Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish.

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life-death balance and cell fate determination during normal homeostasis.

[Application of the blastomere count variations "skip value" in the embryo AI assessment].

Objective: To explore the correlation between blastomere count variations "skip value" which extracted from by time-lapse technology (TLT) combined with artificial intelligence (AI) and morphological features of in vitro fertilization (IVF) embryo, and to test its feasibility in clinical applications. Methods: This study was a diagnostic experiment (AI reassessment of embryo transferred patients), a total of 6 545 embryos from 1 226 patients who underwent IVF at the Women and Children's Hospital of Chongqing Medical University from December 2020 to December 2021 were retrospectively analyzed, of which 2 869 embryos were attempted to cultured to blastocyst stage by TLT. The embryo dynamic map (EDM) was drawn by Embryo Viewer, a TLT recording software, based on embryo developmental kinetics. The self-developed AI embryo evaluation software identified and recorded the number of cleavages in real time during embryonic development, and compared with the EDM, the correlation between the skip value formed by the change of cleavage sphere counts and the outcomes of the embryos was analyzed. The correlation among skip value, morphological score of embryo, implantation rate and live birth rate were performed by Spearman and step-up logistic regression. The receiver operating characteristic (ROC) curve was selected for reporting there relationship of skip value and morphology. Finally, predicting power of skip value for implantation and live birth rate were performed by ROC analysis. Results: The total skip values extracted from the blastomere count of embryos (72 hours post-fertilization) were negatively correlated with abnormal cleavage, blastocyst formation rate, day 3 (D3)-cell score, uneven size and fragmentation (the ß values were -0.268, -0.116, -0.213, -0.159 and -0.222, respectively; all P<0.001); positively correlated with D3-cell number (ß=0.034; P<0.001); negatively correlated with blastocyst formation rate and implantation rate (OR=0.97, 95%CI: 0.93-0.99, P=0.034; OR=0.96, 95%CI: 0.93-0.98, P=0.044). The power of predicting implantation were similar between the order selection of skip values and traditional morphology criteria [area under curve (AUC): 0.679 vs 0.620]. Live birth rate were negatively correlated with female age (OR=0.91, 95%CI: 0.88-0.93; P<0.001), D3 general score (OR=0.77, 95%CI: 0.59-0.99; P=0.045) and order selection of skip values (OR=0.98, 95%CI: 0.96-0.99; P=0.038), while positively correlated with retrieved oocyte number and endometrial thickness in embryo transferred (OR=1.08, 95%CI:1.05-1.11, P<0.001; OR=1.09, 95%CI:1.06-0.12, P<0.001, respectively) from multivariate regression analysis, and the power of predicting live birth was 0.666 for AUC. Conclusions: The skip value and its order form is a systematic quantification of embryo development, correlated with embryo developmental quality and clinical outcome. It could be an addition parameter for embryo culture and selection.

Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

The pluripotency state of human embryonic stem cells derived from single blastomeres of eight-cell embryos.

Human embryonic stem cells (hESCs) derived from blastocyst stage embryos present a primed state of pluripotency, whereas mouse ESCs (mESCs) display naïve pluripotency. Their unique characteristics make naïve hESCs more suitable for particular applications in biomedical research. This work aimed to derive hESCs from single blastomeres and determine their pluripotency state, which is currently unclear. We derived hESC lines from single blastomeres of 8-cell embryos and from whole blastocysts, and analysed several naïve pluripotency indicators, their transcriptomic profile and their trilineage differentiation potential. No significant differences were observed between blastomere-derived hESCs (bm-hESCs) and blastocyst-derived hESCs (bc-hESCs) for most naïve pluripotency indicators, including TFE3 localization, mitochondrial activity, and global DNA methylation and hydroxymethylation, nor for their trilineage differentiation potential. Nevertheless, bm-hESCs showed an increased single-cell clonogenicity and a higher expression of naïve pluripotency markers at early passages than bc-hESCs. Furthermore, RNA-seq revealed that bc-hESCs overexpressed a set of genes related to the post-implantational epiblast. Altogether, these results suggest that bm-hESCs, although displaying primed pluripotency, would be slightly closer to the naïve end of the pluripotency continuum than bc-hESCs.

