Mitochondria structural reorganization during mouse embryonic stem ...

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ORIGINAL ARTICLE

Mitochondria structural reorganization during mouse embryonic stem cell derivation Lyubov A. Suldina 1 & Ksenia N. Morozova 1,2

&

Aleksei G. Menzorov 1,2 & Elena A. Kizilova 1 & Elena Kiseleva 1

Received: 4 October 2017 / Accepted: 2 March 2018 # Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract Mouse embryonic stem (ES) cells are widely used in developmental biology and transgenic research. Despite numerous studies, ultrastructural reorganization of inner cell mass (ICM) cells during in vitro culture has not yet been described in detail. Here, we for the first time performed comparative morphological and morphometric analyses of three ES cell lines during their derivation in vitro. We compared morphological characteristics of blastocyst ICM cells at 3.5 and 4.5 days post coitum on feeder cells (day 6, passage 0) with those of ES cells at different passages (day 19, passage 2; day 25, passage 4; and passage 15). At passage 0, there were 23–36% of ES-like cells with various values of the medium cross-sectional area and nucleocytoplasmic parameters, 55% of fibroblast-like (probably trophoblast derivatives), and ~ 19% of dying cells. ES-like cells at passage 0 contained autolysosomes and enlarged mitochondria with reduced numerical density per cell. There were three types of mitochondria that differed in matrix density and cristae width. For the first time, we revealed cells that had two and sometimes three morphologically distinct mitochondria types in the cytoplasm. At passage 2, there were mostly ES cells with a high nucleocytoplasmic ratio and a cytoplasm depleted of organelles. At passage 4, ES cell morphology and morphometric parameters were mostly stable with little heterogeneity. According to our data, cellular structures of ICM cells undergo destabilization during derivation of an ES cell line with subsequent reorganization into the structures typical for ES cells. On the basis of ultrastructural analysis of mitochondria, we believe that the functional activity of these organelles changes during early stages of ES cell formation from the ICM. Keywords Embryonic stem cell . Mitochondria . Electron microscopy . Cell ultrastructure

Introduction The ability to retain pluripotency during prolonged cell culture in vitro and to differentiate into derivatives of all three germ layers makes embryonic stem (ES) cells an ideal model for the study of pluripotency and differentiation (Wobus et al. 1994; Rohwedel et al. 1999). Mouse ES cells were obtained from the blastocyst inner cell mass (ICM) (Evans and Kaufman 1981; Martin 1981) form compact colonies with a high Lyubov A. Suldina and Ksenia N. Morozova contributed equally to this work. Handling Editor: Douglas Chandler * Ksenia N. Morozova [email protected]; [email protected] 1

Institute of Cytology and Genetics SB RAS, Russian Academy of Sciences, Lavrentiev ave., 10, Novosibirsk, Russia 630090

2

Novosibirsk State University, Novosibirsk 630090, Russia

nucleocytoplasmic ratio, large nuclei, and the cytoplasm depleted of organelles (Baharvand and Matthaei 2003). Similar ultrastructure of trypsinized mouse ES cells has been described in different cell lines (Alharbi et al. 2014). Commonly used ES cell lines have a naïve state of pluripotency as compared to the primed state of epiblast stem cells (Hanna et al. 2010). Recent additional data based on the similarities between human ES cells and mouse epiblast stem cells suggest that human ES cells are in a primed state of pluripotency (Guo et al. 2016). ES cells are widely used for production of transgenic mice: complex genomic modifications are introduced into ES cells, which are then injected into blastocysts; chimeric mice develop, and the ES cell-derived gametes are employed to transmit the modifications. In light of the ES cell participation in early embryonic development, it is important to analyze changes in ICM cells during in vitro development into ES cells. More than 6000 genes change their expression between ICM and ES cells. For instance, early-differentiation genes and differentiation marker genes of the three germ layers (Pax6, Otx1, and Neurod1 [ectoderm]; Tbx2, Nkx2, and Myod1

