Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies

G. V. Budash, D. I. Bilko, N. M. Bilko © 2016 G. V. Budash et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 576.364


Introduction
The ability of pluripotent stem cells (PSC) to proliferate in vitro and differentiate into any cell of three germ layers [1], such as hepatocytes [2], beta cells of the pancreas [3], cardiomyocytes [4], osteoblasts [5], endothelial cells [6], neurons [7] and other makes them a target of up-to-date research.Moreover, PSC can be used for disease modeling, drug toxicity screening or drug discovery, gene therapy and cell replacement therapy [8].One of the main hurdles to overcome for achieving these goals is the determination of controlled conditions required for the process of differentiation.
It is known that the process of differentiation is affected by various environmental stimuli such as matrix components and cell-matrix interactions, growth factors, cytokines, signaling molecules providing intercellular contacts [9,10].During embryogenesis, the cell-cell interactions and cell position are the key factors which direct the differentiation.The same aspects influence the process in vitr o [11].
The Embryoid body (EB) formation is often used as a method for initiating differentiation.EBs are three-dimensional aggregates, representing the early stages of embryo development.However, because the EB formation depends on various factors, it is

Molecular and Cell Biotechnologies
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print) Biopolymers and Cell. 2016. Vol. 32.N 2. P 119-125 doi: http://dx.doi.org/10.7124/bc.000914much more disorganized in comparison with real embryo.Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) form the aggregates covered with a layer of visceral endoderm on the surface and an inner layer filled with primitive ectoderm cells, when the factors supporting PSC in undifferentiated state are removed from the media.This arrangement is similar to the early stages of embryonic development, where the endoderm tissue, which is in direct contact with the mesoderm, stimulates its development into cardiomyocytes [12].It was proven that despite the differences in spatial organization most of the cell types are formed inside EBs, including nerve cells, cardiomyocytes, hematopoietic cells and others.Due to the similarities between embryogenesis and EB formation, many of the same growth factors are involved at the same stages [13,14].
In order to improve the efficiency of differentiation of PSCs two main approaches are used.The first one is to supply cultivation medium with the factors involved in the process of formation, maturation and proliferation of the certain cell type.The second strategy is to adjust the physical parameters that influence the formation of cell aggregates, such as the size and shape of EBs [10,8].S. M. Dang et al [12] demonstrated that homogenization of EB population can significantly improve the efficiency of PSC differentiation into cardiomyocytes.Little is known about the basis of interconnection between EB size and shape and PSC direct differentiation.In order to reveal this mechanism, the profound molecular and cell research is required.Therefore it is important to find the relationship between the size of embryoid bodies and the efficiency of PSC differentiation into cardiomyocyte.

