NSUN5 is essential for proper cell proliferation and differentiation of mouse preimplantation embryos.

In brief: Proper early embryonic development in mammals relies on precise cellular signaling pathways. This study reveals that NSUN5 is crucial for the regulation of the Hippo pathway, ensuring normal proliferation and differentiation in mouse preimplantation embryos. Abstract: NOL1/NOP2/Sun domain family, member 5 (NSUN5) is an enzyme belonging to the 5-methylcytosine (m5C) writer family that modifies rRNA and mRNA. Our data revealed an upregulation of Nsun5 at the two-cell stage of mouse preimplantation development, suggesting its significance in early embryonic development. Given m5C's important role in stabilizing rRNA and mRNA and the Hippo signaling pathway's critical function in lineage segregation during embryogenesis, we hypothesized that NSUN5 controls cell differentiation by regulating the expression of components of the Hippo signaling pathway in mouse early embryos. To examine this hypothesis, we employed Nsun5-specific small interfering RNAs for targeted gene silencing in mouse preimplantation embryos. Nsun5 knockdown resulted in significant developmental impairments including reduced blastocyst formation, smaller size of blastocysts, and impaired hatching from the zona pellucida. Nsun5 knockdown also led to decreased cell numbers and increased apoptosis in embryos. We also observed diminished nuclear translocation of yes-associated protein 1 (YAP1) in Nsun5 knockdown embryos at the morula stage, indicating disrupted cell differentiation. This disruption was further evidenced by an altered ratio of CDX2-positive to OCT4-positive cells. Furthermore, Nsun5 depletion was found to upregulate the Hippo signaling-related key genes, Lats1 and Lats2 at the morula stage. Our findings underscore the essential role of Nsun5 in early embryonic development by affecting cell proliferation, YAP1 nuclear translocation, and the Hippo pathway.

Chimeric PRMT6 protein produced by an endogenous retrovirus promoter regulates cell fate decision in mouse preimplantation embryos†.

Murine endogenous retrovirus with leucine tRNA primer, also known as MERVL, is expressed during zygotic genome activation in mammalian embryos. Here we show that protein arginine N-methyltransferase 6 (Prmt6) forms a chimeric transcript with MT2B2, one of the long terminal repeat sequences of murine endogenous retrovirus with leucine tRNA primer, and is translated into an elongated chimeric protein (PRMT6MT2B2) whose function differs from that of the canonical PRMT6 protein (PRMT6CAN) in mouse preimplantation embryos. Overexpression of PRMT6CAN in fibroblast cells increased asymmetric dimethylation of the third arginine residue of both histone H2A (H2AR3me2a) and histone H4 (H4R3me2a), while overexpression of PRMT6MT2B2 increased only H2AR3me2a. In addition, overexpression of PRMT6MT2B2 in one blastomere of mouse two-cell embryos promoted cell proliferation and differentiation of the blastomere into epiblast cells at the blastocyst stage, while overexpression of PRMT6CAN repressed cell proliferation. This is the first report of the translation of a chimeric protein (PRMT6MT2B2) in mouse preimplantation embryos. Our results suggest that analyzing chimeric transcripts with murine endogenous retrovirus with leucine tRNA primer will provide insight into the relationship between zygotic genome activation and subsequent intra- and extra-cellular lineage determination.

Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods.

In ruminants, the overgrowth of offspring produced by in vitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an aetiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in foetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215 and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on foetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine foetal placentae and reveal their differential characteristics between different calf-production methods.

Evaluating histone modification analysis of individual preimplantation embryos.

BACKGROUND: We previously reported a modification of the CUT&Tag method (NTU-CAT) that allows genome-wide histone modification analysis in individual preimplantation embryos. In the present study, NTU-CAT was further simplified by taking advantage of the Well-of-the-Well (WOW) system, which enables the processing of multiple embryos in a shorter time with less reagent and cell loss during the procedure (WOW-CUT&Tag, WOW-CAT). RESULTS: WOW-CAT allowed histone modification profiling from not only a single blastocyst but also from a portion of it. WOW-CAT generated similar H3K4me3 profiles as NTU-CAT, but they were closer to the profiles produced by chromatin immunoprecipitation-sequencing, such as a valley-like trend and relatively lower false positive rates, indicating that WOW-CAT may attenuate the bias of Tn5 transposase to cut open chromatin regions. Simultaneous WOW-CAT of two halves of single blastocysts was conducted to analyze two different histone modifications (H3K4me3 and H3K27ac) within the same embryo. Furthermore, trophectoderm cells were biopsied and subjected to WOW-CAT in anticipation of preimplantation diagnosis of histone modifications. WOW-CAT allowed the monitoring of epigenetic modifications in the main body of the embryo. For example, analysis of H3K4me3 modifications of XIST and DDX3Y in trophectoderm biopsies could be used to sex embryos in combination with quantitative PCR, but without the need for deep sequencing. CONCLUSIONS: These results suggest the applicability of WOW-CAT for flexible epigenetic analysis of individual embryos in preimplantation epigenetic diagnosis.

