genome editing as therapeutic strategies for dysferlinopathy

 

 

Research

Gene Editing in Muscular Dystrophies

Helena Escobar Fernandez, Stefanie Müthel, Christian Stadelmann, Haicui Wang,
Alexej Zhogov

Precise gene editing of muscular dystrophy-causing mutations in patient-derived induced pluripotent stem cells

Helena Escobar Fernandez

Muscular dystrophies are devastating diseases for which specific treatments are still lacking. They cause a progressive loss, degeneration and weakness of skeletal muscle and are often monogenic (caused by mutations in a single gene). A possible therapeutic avenue is thus to repair the genetic defect in patient-derived cells and later use those for autologous transplantation. Muscle stem cells (MSC) are responsible for muscle regeneration and would be the cell type of choice for repairing dystrophic muscles in a cell therapy context. However, they are scarce within the muscle tissue, have a limited proliferative potential and are difficult to manipulate genetically. Therefore, the number of MSC that could be taken from a patient and re-infused following gene repair would likely not suffice to treat large groups of muscles. Induced pluripotent stem cells (iPSC), on the other hand, can be generated from the patient’s adult somatic cells, widely expanded ex vivo, genetically corrected and differentiated back into cells displaying some of the characteristics of MSC. Our work focuses on developing a platform for precise gene editing of muscular dystrophy-causing mutations in patient-derived iPSC and the subsequent generation of gene-corrected MSC-like cells from each individual donor. We are developing methods to reliably assess the phenotypic rescue of the genetic defect in vitro as well as the biosafety features and in vivo regenerative potential of the gene-corrected iPSC-derived MSC through transplantation studies into mouse skeletal muscle.

 

 

Precise gene editing of LGMD2A causing mutations

Stefanie Müthel

Gene editing is a powerful tool to repair disease-causing mutations in patient-derived primary cells. In this project we aim to repair mutations in the CAPN3 gene. Calpain 3, the protein encoded by CAPN3, is a cysteine protease predominantly expressed in the skeletal muscle. Mutations in CAPN3 lead to Limb Girdle Muscular Dystrophy Type 2A, a progressive skeletal muscle disorder without treatment and the most common LGMD worldwide. 

In our Outpatient Clinic we have several patients suffering from LGMD2A with different mutations in CAPN3. The project aims to establish an efficient gene editing platform for precise gene correction of disease-causing mutations in primary human muscle stem cells from patients. To assess a successful gene repair, we are also developing methods to determine reliably the rescue of the phenotype in vitro and in vivo. Due to the regenerative potential of primary muscle stem cells, we are confident to use repaired muscle stem cells from LGMD2A patients for an autologous cell therapy treatment. 

Gene Edited Primary Human Muscle Stem Cells for Treatments in Muscular Dystrophy

Christian Stadelmann

Muscle-specific stem cells populate human skeletal muscle and hold great regenerative potential. They can be isolated from patients, expanded, and genetically manipulated in cell culture. These qualities make them a promising candidate for the development of an autologous cell therapy for the treatment of hereditary Muscular Dystrophies.

The heterogeneous group of untreatable muscle wasting disorders is often caused by monogenic mutations that can be targeted with precise CRISPR/Cas-based gene editing tools such as traditional CRISPR/Cas9 editing or the newly developed base editors.

This project aims to develop gene editing strategies that may become suitable for clinical use. This entails optimising and validating the ex-vivo gene editing step to establish a safe and efficient protocol to be translated to the clinic.

Gene editing in LMNA related muscular dystrophy patient-derived cells

Haicui Wang

Classical laminopathy refers to diseases caused by mutations in gene LMNA coding for lamin A/C, key components of nuclear lamina on the interior of the nuclear envelope. The majority of classical laminopathy are caused by autosomal dominant LMNA mutations and the clinical phenotypes can vary from muscular dystrophy, dilated cardiomyopathy, Charcot-Marie-Tooth type 2B, to aging phenotype progeria.

We aim to use the state of the art gene editing tools including CRIPSR-Cas9 with double strand breaks or CRISPR base editing without double strand breaks to correct the mutations in cells derived from LMNA related muscular dystrophy patients. The screening for the efficient editing tool is carried on with patient derived induced pluripotent stem cells (iPSC). The patient derived muscle stem cells corrected with the validated editing strategy from iPSC are prepared further for the transplantation therapy.