Materials and methods
Transgenic murine iPSC line AT25 was engineered from murine iPSC line TiB7.4 in Center for Physiology and Pathophysiology, Institute for Neurophysiology, Medical Faculty, University of Cologne, Cologne, Germany (Fatima et al, manuscript in preparation).iPSC line TiB7.4 was kindly provided by Rudolf Jaenisch and Alexander Meiss ner [15].Transgenic murine D3 ESC line αPIG (clone 44) was described earlier [16].Both cell lines were genetically modified and express the puromycin resistance gene N-acetyl-aminotransferase and the IRESflanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter (αMHC).The ability of cardiomyocytes to express eGFP under the control of cardiacα-MHC promoter gave us the opportunity to apply flow cytometry and fluorescence microscopy to test the efficiency of the differentiation processes [16].These cells were maintained on irradiated mouse embryonic fibroblasts (MEF) in Dulbe cco's minimal essential medium containing 15 % fetal bovine serum (FBS), 1x non-essential amino acids (NEAA), 2 mM L-glutamine, 50 µM β-merca ptoethanol (β-ME) and 1000 U/ml leukemia inhibitory factor (LIF, ESGRO, Chemicon/Merck Milli pore, Billerica, MA, USA).Unless otherwise specified, all cell culture reagents were obtained from Life Technologies (Carlsbad, CA, USA).Both stem cell lines iPSC and ESC were used in the experiments starting from the passage 5.
AggreWell TM 400 (Stem Cell Technologies) plates were used to start cardiomyocyte differentiation in differentiation medium.It consists of Iscove's modified Dulbecco's medium containing 20 % FBS, 1x NEAA, 50 µM β-ME.These plates contain microwells (400 µm in size) which cause the pluripotent stem cells (iPSC and ESC) to aggregate into EBs.The procedure was done accordantly to the manufacturer's instructions from the AggreWell manual.Briefly 0.5 ml of differentiation medium was placed into each well of an AggreWell TM 400 plate, centrifuged at 2000g for 10 min in a swinging bucket rotor that was fitted with a plate holder to remove any small bubbles from the AggreWell plates.PSC were added to each well at concentrations of 3×10 5 cells/ ml, 6×10 5 cells/ml, 9×10 5 cells/ml, 1.2×10 6 cells/ml and 2.4×10 6 cells/ml to make EBs with 250, 500, 750, 1000, and 2000 cells respectively.From each well of the AggreWell TM 400 plate we obtained 1200 EBs.The AggreWell TM 400 plate was centri-fuged at 200 g for 3 min to capture the cells in the wells.EBs were maintained under standard CO 2 incubator conditions (37 °C, 5 % CO 2 ) for 48 h.
EBs were counted under a light microscope Axiovert 10 (ZEISS, Germany) after 2 days of cultivation.Then they were transferred into Petry dishes with fresh differentiation medium and maintained on a shaker GFL 3006 (GFL, Braunschweig, Germany) under continuous horizontal agitation.Differentiation was continued without medium change until the 9 th day and afterwards the medium was changed every 2-3 days.
Efficiency of cardiac differentiation was analyzed by determining beating EBs and the fraction of eGFP-positive cardiomyocytes on days 6, 9, 11, 13 and 15 of differentiation by flow cytometry and fluorescence microscopy.EBs were examined using Zeiss Axiovert 200M fluorescence microscope and analyzed with Zeiss Axiovision 4.5 software (Carl Zeiss, Jena, Germany).Single cell suspension was prepared for flow cytometry.Cells were analyzed by FACScan (BD Pharmingen).Cell debris was gated out and 10000 events were acquired for analysis.The presence of dead cells was determined by propidium iodide staining (Sigma, Germany).Data analysis was performed using FSC Express 4 Flow Research Edition (De Novo Software, USA) software.
The data on cell numbers are represented as mean ± standard deviation for 3 samples.Statistical significance was determined using the Student t-test at P < 0.05.