L. A. Suldina et al.

[mesoderm]; Onecut1, Gata4, Gata5, and Gata6 [endoderm]; and Cdx2 and Tpbpa [extraembryonic tissues]) are silenced during this process (Tang et al. 2010). Expression of Вmp4 decreases while expression of its receptor Bmpr1a increases: the BMP4 protein along with LIF is important for ES cell pluripotency in vitro. Genes related to ES cell pluripotency are activated (Nodal, Eras, Lin28, and Smad1), while those related to ICM development (paramel 7, Tbx3, Bmi1, Nr5a2, and Amhr2) are silenced. The mitochondria have evolved multiple processes in cell and are responsible for energy production, calcium homeostasis, the signal transduction, and apoptosis (Pickles et al. 2018). It was shown that mitochondrial dysfunction affects for the vitality of oocytes, and their dynamics in these cells is important for early embryo development (Dalton and Carroll 2013). The morphology of these organelles changes during the oocyte and early development in mice (Stern et al. 1971). They are more diverse in shape and contain vacuole-like inclusions at the 4- and 8-cell stages of mouse embryonic development, while their majority in the morula stage and blastocyst stage contain transverse cristae. It has been suggested that mitochondria play an important functional role in the regulation of stem cell differentiation and metabolic plasticity of stem cell reprogramming (Rehman 2010; Xu et al. 2013). Indeed, it was shown that the product of PHB2, an essential gene for mitochondrial homeostasis, functions as a crucial protein that regulates proliferation during stem cell differentiation (Kowno et al. 2014). Pluripotent ES cells are highly dependent on anaerobic glycolytic metabolism—rather than more efficient mitochondrial oxidative metabolism—for energy production (Chen et al. 2012). Studying mitochondria in ES and induced pluripotent stem cells is important because mitochondria may regulate development by a number of means, including modulating Ca2+ signaling and the production of ATP, reactive oxygen species, and intermediary metabolites (Dumollard et al. 2007; Xu et al. 2013). Despite the progress in molecular biological methods, there are virtually no data on the morphological reorganization of ICM cells at early stages of ES cell derivation in vitro. Here, we for the first time analyzed changes in cellular and organelle ultrastructure and morphometric parameters at early stages of ES cell formation from the ICM. Three pluripotent ES cell lines (MA13, MA14, and MA15) were derived from outbred mice (129 × BALB) (Menzorov et al. 2016) and analyzed by light microscopy and transmission electron microscopy (TEM). We found that ES-like cells are detectable as early as passage 0. ES cell line formation was found to be accompanied by structural changes in the nucleus and in cytoplasmic organelles, especially mitochondria reorganization. At later passages, heterogeneity of morphometric parameters between cell lines decreases.

Materials and methods ES cell culture We analyzed three mouse ES cell lines, MA13, MA14, and MA15 (Menzorov et al. 2016), produced from blastocysts of outbred mice (129 × BALB) according to the protocol developed by Bryja et al. (2006). These ES cell lines are available at a Collective Center of ICG SB RAS Collection of Pluripotent Human and Mammalian Cell Cultures for Biological and Biomedical Research (http://ckp.icgen.ru/cells/). Briefly, blastocysts at 3.5 days post coitum (dpc) without a zona pellucida were plated on mitomycin C-inactivated 13.5 dpc mouse embryonic fibroblasts (feeder) from strain ICR in the ES culture medium supplemented with 20% of KSR (knockout serum replacement; Invitrogen, Carlsbad, USA). At passage 0 (day 6 in vitro), the embryos were divided into two equal parts, one for ES cell derivation and the other for light microscopy and TEM. The embryos were disaggregated with 2.5% Trypsin-EDTA (Invitrogen, USA) and plated on feeder cells in the culture medium supplemented with 20% of fetal bovine serum (FBS). The next day, the medium was changed to the one containing 20% of KSR, and this medium was used before passaging. Colonies were trypsinized with 0.05% Trypsin-EDTA and plated in the 20% FBS-containing medium with incubation for 1 day, and the next day, the medium was changed to the one containing 20% of KSR. After passage 4, we applied the ES cell culture medium: DMEM (Invitrogen, USA) supplemented with 15% of ES cell-qualified FBS, 1× nonessential amino acids (Invitrogen, USA), 1× GlutaMAX (Invitrogen, USA), 0. 1 mM β-mercaptoethanol (Sigma, USA), 1× penicillin–streptomycin solution (Invitrogen, USA), and 1.000 U/ml LIF ESGRO (Chemicon, USA). The cells were cultured on feeder cells in 12-well plates that were coated with 0.1% gelatin. We used mitomycin C-inactivated 13.5 dpc mouse embryonic fibroblasts from strain ICR as feeder cells (Menzorov et al. 2016). ES cell lines were analyzed by light microscopy and TEM at passages 0 (day 6), 2 (day 19), 4 (day 25), and 15.

