Generation of induced pluripotent stem cells from human β-thalassemia fibroblast cells

Dear Editor, Induced pluripotent stem （iPS） cells have recently been generated by directly introducing several transcription factors into differentiated human somatic cells, and these iPS cells show great similarities to embryo-derived ES cells. Moreover, patient-specific iPS cells have recently been generated, and these studies provided hopes for patients with genetic and degenerative diseases . β-thalassemia is an inherited blood disorder that is characterized by reduced synthesis of hemoglobin subunit beta （hemoglobin β-chain）. Individuals with thalassemia major （also called Cooley＇s anemia） have severe anemia and hepatosplenomegaly; without treatment, affected children have severe failure to thrive and a shortened life expectancy. Even with transfusion and chelation therapy treatments, the life span of patients with thalassemia major can only be extended for a limited time.

Pins homolog LGN regulates meiotic spindle organization in mouse oocytes

Mouse oocytes undergo polarization during meiotic maturation, and this polarization is essential for asymmetric cell divisions that maximize retention of maternal components required for early development. Without conventional centrosomes, the meiotic spindle has less focused poles and is barrel-shaped. The migration of meiotic spindles to the cortex is accompanied by a local reorganization and polarization of the cortex. LGN is a conserved protein involved in cell polarity and regulation of spindle organization. In the present study, we characterized the localization dynamics of LGN during mouse oocyte maturation and analyzed the effects of LGN upregulation and downregulation on meiotic spindle organization. At the germinal vesicle stage, LGN is distributed both cytoplasmically and at the cortex. During maturation, LGN localizes to the meiotic spindle apparatus and cortical LGN becomes less concentrated at the actin cap region. Excessive LGN induces meiotic spindle organization defects by elongating the spindle and enhancing pole focusing, whereas depletion of LGN by RNA interference results in meiotic spindle deformation and chromosome misalignment. Furthermore, the N-terminus of LGN has the ability of full-length LGN to regulate spindle organization, whereas the C-terminus of LGN controls cortical localization and polarization. Our results reveal that LGN is cortically polarized in mouse oocytes and is critical for meiotic spindle organization.

Pluripotency of induced pluripotent stem cells

Recent studies have demonstrated that differentiated somatic cells from various mammalian species can be reprogrammed into induced pluripotent stem （iPS） cells by the ectopic expression of four transcription factors that are highly expressed in embryonic stem （ES） cells. The generation of patient-specific iPS cells directly from somatic cells without using oocytes or embryos holds great promise for curing numerous diseases that are currently unresponsive to traditional clinical approaches. However, some recent studies have argued that various iPS cell lines may still retain certain epigenetic memories that are inherited from the somatic cells. Such observations have raised concerns regarding the safety and efficacy of using iPS cell derivatives for clinical applications. Recently, our study demonstrated full pluripotency of mouse iPS cells by tetraploid complementation, indicating that it is possible to obtain fully reprogrammed iPS cells directly from differentiated somatic cells. Therefore, we propose in this review that further comprehensive studies of both mouse and human iPS cells are required so that additional information will be available for evaluating the quality of human iPS cells.

Effect of SARS-CoV-2 infection in early pregnancy on placental development

Dear Editor,Since the COVID-19 pandemic, the potential risks associated with maternal SARS-CoV-2 infection and its effect on fetal development have been a subject of considerable public concern. Previous studies have shown that SARS-CoV-2 infection during pregnancy may increase the incidence of adverse outcomes.

Pivotal role for long noncoding RNAs in zygotic genome activation in mice

Vertebrate life begins with fertilization,and then the zygote genome is activated after transient silencing,a process termed zygotic genome activation(ZGA).Despite its fundamental role in totipotency and the initiation of life,the precise mechanism underlying ZGA initiation remains unclear.The existence of minor ZGA implies the possible critical role of noncoding RNAs in the initiation of ZGA.Here,we delineate the expression profile of long noncoding RNAs(lncRNAs)in early mouse embryonic development and elucidate their critical role in minor ZGA.Compared with protein-coding genes(PCGs),lncRNAs exhibit a stronger correlation with minor ZGA.Distinct H3K9me3 profiles can be observed between lncRNA genes and PCGs,and the enrichment of H3K9me3 before ZGA might explain the suspended expression of major ZGA-related PCGs despite possessing PolII pre-configuration.Furthermore,we identified the presence of PolII-enriched MuERV-L around the transcriptional start site of minor ZGA-related lncRNAs,and these repeats are responsible for the activation of minor ZGA-related lncRNAs and subsequent embryo development.Our study suggests that MuERV-L mediates minor ZGA lncRNA activation as a critical driver between epigenetic reprogramming triggered by fertilization and the embryo developmental program,thus providing clues for understanding the regulatory mechanism of totipotency and establishing bona fide totipotent stem cells.

