Targeted conditional gene knockout in human embryonic stem cells

We report our application of a promoterless knockout （KO） strategy combined with Flpase- and Cre-mediated recombination to efficiently ablate gene function in hu- man embryonic stem （hES） cells. Using this strategy, we modified and deleted both alleles of BAF250a （ARID 1A）, a signature component of the ATP-dependent SWI/ SNF chromatin remodeling complex BAF. This strategy should facilitate the functional studies of genes essential for hES cell self-renewal and differentiation, and be instrumental for human therapeutic applications.

Low complexity domains, condensates, and stem cell pluripotency

Biological reactions require self-assembly of factors in the complex cellular milieu.Recent evidence indicates that intrinsically disordered,low-complexity sequence domains(LCDs)found in regulatory factors mediate diverse cellular processes from gene expression to DNA repair to signal transduction,by enriching specific biomolecules in membraneless compartments or hubs that may undergo liquidliquid phase separation(LLPS).In this review,we discuss how embryonic stem cells take advantage of LCD-driven interactions to promote cell-specific transcription,DNA damage response,and DNA repair.We propose that LCDmediated interactions play key roles in stem cell maintenance and safeguarding genome integrity.

Molecular basis of the first cell fate determination in mouse embryogenesis

Through proliferation and differentiation, a single cell, the zygote, can give rise to a complex organism composed of many types of cells. Up to the eight-cell embryo stage, the blastomeres are morphologically identical and distributed symmetrically in the mammalian embryo. Functionally, in some species, they are all totipotent. However, due to the compaction of blastomeres and the asymmetrical cell division at the late phase of the eight-cell embryo, the blastomeres of the morula are no longer identical. During the transition from morula to blastocyst, blastomeres differentiate, resulting in the first cell fate decision in embryogenesis, namely, the segregation of the inner cell mass and the tropheetoderm. In this review, we will discuss the regulatory mechanisms essential for the cell fate choice during blastocyst development, including transcriptional regulation, epigenetic regulation, mieroRNAs, and signal transduction.

Yap Controls Notochord Formation and Neural Tube Patterning by Integrating Mechanotransduction with FoxA2 and Shh Expression

目的正确的脊索和神经管(NT)形成对于中枢神经系统的发育至关重要。在胚胎发育中,生物力信息不断被胚胎细胞收集,生化和生物物理信号控制着胚胎的生长和模式化。然而,目前对生物信号的功能以及其与生物物理刺激是如何相互作用的仍知之甚少。方法本试验利用原子力显微镜量化神经管发育过程中的组织硬度,通过构建基质胶外植体培养体系、多种转基因小鼠模型、胚胎原位杂交技术、免疫荧光、萤光素酶报告基因检测等实验,探究Yap在神经形成中的作用。结果在胚胎形态发生过程中,脊索和神经管中存在机械应力和组织硬度梯度。脊索和神经管腹侧中最高的机械应力诱导Yap转录因子在脊索和底板中的激活,从而直接促进腹侧组织中心形成及其中Fox A2和Shh的表达。在底板中,Yap通过与Fox A2相互作用,是促进神经管中Shh表达的必要和充分条件。Hedgehog信号激活可挽救由Yap缺乏引起的神经管模式化缺陷,但不能挽救脊索形成,因此,Yap是独立于Hedgehog信号传导的脊索形成所必需的。结论胚胎形态发生过程中产生的生物物理刺激对包括神经管在内的中线结构的生长及形态范式发生起着指导作用,本研究揭示了Yap作为胚胎生长和编排的信号中心,将机械刺激转化为生化信号激活的一项极其重要的功能。

Cross-regulation of the Nanog and Cdx2 promoters

The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass （ICM） and trophectoderm （TE）, is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other＇s promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.

The emerging role of autophagic-lysosomal dysfunction in Gaucher disease and Parkinson's disease

Gaucher disease（GD）,the commonest lysosomal storage disorder,results from the lack or functional deficiency of glucocerebrosidase（GCase） secondary to mutations in the GBA1 gene.There is an established association between GBA1 mutations and Parkinson＇s disease（PD）,and indeed GBA1 mutations are now considered to be the greatest genetic risk factor for PD.Impaired lysosomal-autophagic degradation of cellular proteins,including α-synuclein（α-syn）,is implicated in the pathogenesis of PD,and there is increasing evidence for this also in GD and GBA1-PD.Indeed we have recently shown in a Drosophila model lacking neuronal GCase,that there are clear lysosomal-autophagic defects in association with synaptic loss and neurodegeneration.In addition,we demonstrated alterations in mechanistic target of rapamycin complex 1（mTORC1） signaling and functional rescue of the lifespan,locomotor defects and hypersensitivity to oxidative stress on treatment of GCase-deficient flies with the mT OR inhibitor rapamycin.Moreover,a number of other recent studies have shown autophagy-lysosomal system（ALS） dysfunction,with specific defects in both chaperone-mediated autophagy（CMA）,as well as macroautophagy,in GD and GBA1-PD model systems.Lastly we discuss the possible therapeutic benefits of inhibiting mT OR using drugs such as rapamycin to reverse the autophagy defects in GD and PD.

