Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies
Aim. To find the relationship between the size of embryoid bodies and the efficiency of pluripotent stem cells differentiation into cardiomyocytes. Methods. Transgenic murine iPSC line AT25 and D3 ESC line αPIG (clone 44) were differentiated into cardiomyocytes in AggreWell plates containing microwe...
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irk-123456789-1528132019-06-14T01:26:27Z Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies Budash, G.V. Bilko, D.I. Bilko, N.M. Molecular and Cell Biotechnologies Aim. To find the relationship between the size of embryoid bodies and the efficiency of pluripotent stem cells differentiation into cardiomyocytes. Methods. Transgenic murine iPSC line AT25 and D3 ESC line αPIG (clone 44) were differentiated into cardiomyocytes in AggreWell plates containing microwells which cause the pluripotent stem cells to aggregate into EBs of an appropriate size. Both cell lines were genetically modified and expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter. We applied flow cytometry and fluorescence microscopy to test the efficiency of the differentiation processes. Results. The efficiency of differentiation of embryoid bodies formed from iPSC line AT25 and containing 250 and 1000 cells was found to be lower as compared to embryoid bodies formed of 500 and 750 cells. The number of eGFP+ cells derived from embryoid bodies of 500 cells was 8.5 times higher compared to embryoid bodies of 250 cells (2.86 ± 0.30 % cardiomyocytes per embryoid bodies of 500 cells vs. only 0.34 % cardiomyocytes per embryoid bodies containing 250 cells); the difference was 4.7 times higher in comparison with embryoid bodies formed from 1000 cells. Conclusions. The size of embryoid bodies can affect differentiation of pluripotent stem cells into cardiomyocytes. Among the embryoid bodies formed from 250 to 2000 cells per embryoid body, the highest percentage of eGFP+ cells was obtained from 500-cell embryoid bodies. Мета. Знайти зв’язок між розміром ембріоїдних тілець та ефективністю диференціювання плюрипотентних стовбурових клітин в кардіоміоцити. Методи. Трансгенні клітинні лінії індукованих плюрипотентних клітин AT25 та ембріональних стовбурових клітин D3 αPIG44 диференціювали в кардіоміоцити в AggreWell планшетах. Вище згадані планшети містять мікролунки, які дають можливість сформувати ембріоїдні тільця з плюрипотентних стовбурових клітини певного заданого розміру. Обидві клітинні лінії були генетично модифіковані і експресували IRES-фланкований зелений флуоресцентний білок (еGFP), під контролем кардіоспецифічного α-MHC промоутера. Для перевірки ефективність процесів диференціювання було застосовано методи проточної цитометрії та флуоресцентної мікроскопії. Результати. Встановлено, що ефективність диференціювання ембріоїдних тілець отриманих з лінії індукованих плюрипотентних клітин лінії AT25 розміром 250 і 1000 клітин менша в порівнянні з ембріональними тільцями сформованими з 500 і 750 клітин. Кількість eGFP+ клітин, отриманих з ембріоїдних тілець розміром 500 клітин була 8,5 разів більшою ніж з ембріональних тілець розміром 250 клітин (що становило 2,86 ± 0,30 % кардіоміоцитів для ембріоїдних тілець розміром 500 клітин, і лише 0,34 % eGFP+ клітин для ембріоїдних тілець розміром 250 клітин). Висновки. Впливати на ефективність диференціювання плюрипотентних стовбурових клітин в кардіоміоцити можна змінюючи початковий розмір ембріоїдних тілець. Серед ембріоїдних тілець, утворених в діапазоні від 250 до 2000 клітин, найвищий відсоток eGFP + клітин отримували з ембріоїдних тілець, утворених 500 клітинами. Цель. Найти связь между размером эмбриоидных телец и эффективностью дифференцировки плюрипотентных стволовых клеток в кардиомиоциты. Методы. Трансгенные клеточные линии индуцированных плюрипотентных клеток AT25 и эмбриональных стволовых клеток D3 αPIG44 дифференцировали в кардиомиоциты в AggreWell планшетах. Упомянутые планшеты содержат микролунки, дающие возможность сформировать эмбриоидные тельца из плюрипотентных стволовых клеток определенного заданного размера. Обе клеточные линии были генетически модифицированы и экспрессировали IRES-фланкированый зеленый флуоресцентный белок (еGFP), под контролем кардиоспецифического α-MHC промотера. Для проверки эффективности процессов дифференцировки были применены методы проточной цитометрии и флуоресцентной микроскопии. Результаты. Было установлено, что эффективность дифференцировки эмбриоидных телец полученных из линии индуцированных плюрипотентных клеток AT25 размером 250 и 1000 клеток меньше по сравнению с эмбриональными тельцами сформированными из 500 и 750 клеток. Количество eGFP + клеток, полученных из эмбриоидных телец размером 500 клеток была 8,5 раз больше чем по сравнению с эмбриональными тельцами размером 250 клеток (что составляло 2,86 ± 0,30 % кардиомиоцитов для эмбриоидных телец размером 500 клеток, и только 0,34 % eGFP+ клеток для эмбриоидных телец размером 250 клеток). Выводы. Изменение первоначального размера эмбриоидных телец влияет на эффективность дифференцировки плюрипотентных стволовых клеток в кардиомиоциты. Среди эмбриоидных телец, образованных в диапазоне от 250 до 2000 клеток, высокий процент eGFP + клеток получали из эмбриоидных телец, образованных 500 клетками. 2016 Article Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies / G.V. Budash, D.I. Bilko, N.M. Bilko // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 119-125. — Бібліогр.: 18 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000914 http://dspace.nbuv.gov.ua/handle/123456789/152813 576.364 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies |
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Molecular and Cell Biotechnologies Molecular and Cell Biotechnologies Budash, G.V. Bilko, D.I. Bilko, N.M. Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies Вiopolymers and Cell |
| description |
Aim. To find the relationship between the size of embryoid bodies and the efficiency of pluripotent stem cells differentiation into cardiomyocytes. Methods. Transgenic murine iPSC line AT25 and D3 ESC line αPIG (clone 44) were differentiated into cardiomyocytes in AggreWell plates containing microwells which cause the pluripotent stem cells to aggregate into EBs of an appropriate size. Both cell lines were genetically modified and expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter. We applied flow cytometry and fluorescence microscopy to test the efficiency of the differentiation processes. Results. The efficiency of differentiation of embryoid bodies formed from iPSC line AT25 and containing 250 and 1000 cells was found to be lower as compared to embryoid bodies formed of 500 and 750 cells. The number of eGFP+ cells derived from embryoid bodies of 500 cells was 8.5 times higher compared to embryoid bodies of 250 cells (2.86 ± 0.30 % cardiomyocytes per embryoid bodies of 500 cells vs. only 0.34 % cardiomyocytes per embryoid bodies containing 250 cells); the difference was 4.7 times higher in comparison with embryoid bodies formed from 1000 cells. Conclusions. The size of embryoid bodies can affect differentiation of pluripotent stem cells into cardiomyocytes. Among the embryoid bodies formed from 250 to 2000 cells per embryoid body, the highest percentage of eGFP+ cells was obtained from 500-cell embryoid bodies. |
| format |
Article |
| author |
Budash, G.V. Bilko, D.I. Bilko, N.M. |
| author_facet |
Budash, G.V. Bilko, D.I. Bilko, N.M. |
| author_sort |
Budash, G.V. |
| title |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
| title_short |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
| title_full |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
| title_fullStr |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
| title_full_unstemmed |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
| title_sort |
differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies |
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Інститут молекулярної біології і генетики НАН України |
| publishDate |
2016 |
| topic_facet |
Molecular and Cell Biotechnologies |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152813 |
| citation_txt |
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies / G.V. Budash, D.I. Bilko, N.M. Bilko // Вiopolymers and Cell. — 2016. — Т. 32, № 2. — С. 119-125. — Бібліогр.: 18 назв. — англ. |
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Вiopolymers and Cell |
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2025-07-14T04:18:08Z |
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118
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
Differentiation of pluripotent stem cells into cardyomyocytes is influenced
by size of embryoid bodies
G. V. Budash, D. I. Bilko, N. M. Bilko
National University of Kyiv-Mohyla Academy
2, Skovorody Str, Kyiv, Ukraine, 04655
Galina19@ukr.net
Aim. To find the relationship between the size of embryoid bodies and the efficiency of pluripotent stem cells
differentiation into cardiomyocytes. Methods. Transgenic murine iPSC line AT25 and D3 ESC line αPIG
(clone 44) were differentiated into cardiomyocytes in AggreWell plates containing microwells which cause the
pluripotent stem cells to aggregate into EBs of an appropriate size. Both cell lines were genetically modified
and expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha
myosin heavy chain promoter. We applied flow cytometry and fluorescence microscopy to test the efficiency
of the differentiation processes. Results. The efficiency of differentiation of embryoid bodies formed from
iPSC line AT25 and containing 250 and 1000 cells was found to be lower as compared to embryoid bodies
formed of 500 and 750 cells. The number of eGFP+ cells derived from embryoid bodies of 500 cells was
8.5 times higher compared to embryoid bodies of 250 cells (2.86 ± 0.30 % cardiomyocytes per embryoid bod-
ies of 500 cells vs. only 0.34 % cardiomyocytes per embryoid bodies containing 250 cells); the difference was
4.7 times higher in comparison with embryoid bodies formed from 1000 cells. Conclusions. The size of em-
bryoid bodies can affect differentiation of pluripotent stem cells into cardiomyocytes. Among the embryoid
bodies formed from 250 to 2000 cells per embryoid body, the highest percentage of eGFP+ cells was obtained
from 500-cell embryoid bodies.
