General note: We are proud and happy that the laboratory is attracting many excellent and motivated students. Due to Corona crisis as well as the high number of requests for internships, doctoral thesis projects and Research Track/WissPro projects we are currently not able to accept any further applications in winter semester 2020/21 and in summer semester 2021.
Denise Philipp, Michelle Holthaus, Vida Basoah, Kurt Pfannkuche, Laura Suhr, Thorsten Wahlers, and Adnana Paunel-Görgülü
Myocardial hypertrophy is present in many heart diseases, representing a strong predictor of adverse cardiovascular outcomes. Regarding therapeutic intervention, mesenchymal stem cells (MSCs) have been suggested to significantly reduce cardiac hypertrophy and progression to heart failure. Preconditioning of MSCs was previously demonstrated to highly improve their paracrine activity resulting in modulation of immune responses and the progression of diseases. Here, we studied the effects of bone marrow-derived preconditioned MSCs on hypertrophied induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) and also sought to identify MSC-derived antihypertrophic molecules. Phenylephrine (PE) was used to induce hypertrophy in murine iPS-CM, and markers of hypertrophy were identified by microarray analysis. Murine MSCs were treated with IFN-γ and IL-1β to enhance their paracrine activity, and transcriptional profiling was performed by microarray analysis. Hypertrophied iPS-CM were subsequently cocultured with preconditioned MSCs or MSC-conditioned medium (CM), respectively. Effects on hypertrophied iPS-CM were studied by cell area quantification, real-time PCR, and western blot. In some experiments, cells were incubated with fractions of MSC-CM obtained by ultrafiltration or by MSC-CM supplemented with inhibitory antibodies. Intracellular and extracellular levels of vascular endothelial growth factor (VEGF) were evaluated by western blot and ELISA. PE-induced hypertrophy in iPS-CM was associated with an upregulation of neuron-derived orphan receptor (Nor1) expression, activation of Akt, and inhibition of both strongly prevented hypertrophy induction in iPS-CM. VEGF secreted by preconditioned MSCs provoked hypertrophy regression in iPS-CM, and a negative correlation between Nor1 expression and hypertrophic growth could be evidenced. Our results demonstrate that Nor1 expression strongly supports hypertrophy in iPS-CM. Moreover, the secretome of preconditioned MSCs triggered regression of hypertrophy in iPS-CM in a VEGF-dependent manner. We suggest that the delivery of the MSC-derived secretome may represent a therapeutic strategy to limit cardiac hypertrophy. However, additional in vivo studies are needed to prove this hypothesis.
Read the full text HERE
Cardiomyocytes, differentiated from induced pluripotent stem cells (iPSCs), have the potential to produce patient- and disease-specific pharmacological and toxicological platforms, in addition to their cardiac cell therapy applications. However, the lack of both a robust and a simple procedure for scalable cell substrate production is one of the major limitations in this area. Mimicking the natural healthy myocardium extracellular matrix (ECM) properties by altering the cell substrate properties, such as stiffness and chemical/biochemical composition, can significantly affect cell substrate interfacial characteristics and potentially influence cellular behavior and differentiation of iPSCs to cardiomyocytes. Here, we propose a systematic and biomimetic approach, based on the preparation of poly(dimethylsiloxane) (PDMS) substrates having the similar stiffness as healthy heart tissue and a well-defined surface chemistry obtained by conventional [(3-aminopropyl)triethoxysilane (APTES) and octadecyltrimethoxysilane (OTS)] and amino acid (histidine and leucine)-conjugated self-assembled monolayers (SAMs). Among a wide range of different concentrations, the 50:1 prepolymer cross-linker ratio of PDMS allowed adaptation of the myocardium stiffness with a Young’s modulus of 23.79 ± 0.61 kPa. Compared with conventional SAM modification, amino acid-conjugated SAMs greatly improved iPSC adhesion, viability, and cardiac marker expression by increasing surface biomimetic properties, whereas all SAMs enhanced cell behavior, with respect to native PDMS. Furthermore, leucine-conjugated SAM modification provided the best environment for cardiac differentiation of iPSCs. This optimized approach can be easily adapted for cardiac differentiation of iPSCs in vitro, rendering a very promising tool for microfluidics, drug screening, and organ-on-chip platforms.