TEM Intact blastocysts of outbred mice (129 × BALB) at 3.5 and 4.5 dpc were flushed in the M2 medium (Sigma, USA) and immediately fixed. ES cells were cultured on disks of Melinex polyester film (175 μm thickness, Agar Scientific, UK; Kruglova et al. 2010) and remained attached to the Melinex film for all treatments and embedding. Primary fixation of the cells was done with 2.5% glutaraldehyde in the culture medium for 15 min, post fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, for 1 h at room temperature. The samples were washed three times in the same buffer and additionally fixed in 1% osmium tetroxide for 1 h, washed twice in double-distilled (dd) H2O, and incubated in 1%

Mitochondria structural reorganization during mouse embryonic stem cell derivation

uranyl acetate for 12 h at 4 °C. Next, the samples were dehydrated in a graded series of ethanol solutions (10, 30, 50, 70, 96, 100%, 10 min in each) and acetone (twice, 10 min), embedded in epoxy resin Epon 812 (Sigma, USA), and polymerized for 2 days at 60 °C (Morozova and Kiseleva 2006). Semithin sections for light microscopy and ultrathin sections for TEM (50 nm thickness) were cut in parallel to the plane of the substrate by using a Leica EM UC7 ultramicrotome (Leica, Austria). For studies in the TEM, sections were contrasted with 2% uranyl acetate (EMS, Hatfield, USA) and with 1% lead citrate (EMS, USA) according to Reynolds (1963) for 1 min and rinsed with ddH2O. Finally, the contrasted ultrathin sections were examined and photographed under a transmission electron microscope, JEOL-1400 (JEOL, Japan), with a Veleta camera (Olympus, USA) and iTEM 5.1 software (Olympus, USA).

Morphometric analysis Morphometric analysis was carried out on semithin sections stained with methylene blue under the light microscope AxioVision-40 (Carl Zeiss, Germany) and on ultrathin sections. The mean cross-sectional area of the nucleus and ICM cells was measured in 25 randomly selected sections passing through the blastocyst central region; blastocysts were oriented toward the long axis before mounting. ES cell parameters were assessed in a similar manner in 30 sections from 5 to 6 randomly selected colonies. In total, we analyzed three early (3.5 dpc) and three 4.5 dpc blastocysts, as well as three samples from each passage of the cell lines. The nucleocytoplasmic ratio was defined as a ratio of the nucleus area to the cytoplasm area in a semithin section using ImageJ freeware (ImageJ, USA, https://imagej. nih.gov/ij/). Ninety cells were analyzed for each cell type: ICM and ES cells of three lines. The percentages of cells with different morphology at passage 0 and of cells containing mitochondria with different morphology were assessed by direct counting of 100 cells in random sections in TEM images. The mean cross-sectional area of mitochondria and their volume and numerical density per 1 μm2 of cytoplasmic area were assessed in randomly selected cell sections. In total, 50 mitochondria from each cell type and each sample were analyzed. The volume density and numerical density of cristae were evaluated in 20 longitudinal mitochondrial sections for each of the three types of organelles in cell sections from different cell lines. Area measurements were conducted in the ImageJ freeware. The mitochondria and cristae volume densiV ties were calculated as V v ¼ Vpt , where Vp is total mitochondrial area or area of cristae, and Vt is the cytoplasmic or mitochondrial area, respectively (Weibel 1969). The numerical density of 3=2

mitochondria and cristae was calculated as Nv ¼ βk NV va 1=2 , where Na is the number of mitochondria or cristae, and Vv is volume density of mitochondria in the cytoplasm or cristae in

the mitochondria. Coefficient β characterizes the shape, and k characterizes size distribution; they were set as follows: k = 1. 07 and β = 2.2 for mitochondria and β = 3 for cristae (Weibel 1969). Three randomly selected samples were analyzed for each passage of different ES cell lines. The Student’s t test was conducted to determine statistical significance of differences between the average values.

Results and discussion Differences in cell morphometric parameters among cell lines at light-microscopy level Characteristics of cell populations at different passages Passage 0 ES-like cells with large nuclei were detected at passage 0 (day 6 after the blastocyst was placed on the feeder) in semithin sections under a light microscope and constituted 17, 18, and 19% in MA13, MA14, and MA15 cell cultures, respectively. We also observed dying fragmented cells identified by hallmarks such as pyknotic nuclei, morphological defects in the cytoplasm (electron-lucent areas), and the presence of big empty vacuoles. The dying cells constituted 36, 23, and 26%, and fibroblast-like elongated cells constituted 47, 59, and 55%, of the total number of cells among МА13, МА14, and МА15 cells, respectively (Fig. 1). The latter cells may represent polar and mural trophoblast cells. These cells produce bone morphogenetic protein (BMP) 4, which promotes proper growth of early mouse embryos (Murohashi et al. 2010) and can induce a Smad-dependent apoptosis of stem cell-derived neural precursors (Gambaro et al. 2006). Changes of Bmp-4 expression may be one of the possible reasons for the appearance of dying cells in the culture at passage 0. The mean cross-sectional area of cell in late blastocyst ICM decreased 2.3-fold as compared to the early blastocyst (Fig. 2a), and as the average cross-sectional area of the nucleus also decreased 2.5-fold (Fig. 2b), nucleocytoplasmic ratios did not change significantly (Fig. 2c). This finding is consistent with the decrease in cell size during cell divisions in early embryonic development (Hogan et al. 1994). The mean ES-like cells’ cross-sectional area was 1.3, 1.8, and 2.3 times that of ICM cells in МА13, МА14, and МА15 cell lines, respectively (Fig. 2a). We measured an increase in the mean cross-sectional area of nuclei: 1.5-fold in MA13 cells and 2.5-fold in MA14 cells at passage 0 as compared to the late blastocyst (Fig. 2b). As a result, the nucleocytoplasmic ratio significantly increased in МА13 and МА14 cell lines (1.4- and 1.5-fold, respectively, P > 0.01), as the relative increase in the average cross-sectional area of nuclei surpassed that of cells (Fig. 2c). In contrast, mean cross-sectional area of cell in the MA15 cell line increased 2.3-fold while that of the nuclei— 2.4-fold, thus maintaining the nucleocytoplasmic ratio at the