K e y w o r d s: pluripotent stem cells, induced pluripotent stem cells, embryoid bodies, cardiomyocyte, dif-
ferentiation.
Introduction
The ability of pluripotent stem cells (PSC) to prolif-
erate 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 ther-
apy and cell replacement therapy [8]. One of the
main hurdles to overcome for achieving these goals
is the determination of controlled conditions re-
quired 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 pro-
viding intercellular contacts [9, 10]. During em-
bryogenesis, the cell-cell interactions and cell posi-
tion are the key factors which direct the differentia-
tion. 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.000914
mailto:Galina19@ukr.net
119
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies
much 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 ecto-
derm cells, when the factors supporting PSC in un-
differentiated state are removed from the media.
This arrangement is similar to the early stages of em-
bryonic development, where the endoderm tissue,
which is in direct contact with the mesoderm, stimu-
lates its development into cardiomyocytes [12]. It
was proven that despite the differences in spatial or-
ganization most of the cell types are formed inside
EBs, including nerve cells, cardiomyocytes, hemato-
poietic cells and others. Due to the similarities be-
tween 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 differentia-
tion 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 in-
fluence 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 dif-
ferentiation 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 Physio-
logy and Pathophysiology, Institute for Neurophy-
siology, 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 ge-
netically modified and express the puromycin resis-
tance gene N-acetyl-aminotransferase and the IRES-
flanked enhanced green fluorescent protein (eGFP)
under the control of cardiac alpha myosin heavy
chain promoter (αMHC). The ability of cardiomyo-
cytes to express eGFP under the control of cardiac-
α-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 se-
rum (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 cul-
ture 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.
AggreWellTM400 (Stem Cell Technologies) plates
were used to start cardiomyocyte differentiation in
differentiation medium. It consists of Iscove’s modi-
fied Dulbecco’s medium containing 20 % FBS, 1x
NEAA, 50 µM β-ME. These plates contain microw-
ells (400 µm in size) which cause the pluripotent
stem cells (iPSC and ESC) to aggregate into EBs.
The procedure was done accordantly to the manu-
facturer’s instructions from the AggreWell manual.
Briefly 0.5 ml of differentiation medium was placed
into each well of an AggreWellTM400 plate, centri-
fuged 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 3×105 cells/
ml, 6×105 cells/ml, 9×105 cells/ml, 1.2×106 cells/ml
and 2.4×106 cells/ml to make EBs with 250, 500,
750, 1000, and 2000 cells respectively. From each
well of the AggreWellTM400 plate we obtained
1200 EBs. The AggreWellTM400 plate was centri-
120
G. V. Budash, D. I. Bilko, N. M. Bilko
fuged at 200 g for 3 min to capture the cells in the
wells. EBs were maintained under standard CO2 in-
cubator conditions (37 °C, 5 % CO2) for 48 h.
EBs were counted under a light microscope
Axiovert 10 (ZEISS, Germany) after 2 days of culti-
vation. 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 9th
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 fluo-
rescence 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 prop-
idium iodide staining (Sigma, Germany). Data anal-
ysis was performed using FSC Express 4 Flow
Research Edition (De Novo Software, USA) soft-
ware.
