Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stern cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, ~lll-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neuregenesis related genes （Sox-1, Nestin, GABA, Pax6, Zic5 and Pitxl） in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.

Intravenous transplantation of mouse embryonic stem cells attenuates demyelination in an ICR outbred mouse model of demyelinating diseases

Induction of demyelination in the central nervous system （CNS） of experimental mice using cuprizone is widely used as an animal model for studying the pathogenesis and treatment of demyelination. How- ever, different mouse strains used result in different pathological outcomes. Moreover, because current medicinal treatments are not always effective in multiple sclerosis patients, so the study of exogenous cell transplantation in an animal model is of great importance. The aims of the present study were to establish an alternative ICR outbred mouse model for studying demyelination and to evaluate the effects of intrave- nous cell transplantation in the present developed mouse model. Two sets of experiments were conducted. Firstly, ICR outbred and BALB/c inbred mice were fed with 0.2% cuprizone for 6 consecutive weeks; then demyelinating scores determined by luxol fast blue stain or immunolabeling with CNPase were evaluated. Secondly, attenuation of demyelination in ICR mice by intravenous injection of mES cells was studied. Scores for demyelination in the brains of ICR mice receiving cell injection （mES cells-injected group） and vehicle （sham-inoculated group） were assessed and compared. The results showed that cuprizone signifi- cantly induced demyelination in the cerebral cortex and corpus callosum of both ICR and BALB/c mice. Additionally, intravenous transplantation of mES cells potentially attenuated demyelination in ICR mice compared with sham-inoculated groups. The present study is among the earliest reports to describe the cuprizone-induced demyelination in ICR outbred mice. Although it remains unclear whether mES cells or trophic effects from mES cells are the cause of enhanced remyelination, the results of the present study may shed some light on exogenous cell therapy in central nervous system demyelinating diseases.

Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

MM： To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes, METHODS： Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem （ES） cells were obtained in a sequential manner, induced by valproic acid （VPA） and cytokines （hepatocyte growth factor, epidermal growth factor and insulin）, Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy, Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepa- tocytes, Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells, Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells.RESULTS： Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The dif- ferentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers,in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells to-wards hepatic lineages. CONCLUSION： Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.

Differentiation of mouse embryonic stem cells into insulin-secreting cells in vitro

Regenerative medicine, including cell-replacement strategies, may have an important role in the treatment of type 1 diabetes which is associated with decreased islet cell mass. To date, significant progress has been made in generating insulin-secreting 13 cells from pluripotent mouse embryonic stem cells （ESCs）.The aim of this study is to explore the potential of regulating the differentiation of ESCs into pancreatic endocrine cells capable of synthesizing the pancreatic hormones including insulin, glucagon, somatostatin and pancreatic polypeptide under proper conditions. Undifferentiated ES cell line was stably transfected with mouse RIP-YFP plasmid construction in serum-free medium using LipofectamineTM 2000 Reagents. We tested pancreatic specific gene expression and characterized these ESC-derived pancreatic endocrine cells. Most of these insulin-secreting cells co-expressed many of the phenotypic markers characteristic of 13 cells such as insulinl, insulin2, Isletl, MafA, insulinoma-associated antigen 1 （IA1） and so on, indicating a similar gene expression pattern to adult islet 13 cells in vivo. Characterization of this population revealed that it consisted predominantly of pancreatic endocrine cells that were able to undergo pancreatic specification under the appropriate conditions. We also demonstrated that zinc supplementation mediated up-regulation of insulin-secreting cells as an effective inducer promoted the development of ESC-derived diabetes therapy. In conclusion, this work not only established an efficient pancreatic differentiation strategy from ESCs to pancreatic endocrine lineage in vitro, but also leaded to the development of new strategies to derive transplantable islet-replacement 13 cells from embryonic stem cells for the future applications of a stem cell based therapy of diabetes.

Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells

BACKGROUND Embryonic stem cells(ESCs)serve as a crucial ex vivo model,representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos.ESCs exhibit a unique combination of self-renewal potency,unlimited proliferation,and pluripotency.The latter is evident by the ability of the isolated cells to differ-entiate spontaneously into multiple cell lineages,representing the three primary embryonic germ layers.Multiple regulatory networks guide ESCs,directing their self-renewal and lineage-specific differentiation.Apoptosis,or programmed cell death,emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development.How-ever,the molecular mechanisms underlying the dynamic interplay between diffe-rentiation and apoptosis remain poorly understood.AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells,using mouse ESC(mESC)models-mESC-B-cell lym-phoma 2(BCL-2),mESC-PIM-2,and mESC-metallothionein-1(MET-1)-which overexpress the anti-apoptotic genes Bcl-2,Pim-2,and Met-1,respectively.METHODS mESC-T2(wild-type),mESC-BCL-2,mESC-PIM-2,and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation.The hanging drop method was adopted to generate embryoid bodies(EBs)and induce terminal differentiation of mESCs.The size of the generated EBs was measured in each condition compared to the wild type.At the functional level,the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control.At the molecular level,quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers:Troponin T,GATA4,and NKX2.5.Additionally,troponin T protein expression was evaluated through immunofluorescence and western blot assays.RESULTS Our findings showed that the upregulation of Bcl-2,Pim-2,and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs,in comparison with their wild-type counterpart.Additionally,a decrease in the count of beating cardiomyocytes among differentiated cells was observed.Furthermore,the mRNA expression of three cardiac markers-troponin T,GATA4,and NKX2.5-was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line.Moreover,the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression.CONCLUSION Our findings revealed that the upregulation of Bcl-2,Pim-2,and Met-1 genes altered cardiac differentiation,providing insight into the intricate interplay between apoptosis and ESC fate determination.

Mouse A6-positive Hepatic Oval Cells Derived from Embryonic Stem Cells

Summary： Oval cells have a potential to differentiate into a variety of cell lineages including hepatocytes and biliary epithelia. Several models have been established to activate the oval cells by incorporating a variety of toxins and carcinogens, alone or combined with surgical treatment. Those models are obviously not suitable for the study on human hepatic oval cells. It is necessary to establish a new and efficient model to study the human hepatic oval cells. In this study, the hepatocyte growth factor（HGF） and epidermal growth factor（EGF） were used to induce differentiation of mouse embryonic stem（ES） cells into hepatic oval cells. We first confirmed that hepatic oval cells derived from ES cells, which are bipotential, do exist during the course of mouse ES cells＇ differentiation into hepatic parenchymal cells. RT-PCR and transmission electron microscopy were applied in this study. The ratio of Sca-1＋/CD34＋ cells sorted by FACS in the induction group was increased from day 4 and reached the maximum on the day 8, whereas that in the control group remained at a low level. The differentiation ratio of Sca-1＋/CD34＋ cells in the induction group was significantly higher than that in the control group. About 92.48% of the sorted Sca-1＋/CD34＋ cells on the day 8 were A6 positive. Highly purified A6＋/Sca-1＋/CD34＋ hepatic oval cells derived from ES cells could be obtained by FACS. The differentiation ratio of hepatic oval cells in the induction group（up to 4.46%） was significantly higher than that in the control group. The number of hepatic oval cells could be increased significantly by HGF and EGF. The study also examined the ultrastructures of ES-derived hepatic oval cells＇ membrane surface by atomic force microscopy. The ES-derived hepatic oval cells cultured and sorted by our protocols may be available for the future clinical application.

