| Placenta is the first organ formed in the embryogenesis of mammals,which plays an important role in supporting the normal development of the fetus in the womb environment.Placenta regulates the transport of nutrients between mother and fetus,the exchange of metabolic waste and other basic functions.At the same time,it can synthesize and secrete a variety of hormones,enzymes,cytokines to maintain normal pregnancy.At present,many maternal pregnancy-related diseases and fetal developmental abnormalities are related to developmental disorders of the placenta,such as placenta previa,pregnancyinduced hypertension syndrome,fetal growth restriction and so on,which directly threaten the health and even life of the mother and fetus.In the process of placental development,a variety of signaling pathways and imprinted genes are involved in regulating the differentiation of placental cells,the formation of placental structure and placental functions.However,the current understanding of the overall pattern of placental development and the causes of abnormal placental development are still limited.Somatic cell nuclear transfer(SCNT)technology has wide application and development potential in life science research,agriculture and animal breeding,endangered species resource protection and so on.However,the very low efficiency makes this technique difficult to use in practice.Existing studies have proved that somatic cell reprogramming exists a variety of epigenetic disorders,such as abnormal DNA methylation level,abnormal histone modification(histone acetylation,H3K4me3,H3K9me3,H3K27me3,etc.),abnormal imprinted genes and abnormal X chromosome activity.These epigenetic disorders can lead to defects in the development of the reprogrammed embryo(e.g.,placenta,umbilical cord,etc.),or defects accompanying the growth and development of fetal tissue,resulting in low efficiency.At the same time,using small molecule compounds,gene editing and other techniques to modify the above epigenetic disorders can improve the efficiency of nuclear transfer to a certain extent.For example,studies have shown that placental imprinting gene disorder caused by somatic cell nuclear transfer is a major cause of placenta expansion in mice at middle and late gestation,and this defect can be ameliorated by modifying the abnormal gene to a certain extent.However,the efficiency of nuclear transfer is still low and placental defects have not been completely improved,suggesting that there are still many unknown factors involved.In conclusion,in-depth and comprehensive analysis of the mechanism of placenta abnormalities in nuclear transfer is very necessary to improve the cloning efficiency and ensure the normal development of the placenta.With the development of single cell sequencing technology,people can study the gene expression pattern at the level of individual cells,so that the heterogeneity between cells can be studied more accurately.Single cell sequencing technology has been widely used in a variety of studies,such as cell differentiation,tissue and embryonic development,cancer development and so on.In terms of placenta development,it has been reported that single cell RNA sequencing technology has been used to perform high-throughput sequencing on the placenta of multiple species,so as to fill the gaps in the placenta development process and placental constituent cells of corresponding species.However,due to the existence of polyploid cells and multinucleated cells in placenta,the current single cell RNA sequencing technology based on microfluidic technology cannot isolate all types of placental cells.At the same time,single cell RNA sequencing technology has certain limitations due to the easy degradation of m RNA in cells and the influence of gene expression by dissociation.Compared with single cell RNA sequencing technology,single nuclear RNA sequencing technology is not restricted by dissociation conditions,can better reflect the cell composition and gene expression in the original tissue,and can be used for frozen samples,so it is more suitable for sequencing analysis of placental tissue.In this study,in order to investigate the abnormality of placenta derived from mouse somatic cell nuclear transfer and explore potential strategies to improve the efficiency of nuclear transfer,we conducted high-throughput sequencing analysis of placentas derived from in vitro fertilization(IVF)and placentas derived from somatic cell nuclear transfer using single nuclei RNA sequencing technology.Firstly,we compared the survival rate of transplanted embryos after somatic cell nuclear transfer with that after in vitro fertilization.The results showed that SCNT embryo had significant developmental arrest at E9.5(Embryonic day).We extracted nuclei from the placental tissues,and then conducted quality control and downstream analysis of the sequencing data.Based on the reported marker genes for cell types,we have identified 7 categories of placental cell types in these nuclei and 12 subtypes in trophoblast cells.Then,we enriched the nuclei of maternal origin by using the strain-specific SNP site information,and then calculated the cell proportion of fetal origin.The results showed that the proportion of immune cells and erythrocyte of E9.5 SCNT placenta was slightly different from IVF placenta.There was no significant difference in the total amount of trophoblast cells,but there was a difference in the composition of trophoblast cell subtypes.As an important mother-to-fetus interface,the placenta plays an important role in cellular interactions.Therefore,we performed cell-to-cell communication analysis and found that decidual stromal cells in SCNT placenta enhanced their interaction with pericytes and endothelial cells by regulating cell number and interaction intensity.At the same time,the up-regulation of VISFATIN pathway and NRG pathway related genes in SCNT group also indicated their influence on interaction differences.Then we analyzed the expression levels of non-canonical imprinted genes and canonical imprinted genes,and the results showed that non-canonical imprinted genes were generally up-regulated in SCNT placenta,while the expression patterns of various canonical imprinted genes were inconsistent between different cell types and cell subtypes.At the same time,we found that in the components derived from fetal cells,the X chromosome of SCNT group showed decreased activity.In order to explore the reasons for the lower activity,we also analyzed the expression levels of X chromosome associated genes,and the results showed that in SCNT placental cells and trophoblast cell subtypes,there were a variety of abnormal expression patterns of genes related to placental development.Analysis of the locations of these differentially expressed genes on the X chromosome showed that there was uniform inactivation of X chromosome in the placenta from SCNT.In conclusion,this study for the first time conducted high-throughput sequencing analysis on placenta from somatic cell nuclear transfer at the single nuclei level,and deciphered many abnormalities of placenta from nuclear transfer by comparing it with placenta from in vitro fertilization,including abnormal placental cell composition ratio,abnormal expression level of histone modification related genes,abnormal intercellular interaction and cell communication level,abnormal proportion of trophoblast cell subtypes,abnormal expression level of non-canonical imprinted genes and canonical imprinted genes,abnormal expression level of X chromosome associated genes and abnormal X chromosome activity and so on,which provides a potential strategy for improving the efficiency of nuclear transfer. |
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