Read the full manuscript here:
Transposon for fluorescent labelling and puromycin selection of human stem cell derived cardiomyocytes
As a service to our colleagues working in the field of human pluripotent stem cell derived cardiomyocytes the Marga and Walter Boll Laboratory for Cardiac Tissue Engineering releases a transposon free of charge via Addgene.org.
To access and order the plasmid go to https://www.addgene.org/163755/
You will receive a plasmid that contains a transposable element. A piggyBac transposase needs to be co-transfected for active insertion of the transposon in the genome. Selection of transfected human iPS cells is performed by Zeocin. Resistant clones need to be differentiated and screened for lines that show bright expression of the red fluorescent protein mCherry. As the coding sequence of mCherry is linked to the coding sequence of puromycin-N-acetyltransferase, red fluorescent cardiomyocytes exhibit resistance to puromycin and can be selected for experiments that require pure cells. Contact us if you need assistance.
10th International Meeting of the Stem Cell Network NRW
On March 29-30, 2021 the 10th International Meeting of the Stem Cell Network NRW “From Fundamental Biology to Translational Concepts” will take place – this year for the first time as a virtual event! The congress chairman Professor Oliver Bruestle of Bonn University/ University Hospital Bonn is looking forward to welcoming inspiring international stem cell experts, basic and clinical researchers as well as young talents and would like to invite you to join the conference.
The highlights include:
The deadline for abstract submission is February 15, 2021!
The preliminary program and further information about the event can be found on our homepage.
We look forward to seeing you on March 29 & 30, 2021!
Best regards from the Stem Cell Network NRW
Burkert K, Taheri H, Hamad S, Oliverio M, Peinkofer G, Kornfeld JW, Harnying W, Pfannkuche K, Hescheler J, Berkessel A, Šarić T.
Salicylic diamines selectively eliminate residual undifferentiated cells from pluripotent stem cell-derived cardiomyocyte preparations
Sci Rep. 2021 Jan 27;11(1):2391. doi: 10.1038/s41598-021-81351-z.
Clinical translation of pluripotent stem cell (PSC) derivatives is hindered by the tumorigenic risk from residual undifferentiated cells. Here, we identified salicylic diamines as potent agents exhibiting toxicity to murine and human PSCs but not to cardiomyocytes (CMs) derived from them. Half maximal inhibitory concentrations (IC50) of small molecules SM2 and SM6 were, respectively, 9- and 18-fold higher for human than murine PSCs, while the IC50 of SM8 was comparable for both PSC groups. Treatment of murine embryoid bodies in suspension differentiation cultures with the most effective small molecule SM6 significantly reduced PSC and non-PSC contamination and enriched CM populations that would otherwise be eliminated in genetic selection approaches. All tested salicylic diamines exerted their toxicity by inhibiting the oxygen consumption rate (OCR) in PSCs. No or only minimal and reversible effects on OCR, sarcomeric integrity, DNA stability, apoptosis rate, ROS levels or beating frequency were observed in PSC-CMs, although effects on human PSC-CMs seemed to be more deleterious at higher SM-concentrations. Teratoma formation from SM6-treated murine PSC-CMs was abolished or delayed compared to untreated cells. We conclude that salicylic diamines represent promising compounds for PSC removal and enrichment of CMs without the need for other selection strategies.
Read the full text HERE
Dr. Sarkawt Hamad from our group has contributed to a recent manuscript about the interpretation of statistic data.