Revisiting trophectoderm-inner cell mass lineage segregation in the mammalian preimplantation embryo.

In the first days of life, cells of the mammalian embryo segregate into two distinct lineages, trophectoderm and inner cell mass. Unlike nonmammalian species, mammalian development does not proceed from predetermined factors in the oocyte. Rather, asymmetries arise de novo in the early embryo incorporating cues from cell position, contractility, polarity, and cell-cell contacts. Molecular heterogeneities, including transcripts and non-coding RNAs, have now been characterized as early as the 2-cell stage. However, it's debated whether these early heterogeneities bias cells toward one fate or the other or whether lineage identity arises stochastically at the 16-cell stage. This review summarizes what is known about early blastomere asymmetries and our understanding of lineage allocation in the context of historical models. Preimplantation development is reviewed coupled with what is known about changes in morphology, contractility, and transcription factor networks. The addition of single-cell atlases of human embryos has begun to reveal key differences between human and mouse, including the timing of events and core transcription factors. Furthermore, the recent generation of blastoid models will provide valuable tools to test and understand fate determinants. Lastly, new techniques are reviewed, which may better synthesize existing knowledge with emerging data sets and reconcile models with the regulative capacity unique to the mammalian embryo.

The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Should we should consider day 3 blastomere number during single vitrified-warmed blastocyst transfer cycle? A retrospective study.

The present study investigated whether day 3 blastomere number has an effect on the clinical outcomes during single vitrified-warmed blastocyst transfer cycles. A total of 3294 vitrified-warmed single day 5 blastocyst transferred cycles were analyzed in this retrospective study from January 2018 to December 2021. The cycles were divided into ≥ 7 and < 7 blastomere groups depending on the day 3 embryo blastomere number. The clinical outcomes were compared between the two groups, moreover multivariate logistic regression analysis was conducted to investigate the correlation between the number of day 3 blastomeres and clinical outcomes. The chi-square test demonstrated that the rates of clinical pregnancy and live birth were significantly higher in the ≥ 7 blastomere group compared to the < 7 blastomere group with respect to single high-quality blastocyst transfer cycles. Conversely, these rates were similar in the two groups with respect to single low-quality blastocyst transfer cycles. These results were confirmed by multivariate logistic regression analysis. However, the miscarriage rate was higher in the < 7 blastomere group than in ≥ 7 group during low-quality blastocyst transfer cycles. These results suggested that day 3 blastomere number should be considered during single vitrified-warmed blastocyst transfer cycles. Thus, blastocsyts derived from ≥ 7 blastomere embryos are preferred when choosing the same quality blastocysts.

Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.

Cleavage-stage human embryo arrest, is it embryo genetic composition or others?

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle.

Morphometric and morphokinetic differences in the sperm- and oocyte-originated pronuclei of male and female human zygotes: a time-lapse study.

PURPOSE: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. METHOD(S): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. RESULTS: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). CONCLUSION(S): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring.

Retrospective comparison of pregnancy outcomes of fresh and frozen-warmed single blastocyst transfer: a 5-year single-center experience.

