scholarly journals Influence of co-culture during maturation on the developmental potential of equine oocytes fertilized by intracytoplasmic sperm injection (ICSI)

Reproduction ◽  
2001 ◽  
pp. 925-932 ◽  
Author(s):  
X Li ◽  
LH Morris ◽  
WR Allen
Keyword(s):  
Epithelial Cells ◽  
Intracytoplasmic Sperm Injection ◽  
Fibroblast Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Nuclear Maturation ◽  
Fetal Fibroblasts ◽  
Oviduct Epithelial Cells

The influence of co-culture with either oviduct epithelial cells or fetal fibroblast cells on in vitro maturation of equine oocytes and their potential for development to blastocysts and fetuses after intracytoplasmic sperm injection (ICSI) was investigated. The oocytes were obtained from ovaries from abattoirs and were matured in vitro for 28-30 h in TCM-199 only, or in TCM-199 co-culture with oviduct epithelial cells or fetal fibroblast cells. Metaphase II oocytes were subjected to ICSI with an ionomycin-treated spermatozoon. The injected oocytes were cultured for 7-9 days in Dulbecco's modified Eagle's medium. Morphologically normal early blastocysts were transferred to the uteri of recipient mares. Nuclear maturation rates and the rates of cleavage to the two-cell stage for injected oocytes were similar in the groups of oocytes that were matured in TCM-199 (49 and 63%), in co-culture with oviduct epithelial cells (53 and 65%) or in co-culture with fetal fibroblasts (51 and 57%). There were no significant differences in the proportions of blastocysts that developed from the two-cell embryos derived from oocytes matured by co-culture with either oviduct epithelial cells (30%) or fetal fibroblasts (17%). However, significantly higher proportions of blastocysts were produced from both these co-culture groups than from the groups of oocytes matured in TCM-199 only (P < 0.05). Six of the blastocysts that had developed from oocytes co-cultured with oviduct epithelial cells were transferred into recipient mares and four pregnancies resulted. These results demonstrate a beneficial influence of co-culture with either oviduct epithelial cells or fetal fibroblasts for maturation of oocytes in vitro.

83 IN VITRO DEVELOPMENTAL ABILITIES OF PORCINE CLONED EMBRYOS RECONSTITUTED WITH CELL NUCLEI OF GENETICALLY TRANSFORMED FETAL FIBROBLASTS CYTOMETRICALLY DIAGNOSED ON CELL CYCLE AND APOPTOSIS

2007 ◽  
Vol 19 (1) ◽  
pp. 159
Author(s):  
M. Samiec ◽  
M. Skrzyszowska ◽  
M. Bochenek ◽  
D. Lipinski ◽  
R. Slomski
Keyword(s):  
Cell Cycle ◽  
High Efficiency ◽  
Fibroblast Cells ◽  
Apoptotic Cells ◽  
Facs Analysis ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Donor Cells ◽  
Fetal Fibroblasts

The important factor that determines the development of mammalian cloned embryos is structuro-functional quality of nuclear donor cells. Analysis of nuclear DNA (nDNA) content of somatic cells undergoing apoptosis has become one of the most common methods for single-parameter flow cytometric measurement of this process. Apoptosis assessment is performed by quantification of hypodiploid cells. The aim of our study was to examine the in vitro developmental potential of porcine nuclear transfer (NT) embryos reconstituted with non-apoptotic fetal fibroblast cells expressing the eGFP transgene. The nuclear donor cells were derived from cell line populations whose representative random samples had been analyzed on both cell cycle and apoptosis through non-vital nDNA fluorescent dyeing and flow cytometry (FACS). Frozen-thawed fibroblast cells, which had been cultured up to a total confluency after 2–4 passages, were used for the diagnostics. The cells were fixed in ice-cold 70% ethanol. Then, the fetal fibroblasts were exposed to nDNA extraction buffer for 5 min at room temperature, and incubated in DNA staining solution (propidium iodide and RNAse) for 30 min. After fluorescent labeling, the cells were analyzed in the flow cytometer by reading nDNA fluorescence in the red band. In vitro-matured oocytes were the source of recipient cells. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. FACS analysis revealed that, out of all of the diagnosed fetal fibroblast cells, 54.7% were cycling, and up to 45.3% were late-apoptotic. In turn, from among the normal (i.e. non-apoptotic) cells, 82.2% were at G0/G1 stages of cell cycle, 17.0% at the S stage, and 0.8% at G2/M stages. A total of 150 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells. Out of 150 cultured NT embryos, 123 (82.0%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages yielded 53/150 (35.3%) and 37/150 (24.7%), respectively. In conclusion, the FACS analysis for mitotic cycle of 100%-confluent transgenic fetal fibroblasts confirmed the high efficiency of the cell cycle synchronization at G0/G1 phases. However, a contact inhibition method induced the high frequency of late-apoptotic cells. Moreover, the relatively high percentage of NT blastocysts was developed from oocytes reconstructed with eGFP transgenic fetal fibroblast cells. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.