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Muscle Stem Cells

Silvia Di Francescantonio, Andreas Marg, Eric Metzler

Bacterial Nano-Cellulose: Human Muscle Stem Cells Culture Strategy For Improving Gene-Editing Approaches

Silvia Di Francescantonio

 

Satellite cells and heterogeneity

Andreas Marg

Muscle repair and regeneration require activation of satellite cells. These rare muscle precursor cells are located in a specific niche and are probably mitotically quiescent in healthy muscle. It is unclear to what extent satellite cells are heterogeneous in respect to their gene expression profiles, their myogenic differentiation potential and their stemness. Today, our understanding of human satellite cell heterogeneity is fragmentary, but clinical applications of stem cell populations require extensive knowledge in this field.

In collaboration with the "Berlin Institute for Medical Systems Biology" (BIMSB) we used the drop-seq method for the rapid profiling of single cells. After sequencing, we obtained the mRNA expression profile of thousands of satellite cells. Based on these data, we are now trying to isolate the cells with different cultivation and selection methods that have the best requirements for a successful therapy of muscular dystrophies.

Human activated human satellite cells show heterogeneity.
 

Development of a cell therapy concept based on the generation of human induced Pluripotent Stem Cells (hiPSCs) and their differentiation into induced myogenic cells

Eric Metzler

Human induced pluripotent stem cells (hiPSCs) are a keystone to unrestricted cell numbers which are necessary for gene correction and repopulation of large organs such as skeletal muscle in genetic muscular dystrophies. hiPSCs have been generated from many different cell types and several protocols have been established to differentiate them into muscle cells or dedicated muscle stem cells. However, the biotechnological and therapeutic capabilities of these induced myogenic cells remain unclear.

This project aims to develop an efficient in vitro myogenic differentiation strategy, including the comparison of hiPSCs generated from different somatic sources, to finally produce a pure population of induced myogenic cells with a high potential to contribute to myofibre formation in vivo. 

Toxic Myopathies

Joanna Schneider

Epigenetic changes and repair of the DNA breaks in skeletal muscle/or muscle stem cells in critical illness myopathy

Joanna Schneider

Critical illness myopathy (CIM) is a devastating acquired skeletal muscle disease characterized by atrophy, flaccid paralysis and respiratory failure. It develops in severely ill patients during the course of critical illness and is a frequent complication of intensive care unit (ICU) treatment. It is a very peculiar aspect of CIM that, in some patients, skeletal muscle atrophy and weakness last for a prolonged period of time, often lifelong, although all identified risk factors like inflammation, hyperglycemia, medications etc. have been removed. We hypothesize that the acute phase of severe critical illness leads to epigenetic changes in skeletal muscle stem cells or early myoblasts, which results in an impaired ability of muscles to regenerate, a long lasting myopathy and an increase of DNA double-strand breaks in the muscle cells. Our project aims to identify and characterize the epigenetic modifications in muscle stem cells derived from acute-onset CIM patients within the first days after admission to the ICU. We analyze the epigenome and transcriptome as well as the DNA double-strand breaks process of activated satellite cells and early myoblasts. This project is part of the Clinical Scientist Program of the Berlin Institute of Health and Charité - Universitätsmedizin Berlin.

Application oriented projects

Biniam Bekele, Janine Kieshauer, Hans-Jürgen Peter, Verena Schöwel

Validation of processing and manufacturing human muscle stem cells as an ATMP

Janine Kieshauer

Skeletal muscle, the largest organ of the human body, possesses an own stem cell population, the satellite cells (SCs). Their high regenerative capacity makes SCs a perfect source of cells for cell-based therapies of muscle diseases. We invented a new technology that allows for the first time million-fold expansion of human satellite cells and at the same time the delay of their differentiation. We name our product PHSat (primary human satellite cell product). Hypothermia pretreatment eliminates otherwise co-isolated contaminating fibroblasts. Without the need of a cell-sorting procedure our cell colonies are >98% myogenic (desmin-positive). The muscle tissue is gently mechanically prepared, no enzymatic digestion is performed. By this, we generate native (not activated) and highly regenerative satellite cell populations. The regenerative potential of PHSats has been demonstrated in preclinical efficacy studies: 1. Injected PHSats build muscle fibers, 2. They re-populate the satellite cell niche and 3. They regenerate muscle also after re-injury. The aim of this project is to develop a pharmaceutical manufacturing process to transfer the product PHSat into a clinical study. This process must be carried out under a specific infrastructure, whereby highly standardized parameters must be established in order to characterize the product for regulatory approval in the best possible way. This was enabled by the funding of Pregobio.

