Mouse and human pluripotent stem cells and the means of their myogenic differentiation.

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, are an important tool in the studies focusing at the differentiation of various cell types, including skeletal myoblasts. They are also considered as a source of the cells that due to their pluripotent character and availability could be turned into any required tissue and then used in future in regenerative medicine. However, the methods of the derivation of some of cell types from pluripotent cells still need to be perfected. This chapter summarizes the history and current advancements in the derivation and testing of pluripotent stem cells-derived skeletal myoblasts. It focuses at the in vitro methods allowing the differentiation of stem cells grown in monolayer or propagated as embryoid bodies, and also at in vivo tests allowing the verification of the functionality of obtained skeletal myoblasts.

Cell cycle regulation in early mouse embryos.

For a long time it has been thought that the cell cycles of the early mouse embryo do not differ from the somatic cell cycle. They are long and are composed of classical G1, S, G2 and M phases and have functional checkpoint controls. However, a few characteristics observed during the earliest mitotic cleavage divisions suggest that the embryonic cell cycle could differ significantly from the somatic ones. Understanding these differences could have an important impact on our understanding of both general cell cycle mechanisms as well as the developmental programme of the early mouse embryo. Over the last few years our laboratories have undertaken a project focused on describing the differences in the first two cell cycles of the mouse embryo. We discuss here the results concerning (1) the way mouse oocytes switch from the meiotic to the mitotic cell cycle upon activation of development (inactivation of the cytostatic factor, CSF); (2) how the entry into the first and the second mitotic M phase is regulated (nucleus-independent activation of M phase-promoting factor, MPF); and (3) how the duration of the early embryonic mitoses is regulated. These data show that developmentally regulated phenomena are superimposed on and highly coordinated with the cell cycle machinery.

Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development.

Recent studies suggest early (preimplantation) events might be important in the development of polarity in mammalian embryos. We report here lineage tracing experiments with green fluorescent protein showing that cells located either near to or opposite the polar body at the 8-cell stage of the mouse embryo retain their same relative positions in the blastocyst. Thus they come to lie on either end of an axis of symmetry of the blastocyst that has recently been shown to correlate with the anterior-posterior axis of the postimplantation embryo (see R. J. Weber, R. A. Pedersen, F. Wianny, M. J. Evans and M. Zernicka-Goetz (1999). Development 126, 5591-5598). The embryonic axes of the mouse can therefore be related to the position of the polar body at the 8-cell stage, and by implication, to the animal-vegetal axis of the zygote. However, we also show that chimeric embryos constructed from 2-cell stage blastomeres from which the animal or the vegetal poles have been removed can develop into normal blastocysts and become fertile adult mice. This is also true of chimeras composed of animal or vegetal pole cells derived through normal cleavage to the 8-cell stage. We discuss that although polarity of the postimplantation embryo can be traced back to the 8-cell stage and in turn to the organisation of the egg, it is not absolutely fixed by this time.

Early development of mouse embryos null mutant for the cyclin A2 gene occurs in the absence of maternally derived cyclin A2 gene products.

Progression through the mammalian cell cycle is regulated by the sequential activation and inactivation of the cyclin-dependent kinases. In adult cells, cyclin A2-dependent kinases are required for entry into S and M phases, completion of S phase, and centrosome duplication. However, mouse embryos lacking the cyclin A2 gene nonetheless complete preimplantation development, but die soon after implantation. In this report, we investigated whether a contribution of maternal cyclin A2 mRNA and protein to early embryonic cell cycles might explain these conflicting observations. Our data show that a maternal stock of cyclin A2 mRNA is present in the oocyte and persists after fertilization until the second mitotic cell cycle, when it is degraded to undetectable levels coincident with transcriptional activation of the zygotic genome. A portion of maternally derived cyclin A2 protein is stable during the first mitosis and persists in the cytoplasm, but is completely degraded at the second mitosis. The ability of cyclin A2-null mutants to develop normally from the four-cell to the postimplantation stage in the absence of detectable cyclin A2 gene product indicates therefore that cyclin A2 is dispensable for cellular progression during the preimplantation nongrowth period of mouse embryo development.

Control of duration of the first two mitoses in a mouse embryo.

The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.

Transient reactivation of CSF in parthenogenetic one-cell mouse embryos.

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min post-activation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases.

Cytostatic activity develops during meiosis I in oocytes of LT/Sv mice.

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that during in vitro maturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H x C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.

Autonomous activation of histone H1 kinase, cortical activity and microtubule organization in one- and two-cell mouse embryos.

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one- and two-cell embryos.

Transcription and DNA replication of sperm nuclei introduced into blastomeres of 2-cell mouse embryos.