Loss of Tet hydroxymethylase activity causes mouse embryonic stem cell differentiation bias and developmental defects

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription,the epigenome and development.Nevertheless,previous studies using knockdown(KD)or knockout(KO)models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles,as well as compensatory effects among TET family members,which has made the understanding of the enzymatic role of TET not accurate enough.To solve this problem,we successfully generated mice catalytically inactive for specific Tet members(Tetm/m).We observed that,compared with the reported KO mice,mutant mice exhibited distinct developmental defects,including growth retardation,sex imbalance,infertility,and perinatal lethality.Notably,Tetm/mmouse embryonic stem cells(mESCs)were successfully established but entered an impaired developmental program,demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation.Intriguingly,Tet3,traditionally considered less critical for m ESCs due to its lower expression level,had a significant impact on the global hydroxymethylation,gene expression,and differentiation potential of mESCs.Notably,there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation.In summary,our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.

Comprehensive transcriptional atlas of human adenomyosis deciphered by the integration of single-cell RNA-sequencing and spatial transcriptomics

Adenomyosis is a poorly understood gynecological disorder lacking effective treatments.Controversy persists regarding“invagination”and“metaplasia”theories.The endometrial-myometrial junction(EMJ)connects the endometrium and myometrium and is important for diagnosing and classifying adenomyosis,but its in-depth study is just beginning.Using single-cell RNA sequencing and spatial profiling,we mapped transcriptional alterations across eutopic endometrium,lesions,and EMJ.Within lesions,we identified unique epithelial(LGR5+)and invasive stromal(PKIB+)subpopulations,along with WFDC1+progenitor cells,supporting a complex interplay between“invagination”and“metaplasia”theories of pathogenesis.Further,we observed endothelial cell heterogeneity and abnormal angiogenic signaling involving vascular endothelial growth factor and angiopoietin pathways.Cell-cell communication differed markedly between ectopic and eutopic endometrium,with aberrant signaling in lesions involving pleiotrophin,TWEAK,and WNT cascades.This study reveals unique stem cell-like and invasive cell subpopulations within adenomyosis lesions identified,dysfunctional signaling,and EMJ abnormalities critical to developing precise diagnostic and therapeutic strategies.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development

The development and maturation of follicles is a sophisticated and multistage process.The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process.In this study,we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells.The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells.Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells.Moreover,the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage,most of which persistently passed on to the later stages.Strikingly,cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward.Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners.TGFB2/3,TGFBR2/3,INHBA/B,and ACVR1/1B/2B of TGF-βsignaling pathway functioned in the follicle development.NOTCH signaling pathway regulated the development of granulosa cells.Additionally,many maternally DNA methylation-or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis.Collectively,Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells,and the cell dialogs become active to assure follicle development.These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

Immune rebalancing at the maternal-fetal interface of maternal SARS-CoV-2 infection during early pregnancy

The current coronavirus disease 2019(COVID-19)pandemic caused by severe acute respiratory syndrome coronavirus(SARS-CoV-2)remains a threat to pregnant women.However,the impact of early pregnancy SARS-CoV-2 infection on the maternal-fetal interface remains poorly understood.Here,we present a comprehensive analysis of single-cell transcriptomics and metabolomics in placental samples infected with SARS-CoV-2 during early pregnancy.Compared to control placentas,SARS-CoV-2 infection elicited immune responses at the maternal-fetal interface and induced metabolic alterations in amino acid and phospholipid profiles during the initial weeks post-infection.However,subsequent immune cell activation and heightened immune tolerance in trophoblast cells established a novel dynamic equilibrium that mitigated the impact on the maternal-fetal interface.Notably,the immune response and metabolic alterations at the maternal-fetal interface exhibited a gradual decline during the second trimester.Our study underscores the adaptive immune tolerance mechanisms and establishment of immunological balance during the first two trimesters following maternal SARS-CoV-2 infection.

Lipidomic remodeling during mammalian preimplantation embryonic development

The dynamic changes in lipids during early embryonic development in mammals have not yet been comprehensively investigated.In a recent paper published in Nature Cell Biology,Zhang et al.reported the dynamic lipid landscapes during preimplantation embryonic development in mice and humans.They highlight the crucial role of lipid unsaturation in regulating embryogenesis.

Insights into epigenetic patterns in mammalian early embryos

Mammalian fertilization begins with the fusion of two specialized gametes,followed by major epigenetic remodeling leading to the formation of a totipotent embryo.During the development of the pre-implantation embryo,precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality,but the underlying molecular mechanisms remain elusive.For the past few years,unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development,taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies.The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals,including DNA methylation,histone modifications,chromatin accessibility and 3D chromatin organization.