MYC-MAX heterodimerization is essential for the induction of major zygotic genome activation and subsequent preimplantation development.

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, begins in the S phase at the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligonucleotides targeting Myc or MYC-specific inhibitors from the one-cell stage. To identify when MYC inhibition affects development, we applied time-limited inhibitor treatment and found that inhibition of MYC at the one-cell, four-cell, and morula stages had no effect on preimplantation development, whereas inhibitor treatment at the two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that MYC is essential for the induction of major ZGA and subsequent preimplantation development. Revealing the function of MYC in preimplantation development is expected to contribute to advances in assisted reproductive technology.

A more accurate analysis of maternal effect genes by siRNA electroporation into mouse oocytes.

Maternal RNA and proteins accumulate in mouse oocytes and regulate initial developmental stages. Sperm DNA combines with protamine, which is exchanged after fertilization with maternal histones, including H3.3; however, the effect of H3.3 on development post-fertilization remains unclear. Herein, we established an electroporation method to introduce H3.3 siRNA into germinal vesicle (GV)-stage oocytes without removing cumulus cells. Oocyte-attached cumulus cells need to be removed during the traditional microinjection method; however, we confirmed that artificially removing cumulus cells from oocytes reduced fertilization rates, and oocytes originally free of cumulus cells had reduced developmental competence. On introducing H3.3 siRNA at the GV stage, H3.3 was maintained in the maternal pronucleus and second polar body but not in the paternal pronucleus, resulting in embryonic lethality after fertilization. These findings indicate that H3.3 protein was not incorporated into the paternal pronucleus, as it was repeatedly translated and degraded over a relatively short period. Conversely, H3.3 protein incorporated into the maternal genome in the GV stage escaped degradation and remained in the maternal pronucleus after fertilization. This new method of electroporation into GV-stage oocytes without cumulus cell removal is not skill-intensive and is essential for the accurate analysis of maternal effect genes.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag).

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

Comparative analysis of histone H3K27me3 modifications between blastocysts and somatic tissues in cattle.

Epigenetic modifications established in the early developmental stages can have long-term consequences throughout life. This concept encompasses the possibility of controlling livestock health and diseases by epigenetic regulation during early development. To explore the candidates of epigenetic modifications in early embryos that might exert long-lasting effects in adulthood, we aimed to obtain genome-wide histone H3 lysine 27 trimethylation (H3K27me3) profiles of bovine blastocysts and compare these data with those from adult somatic tissues in order to extract common and typical features between them. Bovine blastocysts were produced in vitro and subjected to chromatin immunoprecipitation-sequencing analysis of H3K27me3. Comparative analysis of the blastocyst-derived H3K27me3 profile performed using publicly available data from adult muscle, fat, and liver tissues revealed that (1) blastocyst-specific modifications against somatic tissues were enriched in immune function-related genes, (2) somatic modifications "sieved" by blastocyst modifications were enriched in biological processes in tissue-specific trends, (3) the modifications common in blastocyst and each somatic tissue were largely overlapped and enriched in developmentally important genes, including homeobox and imprinted genes. The results of this study produced a genome-wide H3K27me3 profile of bovine blastocysts and revealed its common and typical features in relation to the profiles of adult somatic tissues.

Comparative analysis of histone H3K4me3 modifications between blastocysts and somatic tissues in cattle.

Epigenetic changes induced in the early developmental stages by the surrounding environment can have not only short-term but also long-term consequences throughout life. This concept constitutes the "Developmental Origins of Health and Disease" (DOHaD) hypothesis and encompasses the possibility of controlling livestock health and diseases by epigenetic regulation during early development. As a preliminary step for examining changes of epigenetic modifications in early embryos and their long-lasting effects in fully differentiated somatic tissues, we aimed to obtain high-throughput genome-wide histone H3 lysine 4 trimethylation (H3K4me3) profiles of bovine blastocysts and to compare these data with those from adult somatic tissues in order to extract common and typical features between these tissues in terms of H3K4me3 modifications. Bovine blastocysts were produced in vitro and subjected to chromatin immunoprecipitation-sequencing analysis of H3K4me3. Comparative analysis of the blastocyst-derived H3K4me3 profile with publicly available data from adult liver and muscle tissues revealed that the blastocyst profile could be used as a "sieve" to extract somatic tissue-specific modifications in genes closely related to tissue-specific functions. Furthermore, principal component analysis of the level of common modifications between blastocysts and somatic tissues in meat production-related and imprinted genes well characterized inter- and intra-tissue differences. The results of this study produced a referential genome-wide H3K4me3 profile of bovine blastocysts within the limits of their in vitro source and revealed its common and typical features in relation to the profiles of adult tissues.