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC_3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 × 10~6 smooth muscle cells (SMCs) obtained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biodegradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC_3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6～8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells

Yamanaka factors （Oct3/4, Sox2, Klf4, c-Myc） are highly expressed in embryonic stem （ES） cells, and their overexpression can induce pluripotency in both mouse and human somatic cells, indicating that these factors regulate the developmental signaling network necessary for ES cell pluripotency. However, systemic analysis of the signaling pathways regulated by Yamanaka factors has not yet been fully described. In this study, we identified the target promoters of endogenous Yamanaka factors on a whole genome scale using ChIP （chromatin immunoprecipitation）- on-chip in El4.1 mouse ES cells, and we found that these four factors co-occupied 58 promoters. Interestingly, when Oct4 and Sox2 were analyzed as core factors, Klf4 functioned to enhance the core factors for development regulation, whereas c-Myc seemed to play a distinct role in regulating metabolism. The pathway analysis revealed that Yamanaka factors collectively regulate a developmental signaling network composed of 16 developmental signaling pathways, nine of which represent earlier unknown pathways in ES cells, including apoptosis and cellcycle pathways. We further analyzed data from a recent study examining Yamanaka factors in mouse ES ceils. Interestingly, this analysis also revealed 16 developmental signaling pathways, of which 14 pathways overlap with the ones revealed by this study, despite that the target genes and the signaling pathways regulated by each individual Yamanaka factor differ significantly between these two datasets. We suggest that Yamanaka factors critically regulate a developmental signaling network composed of approximately a dozen crucial developmental signaling pathways to maintain the pluripotency of ES cells and probably also to induce pluripotent stem cells.

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice, the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF). Compared with the feeder layer of MEF cells, medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyo-typic normality of ES cells only in short term cell propagation. Besides, ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras. Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more convenient and efficient than the conventional microdrop method.

Smad2 mediates Activin/Nodal signaling in mesendoderm differentiation of mouse embryonic stem cells

Although Activin/Nodal signaling regulates pluripotency of human embryonic stem （ES） cells, how this signaling acts in mouse ES cells remains largely unclear. To investigate this, we confirmed that mouse ES cells possess active Smad2-mediated Activin/Nodal signaling and found that Smad2-mediated Activin/Nodal signaling is dispensable for self-renewal maintenance but is required for proper differentiation toward the mesendoderm lineage. To gain insights into the underlying mechanisms, Smad2-associated genes were identified by genome-wide chromatin immu- noprecipitation-chip analysis. The results showed that there is a transcriptional correlation between Smad2 binding and Activin/Nodal signaling modulation, and that the development-related genes were enriched among the Smad2- bound targets. We further identified Tapbp as a key player in mesendoderm differentiation of mouse ES cells acting downstream of the Activin/Nodal-Smad2 pathway. Taken together, our findings suggest that Smad2-mediated Activin/Nodal signaling orchestrates mesendoderm lineage commitment of mouse ES cells through direct modulation of corresponding developmental regulator expression.

Development of neural precursor cells from mouse embryonic stem cells

Objective:To exploretheserum-freecultureconditionsfordifferentiatingmouseembryonicstemcells(ES cells)intoneuralprecursorcells(NPC)andcomparetheeffectsof humanembryonicfibroblasts(HEF)as thefeederlayer of ES withthatof mouseembryonicfibroblasts(MEF)in vitro.Methods:MouseES cellswereculturedin or notin feederlayer cellsmediumcontainingor notleukemiainhibitoryfactorto suppresstheirdifferentiation.Immunocytochemicalmethod was usedto identifyNPCby detectingnestinantigenandalkalinephosphatase.Results: TheES cellsculturedin HEF werepositiveto alkalinephosphatase.Serum-freemediumallowedthedifferentiationof ES cellsintoNPC.Conclusion:HEFcouldreplaceMEFandkeeptheundifferentiatedconditionof ES cellswithmorebenefits.NPCof highpuritycould be culturedfromEScellsby serum-freeculturemethod.

Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from ferti-lized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver- stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.

