Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells

人的导致的 pluripotent 茎(iPS ) 房间类似于胚胎的茎(ES ) 房间，和罐头强烈地增殖并且区分进许多房间类型。然而，人的 iPS 房间的肝的区别还没被报导了。在这份报告，人的 iPS 房间被导致由一个逐步的协议区分进肝的房间。肝房间标记的表达式和人的 iPS 的肝相关的函数导出房间的房间被监视并且与区分的人的 ES 房间和主要人的 hepatocytes 的相比。在白天 7 点的约 60% 区分的人的 iPS 房间表示了肝的标记 alpha fetoprotein 和白长袍的。在白天 21 点的区分的房间包括白朊 Asecretion，肝糖合成，脲生产和可诱导的细胞色素 P450 活动展出了肝房间功能。肝的标记的表示和 iPS 的肝相关的功能导出房间的肝的房间比得上人的 ES 导出房间的肝的房间的。这些结果证明人的 iPS 房间，类似于人的 ES 房间，能高效地被导致区分房间进象 hepatocyte 一样。

Pericentrin contains five NESs and an NLS essential for its nucleocytoplasmic trafficking during the cell cycle

Pericentrin，一个保存 centrosomal 部件，提供结构的支架抛锚众多的 centrosomal 蛋白质，并且因此在中心体和有丝分裂的锭子的组织和功能起一个必要作用。尽管 pericentrin 被显示在细胞质本地化并且报导对 leptomycin B (LMB ) 敏感， Crm1 的一个特定的禁止者，在 pericentrin 以内的区域当为搬运进出原子核的信号还没被识别了，服务。在这研究，我们与多样的出口活动在 pericentrin 识别了五个新奇原子出口信号(海角) 。当调停时，所有能以一个 LMB 敏感的方法五只海角绑在 Crm1 pericentrin 的原子出口。我们也证明在 pericentrin 的氨基酸 8-42 的区域包含由基本氨基酸的三簇组成的一个分成三部分的原子本地化信号(NLS ) 。pericentrin 的 NLS 直接或经由适配器 importin 伪绑在 importin 尾形成进口建筑群，它能被 RanQ69L 破坏，一主导否定运用了为 importin 尾拥有高亲密关系的 GTPase。而且，我们发现在全身的 pericentrin 的海角的那个变化导致原子、细胞质的本地化，并且 NLS 的变化废除 pericentrin 的原子进口。根据这些结果，我们建议 pericentrin 的海角和 NLS 在房间周期期间为它的 subcellular 本地化和 nucleocytoplasmic trafficking 是必要的。

Cyclin B1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis

Cyclin B1 是在脊椎动物控制房间周期前进的关键规章的蛋白质。Cyclin B1 绑 CDK1， cyclin 依赖的 kinase 催化子单元，形成通过关键蛋白质的磷酸化安排有丝分裂的建筑群。Cyclin B1 两个都调整 CDK1 和它的潜水艇的激活细胞的本地化，它可能为底层选择批评。这里，我们证明 cyclin B1 被专注于 kinetochore 的外部板在专业版期间中期。这本地化要求蛋白质的 cyclin 盒子区域。Cyclin B1 被微导管附件从单个 kinetochores 代替到锭子杆到 kinetochores，并且这排水量依赖于 dynein/dynactin 建筑群。由基于向量的 siRNA 的 B1 引起在 kinetochores 和微导管之间的低效的附件的 cyclin 的弄空，和染色体排列背叛，并且推迟后期的发作。在它贡献微导管的正确附件到 kinetochores 和染色体的有效排列的地方，我们断定 cyclin B1 在职业人员中期期间在 kinetochores 积累，通过由 cyclin B1-CDK1 的特定的底层的局部性的磷酸化最可能。当微导管附件被完成， Cyclin B1 然后从每 kinetochore 被搬运，并且这本地化可以重定向 cyclin B1-CDK1 的活动并且在锭子集会检查点的激活贡献。

Corrigendum to“Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration”[Journal of Genetics and Genomics(2022)49,1114-1126]

The authors regret that the grant number“21CJ1402200”in the Acknowledgments session should be replaced as“21JC1402200”.The corrected contents areprovided as follows.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and noncarriers.Susceptibility to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies.Here,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander effects.In particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying embryos.Stringent control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells.Furthermore,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage.Finally,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia.Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.