The data on cell numbers are represented as mean
± standard deviation for 3 samples. Statistical sig-
nificance 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, meso-
derm and endoderm. A lot of differentiation proto-
cols start with the formation of 3-dimensional ag-
gregates of cells called embryoid bodies. The forma-
tion 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 opportu-
nity to get EBs of different sizes - from 250 to 2000
cells per EB. At the first stage the efficiency of dif-
ferentiation 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 irregu-
lar form, without clearly defined smooth edges, with
dark opaque color. On the 3d 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 endoder-
mal 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 differ-
entiation. The flow cytometry analysis showed that
on the 9-th day of differentiation the amount of car-
diomyocytes 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 differentia-
tion. It was 0.60 ± 0.03 % of eGFP+ cells. The
Fig. 1. AggreWellTM contains microwells to make uniform cell
aggregates
121
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies
amount of cardiomyocytes identified in EBs formed
with 250 cells was growing from the 9-th until the
13-th day of differentiation: 0.04 ± 0.01 % of eGFP+
cells were identified on the 9-th day, 0.19 ± 0.01 % –
on the 11-th day and 0.34 ± 0.02 % – on the 13-th
day. There were no eGFP+ cells in EBs formed with
2000 cells.
Differentiation of PSC into cardiomyocytes ob-
tained 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 car-
diomyocytes 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 high-
er 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
250 cells 1000 cells 2000 cells
Fig. 2. Embryoid bodies of
different size in AggreWell
plates on the 2d and 3d days of
differentiation, x4.
D
ay
2
D
ay
3
Fig. 3. The efficiency of car-
diomyocyte differentiation
depending on the size of EBs.
122
G. V. Budash, D. I. Bilko, N. M. Bilko
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 car-
diomyocytes 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 differentia-
tion. The highest percentage of eGFP+ cells was ob-
tained 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 cardio-
myocyte by a “hanging drop” method (EBs are at-
tached 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 culti-
vated in suspension likely to the mass culture meth-
od. Therefore, we assume that the AgreWell method
is linespecific and is insufficient to produce cardio-
myocytes from the applied ESC cell line.
250 cells/EB 500 cells/EB 750 cells/EB 1000 cells/EB
D
ay
2
D
ay
9
D
ay
1
1
D
ay
1
3
Fig. 4. iPSCs differentiation into cardiomyocytes with AggreWell plates (fluorescence microscopy, x4)
123
Differentiation of pluripotent stem cells into cardyomyocytes is influenced by size of embryoid bodies
According to the outcome of our work it is possi-
ble 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 signal-
ing molecules are required. For example, the factors
secreted from neighboring lateral endoderm, such as
BMP, stimulate cardiogenesis, whereas the canoni-
cal 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, con-
taining nucleus formed from ectodermal cells in the
middle of it, and a layer of mesodermal cells be-
tween 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 endo-
derm cells we increase the number of mesoderm
250 cells/EB 500 cells/EB 750 cells/EB 1000 cells/EB
D
ay
2
D
ay
9
D
ay
1
1
D
ay
1
3
Fig. 5. ESC differentiation into cardiomyocytes with AggreWell plates
124
G. V. Budash, D. I. Bilko, N. M. Bilko
cells. In turn mesoderm cells are transformed into
cardiomyocytes by the factors secreted by endoderm
cells. So we modulate cell position relatively to each
other by changing the seeding density of cells.
Moreover EB size, which depends mainly on the ini-
tial amount of PSC, forming EB, influences such pa-
rameters as diffusion of soluble adhesion molecules
and intercellular interactions. Therefore, it is possi-
ble that EBs, containing 500 cells have the most ap-
propriate 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 be-
come unable to support diffusion of cardiogenic fac-
tors, at the sufficient level for differentiation.
Conclusions
Modifying such parameter as EB size can beneficial-
ly affect the differentiation of PSC into cardiomyo-
cytes. 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.
Acknowledgement
We would like to thank professor J. Hecheler and
doctor T.Saric (Institute of Neurophysiology,
University of Cologne, Germany) for coordination
of key stages of the project and for providing the
possibilities for productive cooperation.