Fig. 1 Structural organization and cell type diversity of early and late blastocysts and ES cell lines MA13, MA14, and MA15 at different passages. a Semithin sections of early and late blastocysts and of ES cell colonies at passages 0, 2, 4, and late (15) passage, methylene blue

staining. b The percentage of ICM and trophoblast cells in early and late blastocysts and fibroblast-like, ES-like, and dying cells among MA13, MA14, and MA15 ES cells at passage 0. Scale bars, 10 μm

late ICM level. Thus, in most of investigated cell lines, the nucleocytoplasmic ratio increased due to the enlargement of the mean cross-sectional area of nuclei with a virtually stable area of the cytoplasm at passage 0 as compared to the late blastocyst.

passages to 205.3 ± 12 μm2. In contrast, these parameters increased in MA14 and MA15 cell lines at passage 0 (249.6 ± 19.2 and 134.8 ± 13.5 μm2 in МА14 and 315.1 ± 28.3 and 131.6 ± 19.8 μm2 in МА15) as compared to ICM cells, followed by a decrease at late passages. For example, the average area of MA15 cells was not significantly different from that of the late blastocyst ICM at passage 15. The nucleocytoplasmic ratio showed an opposite tendency (Fig. 2c). It decreased after a peak at passage 2 in МА13 and МА14 cell lines and increased in MA15 cells during in vitro culture. Thus, we observed variability of morphometric parameters, especially at passage 0. There are several possible explanations of these variations. First, the gene expression pattern of differentiation and pluripotency marker genes may change stochastically. Second, ICM cells manifest variability of expression of pluripotency genes (Halbisen and Ralston 2014), and this variability may cause differences in the rate of ES cell formation. The heterogeneity in the expression of such genes as Nanog, Tbx3, Klf4, and Rex1 was demonstrated even at the level of single cells in ES cell colonies (Niwa et al. 2009). In addition, Oct4 and Sox2 expression levels vary with time in cultivated ES cells (Kalmar et al. 2009). There is evidence that ES cell cultures contain cell populations with a different differentiation potential (Martinez et al. 2012). Nonetheless, our statistical data revealed that despite the differences in cell parameters at passage 0, heterogeneity among all three cell lines decreased by passage 4.

Passage 2 At passage 2, the mean cross-sectional cell area of all three cell lines decreased though only MA15 cells differed significantly from passage 0 in that parameter. The mean cross-sectional nuclear area remained at a similar level, whereas the nucleocytoplasmic ratio increased in all three cell lines, probably due to a decrease in cytoplasmic volume (Fig. 2). During subsequent passages (4 and 15), morphometric parameters of the cells remained stable. Thus, we conclude that nucleus size has been stabilized in ES-like cells at passage 0 (day 6 in vitro) after an increase in size, whereas cytoplasmic size continued to shrink until passage 2. Passage 4 and 15 During subsequent passages (4 and 15), morphometric parameters of both the nucleus and cytoplasm remained stable. Of note, the blastocyst pattern of gene expression is comparable to that of ES-like cells by day 5 after the blastocyst attaches to the surface (Tang еt al. 2010); this finding is consistent with our observation of morphological stability of nuclei since day 6 in vitro. Comparison of ES cell parameters of the three cell lines at different stages of their derivation We showed that the ES cell lines under the same conditions differed in their morphometric parameters at the early stages of in vitro culture (Fig. 2). MA13 ES-like cells had smaller mean cross-sectional areas of cells and nuclei (176.1 ± 13.9 and 81.5 ± 6.9 μm2, respectively) than the other cell lines at passage 0, but these values gradually increased at late

Ultrastructural organization of cells during ES cell line derivation Early and late blastocysts TEM analysis revealed that early blastocyst ICM cells varied in shape, possessed large nuclei with dense nucleoli, and are joint together with tight junctions (Fig. 3a). The cytoplasm