Results and Discussion
Pluripotent stem cells, which include ESCs and iP-SCs, have the ability to differentiate into the cells of all three germ lineages including ectoderm, mesoderm and endoderm.A lot of differentiation protocols start with the formation of 3-dimensional aggregates of cells called embryoid bodies.The formation of EBs heterogeneous in size and shape is a cause of inefficient and uncontrolled differentiation.In order to get homogeneous population of EBs, the AggreWell plates were used, each well of which contains a certain number of microwells (Fig. 1).After transferring the single cell suspension, EBs of a defined size are formed.
The plates mentioned above provide the opportunity to get EBs of different sizes -from 250 to 2000 cells per EB.At the first stage the efficiency of differentiation into cardiomyocytes was tested with EBs of 250, 100 and 2000 cells/EB.The iPSC cell line AT25 was used.Homogeneous EBs of identical size were formed in each well of AggreWell plates on the second day of differentiation (Fig. 2).
However, EBs formed from 2000 cells had irregular form, without clearly defined smooth edges, with dark opaque color.On the 3 d day of differentiation it was clear that most of EBs formed from 2000 cells were destroyed.The amount of nonviable cells was more than 95 %.
EBs formed with 250 and 1000 cells had typical round shape.They had the surface layer formed with ectodermal cells and the inner layer with endodermal cells.The amount of differentiated cells was higher in EBs formed with 1000 cells.The first GFP+ cells were observed on the 8-th day of differentiation.The flow cytometry analysis showed that on the 9-th day of differentiation the amount of cardiomyocytes was 0.19 ± 0.02 % of cell population, on the 11-th day the amount was 0.58 ± 0.01 % of GFP+ cells.The highest number of differentiated cells was observed on the 13-th day of differentiation.It was 0.60 ± 0.03 % of eGFP+ cells.The Differentiation of PSC into cardiomyocytes obtained from EBs in the range from 500 cells to 750 cells per EB was more efficient (Fig. 3, 4).
It was found that the efficiency of differentiation of EBs with 250 and 1000 cells is lower compared with EBs of 500 and 750 cells.So, on the 13-th day of differentiation when the highest amount of cardiomyocytes was obtained, the number of eGFP+ cells derived from EBs of 500 cells was 8.5 times higher compared to EBs of 250 cells (2.86 ± 0.30 % of the cardiomyocytes per 500 cells EBs versus 0.34 % of eGFP+ cells per EBs containing 250 cells); when compared with 1000 cells EBs the difference was 4.7 times higher (the amount was 0.60 ± 0.03 % of differentiated cells).The difference between EBs of 750 cells and EBs of 250 and 1000 cells was higher by 6.4 and 3.6 times, respectively (the number of eGFP+ cells of the first EB size was 2.16 ± 0.02 %, for 250 cells EBs -0.34 ± 0.02 %, and for 1000 cells EBs -0.60 ± 0.03 %).
Comparison of the efficiency of differentiation of EBs formed with 500 and 750 cells per EB revealed that the difference between them varied from 1.3 times on the 13-th day of differentiation to 1.8 times on the 15-th day.For EBs formed with 500 cells the amount of cardiomyocytes was 2.07 ± 0.01 % on the 11-th day, 2.86 ± 0.30 % on the 13th day and 2.53 ± 0.04 % on the 15th day of differentiation.The number of cardiomyocytes derived from EBs formed with 750 cells was significantly lower (P <0.05).It was 1.25 ± 0.01 % on 11-th day, 2.16 ± 0.02 % on the 13th day and 1.41 ± 0.03 % on the 15th day of differentiation.
Thus, the experiments show that the size of EBs significantly affects the efficiency of cell differentiation.The highest percentage of eGFP+ cells was obtained from EBs formed with 500 cells.
No eGFP+ cells or beating areas were observed when AggreWell plates were used for the ESCs line differentiation.EBs of different size and shape were identified on the 2-d day of differentiation (Fig. 5).According to our previous results [17] the murine ESC line had high ability to differentiate into cardiomyocyte by a "hanging drop" method (EBs are attached to the gelatin-covered Petry dishes) and by a mass culture method (EBs aggregate spontaneously by cultivation in nonadherent Petry dishes).During the first 2 days EBs in AgreWell plates form regular EBs similar to those obtained by the "hanging drop" method; starting from the third day they are cultivated in suspension likely to the mass culture method.Therefore, we assume that the AgreWell method is linespecific and is insufficient to produce cardiomyocytes from the applied ESC cell line.According to the outcome of our work it is possible to improve the efficiency of differentiation in cardio direction if the size of EB changes.It is known that for the normal embryonic development of the cardiovascular system different gradients of signaling molecules are required.For example, the factors secreted from neighboring lateral endoderm, such as BMP, stimulate cardiogenesis, whereas the canonical Wnt signals from neighboring neuroectoderm inhibit cardiogenesis of mesoderm [18].Therefore, the ratio of the endoderm and neuroectoderm can af-fect the processes of differentiation.The size of EBs, in turn, can affect the value formed by different cell layers.
EBs are the aggregates formed from PSC that are covered with endodermal cells on the surface, containing nucleus formed from ectodermal cells in the middle of it, and a layer of mesodermal cells between two other layers.Modifying the EB size we change the amount of endoderm on its surface.So we can assume that increasing the number of endoderm cells we increase the number of mesoderm Moreover EB size, which depends mainly on the initial amount of PSC, forming EB, influences such parameters as diffusion of soluble adhesion molecules and intercellular interactions.Therefore, it is possible that EBs, containing 500 cells have the most appropriate ratio of the surface covered with endoderm cells to the surface of mesoderm cells with which they can interact.At the same time larger EBs become unable to support diffusion of cardiogenic factors, at the sufficient level for differentiation.

Conclusions
Modifying such parameter as EB size can beneficially affect the differentiation of PSC into cardiomyocytes.Among EBs formed within the range from 250 cells to 2000 cells per EB the highest percentage of eGFP + cells was obtained from EBs formed with 500 cells.