Read the full text HERE
Salem Alawbathani, Mehreen Batool, Jan Fleckhaus, Sarkawt Hamad, Floyd Hassenrück, Yanhong Hou, Xia Li, Jon Salmanton-García, Sami Ullah, Frederique Wieters & Martin C. Michel
Naunyn-Schmiedeberg's Archives of Pharmacology (2021)
A teaching tool about the fickle p value and other statistical principles based on real-life data
A poor understanding of statistical analysis has been proposed as a key reason for lack of replicability of many studies in experimental biomedicine. While several authors have demonstrated the fickleness of calculated p values based on simulations, we have experienced that such simulations are difficult to understand for many biomedical scientists and often do not lead to a sound understanding of the role of variability between random samples in statistical analysis. Therefore, we as trainees and trainers in a course of statistics for biomedical scientists have used real data from a large published study to develop a tool that allows scientists to directly experience the fickleness of p values. A tool based on a commonly used software package was developed that allows using random samples from real data. The tool is described and together with the underlying database is made available. The tool has been tested successfully in multiple other groups of biomedical scientists. It can also let trainees experience the impact of randomness, sample sizes and choice of specific statistical test on measured p values. We propose that live exercises based on real data will be more impactful in the training of biomedical scientists on statistical concepts.
Effects of physicochemical properties of polyacrylamide (PAA) and (polydimethylsiloxane) PDMS on cardiac cell behavior
Karim Daliri , Kurt Pfannkuche, Bora Garipcan
In vitro cell culture is commonly applied in laboratories around the world. Cultured cells are either of primary origin or established cell lines. Such transformed cell lines are increasingly replaced by pluripotent stem cell derived organotypic cells with more physiological properties. The quality of the culture conditions and matrix environment is of considerable importance in this regard. In fact, mechanical cues of the extracellular matrix have substantial effects on the cellular physiology. This is especially true if contractile cells such as cardiomyocytes are cultured. Therefore, elastic biomaterials have been introduced as scaffolds in 2D and 3D culture models for different cell types, cardiac cells among them. In this review, key aspects of cell-matrix interaction are highlighted with focus on cardiomyocytes and chemical properties as well as strengths and potential pitfalls in using two commonly applied polymers for soft matrix engineering, polyacrylamide (PAA) and polydimethylsiloxane (PDMS) are discussed.
Soft Matter. 2021 Jan 11.
Link to full text: LINK
New Technology in the lab: PlasmaFecto 30
Our lab equipment has just been upgraded with a vacuum plasma device from Plasma Technologie. We are using argon and oxygen plasmas for plasma cleaning, plasma sterilization and surface activation and modification.
University Hospital press release
August-24 Congratulation Sarkawt for obtaining the PhD in Health Sciences.
Today, Sarkawt has defended his PhD thesis in the graduate school PhD Health Sciences at the Medical Faculty of the University of Cologne. "An outstanding presentation for an outstanding thesis" as one of the reviewers told. That´s definitively true.
April-28 Congratulation Raja for obtaining your doctorate!
Today my team member Raja Sahito has successfully defended his thesis and obtained the doctor title.
Congratulations Raja and good luck with your next projects!
The laboratory is almost down due to the restrictions associated with the spreading of the Corona virus. However, we are still making progress and Daniel has succeeded to derive a seahorse from human stem cells.
Supercritical carbondioxide sterilizer from Novasterilis
New technology has arrived! Thanks to funding by the Marga-and-Walter-Boll Foundation we are now installing a Novasterilis device in our laboratory. The device can be used to sterilize sensitive biological materials with supercritical carbon dioxide. Furthermore, it can decellularize tissues by the same approach but at higher pressures.
Marga-and-Walter-Boll Laboratory for Cardiac Tissue Engineering
The Marga-and-Walter-Boll Foundation has decided to support my group with a new laboratory for cardiac tissue engineering. A room at the Center for Physiology and Pathophysiology is already found and we will soon start with renovations and purchasing the equipment. The Marga and Walter Boll Laboratory for Cardiac Tissue Engineering will allow us to intensify research on human iPS cell-derived cardiomyocytes for cardiac tissue engineering and cell therapy. Thanks a lot for this great opportunity!