Nuclear m^(6)A reader YTHDC1 regulates the scaffold function of LINE1 RNA in mouse ESCs and early embryos

N^(6)-methyladenosine(m^(6)A)on chromosome-associated regulatory RNAs(carRNAs),including repeat RNAs,plays important roles in tuning the chromatin state and transcription,but the intrinsic mechanism remains unclear.Here,we report that YTHDC1 plays indispensable roles in the self-renewal and differentiation potency of mouse embryonic stem cells(ESCs),which highly depends on the m^(6)A-binding ability.Ythdcl is required for sufficient rRNA synthesis and repression of the 2-cell(2C)transcriptional program in ESCs,which recapitulates the transcriptome regulation by the LINE1 scaffold.Detailed analyses revealed that YTHDC1 recognizes m^(6)A on LINE1 RNAs in the nucleus and regulates the formation of the LINE1-NCL partnership and the chromatin recruitment of KAP1.Moreover,the establishment of H3K9me3 on 2C-related retrotrans-posons is interrupted in Ythdcl-depleted ESCs and inner cell mass(ICM)cells,which consequently increases the transcriptional activities.Our study reveals a role of m^(6)A in regulating the RNA scaffold,providing a new model for the RNA-chromatin cross-talk.

BMP4 preserves the developmental potential of mESCs through Ube2s-and Chmp4b-mediated chromosomal stability safeguarding

Chemically defined medium is widely used for culturing mouse embryonic stem cells(mESCs),in which N2B27 works as a substitution for serum,and GSK3βand MEK inhibitors(2i)help to promote ground-state pluripo-tency.However,recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs.Here,we demon-strated the deficient bone morphogenetic protein(BMP)signal in the chemically defined condition is one of the main causes for the impaired pluripotency.Mechanisti-cally,activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream tar-gets Ube2s and Chmp4b.More importantly,BMP4 pro-motes a distinct in vivo developmental potential and a long-term pluripotency preservation.Besides,the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression.Taken together,our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.

Esrrb plays important roles in maintaining self-renewal of trophoblast stem cells (TSCs) and reprogramming somatic cells to induced TSCs

Trophoblast stem cells (TSCs), which can be derived from the trophoectoderm of a blastocyst, have the ability to sustain self-renewal and differentiate into various placental trophoblast cell types. Meanwhile, essential insights into the molecular mechanisms controlling the placental development can be gained by using TSCs as the cell model. Esrrb is a transcription factor that has been shown to play pivotal roles in both embryonic stem cell (ESC) and TSC, but the precise mechanism whereby Esrrb regulates TSC-specific transcriptome during differentiation and reprogramming is still largely unknown. In the present study, we elucidate the function of Esrrb in self-renewal and differentiation of TSCs, as well as during the induced TSC (iTSC) reprogramming. We demonstrate that the precise level of Esrrb is critical for stem state maintenance and further trophoblast differentiation of TSCs, as ectopically expressed Esrrb can partially block the rapid differentiation of TSCs in the absence of fibroblast growth factor 4. However, Esrrb depletion results in downregulation of certain key TSC-specific transcription factors, consequently causing a rapid differentiation of TSCs and these Esrrb-deficient TSCs lose the ability of hemorrhagic lesion formation in vivo. This function of Esrrb is exerted by directly binding and activating a core set of TSC-specific target genes including Cdx2, Eomes, Sox2, Fgfr4, and Bmp4. Furthermore, we show that Esrrb overexpression can facilitate the MEF-to-iTSC conversion. Moreover, Esrrb can substitute for Eomes to generate GEsTM-iTSCs. Thus, our findings provide a better understanding of the molecular mechanism of Esrrb in maintaining TSC self-renewal and during iTSC reprogramming.

IP3R-mediated Ca2＋ signals govern hematopoietic and cardiac divergence of Flk1＋ cells via the calcineurin-N FATc3-Etv2 pathway

Ca2＋ signals participate in various cellular processes with spatial and temporal dynamics, among which, inositol 1,4,5-trisphosphate receptors （IP3Rs）-mediated Ca2＋ signals are essential for early development. However, the underlying mechanisms of IP3R- regulated cell fate decision remain largely unknown. Here we report that IP3Rs are required for the hematopoietic and cardiac fate divergence of mouse embryonic stem cells （mESCs）. Deletion of IP3Rs （IP3R-tKO） reduced FIkl＋/PDGFRα- hematopoietic mesoderm, c-Kit＋/CD41＋ hematopoietic progenitor ceil population, and the colony-forming unit activity, but increased cardiac progenitor markers as well as cardiomyocytes. Concomitantly, the expression of a key regulator of hematopoiesis, Ely2, was reduced in IP3R-tKO cells, which could be rescued by the activation of Ca2＋ signals and calcineurin or overexpression of constitutively active form of NFATc3. Furthermore, IP3R-tKO impaired specific targeting of Ely2 by NFATc3 via its evolutionarily conserved cis-element in differentiating ESCs. Importantly, the activation of Ca2＋-calcineurin-NFAT pathway reversed the phenotype of IP3R-tKO cells. These findings reveal an unrecognized governing role of IP3Rs in hematopoietic and cardiac fate commitment via IP3Rs-Ca2＋-calcineurin-NFATc3- Etv2 pathway.