Modulating microRNAs in cancer: next-generation therapies

MicroRNAs(miRNAs)are a class of endogenously expressed non-coding regulators of the genome with an ability to mediate a variety of biological and pathological processes.There is growing evidence demonstrating frequent dysregulation of microRNAs in cancer cells,which is associated with tumor initiation,development,migration,invasion,resisting cell death,and drug resistance.Studies have shown that modulation of these small RNAs is a novel and promising therapeutic tool in the treatment of a variety of diseases,especially cancer,due to their broad influence on multiple cellular processes.However,suboptimal delivery of the appropriate miRNA to the cancer sites,quick degradation by nucleases in the blood circulation,and off target effects have limited their research and clinical applications.Therefore,there is a pressing need to improve the therapeutic efficacy of miRNA modulators,while at the same time reducing their toxicities.Several delivery vehicles for miRNA modulators have been shown to be effective in vitro and in vivo.In this review,we will discuss the role and importance of miRNAs in cancer and provide perspectives on currently available carriers for miRNA modulation.We will also summarize the challenges and prospects for the clinical translation of miRNAbased therapeutic strategies.

AID for reprogramming

Five decades of nuclear transfer （NT） experiments have established a key principle in developmental genetics - despite vast functional differences, virtually all of the cells in an adult organ- ism maintain an identical genome . Studies in several species have shown that the nucleus of a differentiated cell can be reprogrammed by exposure to egg cytoplasm, which re-initiates an embryonic genetic program in the trans-ferred genome and permits the development of an identical adult organism （i.e. cloning）.

Role of the immune microenvironment in bone,cartilage,and soft tissue regeneration:from mechanism to therapeuticopportunity

Bone,cartilage,and soft tissue regeneration is a complex spatiotemporal process recruiting a variety of cell types,whose activity and interplay must be precisely mediated for effective healing post-injury.Although extensive strides have been made in the understanding of the immune microenvironment processes governing bone,cartilage,and soft tissue regeneration,effective clinical translation of these mechanisms remains a challenge.Regulation of the immune microenvironment is increasingly becoming a favorable target for bone,cartilage,and soft tissue regeneration;therefore,an in-depth understanding of the communication between immune cells and functional tissue cells would be valuable.Herein,we review the regulatory role of the immune microenvironment in the promotion and maintenance of stem cell states in the context of bone,cartilage,and soft tissue repair and regeneration.We discuss the roles of various immune cell subsets in bone,cartilage,and soft tissue repair and regeneration processes and introduce novel strategies,for example,biomaterial-targeting of immune cell activity,aimed at regulating healing.Understanding the mechanisms of the crosstalk between the immune microenvironment and regeneration pathways may shed light on new therapeutic opportunities for enhancing bone,cartilage,and soft tissue regeneration through regulation of the immune microenvironment.

How to make insulin-producing pancreatic β cells for diabetes treatment

Around 400 million people worldwide suffer from diabetes mellitus.The major pathological event for Type 1 diabetes and advanced Type 2 diabetes is loss or impairment of insulin-secreting β cells of the pancreas.For the past 100 years,daily insulin injection has served as a life-saving treatment for these patients.However,insulin injection often cannot achieve full glucose control,and over time poor glucose control leads to severe complications and mortality.As an alternative treatment,islet transplantation has been demonstrated to effectively maintain glucose homeostasis in diabetic patients,but its wide application is limited by the scarcity of donated islets.Therefore,it is important to define new strategies to obtain functional human β cells for transplantation therapies.Here,we summarize recent progress towards the production of β cells in vitro from pluripotent stem cells or somatic cell types including a cells,pancreatic exocrine cells,gastrointestinal stem cells,fibroblasts and hepatocytes.We also discuss novel methods for optimizing β cell transplantation and maintenance in vivo.From our perspective,the future of βcell replacement therapy is very promising although it is still challenging to control differentiation of β cells in vitro and to protect these cells from autoimmune attack in Type 1 diabetic patients.Overall,tremendous progress has been made in understanding βcell differentiation and producing functional β cells with different methods.In the coming years,we believe more clinical trials will be launched to move these technologies towards treatments to benefit diabetic patients.

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells （CPCs） provides a new paradigm for studying and treating heart disease. To realize the full potential of CPCs for therapeutic purposes, it is essential to understand the genetic and epigenetic mechanisms guiding CPC differentiation into cardiomyocytes, smooth muscle, or endothelial cells. ATP-dependent chromatin remodelers mediate one critical epigenetic mechanism. These large multiprotein complexes open up chromatin to modulate transcription factor access to DNA. SWI/SNF, one of the major types of chromatin remodelers, plays a key role in various aspects of development （de la Serna et al., 2006; Wu et al., 2009）, including heart development and disease （Lickert et al., 2004; Wang et al., 2004; Huang et al., 2008; Stankunas et al., 2008; Hang et al., 2010）. In this review, we describe the specific function of various SWI/SNF components in cardiogenesis and cardiac progenitor cell （CPC） self-renewal and differentiation. We envision that a detailed understanding of the SWI/SNF in heart development and CPC formation and differentiation will generate novel insights into epigenetic mechanisms that govern CPC differentiation and may have significant implications in understanding and treating heart disease.