Fig. 2

Morphometric parameters of early and late blastocysts and of MA13, MA14, and MA15 ES cells at different passages. a The mean cross-sectional cell area of cells. b The mean cross-sectional area of nuclei in semithin sections of the ICM and of MA13, MA14, and MA15 ES cells at different passages. c The mean nucleocytoplasmic ratio in ICM and ES cells. Bars indicate standard error, *P < 0.05, **P < 0.01, ***P < 0.001

matrix, and numerous 15 nm fibrous lattices proximal to the rough endoplasmic reticulum (ER) membranes (Fig. 3a–c). It has been suggested that these lattices play a role in ribosomal storage and are required for protein synthesis during early development (Yurttas et al. 2008). Late ICM cells laid in several layers and were often separated by an enlarged extracellular space. In our study, the majority of mitochondria were oval in shape and contained evenly distributed cristae. The number of organelles with vacuoles as well as the fibrous lattices drastically decreased (Fig. 3d–f). The differences between cells of early and late blastocysts corresponded to different stages of mouse embryonic development and are in agreement with other studies (Cech and Sedlácková 1983; Dumollard et al. 2007). ES-like cells are identified in cell populations at passage 0 At passage 0, round ES-like cells with large nuclei and the cytoplasm depleted of organelles were present in all three cell lines (Fig. 4a). Despite the low organelle density, the cytoplasm contained large mitochondria with narrow or swollen cristae (Fig. 5a, b, d), branching ER cisternae (Fig. 5b), a welldeveloped Golgi apparatus (Fig. 5c), and small or sometimes large autolysosomes (Fig. 5d). Close contacts between swollen mitochondria and ER membranes were often observed (Fig. 5a, b). Fibroblast-like cells were elongated and contained organelle-rich cytoplasm and specific electron-dense granules (Fig. 4b). The cell structural organization was strongly abnormal in the dying cells: pyknotic nuclei with enlarged electrondense nucleoli, deep invaginations, and a broken nuclear membrane, as well as big empty vacuoles and numerous autophagosomes (Fig. 4c). Ultrastructure of ES cells in three cell lines at passages 2, 4, and 15

contained a lot of free ribosomes, bottle-shaped mitochondria with large electron-lucid vacuole-like omissions within the

At passages 2 (Fig. 6a–c), 4 (Fig. 6d–f), and 15 (Fig. 6g–i), cell morphology in colonies was typical for ES cells. The cells formed round-shaped colonies and had a large nucleus, a narrow band of the cytoplasm with low density of organelles and numerous ribosomes. At passage 2, the number of lysosomes was negligible, and rough ER was represented by short cisternae. Contacts between ER membranes and mitochondria were observed in ES cells at all passages; however, they were less numerous at passages 4 and 15. Comparative ultrastructural


Fig. 3 Ultrastructural organization of early (a–c) and late (d–f) blastocyst cells. a, d ICM cells (overview). b, e ICM cell mitochondria (mt). b Early blastocyst mitochondria have vacuole-like omissions within the

mitochondrial matrix (black asterisks). c, f Fifteen nanometers of fibrous lattices (white asterisks) surrounded by ER cisternae (ER). N nucleus. Scale bars, a, d 2 μm, b, c, e 1 μm, f 0.5 μm

analysis revealed that in spite of ES-like structural organization of all three cell lines, MA13 cell line contained more cells with nuclear envelope invaginations (Fig. 6a) than the others. At passages 4 and 15, the ultrastructural features of ES-like cells in all the cell lines were typical of pluripotent cells and similar to those observed in ES cells from other species (Alharbi et al. 2014; Baharvand and Matthaei 2003; Sathananthan et al. 2002).

small increase later. By contrast, the mean mitochondrial numerical density drastically decreased (5.7-fold) as compared to the ICM at passage 0 with a subsequent 2.8-fold increase at passage 2 and then an increase at passage 4, with some decrease at a late passage (Fig. 7b). Therefore, the mitochondrial population in the MA13 cell line is represented by a greater number of smaller organelles as compared to the other cell lines at passage 4. The mitochondrial parameters in MA15 cells showed a trend similar to that in the MA13 cell line. The mean mitochondrial cross-sectional area of MA15 cells increased 2.25-fold at passage 0 and then dropped 3-fold at passage 2, with a later increase at subsequent passages (Fig. 7a). The mean volume density and numerical density decreased 1.6-fold at passage 0 and increased 1.3-fold at passage 2 (Fig. 7b). It can be concluded that the number of smaller mitochondria increased in MA15 cells at passage 2. The morphometric parameters of mitochondria in MA14 cells revealed less variation between passages (Fig. 7). Overall, these data suggest that changes in mitochondrial organization are line-specific, especially at passage 0. Nevertheless, all the cell lines showed a decrease in mitochondrial volume density, which remained lower than that in ICM cells and did not change much during subsequent passages. This is an important morphological feature indicative of formation of ES-like cells.