The aim of this study was to investigate the behaviour of sperm nuclei in the cytoplasm of the 2-cell mouse embryo. To this end, we produced hybrids between anucleate fertilised oocyte fragments and blastomeres of the 2-cell embryos. When sperm nuclei at the stage of decondensation or recondensation were introduced into blastomeres the development of male pronuclei was usually retarded and they never reached the size of the blastomere nuclei. These abortive male pronuclei were unable to initiate transcription but they were capable of synthesising DNA. The majority of sperm nuclei introduced into blastomeres as early male pronuclei developed normally and reached the size of the blastomere nuclei. They synthesised DNA simultaneously with blastomere nuclei and were transcriptionally active. In addition they participated in the cleavage division of hybrid cells. This shows that the very early male pronucleus when transmitted from the oocyte cytoplasm to the blastomere cytoplasm can respond positively to the new cytoplasmic factors, i.e. it undertakes both DNA replication and transcription according to the time schedule characteristic of the second cell cycle.

Chromatin condensation activity and cortical activity during the first three cell cycles of a mouse embryo.

One-cell parthenogenetic haploid embryos and blastomeres of the 2- and 4-cell diploid mouse embryos were observed in vitro for the occurrence of two cytoplasmic activities: the cortical activity and the chromatin condensation activity. For this purpose anucleated halves (AHs) and nucleated halves (NHs) were produced by bisection of one-cell embryos and of blastomeres. The cortical activity (manifested by surface deformations) was observed only during the first cleavage cycle. In AHs the surface activity began at the same time as in NHs and disappeared before the time of the cleavage division of nucleated halves. Anucleate fragments of blastomeres from 2- and 4-cell embryos did not exhibit any cortical activity. In the absence of the native nucleus the chromatin condensation activity (assayed by premature chromatin condensation of interphase thymocyte nuclei introduced into cytoplasts by cell fusion) could also have been detected only in the first cleavage cycle. In AHs this activity appeared at the time when NHs started to cleave and disappeared after the NHs finished the first cleavage division. AHs obtained from 2-cell and 4-cell stage blastomeres did not reveal condensation activity.

Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs.

Cytostatic factor (CSF) is an activity responsible for the metaphase II arrest in vertebrate oocytes. This activity maintains a high level of maturation promoting factor (MPF) in the oocyte and both activities are destroyed after fertilization or parthenogenetic activation. To study some of the characteristics of the mechanism involved in MPF and CSF destruction, we constructed hybrid cells between metaphase II arrested oocytes and early embryos obtained after fertilization or artificial activation. We found that the behavior of hybrid cells differed depending upon the type of oocyte activation. Initially, the reaction of both types of hybrid cells was similar, the nuclear envelope broke down and chromatin condensation was induced. However, while metaphase II oocytes fused with parthenogenetic eggs remained arrested in M-phase, the oocytes fused with fertilized eggs underwent activation and passed into interphase. This ability of fertilized eggs to induce oocyte activation was still present at the beginning, but not at the end of the second embryonic cell cycle. Oocyte activation induced by fusion with a fertilized egg could be prevented when calcium was chelated by BAPTA. Thus, element(s) of the mechanism involved in calcium release triggered by a sperm component at fertilization remain(s) active until the second cell cycle and is (are) inactivated before the end of the 2-cell stage.

Expression of N-CAM in fertilized pre- and periimplantation and parthenogenetically activated mouse embryos.

The expression of the cell adhesion molecule of the immunoglobulin family, neural cell adhesion molecule (N-CAM), in the pre- and periimplantation embryo was examined by immunocytochemistry. N-CAM is expressed on unfertilized ovulated oocytes, fertilized preimplantation embryos at all stages of development and parthenogenetically activated eggs and embryos. In fertilized embryos, expression from the 4-cell stage can be partially inhibited by blocking embryonic transcription before 38 h post human chorionic gonadotropin (hCG). Expression of N-CAM was reduced on the trophoblast of day 6 blastocysts in culture, weak on the trophoblast of embryonic outgrowths and disappears from invading trophoblast in utero. An antibody against alpha(2-8) linked polysialic acid, mAb2-2B, reacted with embryos from the 8-cell stage, and staining was similarly reduced on the trophoblast of blastocysts at the time of implantation. These results suggest a role for N-CAM in the interactions of cells of the preimplantation mammalian embryo which requires further investigation.

Differential chromatin condensation of female and male pronuclei in mouse zygotes.

Mouse zygotes or halves of zygotes, containing either a female or a male pronucleus, were fused with ovulated metaphase II oocytes. In 59.7% of the resulting hybrid cells, the pronuclei underwent premature chromosome condensation (PCC). In some of these heterokaryons the 2 pronuclei differed in the dynamics of condensation. Detectability of differential PCC of pronuclei (dPCC) depended on the type of preparation. In hybrids with PCC, produced by fusion of intact zygotes with metaphase II oocytes and processed for whole-mount preparations, one pronucleus was more advanced in the condensation process in 47% of cases. In air-dried preparations dPCC was detected in as many as 94% of hybrids. Experiments with the fusion of halves of zygotes with metaphase II oocytes have shown that the differential reaction of pronuclei to condensation factor depended on their parental origin. Maternal chromatin responded faster to the condensation factor and attained more advanced stages of PCC than paternal chromatin. Different responses of the maternal and paternal pronucleus to the condensation factor suggests that the 2 pronuclei are not identical with regard to the organization of chromatin and/or the lamin composition of the nuclear envelope.