2nd Cologne Conference on Cardiac Regeneration and Therapy (26./27.9.2019)
Our conference is coming closer and we have finalized the conference program. Registration is open as well.
Please visit the conference page www.cococare.de
(updated October 2019)
Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations
Sarkawt Hamad, Daniel Derichsweiler, Symeon Papadopolous, Filomain Nguemo, Tomo Saric, Agapios Sachinidis, Jürgen Hescheler, Bastiaan Boukens, Kurt Pfannkuche
Theranostics online date 2019-7-18; doi:10.7150/thno.32058; accepted
To download the PDF version click here
On March 21st the 7th "Forschungsbörse" took place at the Medical Faculty Cologne. This event is organized by the students and allows research groups of the faculty to present their current work and discuss project opportunities with the students.
I had a lot of interesting discussions with our highly motivated students that inquire for information on scientific projects and seek to inform themselves on current progress in cardiac tissue engineering and stem cell technologies.
(Foto: Dorothea Hensen, Medizinfoto Köln)
First Cologne Conference on Cardiac Repair
Save the date: Sept. 26-27, 2019
Organizers: Kurt Pfannkuche and Tomo Saric
Further infos will appear soon on our conference website
Sponsor: Fritz-Thyssen-Foundation, Cologne
Leitmarkt NRW: Vaskularisierter, bioartifizieller Herzmuskel aus induziert-pluripotenten Stammzellen
We proudly announce that the project proposal "Vaskularisierter, bioartifizieller Herzmuskel aus induziert-pluripotenten Stammzellen" has been selected by the "Leitmarkt-Agentur" for funding.
In the frame of this project the project consortium will employ a radically novel approach to generate vascularized engineered tissues for therapeutical approaches.
LightFab GmbH, Aachen
Miltenyi Biotech GmbH, Bergisch Gladbach
Taros Chemicals GmbH und Co KG, Dortmund
Fraunhofer-Institut für Lasertechnik ILT, Aachen (Dr. Elke Bremus-Koebberling, Dr. Nadine Nottrodt, Dr. Martin Wehner)
Universitätsklinik Köln (AG PD Dr. Kurt Pfannkuche)
Universität zu Köln, Institut für physikalische Chemie (AG Prof. Annette Schmidt)
Follow this link to see a list with all granted projects: Pressemitteilung
"In vitro grown micro-tissues for cardiac cell replacement therapy in vivo"
Raja Ghazanfar Ali Sahito, Xiaowu Sheng, Martina Maass, Nelly Mikhael,l Sarkawt Hamad, Carlos O. Heras-Bautista, Daniel Derichsweiler, Dimitry Spitkovsky, Frank Suhr, Markus Khalil, Konrad Brockmeier, Marcel Halbach, Tomo Saric, Jürgen Hescheler, Benjamin Krausgrill, Kurt Pfannkuche
Background/Aims: Different approaches have been considered to improve heart reconstructive medicine and direct delivery of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) appears to be highly promising in this context. However, low cell persistence post-transplantation remains a bottleneck hindering the approach. Here, we present a novel strategy to overcome the low engraftment of PSC-CMs during the early post-transplantation phase into the myocardium of both healthy and cryoinjured syngeneic mice.
Methods: Adult murine bone marrow mesenchymal stem cells (MSCs) and PSC-CMs were co-cultured on thermo-responsive polymers and later detached through temperature reduction, resulting in the protease-free generation of cell clusters (micro-tissues) composed of both cells types. Micro-tissues were transplanted into healthy and cryo-injured murine hearts. Short term cell retention was quantified by real-time-PCR. Longitudinal cell tracking was performed by bioluminescence imaging for four weeks. Transplanted cells were further detected by immunofluorescence staining of tissue sections.