Changes of mitochondrial morphometric parameters during derivation of ES cell lines A comparison of late ICM cells with the derived ES-like cell lines revealed that at passage 0, our cell lines had considerably smaller numbers of mitochondria, and although the average mitochondrial area (i.e., size) grew larger, the overall volume occupied by mitochondria was still lower in these cell lines than in ICM cells. Morphometric analyses revealed a 1.8- to 4fold increase in the mean cross-sectional mitochondrial area in ES-like cells compared to the late ICM at passage 0 (Fig. 7a). The parameters of mitochondria were significantly different among the cell lines at this passage. The mean cross-sectional area per mitochondrion was larger in the MA13 cell line than in the other cell lines. At subsequent passages, it decreased 2.5-fold and reached its minimum at passage 4 with a


Fig. 4 Three cell types at passage 0 (day 6 in vitro) within cell lines MA13, MA14, and MA15. a ES-like round cells with large nuclei and the cytoplasm depleted of organelles. b Fibroblast-like elongated cells with high density of organelles. c Dying cells with dense disturbed

cytoplasm containing large empty vacuoles (black asterisks), electronlucent areas in the cytoplasm, and pyknotic nuclei (N). In dying MA13 cells, agglomeration of ER cisternae is seen between the two large vacuoles. Scale bars, 5 μm

Different types of mitochondria at early ES cell passages

a high membrane potential under hypoxic conditions (Hackenbrock et al. 1971; Hackenbrock 1972). In contrast, mitochondria condense and its membrane potential decrease with increased oxygen because of an ADP/ATP ratio increase (De Martino et al. 1979). Transitional mitochondria are considered to place somewhere between these two functional states. ICM cells in the late blastocyst contain mitochondria most similar to the transitional type: medium matrix density, swollen, and thin cristae. In contrast, as many as 40% of the mitochondria in ES-like cell lines shifted toward the condensed appearance with this proportion increasing further at later passages in MA15 cells. One of the cell lines (MA13), in addition, contained mitochondria that shifted to the orthodox

We observed three morphological types of mitochondria in ES-like and ES cells: (i) orthodox mitochondria with a characteristic light matrix and thin regular cristae; (ii) condensed mitochondria with a dense matrix and swollen cristae; and (iii) transitional mitochondria of medium matrix density, endowed with both thin and swollen cristae (Fig. 8a–c). Moreover, these different types of mitochondria could be found in a single cell (Fig. 8d–f) and could be distinguished by numerical density and a cristae morphology (Fig. 8g–i). It is well known that mitochondria ultrastructural reorganization is linked with a change in a functional state (Perkins and Ellisman 2011). Mitochondria in an orthodox conformation, for instance, have

L. A. Suldina et al. Fig. 5 Ultrastructural organization of ES-like cells at passage 0. a, b Large mitochondria (mt) with thin cristae, one of them is in tight contact with rough ER cisternae (ER—endoplasmic reticulum; the arrow indicates the contact with mitochondria). c The Golgi apparatus (G) with short cisternae and numerous vesicles. d An autolysosome (A) and two mitochondria with swollen cristae (white arrows). Scale bars, 1 μm

conformation at early passages. The observations concerning the morphological types of mitochondria are valid for all three cell lines under study. To our knowledge, this is the first report of the presence of three types of mitochondria in murine pluripotent cell. Previously, Bpoorly developed^ and Bwell-developed mitochondria^ have been described by one research group in ES cells (Alharbi et al. 2014), but they did not provide any morphometric data. In addition, ES cell colonies contained cells with different mitochondrial populations (Fig. 9). At the center of MA13 colonies at passage 0, most of cells (70%) were characterized by the presence of presumably orthodox mitochondria (17 and 13% of cells contained transitional and condensed mitochondria, respectively). Furthermore, the percentage of cells with orthodox mitochondria in this cell line was higher in comparison with the other ES cell lines at passages 0, 2, and 15. At passage 0, more than a half of cells (57%) of МА14 colonies contained transitional mitochondria, and other cells contained orthodox and condensed types of mitochondria (18 and 25% of cells, respectively). Equal numbers of МА15 cells at passage 0 had transitional or condensed mitochondria (48 and

48% of cells, respectively), and only 4% of these cells contained orthodox mitochondria. The proportion of cells with orthodox mitochondria tended to decrease in all the cell lines during passaging (to 21 and 0% in МА13 cells, 7 and 6% in МА14 cells, and 5 and 4% in МА15 cells at passages 2 and 4, respectively). The proportion of cells containing condensed mitochondria among МА13 cells increased at passages 2 and 4 (to 31 and 47%, respectively); however, at passage 15, the cells with this type of mitochondria disappeared from this line for an unclear reason. By contrast, among МА15 cells, the percentage of cells with condensed mitochondria decreased at passage 2 (from 48 to 38%) in comparison with passage 0 and reached the maximum (68%) at passage 15. Thus, at passage 0, the populations of ES-like cells in all three ES cell lines were highly heterogeneous and consisted of three cell variants characterized by the presence one of three types of mitochondria in the cytoplasm. At passage 2, the percentage of ES cells with orthodox mitochondria decreased in all ES lines, and at passage 4, all ES cell populations were composed of two kinds of cells: containing presumably transitional or condensed mitochondria.