scholarly journals Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo

Reproduction ◽  
2019 ◽  
Vol 158 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Meriem Hamdi ◽  
María J Sánchez-Calabuig ◽  
Beatriz Rodríguez-Alonso ◽  
Sandra Bagés Arnal ◽  
Kalliopi Roussi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Metabolic Response ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cell Stage ◽  
Transcriptomic Response ◽  
Early Embryos ◽  
Oviduct Epithelial Cells

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

scholarly journals 65 PRODUCTION OF PORCINE NUCLEAR TRANSFER EMBRYOS USING FETAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

2005 ◽  
Vol 17 (2) ◽  
pp. 182 ◽  
Author(s):  
M. Skrzyszowska ◽  
M. Samiec
Keyword(s):  
Nuclear Transfer ◽  
Fibroblast Cell ◽  
State Committee ◽  
Fibroblast Cells ◽  
Developmental Potential ◽  
Fetal Fibroblast ◽  
Functional Quality ◽  
Donor Cells ◽  
Morphological Transformations

One of the most important factors that determine the developmental potential of mammalian cloned embryos is the structuro-functional quality of nuclear donor cells. Biochemical changes that are some of the earliest symptoms of apoptosis signal transduction are not reflected in the morphological features of somatic cells. Therefore, an appropriate system of cell selection would enable the sorting of donor nuclei with high morphological and biochemical susceptibility to somatic cloning. The aim of our study was to examine the in vitro developmental competencies of porcine nuclear transfer (NT) embryos reconstructed with fetal fibroblast cells that had been analyzed for apoptosis by live-fluorescent labelling. Frozen/thawed fetal fibroblast cells, which had been in vitro-cultured to a confluent state, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, a single cell suspension of nuclear donor cells was subjected to dyeing with live-DNA green fluorochrome YO-PRO-1. The recipient cells were in vitro-matured oocytes. Maternal chromosomes were removed by a chemically assisted microsurgical technique. Then, single nuclear donor cells were inserted into the perivitelline space of enucleated oocytes. Fibroblast cell-ooplast couplets were simultaneously fused and activated with two consecutive DC pulses of 1.2 kV/cm for 60 μs. Reconstructed embryos were in vitro cultured in 50-μL drops of NCSU-23 medium supplemented with 0.4% BSA-V for 6 to 7 days at 38.5°C in a humidified atmosphere of 5% CO2 and 95% air. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of approximately 50 different random samples collected from the population of fetal fibroblast cells, that had been labelled with YO-PRO-1 dye, it was found that a relatively high proportion of donor cells revealed ultrastructural apoptotic changes. The percentage of late apoptotic cells with advanced morphological transformations was about 40% of the total pool of the fibroblast cells. A total of 262/270 (97.0%) enucleated oocytes were subjected to reconstruction and 141/262 (53.8%) were successfully fused with non-apoptotic nuclear donor cells. Following the simultaneous fusion/activation protocol, reconstituted oocytes were selected for in vitro culture. Out of 262, 133 (50.8%) cultured NT embryos cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/133 (36.0%) and 10/133 (7.5%), respectively. In conclusion, morphology is a sufficient selection factor for detection of apoptosis in the cultured (confluent) fetal fibroblast cells to be used for cloning. Moreover, it was found that YO-PRO-1 fluorochrome may be not able to detect the early phases of apoptosis, because only the morphologically abnormal cells emitted the YO-PRO-1-derived fluorescence. This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-KBN-084/P06/2002/4.2 from years 2003 to 2005.

104 DEVELOPMENTAL POTENTIAL OF CLONED EMBRYOS FROM ADULT AND FETAL OVINE SOMATIC CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 169
Author(s):  
H. M. Zhou ◽  
Y. Chen
Keyword(s):  
Skin Fibroblast ◽  
Polar Body ◽  
Subsequent Development ◽  
Fibroblast Cells ◽  
Fetal Skin ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
First Polar Body ◽  
Fetal Fibroblasts