PURPOSE: To assess whether live birth rates (LBR) and maternal/neonatal complications differed following single fresh and frozen-warmed blastocyst transfer. METHODS: The present retrospective observational study analyzed 4,613 single embryo transfers (SET) (646 fresh and 3,967 frozen) from January 1, 2014, to December 31, 2018. Fresh embryo transfer at blastocyst stage was considered according to the age of the patient and her prognosis. In case of the risk of ovarian hyperstimulation syndrome, premature progesterone rise, non-optimal endometrial growth, or supernumerary embryos, cryopreservation with subsequent frozen embryo transfer (FET) was indicated. RESULTS: No differences in LBR were recorded. Fresh embryo transfers yielded an increase both in neonatal complications OR 2.15 (95% CI 1.20-3.86, p 0.010), with a higher prevalence of singletons weighting below the 5th percentile (p 0.013) and of intrauterine growth retardation (p 0.015), as well as maternal complications, with a higher placenta previa occurrence OR 3.58 (95% CI 1.54-8.28, p 0.003), compared to FET. CONCLUSION: LBR appears not to be affected by the transfer procedure preferred. Fresh embryo transfer is associated with higher risk of neonatal complications (specifically a higher prevalence of singletons weighting below the 5th percentile and of intrauterine growth retardation) and placenta previa. Reflecting on the increased practice of ART procedures, it is imperative to understand whether a transfer procedure yields less complications than the other and if it is time to switch to a "freeze-all" procedure as standard practice. TRIAL REGISTRATION: Clinical Trial Registration Number: NCT04310761. Date of registration: March 17, 2020, retrospectively registered.

Are commercial warming kits interchangeable for vitrified human blastocysts? Further evidence for the adoption of a Universal Warming protocol.

PURPOSE: To study whether a new combination of different warming kits is clinically effective for vitrified human blastocysts. METHODS: This is a longitudinal cohort study analysing two hundred fifty-five blastocysts warming cycles performed between January and October 2018. Embryos were vitrified using only one brand of ready-to-use kits (Kitazato), whereas the warming procedure was performed with three of the most widely used vitrification/warming kits (Kitazato, Sage and Irvine) after patient stratification for oocyte source. The primary endpoint was survival rate, while the secondary endpoints were clinical pregnancy, live birth and miscarriage rates. RESULTS: We observed a comparable survival rate across all groups of 100% (47/47) in KK, 97.6% (49/50) in KS, 97.6% (41/42) in KI, 100% (38/38) in dKK, 100% (35/35) in dKS and 100% (43/43) in dKI. Clinical pregnancy rates were also comparable: 38.3% (18/47) in KK, 49% (24/49) in KS, 56.1% (23/ 41) in KI, 47.4% (18/38) in dKK, 31.4% (11/35) in dKS and 48.8% (21/ 43) in dKI. Finally, live birth rates were 29.8% (14/47) in KK, 36.7% (18/49) in KS, 46.3% (19/41) in KI, 36.8% (14/38) in dKK, 25.7% (9/35) in dKS and 41.9% (18/43) in dKI, showing no significant differences. CONCLUSION: This study confirmed the efficacy of applying a single warming protocol, despite what the "industry" has led us to believe, supporting the idea that it is time to proceed in the cryopreservation field and encouraging embryologists worldwide to come out and reveal that such a procedure is possible and safe.

Developmental capacity is unevenly distributed among single blastomeres of 2-cell and 4-cell stage mouse embryos.

During preimplantation development, mammalian embryo cells (blastomeres) cleave, gradually losing their potencies and differentiating into three primary cell lineages: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PE). The exact moment at which cells begin to vary in their potency for multilineage differentiation still remains unknown. We sought to answer the question of whether single cells isolated from 2- and 4-cell embryos differ in their ability to generate the progenitors and cells of blastocyst lineages. We revealed that twins were often able to develop into blastocysts containing inner cell masses (ICMs) with PE and EPI cells. Despite their capacity to create a blastocyst, the twins differed in their ability to produce EPI, PE, and TE cell lineages. In contrast, quadruplets rarely formed normal blastocysts, but instead developed into blastocysts with ICMs composed of only one cell lineage or completely devoid of an ICM altogether. We also showed that quadruplets have unequal capacities to differentiate into TE, PE, and EPI lineages. These findings could explain the difficulty of creating monozygotic twins and quadruplets from 2- and 4-cell stage mouse embryos.

Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos.

GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression.

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells.

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are 'true' totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely 'cluster 3', as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos.

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.

Mouse totipotent stem cells captured and maintained through spliceosomal repression.

Since establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable with in vivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable in vitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.