Research Team of Jiří Pacherník

Keywords

myocard, stem cell, cardiomyogenesis, specification of cardiomyocytes

Head of laboratory: Mgr. Jiří Pacherník, Ph.D.
Office: UKB – A36/111
E-mail: jipa@sci.muni.cz
Phone: 549 49 5578
Teaching
Publications

 

Ongoing projects:

Stem cells are specialized group of cells which are capable of self-renewal and producing same type of cells, but also of differentiating, changing to a different, more specialized type of cell. They play an important role in growth and regulation of homeostasis of animals during their whole development. Study of mechanisms that are responsible for the origin and preservation of stem cells is essential for understanding of basic principles of normal and pathological development of animals.

In our laboratory as a model organism we use lab mouse (Mus musculus domesticus) and cell lines that are derived from it as well as some human cell line (mostly human induced pluripotent stem cells). Our experiments are performed on embryonic stem cells and cells that differentiate from them, where we mainly focus on cardiomyocytes.

According to up-to-date research, adult heart does not contain stem cells and its regeneration capacity is very limited. Therefore in case of big damage to myocardium, possible treatments are either whole heart transplantation or transplantation of cardiomyocytes that were prepared from stem cells in vitro. Ideal source for in vitro preparation of cardiomyocytes would be stem cells of myocardium. Cardiac progenitors, which transiently exist during the embryonic development could be considered as model for these type of stem cell. However, in the case of cardiomyocytes that are prepared in vitro, we must also take into consideration what type of cardiomyocytes were actually prepared.

Our research is therefore mainly focused on preparation of myocardial stem cells, preparation and identification of different cardiomyocytes and providing answers to following problems:

How is differentiation directed by certain factors
Starting from pluripotent cells and later from cells of mesoderm, cardiomyogenesis is not a spontaneous process, but one governed by many factors – structures and chemicals to which the cells are exposed to and many of which they produce themselves. Better understanding of these factors and mechanisms by which they affect the process of cardiomyogenesis leads to increase in efficiency of differentiation and higher yield of cardiomyocytes which can be cultivated in vitro.
In case of structures that can have some influence on cells in vitro, we are mostly focused on observing changes in characteristics and in differentiation of cells that are cultivated on different kinds of polymers. We are also trying to co-cultivate already isolated cardiomyocytes with other types of cells which can be found in heart (fibroblasts) in 3D structures, where we are studying changes in their morphology and function. When it comes to chemical compounds we are mostly interested in different kinds of inhibitors and activators of various signaling pathways and we are trying to study their effect on differentiation into different types of cardiomyocytes.
Flux of calcium in cardiomyocytes
How to prepare cardiac stem cells
First specialized cell types that are derived from mesoderm and which later give rise to most of cells found in heart, cardiomyocytes included, are cardiomyogenic mesodermal cells. These cells are very immature and are quickly proliferating. If these cells would be cultured in vitro and would retain their properties, they could be considered as cardiac stem cells. However, to achieve this, it is necessary to know what mechanisms are responsible for their characteristics. Therefore, we are applying different factors on stem cells and on cells in their early stages of differentiation and we are trying to describe their effects and potential in obtaining cardiac stem cells.
How to distinguish different subtypes of cardiomyocytes
There is a certain variability among cardiomyocytes due to their different location and function in the heart. Specification of individual cardiomyocytes can be detected on gene expression level as well as on differences in their physiological properties. In adult heart individual types of cardiomyocytes are therefore clearly defined by their characteristics such as localization, morphology, expression profile, action potential, dynamics of calcium flux, just to name a few. Since in the in vitro culture the cells are exposed to some conditions, which differ from in utero development, some of the markers, when compared to those found in adult heart, can also be a bit different and therefore it is more difficult to strictly divide these cells to different subtypes. Furthermore, these markers change over time during the development and differentiation of cells both in vivo and in vitro. To be able to determine more precisely what cells were are operating with, subtype and their development stage, we are trying to use many methods (including qPCR, westernblotting, patchclamp, mass spectrometry…) and we are further optimizing them for higher sensitivity and accuracy (using CRISPR-Cas, comparing in vitro derived cardiac cells with embryonal and adult hearts etc.).
Role of signaling pathways in cell differentiation
Although there are many signaling pathways that regulate cell differentiation, our focus is turned mainly to the MAPK pathway.
Mitogen activated protein kinases (MAPK) represent a family of proteins which is a part of MAPK signaling pathway and which is responsible for cells reaction to growth factors, hormones, stress, but also plays an important role in differentiation into many cell types. We have observed that there are changes in this signaling pathway in hypoxic conditions, then when certain chemical compounds are applied to cells as well as changes in phosphorylation of MAPK members during differentiation. MAPK are enzymatically active in their phosphorylated state and inactive in dephosphorylated. This inactivation can be carried out specifically by family of dual specificity phosphatases (DUSPs). Goal of one of our projects is shedding some light on what impact DUSP family has on MAPK pathway and subsequently on differentiation into cardiac cells.
Applied research
Our skills and finding are applicable not only for basic research, but also in applied biotechnical research. Cells derived by us are used for testing of biocompability and physical properties of conductive synthetic polymers. In this area we have two main goals. One is to develop artificial strictures for cells cultivation which could lead to cultivation of tissues or organs for transplantation. The second it development of analytical probes, which could allow to track chemical changes in the tissue in vivo.

Collaborations

  • Doc. Ing. Petr Humpolíček, Ph.D. Tomas Bata University in Zlin, Faculty of Technology, Polymer Centre
  • Doc. Mgr. Petr Vaňhara, Ph.D. Masaryk University, Faculty of Medicine

Research team:

Alumni:

List of grants
• GA19-16861S 01.01.2019 – 31.12.2021 Interakce biomateriálů s kmenovými buňkami v simulovaných in vivo podmínkách, spoluřešitel: J. Pacherník
• GA18-18235S 01.01.2018 – 31.12.2020 Nové možnosti v kardiomyogenní diferenciaci pluripotentních kmenových buněk, rešitel: J. Pacherník
• GA17-05466S 01.01.2017 – 31.12.2019 Role kanonické signální dráhy Wnt v neurogenezi, spoluřešitel: J. Pacherník
• GJ15-13443Y 01.01.2015-31.12.2017 Úloha hypoxií indukovaného faktoru 1 alfa ve vývoji populace neurálních kmenových buněk myši, řešitel: J. Večeřa

Studentské projekty:
• Příprava ventrikulárního kardiomyocytu z pluripotentních kmenových buněk (E. Kohoutková)
• MUNI/C/1392/2017 01.02.2018 Interakce hypoxie a signální dráhy Wnt v neurogenezi (L. Woloszczuková)
• MUNI/C/0836/2011 01.01.2012 Exprese a aktivita ABC transportérů odpovědných za MDR u mES buněk (M. Kohutková Lánová)