An effective pharmacological hydrogel induces optic nerve repair and improves visual function

Irreversible eye lesions, such as glaucoma and traumatic optic neuropathy, can cause blindness;however, no effective treatments exist. The optic nerve, in particular, lacks the capacity to spontaneously regenerate, requiring the development of an effective approach for optic nerve repair, which has proven challenging. Here, we demonstrate that a combination of the small molecules 3BDO and trichostatin A(TSA)—which regulate mTOR and HDAC, respectively—packaged in thermosensitive hydrogel for 4-week-sustained release after intravitreal injection, effectively induced optic nerve regeneration in a mouse model of optic nerve crush injury. Moreover, this combination of 3BDO and TSA also protected axon projections and improved visual responses in an old mouse model(11 months old) of glaucoma. Taken together, our data provide a new, local small molecule-based treatment for the effective induction of optic nerve repair, which may represent a foundation for the development of pharmacological methods to treat irreversible eye diseases.

Unveiling E2F4,TEAD1 and AP-1 as regulatory transcription factors of the replicative senescence program by multi-omics analysis

Senescence,a stable state of growth arrest,affects many physiological and pathophysiological processes,especially aging.Previous work has indicated that transcription factors(TFs)play a role in regulating senescence.However,a systematic study of regulatory TFs during replicative senescence(RS)using multiomics analysis is still lacking.Here,we generated timeresolved RNA-seq,reduced representation bisulfite sequencing(RRBS)and ATAC-seq datasets during RS of mouse skin fibroblasts,which demonstrated that an enhanced inflammatory response and reduced proliferative capacity were the main characteristics of RS in both the transcriptome and epigenome.Through integrative analysis and genetic manipulations,we found that transcription factors E2F4,TEAD1 and AP-1 are key regulators of RS.Overexpression of E2f4 improved cellular proliferative capacity,attenuated SA-β-Gal activity and changed RS-associated differentially methylated sites(DMSs).Moreover,knockdown of Tead1 attenuated SA-β-Gal activity and partially altered the RS-associated transcriptome.In addition,knockdown of Atf3,one member of AP-1 superfamily TFs,reduced Cdkn2a(p16)expression in pre-senescent fibroblasts.Taken together,the results of this study identified transcription factors regulating the senescence program through multi-omics analysis,providing potential therapeutic targets for anti-aging.

Correction to:Efficient derivation of extended pluripotent stem cells from NOD-scid II2rg^-/-mice

Figure 1.Generation of NOD-scid II2rg^-1-extended pluripotent stem cells.(A)Schematic of two approaches used for generating NOD-scid II2rg^-1-extended pluripotent stem cells:de novo derivation from blastocysts(upper panels)and chemical reprogramming from embryonic fibroblasts(lower panels).(B)Phase-contrast images of de novo derived outgrowth and EPS colonies for 17 passages in LCDM medium.Scale bars,100 pm.(C)qRT-PCR analysis of XEN marker genes expression during the chemical induction process(day 16).Error bars indicate SEM(n=2).(D)Co-immunostaining of XEN marker genes during the chemical induction process(day 16).Upper panels:GATA6 and SALL4;lower panels:SOX17 and SALL4.Scale bars,100 pm.

Efficient derivation of extended pluripotent stem cells from NOD-scid II2rg-/-mice