Fig. 6 Ultrastructural organization of MA13 ES cells at passages 2 (a–c), 4 (d–f), and 15 (g–i). a, d, g Overview of cells. b, e, h Transitional mitochondria with medium density of the matrix and thin and swollen cristae. c, f Condensed mitochondria with a dense matrix and swollen

cristae, rough ER (ER) with thin cisternae (c). i Orthodox mitochondria with an electron lucid matrix. White arrows mark swollen mitochondrial cristae. G Golgi apparatus, mt mitochondria, N nucleus. Scale bars, a, d, g 5 μm; b, c, e, f, h, i 1 μm

Dynamics of mitochondrial morphology and behavior during derivation of ES cell lines

matrix of medium density and mostly transverse regular cristae at the late blastocyst stage of mouse development; in contrast, ES cells contain more mitochondria with the condensed configuration (Suhr et al. 2010). Main structural defects of mitochondria, including their swelling, a dispersed matrix and abnormal cristae observed in ES-like cells at passage 0, can point to the dysfunction of these organelles. According to our and other authors’ opinions, these phenomena are related to alteration of a natural microenvironment and to changes in

Mitochondria are highly mobile organelles involved in different cellular processes and have multiple functions in early embryogenesis, as well as in metabolism and in the dynamics of embryonic and somatic stem cells (Wilkerson and Sankar 2011; Chen et al. 2010). In agreement with our data, it was previously shown that the mitochondria of ICM cells have a


Fig. 7

Morphometric parameters of mitochondria in late blastocyst ICM cells, in ES-like cells at passage 0, and in ES cells at passages 2, 4, and 15. a Mean cross-sectional area of mitochondria. b Mean numerical density of mitochondria. c Mean volume density of mitochondria. Bars in the diagram denote standard error, *P < 0.05, **P < 0.01, ***P < 0.001

cell homeostasis and energy metabolism after the transfer of ICM cells from in vivo to in vitro conditions (Prigione et al. 2015). The substantial decrease in mitochondrial number and volume density in ES-like cells correlated with an increase in cell size during this period and is suggestive of their low metabolic activity. Moreover, in spite of structural similarity of ES cells among colonies of different cell lines at passage 2, the mitochondrial parameters continued to vary between cell lines and stabilized only at passage 4. As we mentioned before, expression levels of Bkey^ genes can vary in ES cells (Niwa et al. 2009; Kalmar et al. 2009; Martinez et al., 2012), and this phenomenon may influence mitochondrial activity and morphology as well. For instance, OCT4 directly regulates the expression of glycolysis enzymes HK2 and PKM2 in mouse ES cells (Kim et al. 2015). It has also been demonstrated that expression of genes critical for mitochondrial oxidative phosphorylation is regulated by LIN28, an RNA-binding protein that plays critical roles in embryonic development, tumorigenesis, and pluripotency (Zhang et al. 2016). The elevated oxygen consumption rate in Lin28-knockout murine ES cells is consistent with more densely packed mitochondrial inner membrane cristae. Lin28-knockout ES cells show elevated expression of proteins involved in mitochondrial oxidative phosphorylation complexes (Zhang et al. 2016). It has been suggested that mitochondria perform a crucial function in pluripotency (Xu et al. 2013) and mitochondrial DNA may be involved in the regulation of nuclear gene expression in the pluripotent state (Kelly et al. 2012). A knockdown of mitochondrial DNA polymerase PolG decreased Oct4 expression in murine ES cells (Facucho-Oliveira et al. 2007), and a knockdown of mitochondrial protein GFER (growth factor erv1-like) decreased expression of pluripotency markers NANOG, OCT4, and SSEA1 (Todd et al. 2010). It should be noted that naïve pluripotent cells differ from primed ones in having not only a glycolytic ATP synthesis pathway, but also oxidative phosphorylation. Primed cells rely on glycolysis, which makes them sensitive to its inhibitors (Zhou et al. 2012). Accordingly, we propose that mitochondrial functional activity and ultrastructural modifications may also be influenced by differences in expression of pluripotency genes between different ES cell lines especially at first passages during ES cell derivation. Our analysis shows that stabilization of mitochondrial structural organization and of their morphometric parameters take place at passage 4, that is, later in comparison with emergence of typical ES cells (at passage 2).