Results: We demonstrated that in vitro grown micro-tissues consisting of PSC-CMs and MSCs can increase cardiomyocyte retention by >10-fold one day post-transplantation, but could not fully rescue a further cell loss between day 1 and day 2. Neutrophil infiltration into the transplanted area was detected in healthy hearts and could be attributed to the cellular implantation rather than tissue damage exerted by the transplantation cannula. Injected PSC-CMs were tracked and successfully detected for up to four weeks by bioluminescence imaging.
Conclusion: This approach demonstrated that in vitro grown micro-tissues might contribute to the development of cardiac cell replacement therapies.
Read the full text:
Fig. 5. Histological observation of transplanted micro-tissues. Micro-tissues of low-dose MSC/iPS-CM were transplanted into cryoinjured cardiac tissue. Trichrome staining revealed fibrotic areas of myocardial damage after one week (A) and two weeks (E). Transplanted cells were stained with antibodies against luciferase (green) and nuclei were co-stained by Hoechst dye (blue). Tissue autofluorescence (red) was recorded to visualize the endogenous cardiac tissue. Panel A/B and C/D show results for two individual animals one week after cryoinjury and cell transplantation. Panel E/F show engrafted cells from one animal two weeks after cell transplantation. Panel G/H display magnified regions from the section shown in F.
CCCR: First Cologne Conference on Cardiac Repair: 26./27. September 2019
Together with Tomo Saric at the Institute of Neurophysiology, Cologne we are organizing the first Cologne Conference on Cardiac Repair. The Event will take place on the 26st and 27th Sept. 2019 at the Center of Physiology, Cologne. We gratefully aknowledge support of the Fritz-Thyssen-Foundation to organize the meeting. I am comming back with more detailed information on topics, invited and confirmed speakers soon. This will be a great chance for networking, I am happy to be part of it!
Carlos O.Heras Bautista*, Nelly Mikhael*, Jennifer Lam, Vaibhav Shinde, Alisa Katsen-Globa, Sabine Dieluweit,
Marek Molcanyi, Vladimir Uvarov, Peter Jütten, Raja G.A. Sahito, Francisco Mederos Henry, Alexander Piechot,
Konrad Brockmeier, Jürgen Hescheler, Agapios Sachinidis, Kurt Pfannkuche
Pathophysiological conditions, such as myocardial infarction and mechanical overload affect the mammalian heart integrity, leading to a stiffened fibrotic tissue. With respect to the pathophysiology of cardiac fibrosis but also in the limelight of upcoming approaches of cardiac cell therapy it is of interest to decipher the interaction of cardiomyocytes with fibrotic matrix. Therefore, we designed a hydrogel-based model to engineer fibrotic tissue in vitro as an approach to predict the behavior of cardiomyocytes facing increased matrix rigidity. Here, we generated pure induced pluripotent stem cell-derived cardiomyocytes and cultured them on engineered polyacrylamide hydrogels matching the elasticities of healthy as well as fibrotic cardiac tissue. Only in cardiomyocytes cultured on matrices with fibrotic-like elasticity, transcriptional profiling revealed a substantial up-regulation of a whole panel of cardiac fibrosis-associated transcripts, including collagen I and III, decorin, lumican, and periostin. In addition, matrix metalloproteinases and their inhibitors, known to be essential in cardiac remodeling, were found to be elevated as well as insulin-like growth factor 2. Control experiments with primary cardiac fibroblasts were analyzed and did not show comparable behavior. In conclusion, we do not only present a snapshot on the transcriptomic fingerprint alterations in cardiomyocytes under pathological conditions but also provide a new reproducible approach to study the effects of fibrotic environments to various cell types.
Mobility Grant for MSc. Sarkawt Hamad
Congratulations to Sarkawt for winning a mobility grant (IPaK: Promoting international students at the University of Cologne) supported by the DAAD and selected by the medical faculty, Cologne. The grant will allow Sarkawt to stay for 2 months in the laboratory of Dr. Carolina Galvez-Monton located at Badalona, Spain. The aim of this visit is to initate first experiments of cardiac cell replacement therapy using human induced pluripotent stem cell derived cardiomyocytes in a large animal model.