Recently we have established a new culture condition enabling the derivation of extended pluripotent stem(EPS)cells,which,compared to conventional pluripotent stem cells,possess superior developmental potential and germline competence.However,it remains unclear whether this condition permits derivation of EPS cells from mouse strains that are refractory or non-permissive to pluripotent cell establishment.Here,we show that EPS cells can be robustly generated from non-permissive NOD-sc/d Il2rg 1 mice through de novo derivation from blastocysts.Furthermore,these cells can also be efficiently generated by chemical reprogramming from embryonic NOD-sc/d II2rg-/-fibroblasts.NOD-sc/d II2rg-/-EPS cells can be expanded for more than 20 passages with genomic stability and can be genetically modified through gene targeting.Notably,these cells contribute to both embryonic and extraembryonic lineages in vivo.More importantly,they can produce chimeras and integrate into the E13.5 genital ridge.Our study demonstrates the feasibility of generating EPS cells from refractory mouse strains,which could potentially be a general strategy for deriving mouse pluripotent cells.The generation of NOD-sc/d II2rg-/-Yaqin Du and Ting Wang contributed equally to this work.Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13238-018-0558-z)contains supplementary material,which is available to authorized users.EPS cell lines permits sophisticated genetic modification in NOD-scid II2rg-/-mice,which may greatly advance the optimization of humanized mouse models for biomedical applications.

Metastasis-related methyltransferase 1(Merml)represses the methyltransferase activity of Dnmt3a and facilitates RNA polymerase I transcriptional elongation

Stimulatory regulators for DNA methyltransferase activity,such as Dnmt3L and some Dnmt3b isoforms,affect DNA methylation patterns,thereby maintaining gene body methylation and maternal methylation imprinting,as well as the methylation landscape of pluripotent cells.Here we show that metastasis-related methyltransferase 1(Merm1),a protein deleted in individuals with Williams-Beuren syndrome,acts as a repressive regulator of Dnmt3a.Merm1 interacts with Dnmt3a and represses its methyltransferase activity with the requirement of the binding motif for S-adenosyl-L-methionine.Functional analysis of gene regulation revealed that Merm1 is capable of maintaining hypomethylated rRNA gene bodies and co-localizes with RNA polymerase I in the nucleolus.Dnmt3a recruits Merml,and in return,Merml ensures the binding of Dnmt3a to hypomethylated gene bodies.Such interplay between Dnmt3a and Merml facilitates transcriptional elongation by RNA polymerase I.Our findings reveal a repressive factor for Dnmt3a and uncover a molecular mechanism underlying transcriptional elongation of rRNA genes.

Single-cell Sequencing Reveals Clearance of Blastula Chromosomal Mosaicism in In Vitro Fertilization Babies

Although chromosomal mosaic embryos detected by trophectoderm(TE)biopsy offer healthy embryos available for transfer,high-resolution postnatal karyotyping and chromosome testing of the transferred embryos are insufficient.Here,we applied single-cell multi-omics sequencing for seven infants with blastula chromosomal mosaicism detected by TE biopsy.The chromosome ploidy was examined by single-cell genome analysis,with the cellular identity being identified by single-cell transcriptome analysis.A total of 1616 peripheral leukocytes from seven infants with embryonic chromosomal mosaicism and three control ones with euploid TE biopsy were analyzed.A small number of blood cells showed copy number alterations(CNAs)on seemingly random locations at a frequency of 0%-2.5%per infant.However,none of the cells showed CNAs that were the same as those of the corresponding TE biopsies.The blastula chromosomal mosaicism may be fully self-corrected,probably through the selective loss of the aneuploid cells during development,and the transferred embryos can be born as euploid infants without mosaic CNAs corresponding to the TE biopsies.The results provide a new reference for the evaluations of transferring chromosomal mosaic embryos in certain situations.

Synthetic immunology:T-cell engineering and adoptive immunotherapy

During the past decades,the rapidly-evolving cancer is hard to be thoroughly eliminated even though the radiotherapy and chemotherapy do exhibit efficacy in some degree.However,a breakthrough appeared when the adoptive cancer therapy[1]was developed,especially T cells armed with chimeric antigen receptors(CARs)showed great potential in tumor clinical trials recently.CAR-T cells successfully elevated the efficiency and specificity of cytotoxicity.In this review,we will talk about the design of CAR and CAR-included combinatory therapeutic applications in the principles of systems and synthetic immunology.