The product is currently in a pre-clinical study, where it is being tested for possible harmful effects. These data are essential for the start of the clinical study and the pre-clinical study was funded by Spark.
​​​​​​

Establishment of a GMP-validated serial production of templates and supports made of bacterial nano-cellulose for the long-term cultivation of adult and stem cell lines (e.g. myoblasts)

Hans-Jürgen Peter

The main goal of the “Muscle Research Unit” of Professor Simone Spuler at the Experimental and Clinical Research Centre (ECRC ) is the gene therapy of vegetative muscle dystrophy on myoblasts, which are harvested from patient’s biopsies. Unfortunately, in conventional culture flasks these cells proliferate and differentiate themselves very fast into mature myofibers. Too fast to evaluate an appropriate gene editing system such as CRISPR/Cas9. However, for several cell lines it is described that both, pluripotent stem cells and adult cells can be cultivated on cellulose matrices in a non-dividing stage for many weeks. Also, on bacterial nano-crystalline cellulose (BNC) as culture matrix, the myoblasts remain alive in non-dividing stage for up to 60 days. Apparently mimicking a natural environment BNC stimulates adult cells to a natural physiological behavior as shown as in vivo... ​​​​​

Cellulose, a ß-1, 4, poly-Glucane is the most organic macromolecule on the earth. Usually, Cellulose is harvested from vegetable organisms, in particular from wood. However, the purification processes are involved with severe environment pollutions, because cellulose is integrated as cell wall component and incrustinated by lignin’s etc... In contrast acetic acid bacteria such as Gluconacetobacter do not incorporate the cellulose into their cell walls They synthesize and release cellulose in nearly complete purity into the extracellular environment. One needs only to remove the remains of the culture medium. Furthermore, the molecular structure of bacterial nano-cellulose (BNC) is crystalline (up to 80%) than that of plant cellulose. The only disadvantage of BNC is its long fermentation time to get a sufficient amount and in particular, the fact that many Gluconacetobacter strains are genetically unstable and lose their biosynthetic ability for cellulose. Therefore, BNC for cell culture is not commercially available so far...

Our target is therefore to establish a stable serial production of BNC-discs for cell cultures. We recently managed to establish a stable production of BNC with Gluconacetobacter sucrofermetans by cultivating them into a newly formulated medium based on Green Tea (Carmelia sinensis L.). It is now possible to breed BNC in each 2-dimensional shape and stiffness and up to a size of 25000 cm² (fig.1)

Fig.1 It’s clean! That’s the reason for the smiling BNC face.

Our 1st goal is to up scale the so far limited BNC breeding to a mass production. BNC-dishes for 6-well, 12-well and 24-well plates shall be serially provided (fig.2) as culture matrices for non-dividing cells.

Fig.2 serial assemble of laser cut BNC-discs for 6-well plates.

2nd we want to establish a validation and quality control for the preparative production of cell culture appropriate BNC-sheets according the GLP/GMP-criteria. For that target we elucidate the optimal growth conditions, perform qualified studies to get information about the physical structures and possible endotoxin content.

Furthermore, I want to investigate whether several plant polyphenols of tealeaves might affect and enhance the Cellulose biosynthesis in Gluconacetobacter and how they do that. The focus of this project is lying on the expression and activity of the Cellulose-Synthase enzyme complex.

Finally, our target will also be the development of 3-dimensional site directed BNC-scaffolds for the in vitro creation of muscle organoids and tissues after gene therapeutic repair of defect ipSC. All targets involve the design and construction of special fermenters and machines for the download processes.

Muscle stem cells as ATMP - Creation of a biobank

Verena Schöwel-Wolf

 

Clinical Research

  • International clinical trial on the natural history of dysferlinopathies
  • Muscle metabolism in facioscapulohumeral muscular dystrophy
  • Cardiac involvement in facioscapulohumeral muscular dystrophy

 

Publications

Patients

The university outpatient clinic for muscle disorders offers in close cooperation with primary care physicians or other specialists diagnostic expertise and long-term care for patients suffering from muscle diseases. Transferal is possible from all medical specialties.

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