Nuclear transfer using nonquiescent adult fibroblasts from a bovine ear

The natural reproduction of mammal is sexual reproduction, which needs fertilization involving sperm and oocyte. Nuclear transfer provided an asexual reproduction method for mammal. Donor cells used in previous experiments of nuclear transfer were mostly from undif-ferentiated or non-terminally differentiated cells, such as embryonic or fetal cells. However, since Wilmut et al. obtained a viable lamb by transfer of an adult sheep somatic cell into

Enhancement of Xrcc1-mediated base excision repair improves the genetic stability and pluripotency of iPSCs

诱导多能干细胞(iPSC)因其强大的自我更新和分化能力而具有广阔的应用前景.然而,多个研究表明体细胞诱导重编程过程会引入数以千计的遗传畸变,进而带来临床应用的安全性问题,已有前期研究证明单核苷酸变异(SNVs)的逐渐积累会致使iPSC丧失发育潜能,但致使SNVs产生并积累的原因还尚未探明.本研究通过对不同DNA损伤修复通路的筛选验证后,发现重编程过程早中期碱基切除修复(BER)效率的不足是导致SNVs积累的重要原因,进一步的研究发现XRCC1可以显著增强重编程前中期的BER,从而降低引入的SNVs,提高iPSC基因组的稳定性.同时,XRCC1介导的BER增强也提高了体细胞诱导重编程的效率.更为重要的是,体内嵌合体实验证实高效的BER可以显著提高iPSC的质量.综上,该研究可以有效提高iPSC基因组的稳定性和多能性,为其临床应用的安全性提供了新思路.

Lessons from expanded potential of embryonic stem cells:Moving toward totipotency

Embryonic stem cells possess fascinating capacity of self-renewal and developmental potential,leading to significant progress in understanding the molecular basis of pluripotency,disease modeling,and reprogramming technology.Recently,2-cell-like embryonic stem cells(ESCs)and expanded potential stem cells or extended pluripotent stem cells(EPSCs)generated from early-cleavage embryos display some features of totipotent embryos.These cell lines provide valuable in vitro models to study underlying principles of totipotency,cell plasticity,and lineage segregation.In this review,we summarize the current progress in this filed and highlight the application potentials of these cells in the future.

ELL3通过LINE-1调节小鼠细胞原始多能性

人类基因组中有超过50%的序列是转座子序列(transposon elements,TE).转座子又称跳跃基因,是一类可以在基因组中进行自我复制和粘贴的DNA元件[1].转座子主要分为DNA转座子和反转录转座子,其中反转录转座子会先转录产生RNA,再反转录成DNA来完成转座.长散在重复序列(long interspersed nuclear elements-1,LINE-1)是哺乳动物基因组中最丰富的转座元件家族之一,在人类基因组中占比大约为17%[2].

Cloning non-HUMAN primates by somatic cell nuclear transfer

The recent breakthrough in successful producing cloned non- human primates by somatic cell nuclear transfer （SCNT） has attracted great attention both scientifically and publically （Liu et al., 2018）. Two macaque monkeys, named ＂Zhongzhong＂ and ＂Huahua＂, were cloned from fetal fi- broblast cells by Chinese scientists using the technique that produced the first cloned mammal ＂Dolly＂ 21 years ago （Wilmut et al., 1997）. The first cloned sheep has re- volutionized our thinking that the mammalian somatic cells can be reprogrammed into totipotent embryos by SCNT, although the efficiency is extremely low. Actually, in the same year when ＂Dolly＂ was reported, a paper published in Biology of Reproduction also attracted broad attention in which two rhesus monkeys were produced from blastomeres isolated from 4-cell stage embryos using the same technique （Meng et al., 1997）. However, it remains very challenging if the somatic cells from monkeys can be reprogrammed into totipotent embryos, although many laboratories all over the world have tried very hard in the past 20 years. Indeed, the successful cloning of monkeys today is attributed to both the technical improvement and the progress obtained in me- chanistic studies in reprogramming. Two major techniques are involved in SCNT. The first is called enucleation through which the spindle-chromosome complex （SCC） of an oocyte at metaphase II （MII） stage is removed by an enucleation pipette. The enucleation process is relatively easy for mouse oocytes since the SCC can be visualized under light micro- scope, but the SCC of oocytes from large animals including