Project: METASTEM - Metabolic signaling and energy homeostasis in human embryonic stem cells
Person in Charge: doc. Ing. Petr Dvořák, CSc.
Host institution: Department of biology, Faculty of Medicine, Masaryk University
Country of Origin: Ukraine
Country of scientific activity: Finland
Project duration: 33 months
Scientific panel: Life sciences
Current Proposal offers substantial progress in understanding how maintenance of pluripotency in hESC depends on energy homeostasis. Our research goal is to investigate effects of cellular metabolic parameters on maintaining undifferentiated status of hESCs. Our practical goal is to work out defined metabolic conditions which deliver promoting effects on maintaining undifferentiated status of hESCs, as present cultivation technologies do not allow for reliable stable propagation of xenobiotic-free medical grade hESCs. Our principal hypothesis is: energy homeostasis along with metabolic signaling defines, or at least gives very substantial impact on overall cellular signaling cascades in hESC. Our working hypothesis is: hESCs use glycolysis to obtain their energy. Cells shift their metabolism towards oxidative phosphorylation upon differentiation. We assume that metabolism is gradually shifted towards oxidative phosphorylation also in cells going through a phenomenon of culture adaptation. This implies that by interventions directed towards keeping energy homeostasis in hESC at glycolytic state we will achieve promoting effects on maintenance of their pluripotency. The research goals will be achieved through systematic investigation of main metabolic parameters, in particular intracellular and extracellular NAD+, NADH and their ratio, ATP, lactate, ROS, Plasma Membrane Electron Transport along with cell surface oxygen consumption, etc. in hESC at low, middle and high passage numbers and in their differentiated counterparts. The hypotheses will be confirmed by purposely modifying certain metabolic parameters and investigating phenotypical consequences of such interventions. It was agreed with the host Institution that the Applicant will establish his own research group and build up close collaborative subprojects with research institutions in Finland, Germany, Ukraine etc. based on his own already established connections.
The ongoing project summary:
· a brief summary of the project objectives:
As it is stated in my METASTEM Research Plan, we aimed at investigating how metabolism, especially energy homeostasis, is involved in maintenance of pluripotency and in differentiation of human embryonic stem cells (hESC). In addition, our objective was to establish a new research group and to create a collaborative circle connecting various research labs in different countries, as well as close supportive collaboration within our own lab. To that, we aimed at applying for the external funding and publishing research articles, as well as disseminating our knowledge through other possible means.
· brief summary of activities implemented within the project over the relevant period since the beginning of the project:
At first, I elaborated culture conditions for hESC in monolayers suitable for multi-well assays. Then I went on to substantially improve reproducibility of experiments with hESC in monolayers by a new protocol for freezing cells and storing them in liquid nitrogen. Meanwhile, I invested all possible efforts in order to establish a new independent research group. Together we developed in-house a set of biochemical assays to measure metabolic parameters in hESC and measured certain metabolic parameters. We then established a new and promising cellular model for studying the role of FGF2 in hESC in connection to their metabolic status. In addition, we constructed a series of plasmid vectors for inducible overexpression of various isoforms of human FGF2. We also established conditions for both transient and stable transfection of hESC by various methods and derived stable clones of hESC. Both international and internal collaboration was actively pursued; new international collaborative ties were initiated. During the year under review I participated in two scientific meetings with oral presentations and together with my group we applied for a research grant from IGA ČR in collaboration with University Hospital Brno. In addition, I published one research article where SoMoPro support was acknowledged.
· a brief summary of the main results achieved to date:
- Culture conditions for multi-well assays in monolayers were established:
- Freezing/thawing protocol was adopted to hESC in monolayers; the data are now in preparation for a research publication;
- Conditions for efficient stable transfection of hESC were elaborated;
- Stable hESC clones overexpressing GFP and tetracycline transactivator (rtTA) were derived;
- A series of plasmid vectors for inducible overexpression of various isoforms of human FGF2 was constructed;
- A number of in-house biochemical assays for measurement of various metabolic parameters was developed. These include assays for NAD, lactate, glucose, ATP etc. Importantly, we routinely use these assays for training of our student’s wet lab skills. In addition, our assays will be published;
- A set of metabolic parameters in hESC was measured;
- We showed that lactate is actively produced and exported from hESC into culture medium;
- A promising model for studying the role of FGF2 in connection to metabolic parameters was established;
- In collaboration with Technical University of Berlin a technology for measurement of certain intracellular metabolic parameters directly by HPLC was established;
- A promising collaboration with Rockefeller University in New York was initiated for a project aiming at creating a humanized mouse;
- A new research group was established and began functioning;
- Two oral presentations at international scientific meetings were given;
- One research grant was applied for in collaboration with University Hospital Brno;
- One research article was already published in which SoMoPro support was acknowledged.
· A summary of the envisaged project outputs and their potential impact:
During the year under review my group and myself developed techniques which we transferred to other groups and which are now use in the whole lab. That list includes a novel protocol for freezing hESC in monolayers; the techniques for both stable and transient transfection of hESC, etc. Importantly, our research on the inducible overexpression in hESC is now followed by another group in our lab. In addition, we developed a set in-house of biochemical assays which could be used by other groups. The development of such assays served a very important task of training our student’s pipetting skills. We believe that this will have a broad positive impact in future, as to our understanding the pipetting skills of the young personnel are nowadays not properly addressed and their importance is underestimated. Importantly, we plan to publish our biochemical assays which will have very positive impact on the future carrier prospects of our students.
We also managed to develop a very promising model for studying the role of FGF2 in connection to intracellular metabolic parameters, which we believe will lead to a relatively high impact publication. Then, our international collaboration with Technical University of Berlin already resulted in a technology for direct measurement of certain intracellular parameters of hESC by HPLC, which is going to result in a joint publication. Two lectures at international meetings given by myself disseminated our knowledge in a broad scale. Meanwhile, we managed to establish collaboration with University Hospital Brno and applied for a research grant in a field of cancer stem cell biology. If successful, this project will have good chances to be continued in a spin-off biotech company in South Moravian region.