Preparation of Mouse Embryonic Stem Cells and Cardiomyocyte Differentiation Induced with Retinoic Acid and Ascorbic Acid

The experiment was designed to study effects of retinoic acid and ascorbic acid on differentiation of mouse embryonic stem cells to cardiomyocytes. Embryonic bodies （EB） were developed from mESC in suspension culture, different levels of concentration of retinoic acid and ascorbic acid were used to determine the optimal conditions for EB formation. The results showed that the optimal concentrations were 10.9 mol. L-1 and 0.1 mg. mL-1 for retinoic acid and ascorbic acids, respectively. 50% of EB which was significantly （p〈0.05） different from the control group developed to cardiomyocytes. In conclusion, rctinoic acid and ascorbic acid had strong ability to promote cardiomyocyte differentiation of mouse embryonic stem cells. 10-9 mol. L-1 retinoic acid and 0.10 mg. mL-1 ascorbic acids were recommended to induce differentiation of mouse ES ceUs toward cardiomyocytes.

Similar effects of mouse and human <i>pub</i>gene on proliferation and embryoid bodies formation in mouse embryonic stem cells <i>in vitro</i>

Previously we have demonstrated that pub gene modulates expression of PU.1 transcription factor and plays an important role in differentiation of embryonic stem cells not only at early stages (during formation of embryoid bodies), but also during differentiation into ectodermal, mesodermal and endodermal derivatives in vitro. We have compared the influence of elevated expression of two homological genes mpub and hpub of mouse and human respectively on early stages of differentiation of murine embryonic stem cells. Overexpression of both genes caused an increase in the number of formed embryoid bodies but did not alter the proliferative activity of transfected embryonic stem cells. It was also observed that expression of mpub and hpub led to decreased expression of pu.1 mRNA.

Perfluorinated iodine alkanes promote the differentiation of mouse embryonic stem cells by regulating estrogen receptor signaling

Investigating the development toxicity of perfuorinated iodine alkanes(PFIs)is critical,given their estrogenic effects through binding with estrogen receptors(ERs).In the present study,two PFIs,including dodecafuoro-1,6-diiodohexane(PFHx DI)and tridecafuorohexyl iodide(PFHx I),with binding preference to ERαand ERβ,respectively,were selected to evaluate their effects on proliferation and differentiation of the mouse embryonic stem cells(m ESCs).The results revealed that,similar to E_(2),50μmol/L PFHx DI accelerated the cell proliferation of the m ESCs.The PFI stimulation at the exposure concentrations of 2–50μmol/L promoted the differentiation of the m ESCs as characterized by the upregulation of differentiation-related biomarkers(i.e.,Otx2 and Dnmt3β)and downregulation of pluripotency genes(i.e.,Oct4,Nanog,Sox2,Prdm14 and Rex1).Comparatively,PFHx DI exhibited higher induction effect on the differentiation of the m ESCs than did PFHx I.The tests on ER signaling indicated that both PFI compounds induced exposure concentration-dependent expressions of ER signaling-related biomarkers(i.e.,ERα,ERβand Caveolin-1)in the m ESCs,and the downstream ER responsive genes(i.e.,c-fos,c-myc and c-jun)well responded to PFHx I stimulation.The role of ER in PFI-induced effects on the m ESCs was further validated by the antagonistic experiments using an ER inhibitor(ICI).The findings demonstrated that PFIs triggered ER signaling,and perturbed the differentiation program of the m ESCs,causing the potential health risk during early stage of development.

GPR160 regulates the self-renewal and pluripotency of mouse embryonic stem cells via JAK1/STAT3 signaling pathway