Novel project granted by the Elisabeth and Rudolf Hirsch-Foundation
We have just received the good news that the Elisabeth and Rudolf Hirsch Foundation gives support to the team thereby allowing us to conduct initial experiment of cardiac cell replacement therapy in a porcine model. This project is handled mainly by Daniel Derichsweiler and Msc. Sarkawt Hamad in the laboratory with support by Karina Neumann. Cell production technologies are established and experimental permissions were granted.
Collaboration News Gobio GmbH
Our group has supported the SME Gobio GmbH (Aarbergen, Germany) in the develop of a stem cell-based cell assays for toxicity measurements. The collaborative work was funded by the German Federal Ministry for Economic Affairs and Energy within a "ZIM project" and has now resulted in the application of patents in Europe and US
("Indicator stem cell line/living organism (non human) and a method for detection of the genotoxic potential of aquatic samples or aqueous solutions of test compounds.")
Networking Event Badalona - Spain
In preparation for the upcoming projects in the field of cardiac cell replacement therapy we have visited the IGTP Institute in Badalona, Spain to meet our collaboration partners Dr. Carolina Galvez-Monton and Prof. Bas Boukens (Amsterdam) for a brain storming. The novel facility at IGTP provides the best possible environment for our research with state of the art facilities and instrumentation.
Networking Event Tandojam - Cologne Initiative
In the follow up of our first online course in cell culture technologies for students at our partner University SAU in Tandojam, PK vicechancelor Prof. Memon is guest in Cologne to report on the ongoing initiative to establish a new institute for stem cell research. The building is now ready to use and we are planning the next steps.
Novel project granted by the German Research Foundation (DFG)
Our team has now received a novel grant to optimize the early retention of transplanted stem cell-derived cardiomyocytes in a rodent model of cardiac infarction. In the frame of the project our successful strategy of micro-tissue transplantation will be further developed to enhance the efficiency of the approach. Magnetic targeting is among the strategies that appear attractive to boost the cell transplantation success.
Generation of human induced pluripotent stem cell derived cardiomyocytes (HiPS-CMs) optimized
Sarkawt and Daniel from my team have made a hell of effort to optimize the differentiation of HiPS-CMs for reproducible generation of cardiac differentiation, high differentiation efficiencies of up to 95%, high yields of maximally 100 million cardiomyocytes per 100 ml suspension culture and controlled costs. Alltogether, we are now moving forward to plan preclinical studies in the porcine model together with Dr. Gálvez Montón in Badalona, Spain. Pall Corporation has just offered support for the bioreactor technology - hope to getting started soon.
Tandojam Laboratory of Regenerative Medicine
Our team supports the SAU to rise a own stem cell laboratory at Tandojam, Pakistan. This collaboration has arisen from a project ("KAZE" project) funded by the BMBF in the frame of scientific collaborations with partners in Pakistan in 2014. Raja is busy in networking and keeping everybody in the consortium busy. The Vice-chancellor of SAU Prof. Mujeeb-u-ddin Sahrai has visited Cologne on several occasions to keep the business rolling. Efforts have already payed-off and the Pakistan Ministry has just granted about 3.5 Mio. Euro to raise a stem cell laboratory at SAU. We are involved in the planning and help the SAU to get started in the field of stem cell technology. The online lecture series in April will be followed by further and more specific training measures in the future.
Online course on basic stem cell culturing techniques at Sindh Agricultural University (SAU)
Together with our colleagues at the Institute of Neurophysiology Cologne we have prepared an online course for cell culturing technologies for students at the SAU in Tandojam, Pakistan. The lecture was attended by nearly 50 persons from student to principal investigator level. We are planning further activities in the future to intensify our collaboration with the SAU in the field of Stem Cell Research. Many thanks to the Vice Chancellor Prof. Mujeeb-u-ddin Sahrai for his great support of the project.