This study reconstructed embryos using adult and fetal skin fibroblast cells as donor karyoplasts and ovine enucleated oocytes as recipient cytoplasts for comparing the developmental potential of the reconstructed embryos. Ovine ovaries were collected at a local slaughterhouse and the cumulus–oocyte complexes (COCs) were extracted from antral follicles 2 to 5 mm in diameter. A group of 20 to 30 COCs were matured in a 50-�L microdrop of maturation medium that was composed of TCM-199 supplemented with 20% FBS, 10 �g mL-1 FSH, 20 �g mL-1 LH, and 1.5 �g mL-1 17β-estradiol under mineral oil in a 35-mm petri dish in humidified atmosphere of 5% CO2 in air at 38.5�C for 18–22 h. Then oocytes with extruded first polar body (MII) were selected and enucleated for use as recipient cytoplasm. Adult and fetal ovine skin tissues were cut into small pieces (1 mm3), transferred to a 25-mL culture flask containing 2 mL DMEM-F12 medium supplemented with 10% FBS, and then incubated by using explant tissue culture in humidified atmosphere of 5% CO2 in air at 37�C for 5 to 7 days. The medium and unattached epithelial cells were discarded. The attached fibroblast cells were digested by 0.25% trypsin in D-Hanks solution at 37�C for 5 min and dispersed by pipetting. The cell suspensions were transferred to a centrifuge tube and centrifuged at 100g for 10 min. Subsequently, the recovered cells were subcultured for 4–6 passages and then frozen in DMEM-F12 medium containing 10% dimethyl sulfoxide (DMSO) and 20% FBS in liquid nitrogen. The fibroblast cells were serum-starved in DMEM-F12 supplemented with 0.5% FBS for 3 to 5 days and transferred into a micromanipulation drop consisting of H-M199 supplemented with 10% FBS and 5 �g mL-1 cytochalasin B for use. The adult and fetal skin fibroblast cells were injected into the recipient cytoplasm. The fusion of fibroblast cells into the recipient cytoplasm was induced by electrofusion (1500 V cm-1 for 40 �s two times with an interval of 0.125 s). The fused oocytes were activated by 5 mM mL-1 ionomycin with 2 mM mL-1 6-dimethylaminopurine (6-DMAP). A group of 6–10 of the activated reconstructed embryos were co-cultured with ovine cumulus cells in synthetic oviduct fluid supplemented with amino acid (SOFaa) and 10% FBS in a 20-�L microdrop for 168 h. The results indicated that 76.0% (596/784) and 75.5% (249/330) of the nuclear transfer couplets were successfully fused from adult fibroblasts and fetal fibroblasts, respectively; 76.2% (454/596) and 79.5% (198/249) of the fused oocytes cleaved within 48 h after activation for adult and fetal, respectively; 26.9% (122/454) and 28.3% (56/198) of the cleaved oocytes developed to morula or/and blastocyst embryo stages, respectively. This study demonstrated that the ovine somatic cell transferred embryos were initiated for cell cycle of mitosis and underwent subsequent development to morula/blastocyst embryo stage in vitro, and that there were no statistical differences (P &gt; 0.05) in developmental capacity between the cloned embryos from adult and fetal skin fibroblast cells.

scholarly journals Effects of heat stress during in vitro maturation on cytoplasmic versus nuclear components of mouse oocytes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 181-189 ◽  
Author(s):  
Jun-Zuo Wang ◽  
Hong-Shu Sui ◽  
De-Qiang Miao ◽  
Na Liu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Heat Stress ◽  
Similar Rate ◽  
Blastocyst Formation ◽  
Cortical Granules ◽  
Cell Stage ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Mouse Oocytes

The objectives of this study were to investigate the effect of heat stress duringin vitromaturation on the developmental potential of mouse oocytes and to determine whether the deleterious effect was on the nuclear or cytoplasmic component. While rates of oocyte nuclear maturation (development to the metaphase II stage) did not differ from 37 to 40 °C, rates for blastocyst formation decreased significantly as maturation temperature increased from 38.5 to 39 °C. Chromosome spindle exchange showed that while blastocyst formation did not differ when spindles maturedin vivoorin vitroat 37, 40 or 40.7 °C were transplanted intoin vivomatured cytoplasts, no blastocyst formation was observed whenin vivospindles were transferred into the 40 °C cytoplasts. While oocytes reconstructed between 37 °C ooplasts and 37 or 40 °C karyoplasts developed into 4-cell embryos at a similar rate, no oocytes reconstituted between 40 °C ooplasts and 37 °C spindles developed to the 4-cell stage. Immunofluorescence microscopy revealed impaired migration of cortical granules and mitochondria in oocytes matured at 40 °C compared with oocytes matured at 37 °C. A decreased glutathione/GSSG ratio was also observed in oocytes matured at 40 °C. While spindle assembling was normal and no MAD2 was activated in oocytes matured at 37 or 40 °C, spindle assembling was affected and MAD2 was activated in some of the oocytes matured at 40.7 °C. It is concluded that 1) oocyte cytoplasmic maturation is more susceptible to heat stress than nuclear maturation, and 2) cytoplasmic rather than nuclear components determine the pre-implantation developmental capacity of an oocyte.

Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

2004 ◽  
Vol 16 (6) ◽  
pp. 617 ◽  
Author(s):  
Genevieve M. Magarey ◽  
Karen E. Mate
Keyword(s):  
Glucose Metabolism ◽  
Intracytoplasmic Sperm Injection ◽  
Tammar Wallaby ◽  
Blastocyst Stage ◽  
In Vitro Fertilisation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

23 PRODUCTION OF HANDMADE CLONED GOAT BLASTOCYSTS USING FETAL FIBROBLAST CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 91 ◽  
Author(s):  
Y. S. Akshey ◽  
D. Malakar ◽  
A. K. De
Keyword(s):  
Uv Light ◽  
Light Exposure ◽  
Research Work ◽  
Fibroblast Cells ◽  
Fetal Fibroblast ◽  
Uv Illumination ◽  
Fetal Fibroblasts ◽  
Cone Formation ◽  
Handmade Cloning

Nuclear transfer is a very effective method for propagation of desired, extinct, and endangered animals as well as for the production of 100% transgenic animals. Enucleated oocytes and somatic cells are required for nuclear cloning. For enucleation, DNA-specific stains are used for visualization of the metaphase (MII) plate in matured oocytes under UV illumination in both micromanipulator-based and handmade cloning techniques. The present study was carried out to produce cloned goat embryos using the handmade cloning approach. Fetal fibroblast cells were used as nuclear donors (passages 3–4). Oocytes were collected from slaughterhouse-derived ovaries and matured in maturation medium (TCM-199 (HEPES modified), 5 µg mL–1 FSH, 10 µg mL–1 LH, 1 µg mL–1 estradiol-17β, 50 µg mL–1 gentamicin, 3 mg mL–1 BSA, and 10% inactivated estrus goat serum) at 38.5�C in 5% CO2 in air with maximum humidity for 24 h. We observed that the formation of transparent protrusion cones on the surface of the in vitro-matured goat oocytes was clearly visible under the stereomicroscope after zona digestion with 2 mg mL–1 pronase. The extent of protrusion cone formation in matured oocytes was 95–100% within 20–30 min in handling medium T 20 (TCM-199 + 20% FCS). The MII plate in the protrusion cone was confirmed (100%) after Hoechst 33342 staining and subsequent UV illumination under the inverted microscope. Zona-free oocytes were bisected on the basis of the protrusion cone by a microblade in medium (T 20 + 2.5 µg mL–1 cytochalasin B) for enucleation. Enucleated demi-oocytes were selected which had no protrusion cone and were without staining. Fetal fibroblasts from confluent monolayers were used. Two demi-oocytes were coupled with one trypsinized fetal fibroblast cell using 200 µg mL–1 phytohemagglutinin. The triplets were fused together with a combination of alternating current (7 V) and direct current (2.31 kV cm–1 for 15 µs with a double pulse) in fusion medium (0.3 m mannitol, 0.1 mM MgSO4, 0.05 mm CaCl2, and 3 mg mL–1 BSA). Four h after fusion, reconstructed oocytes were activated by using 2 µm Ca Ionophore for 5 min at room temperature and incubated with 2 mm 6-dimethylaminopurine at 38.5�C in 5% CO2 in air for 3 h. Activated reconstructed embryos were cultured in embryo development medium (TCM-199, 10% FCS, essential and nonessential amino acids, and 10 mg mL–1 BSA) in the well of the well (WOW) culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 258–264) at 38.5�C in 5% CO2 in air. In the present study, fusion, cleavage, and morula and blastocyst formation rates were 180/200 (90%), 72/180 (40%), 56/72 (77%), and 6/56 (11%), respectively. Further studies will be required to optimize blastocyst production. In conclusion, the protrusion cone formation in matured goat oocytes made it convenient for bisection and enucleation without Hoechst staining and UV light exposure, enabling the production of goats from handmade somatic cell cloning. The Council of Scientific and Industrial Research, India, has provided a fellowship to the first author to carry out this research work.

In vitro optimization of the Gallus domesticus oviduct epithelial cells culture

2012 ◽  
Vol 77 (9) ◽  
pp. 1834-1845 ◽  
Author(s):  
K. Kasperczyk ◽  
A. Bajek ◽  
R. Joachimiak ◽  
K. Walasik ◽  
A. Marszalek ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Gallus Domesticus ◽  
Oviduct Epithelial Cells

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

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

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.

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