G-protein-coupled receptors(GPCRs)are the largest family of transmembrane receptors and regulate various physiological and pathological processes.Despite extensive studies,the roles of GPCRs in mouse embryonic stem cells(mESCs)remain poorly understood.Here,we show that GPR160,a class A member of GPCRs,is dramatically downregulated concurrent with mESC differentiation into embryoid bodies in vitro.Knockdown of Gpr160 leads to downregulation of the expression of pluripotency-associated transcription factors and upregulation of the expression of lineage markers,accompanying with the ar-rest of the mESC cell-cycle in the GO/G1 phase.RNA-seq analysis shows that GPR160 participates in the JAK/STAT signaling pathway crucial formaintaining ESC stemness,and the knockdown of Gpr160 results in the downregulation of STAT3 phosphorylation level,which in turn is partially rescued by colivelin,a STAT3 activator.Consistent with these observations,GPR160 physically interacts with JAK1,and co-operates with leukemia inhibitory factor receptor(LIFR)and gp130 to activate the STAT3 pathway.In summary,our results suggest that GPR160 regulates mESC self-renewal and pluripotency by interacting with the JAK1-LIFR-gp130 complex to mediate the JAK1/STAT3 signaling pathway.

Role of cancer stem cell ecosystem on breast cancer metastasis and related mouse models

Breast cancer metastasis is responsible for most breast cancer-related deaths and is influenced by many factors within the tumor ecosystem,including tumor cells and microenvironment.Breast cancer stem cells(BCSCs)constitute a small population of cancer cells with unique characteristics,including their capacity for self-renewal and differentiation.Studies have shown that BCSCs not only drive tumorigenesis but also play a crucial role in promoting metastasis in breast cancer.The tumor microenvironment(TME),composed of stromal cells,immune cells,blood vessel cells,fibroblasts,and microbes in proximity to cancer cells,is increasingly recognized for its crosstalk with BCSCs and role in BCSC survival,growth,and dissemination,thereby influencing metastatic ability.Hence,a thorough understanding of BCSCs and the TME is critical for unraveling the mechanisms underlying breast cancer metastasis.In this review,we summarize current knowledge on the roles of BCSCs and the TME in breast cancer metastasis,as well as the underlying regulatory mechanisms.Furthermore,we provide an overview of relevant mouse models used to study breast cancer metastasis,as well as treatment strategies and clinical trials addressing BCSC-TME interactions during metastasis.Overall,this study provides valuable insights for the development of effective therapeutic strategies to reduce breast cancer metastasis.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid(LPA)is a small molecule glycerophospholipid,which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development.In this study,sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment.At first,the maturation medium containing estrus female sheep serum and synthetic oviduct fluid(SOF)were optimized for sheep IVF,and then the effects of LPA were investigated.From 0.1 to 10μmol L^(–1),LPA had no significant effect on the cleavage rate(P>0.05),but the maturation rate and blastocyst rate increased dependently with LPA concentration(P<0.05),and the blastocyst morphology was normal.When the LPA concentration was 15μmol L^(–1),the maturation rate,cleavage rate and blastocyst rate decreased significantly(P<0.05),and the blastocyst exhibited abnormal morphology and could not develop into highquality blastocyst.Besides,the exogenous LPA increases the expression of LPAR2,LPAR4,TE-related gene CDX-2and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10μmol L^(–1).The expression of LPAR2,LPAR4,CDX-2 and OCT-4 from the LPA-0.1μmol L^(–1)to LPA-10μmol L^(–1)groups in early embryos were extremely significant(P<0.05),while the expression of these genes significantly decreased in 15μmol L^(–1)LPA-treated embryos compared with LPA-10μmol L^(–1)group(P<0.05).The inner cell mass in 15μmol L^(–1)LPA-treated embryos was also disturbed,and the blastocysts formation was abnormal.Secondly,the sheep IVF blastocysts were applied to establish embryonic stem cells.The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells.They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10μmol L^(–1),while 15μmol L^(–1)LPA decreased OCT-4 and CDX-2 expression in the derived cells.The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1μmol L^(–1)group and LPA-10μmol L^(–1)group extremely significantly increased(P<0.05),but there was significant decrease in LPA-15μmol L^(–1)group compared with LPA-10μmol L^(–1)group(P<0.05).Meanwhile,the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10μmol L^(–1)concentration.This study references the IVF embryo production and embryonic stem cell research of domestic animals.