Embryonic MTHFR contributes to blastocyst development.

PURPOSE: Reduction in methylenetetrahydrofolate reductase (MTHFR) activity due to genetic variations in the MTHFR gene has been controversially implicated in subfertility in human in vitro fertilization. However, there is no direct gene-knockdown study of embryonic MTHFR to assess its involvement in mammalian preimplantation development. The purpose of this study is to investigate expression profiles and functional roles of MTHFR in bovine preimplantation development. METHODS: Reverse transcription-quantitative PCR (RT-qPCR) and analysis of publicly available RNA-seq data were performed to reveal expression levels of MTHFR during bovine preimplantation development. We knocked down MTHFR by siRNA-mediated RNA interference from the 8- to 16-cell stage and assessed the effects on preimplantation development. RESULTS: The RT-qPCR analysis showed relatively high MTHFR expression at the GV oocyte stage, which was decreased toward the 8- to 16-cell stage and then slightly restored at the blastocyst stage. Public data-based analysis also showed the similar pattern of expression with substantial embryonic expression at the blastocyst stage. MTHFR knockdown reduced the blastocyst rate (P < 0.01) and the numbers of total (P < 0.0001), trophectoderm (P < 0.0001), and inner cell mass (P < 0.001) cells. CONCLUSION: The results indicate that embryonic MTHFR is indispensable for normal blastocyst development. The findings provide insight into the debatable roles of MTHFR in fertility and may be applicable for the improvement of care for early embryos via modulation of surrounding folate-related nutritional conditions in vitro and/or in utero, depending on the parental and embryonic MTHFR genotype.

Deletion of the PDZ-binding kinase (Pbk) gene does not affect male fertility in mice.

The PDZ-binding kinase (PBK) protein is localised exclusively in spermatogenic cells, such as spermatogonia, spermatocytes and round spermatids, of the adult testis. However, its role in male fertility remains unknown. Analysis of adult Pbk-knockout (KO) male mice showed no significant difference in the weight of the testes, epididymis and seminal vesicle compared with adult wild-type (WT) mice. There were no significant differences in testis morphology, tubule diameter and the number of offspring born to females mated with KO or WT male mice. Sperm number, motility and morphology did not differ significantly between KO and WT mice. The oocyte fertilisation rate and embryo development following IVF were comparable between groups fertilised using spermatozoa from KO versus WT mice (P>0.05). Further analysis revealed that the phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 kinase, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases was dysregulated in the testis of KO mice. In conclusion, Pbk-KO male mice are fertile and their spermatozoa and testis do not show any morphological and functional abnormalities despite the dysregulated phosphorylation of MAPKs. It is likely that functional redundancy of PBK and overlapping substrate specificities of the MAPK superfamily compensated for the loss of PBK from the testis.

H4K20 monomethylation inhibition causes loss of genomic integrity in mouse preimplantation embryos.

Maintaining genomic integrity in mammalian early embryos, which are deficient in DNA damage repair, is critical for normal preimplantation and subsequent development. Abnormalities in DNA damage repair in preimplantation embryos can cause not only developmental arrest, but also diseases such as congenital disorders and cancers. Histone H4 lysine 20 monomethylation (H4K20me1) is involved in DNA damage repair and regulation of gene expression. However, little is known about the role of H4K20me1 during mouse preimplantation development. In this study, we revealed that H4K20me1 mediated by SETD8 is involved in maintaining genomic integrity. H4K20me1 was present throughout preimplantation development. In addition, reduction in the level of H4K20me1 by inhibition of SETD8 activity or a dominant-negative mutant of histone H4 resulted in developmental arrest at the S/G2 phase and excessive accumulation of DNA double-strand breaks. Together, our results suggest that H4K20me1, a type of epigenetic modification, is associated with the maintenance of genomic integrity and is essential for preimplantation development. A better understanding of the mechanisms involved in maintaining genome integrity during preimplantation development could contribute to advances in reproductive medicine and technology.

Synthesis and maintenance of lipid droplets are essential for mouse preimplantation embryonic development.

Lipid droplets (LDs), which are ubiquitous organelles consisting of a neutral lipid core coated with a phospholipid monolayer, play key roles in the regulation of cellular lipid metabolism. Although it is well known that mammalian oocytes and embryos contain LDs and that the amount of LDs varies among animal species, their physiological functions remain unclear. In this study, we have developed a method based on two-step centrifugation for efficient removal of almost all LDs from mouse MII oocytes (delipidation). We found that delipidated MII oocytes could be fertilized in vitro, and developed normally to the blastocyst stage even when the embryos were cultured in the absence of a fatty acid supply. LDs were newly synthesized and accumulated soon after delipidation, but chemical inhibition of long chain acyl-CoA synthetases (ACSLs) blocked this process, resulting in severe impairment of early embryonic development. Furthermore, we found that overabundance of LDs is detrimental to early embryonic development. Our findings demonstrate the importance of synthesis and maintenance of LDs, mediated in part by ACSL activity, during preimplantation embryonic development.