Integrated genomic analyses identify high-risk factors and actionable targets in T-cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia(T-ALL)is an aggressive hematologic malignancy often associated with poor outcomes.To identify high-risk factors and potential actionable targets for T-ALL,we perform integrated genomic and transcriptomic analyses on samples from 165 Chinese pediatric and adult T-ALL patients,of whom 85%have outcome information.The genomic mutation landscape of this Chinese cohort is very similar to the Western cohort published previously,except that the rate of NOTCH1 mutations is significant lower in the Chinese T-ALL patients.Among 47 recurrently mutated genes in 7 functional categories,we identify RAS pathway and PTEN mutations as poor survival factors for non-TAL and TAL subtypes,respectively.Mutations in the PI3K pathway are mutually exclusive with mutations in the RAS and NOTCH1 pathways as well as transcription factors.Further analysis demonstrates that approximately 43%of the high-risk patients harbor at least one potential actionable alteration identified in this study,and T-ALLs with RAS pathway mutations are hypersensitive to MEKi in vitro and in vivo.Thus,our integrated genomic analyses not only systematically identify high-risk factors but suggest that these high-risk factors are promising targets for T-ALL therapies.

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem(ES)cells,which is used to produce gene-targeted mice for wide applications in biomedicine.However,a major bottleneck in this approach is that the robustness of germiine transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing,which impairs the efficiency and robustness of mouse model generation.Recently,we have established a new type of pluripotent cells termed extended pluripotent stem(EPS)cells,which have superior developmental potency and robust germline competence compared to conventional mouse ES cells.In this study,we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage.Based on gene targeting in mouse EPS cells,we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation,Haibo Li and Chaoran Zhao contributed equally to this work.Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13238-018-0556-1)contains supplementary material,which is available to authorized users.which could be accomplished in approximately 2 months.Importantly,using this approach,we successfully constructed mouse models in which the human interleukin 3(IL3)or interleukin 6(IL6)gene was knocked into its corresponding locus in the mouse genome.Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting,which have great application potential in biomedical research.

Lineage Specifers:New Players in the Induction of Pluripotency

Pluripotency-associated factors and their rivals, lineage specifiers, have long been consid- ered the determining factors for the identity of pluripotent and differentiated cells, respectively. Therefore, factors that are employed for cellular reprogramming in order to induce pluripotency have been identified mainly from embryonic stem cell （ESC）-enriched and pluripotency-associated factors. Recently, lineage specifiers have been identified to play important roles in orchestrating the process of restoring pluripotency. In this review, we summarize the latest discoveries regarding cell fate conversion using pluripotency-associated factors and lineage specifiers. We highlight the value of the ＂seesaw＂ model in defining cellular identity, opening up a novel scenario to consider pluri- potency and lineage specification.

Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications.However,it is still infeasible because homologous recombination(HR)is inefficient,especially for nondividing cells.To overcome the challenge,we report that a homology-independent targeted integration(HITI)strategy is used for permanent integration of high-specificity-activity Factor IX variant(F9 Padua,R338L)at the albumin(Alb)locus in a novel hemophilia B(HB)rat model.The knock-in efficiency reaches 3.66%,as determined by droplet digital PCR(dd PCR).The clotting time is reduced to a normal level four weeks after treatment,and the circulating factor IX(FIX)level is gradually increased up to 52%of the normal level over nine months even after partial hepatectomy,demonstrating the amelioration of hemophilia.Through primer-extension-mediated sequencing(PEM-seq),no significant off-target effect is detected.This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.

Exogenous artificial DNA forms chromatin structure with active transcription in yeast

Yeast artificial chromosomes(YACs) are important tools for sequencing,gene cloning,and transferring large quantities of genetic information.However,the structure and activity of YAC chromatin,as well as the unintended impacts of introducing foreign DNA sequences on DNA-associated biochemical events,have not been widely explored.Here,we showed that abundant genetic elements like TATA box and transcription factor-binding motifs occurred unintentionally in a previously reported datacarrying chromosome(d Chr).In addition,we used state-of-the-art sequencing technologies to comprehensively profile the genetic,epigenetic,transcriptional,and proteomic characteristics of the exogenous d Chr.We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels.The d Chr also displayed highly pervasive transcriptional ability and transcribed hundreds of noncoding RNAs.The results demonstrated that exogenous artificial chromosomes formed chromatin structures and did not remain as naked or loose plasmids.A better understanding of the YAC chromatin nature will improve our ability to design better data-storage chromosomes.