Fig. 8 Ultrastructural organization and morphometric parameters of three types of mitochondria in ES cells. a Orthodox mitochondria contain a lucid matrix and thin cristae. b Transitional mitochondria have a matrix of medium density and both thin and swollen cristae. c Condensed mitochondria contain a dense matrix and swollen cristae. d–f Mitochondria with different morphological characteristics in the

cytoplasm of a single cell. g Mean cross-sectional area of mitochondria of different types. h Mean volume density of cristae in different types of mitochondria. i Mean numerical density of cristae in different types of mitochondria. White arrows mark swollen mitochondrial cristae. Scale bars, 1 μm. Bars in the diagram denote standard error. *P < 0.05, **P < 0.01


Fig. 9 The percentage of cells that contain mitochondria of three different types in the ICM of the late blastocyst, in ES-like cells at passage 0, and ES cells at passages 2, 4, and 15: cells with orthodox mitochondria, cell with transitional mitochondria, and cells with condensed mitochondria. Bars in the diagram denote standard error

Several studies have uncovered close proximity of mitochondria and the ER in mouse oocytes (Dumollard et al. 2007), which is important for calcium homeostasis, mitochondrial biogenesis, and mitophagy in ES cells (Xu et al. 2013). We observed similar contacts in the cytoplasm of ICM cells from the late blastocyst; however, ER membranes seemed to be associated with the fibrous lattice structures there. Numerous contacts identified between the ER membranes and the defective mitochondria in the cytoplasm of ES-like cells at passage 0 are evidently associated with the processes of their lysis and destruction by the autophagosome. Autophagy is one of the main processes for elimination of damaged organelles for prevention of cellular damage. Recent studies revealed that autophagy maintains stem cells in relatively undifferentiated states (stemness) (Pan et al. 2013). The ER-mitochondria contacts observed in ES cells at later (2, 4, and 15) passages may participate in mitochondrial biogenesis and support the correct balance of calcium homeostasis between the cytoplasm and the mitochondria.

of their morphological rearrangements at passages 0, 2, 4, and 15. Our study shows that extensive structural reorganization of ICM cells after their transfer to in vitro conditions gives rise to the ES-like cell type in culture—this cell type is detected as early as day 6 (passage 0). The resulting ES-like cells differ from ICM cells in several parameters: the cross-sectional cell area, nucleocytoplasmic ratio, density of organelles in the cytoplasm, and particularly in the mitochondrial crosssectional area, quantity, volume density, and the type of mitochondria. Our examination revealed profound changes of mitochondrial structure in ES-like cells at passage 0, which are induced evidently by the new environment and by remodeling of bioenergetic mechanisms aside from the influence of many other in vitro factors. The variable morphometric parameters of ES cells stabilized in all three cell lines by passage 2, whereas the variations in mitochondrial morphology strongly diminished at passage 4. Mitochondrial volume decreased relative to ICM cells and showed greater stability in all the cell lines at different passages in comparison with numerical density and mitochondrial cross-sectional area. We also demonstrated that cells within each ES line colony can differ in terms of the presence of dissimilar morphological types of mitochondria. Thus, we observed and for the first time quantified three morphological types of mitochondria in a single pluripotent cell identified in all the ES cell lines. We believe that the findings about ES cells’ structural modifications and especially changes in mitochondrial architecture at different stages of ES cell line derivations from the ICM are an important step toward elucidation of murine ES cell pluripotency. Acknowledgments We are thankful to Antonina I. Zhelezova and Aleftina N. Golubitsa for assistance with the experiments on mice. The study was supported by the Federal Agency for Scientific Organizations program for support of bioresource collections. We thank the Collective Center of ICG SB RAS BCollection of Pluripotent Human and Mammalian Cell Cultures for Biological and Biomedical Research^ (http://ckp.icgen.ru/cells/) for providing the cell lines, and we are grateful to the Interinstitutional Shared Center for Microscopic Analysis of Biological Objects (ICG SB RAS, Novosibirsk) for providing the microscopy equipment for this study. The English language was corrected and certified by shevchuk-editing.com.

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conclusion In this study, we conducted ultrastructural and morphometric analyses of three ES cell lines derived from mouse blastocyst ICM cells and characterized them by comparative evaluation

Abbreviations dpc, days post coitum; EDTA, ethylenediaminetetraacetic acid; ER, endoplasmic reticulum; ESC, embryonic stem cell; FBS, fetal bovine serum; ICM, inner cell mass; KSR, knockout serum replacement; LIF ESGRO, leukemia inhibitory factor; NEAA, non-essential amino acid; TEM, transmission electron microscopy


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