Altered hormonal milieu and dysregulated protein expression can cause spermatogenic arrest in ectopic xenografted immature rat testis.

Testis tissue xenografting complemented with cryopreservation is a feasible technique for fertility preservation in children with malignancy receiving gonadotoxic therapy and for endangered species with high neonatal mortality rate. However, xenografted testis of human and most endangered species are known to undergo spermatogenic arrest. In this study, we xenografted immature rat testis onto immunodeficient male mice to investigate the plausible underlying causes of spermatogenic arrest. Histological analysis of xenografted testes collected 8-wk post-grafting showed incomplete spermatogenesis with pachytene-stage spermatocytes as the most advanced germ cells. Although the levels of serum luteinizing hormone and testosterone were normal in recipient mice, those of follicle stimulating hormone (FSH) were significantly high, and specific receptors of FSH were absent in the xenografts. The xenografts demonstrated dysregulated expression of Sertoli cell-transcriptional regulators (WT1 and SOX9) and secretory proteins (SCF and GDNF). In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis. Further stereological analysis of xenografts can demonstrate precise cellular composition of xenografts to decipher interactions between germ and somatic cells to better understand spermatogenic arrest in xenografted testis.

How does the promoter of an oocyte-specific gene function in male germ cells?

Studying gene expression in germ cells is useful for elucidating mechanisms of transcriptional regulation, because different genes are activated in male and female germ cells. The promoter regions of an oocyte-specific gene, Oog1, have been characterized. Driving the expression of green fluorescent protein with these different promoter regions provided us with critical information on the regulation of gene expression. The 3.9 kb long promoter functions in both male and female germ cells in transgenic mice. What is the cause of this sexually dimorphic expression? There may be important factors within and perhaps also outside this 3.9 kb promoter region that are required to maintain proper sex-specific gene expression.

Oocyte-specific gene Oog1 suppresses the expression of spermatogenesis-specific genes in oocytes.

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Forced lipophagy reveals that lipid droplets are required for early embryonic development in mouse.

Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

CHD1 Controls Cell Lineage Specification Through Zygotic Genome Activation.

In mammals, the processes spanning from fertilization to the generation of a new organism are very complex and are controlled by multiple genes. Life begins with the encounter of eggs and spermatozoa, in which gene expression is inactive prior to fertilization. After several cell divisions, cells arise that are specialized in implantation, a developmental process unique to mammals. Cells involved in the establishment and maintenance of implantation differentiate from totipotent embryos, and the remaining cells generate the embryo proper. Although this process of differentiation, termed cell lineage specification, is supported by various gene expression networks, many components have yet to be identified. Moreover, despite extensive research it remains unclear which genes are controlled by each of the factors involved. Although it has become clear that epigenetic factors regulate gene expression, elucidation of the underlying mechanisms remains challenging. In this chapter, we propose that the chromatin remodeling factor CHD1, together with epigenetic factors, is involved in a subset of gene expression networks involved in processes spanning from zygotic genome activation to cell lineage specification.

Immobilized pH in culture reveals an optimal condition for somatic cell reprogramming and differentiation of pluripotent stem cells.

Aim: One of the parameters that greatly affects homeostasis in the body is the pH. Regarding reproductive biology, germ cells, such as oocytes or sperm, are exposed to severe changes in pH, resulting in dramatic changes in their characteristics. To date, the effect of the pH has not been investigated regarding the reprogramming of somatic cells and the maintenance and differentiation of pluripotent stem cells. Methods: In order to investigate the effects of the pH on cell culture, the methods to produce induced pluripotent stem cells (iPSCs) and to differentiate embryonic stem cells (ESCs) into mesendoderm and neuroectoderm were performed at each medium pH from 6.6 to 7.8. Using the cells of the Oct4-GFP (green fluorescent protein) carrying mouse, the effects of pH changes were examined on the timing and colony formation at cell reprogramming and on the cell morphology and direction of the differentiation of the ESCs. Results: The colony formation rate and timing of the reprogramming of the somatic cells varied depending on the pH of the culture medium. In addition, mesendodermal differentiation of the mouse ESCs was enhanced at the high pH level of 7.8. Conclusion: These results suggest that the pH in the culture medium is one of the key factors in the induction of the reprogramming of somatic cells and in the differentiation of pluripotent stem cells.