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Диференціація плюрипотентних стовбурових клітин в
кардіоміоцити залежить від розміру ембріоїдних тілець
Г. В. Будаш, Д. І. Білько, Н. М. Білько
Мета. Знайти зв’язок між розміром ембріоїдних тілець та
ефективністю диференціювання плюрипотентних стовбуро-
вих клітин в кардіоміоцити. Методи. Трансгенні клітинні лінії
індукованих плюрипотентних клітин AT25 та ембріональних
стовбурових клітин D3 αPIG44 диференціювали в кардіоміо-
цити в AggreWell планшетах. Вище згадані планшети містять
мікролунки, які дають можливість сформувати ембріоїдні тіль-
ця з плюрипотентних стовбурових клітини певного заданого
розміру. Обидві клітинні лінії були генетично модифіковані і
експресували IRES-фланкирований зелений флуоресцентний
білок (еGFP), під контролем кардіоспецифічного α-MHC про-
мотера. Для перевірки ефективність процесів диференціюван-
ня були застосовані методи проточної цитометрії та флуорес-
центної мікроскопії. Результати. Було встановлено, що ефек-
тивність диференціювання ембріоїдних тілець отриманих з
лінії індукованих плюрипотентних клітин лінії AT25 розміром
250 і 1000 клітин менша в порівнянні з ембріональними тіль-
цями сформованими з 500 і 750 клітин. Кількість eGFP+ клі-
тин, отриманих з ембріоїдних тілець розміром 500 клітин була
8,5 разів більшою ніж в порівнянні з ембріональними тільцями
розміром 250 клітин (що становило 2,86 ± 0,30 % кардіоміоци-
тів для ембріоїдних тілець розміром 500 клітин, і лише 0,34 %
eGFP+ клітин для ембріоїдних тілець розміром 250 клітин).
Висновки. Впливати на ефективність диференціювання плю-
рипотентних стовбурових клітин в кардіоміоцити можна змі-
нюючи початковий розмір ембріоїдних тілець. Серед ембріоїд-
них тілець, утворених в діапазоні від 250 до 2000 клітин, най-
вищий відсоток eGFP + клітин отримували з ембріоїдних ті-
лець, утворених 500 клітинами.
К л юч ов і с л ов а: плюрипотентні стовбурові клітини, інду-
ковані плюрипотентні стовбурові клітини, ембріоїдні тільця,
кардіоміоцити, диференціювання.
Дифференцировка плюрипотентных стволовых клеток
в кардиомиоциты зависит от размера эмбриоидных
телец
Г. В. Будаш, Д. И. Билько, Н. М. Билько
Цель. Найти связь между размером эмбриоидных телец и эф-
фективностью дифференцировки плюрипотентных стволовых
клеток в кардиомиоциты. Методы. Трансгенные клеточные
линии индуцированных плюрипотентных клеток AT25 и эм-
бриональных стволовых клеток D3 αPIG44 дифференцирова-
ли в кардиомиоциты в AggreWell планшетах. Упомянутые
планшеты содержат микролунки, которые дают возможность
сформировать эмбриоидные тельца из плюрипотентных ство-
ловых клеток определенного заданного размера. Обе клеточ-
ные линии были генетически модифицированы и экспрессиро-
вали IRES-фланкированый зеленый флуоресцентный белок
(еGFP), под контролем кардиоспецифического α-MHC промо-
тера. Для проверки эффективности процессов дифференци-
ровки были применены методы проточной цитометрии и флу-
оресцентной микроскопии. Результаты. Было установлено,
что эффективность дифференцировки эмбриоидных телец по-
лученных из линии индуцированных плюрипотентных клеток
AT25 размером 250 и 1000 клеток меньше по сравнению с эм-
бриональными тельцами сформированными из 500 и 750 кле-
ток. Количество eGFP + клеток, полученных из эмбриоидных
телец размером 500 клеток была 8,5 раз больше чем по сравне-
нию с эмбриональными тельцами размером 250 клеток (что
составляло 2,86 ± 0,30 % кардиомиоцитов для эмбриоидных
телец размером 500 клеток, и только 0,34 % eGFP+ клеток для
эмбриоидных телец размером 250 клеток). Выводы. Изме не-
ние первоначального размера эмбриоидных телец влияет на
эффективность дифференцировки плюрипотентных стволо-
вых клеток в кардиомиоциты. Среди эмбриоидных телец, об-
разованных в диапазоне от 250 до 2000 клеток, высокий про-
цент eGFP + клеток получали из эмбриоидных телец, образо-
ванных 500 клетками.
К л юч е в ы е с л ов а: плюрипотентные стволовые клетки,
индуцированные плюрипотентные стволовые клетки, эмбрио-
идные тельца, кардиомиоциты, дифференцирование.
Received 31.01.2016
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