Metabolic alterations and vulnerabilities in hepatocellular carcinoma

Liver cancer is a serious disease.It is ranked as the cancer with the second highest number of cancer-related deaths worldwide.Hepatocellular carcinoma(HCC),which arises from transformed hepatocytes,is the major subtype of liver cancer.It accounts for 85%of total liver-cancer cases.An important aspect of HCC that has been actively studied is its metabolism.With the liver as the primary site of numerous metabolic processes in the body,it has been shown that the metabolism of HCC cells is highly dysregulated compared to that of normal hepatocytes.It is therefore crucial to understand the metabolic alterations caused by HCC and the underlying mechanisms for these alterations.This deeper understanding will allow diagnostic and therapeutic advancements in the treatment of HCC.In this review,we will summarize the current literature in HCC metabolic alterations,induced vulnerabilities,and potential therapeutic interventions.

Nature:骨髓微环境发生突变可导致造血干细胞异常

白血病骨髓微环境中支持血液发育的某些DNA突变可以驱动周围造血干细胞形成白血病，来自艾默里大学温希普癌症研究所和亚特兰大儿童健康中心的研究人员在国际学术期刊Nature上公布了他们的最新研究进展。许多致癌突变都作用于细胞自身。让细胞自身生长更快。相比之下，努南综合征小鼠模型上可以观察到间接的邻近细胞效应，努南综合征是一种能够增加白血病患病风险的遗传紊乱。

EZH2-, CHD4-, and IDH-linked epigenetic perturbation and its association with survival in glioma patients

GUoma is a complex disease with limited treatment options. Recent advances have identified isocitrate dehydrogenase （IDH） mutations in up to 80% lower grade gUomas （LGG） and in 76% secondary gUoblastomas （GBM）. IDH mutations are also seen in 10%-20% of acute myeloid leukemia （AML）. In AML, it was determined that mutations of IDH and other genes involving epigenetic regulations are early events, emerging in the pre-leukemic stem cells （pre-LSCs） stage, whereas mutations in genes propa- gating oncogenic signal are late events in leukemia. IDH mutations are also early events in gUoma, occurring before TP53 mutation, 1p/19q deletion, etc. Despite these advances in gUoma research, studies into other molecular alterations have lagged considerably. In this study, we analyzed currently available databases. We identified EZH2, KMT2C, and CHD# as important genes in glioma in addition to the known gene IDH1/2. We also showed that genomic alterations of PIK3CA, CDKN2A, CDK#, FIPIL1, or FUBP1 collaborate with IDH mutations to negatively affect patients＇ survival in LGG. In LGG patients with TP53 mutations or IDH1/2 mutations, additional genomic alterations of EZH2, KMC2C, and CHD4 individually or in combination were associated with a markedly decreased disease-free survival than patients without such alterations. Alterations of EZH2, KMT2C, and CHD4, at gen- etic level or protein level could perturb epigenetic program, leading to malignant transformation in glioma. By reviewing current literature on both AML and gUoma and performing bioinformatics analysis on available datasets, we developed a hypothetical model on the tumorigenesis from premaUgnant stem cells to gUoma.

microRNAs in cardiac regeneration and cardiovascular disease

microRNAs(miRNAs)are a class of small non-coding RNAs,which have been shown important to a wide range of biological process by post-transcriptionally regulating the expression of protein-coding genes.miRNAs have been demonstrated essential to normal cardiac development and function.Recently,numerous studies indicate miRNAs are involved in cardiac regeneration and cardiac disease,including cardiac hypertrophy,myocardial infarction and cardiac arrhythmia.These observations suggest miRNAs play important roles in cardiology.In this review,we summarize the recent progress of studying miRNAs in cardiac regeneration and cardiac disease.We also discuss the diagnostic and therapeutic potential of miRNAs in heart disease.

Sensing of cytoplasmic chromatin by cGAS activates innate immune response in SARS-CoV-2 infection

The global coronavirus disease 2019(COVID-19)pandemic is caused by severe acute respiratory syndrome coronavirus 2(SARSCoV-2),a positive-sense RNA virus.How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved.Here,we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway.SARS-CoV-2 infection induces the cellular level of 2′3′-cGAMP associated with STING activation.cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection.We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion.Furthermore,cytoplasmic chromatin-cGAS-STING pathway,but not MAVS-mediated viral RNA sensing pathway,contributes to interferon and pro-inflammatory gene expression upon cell fusion.Finally,we show that cGAS is required for host antiviral responses against SARS-CoV-2,and a STING-activating compound potently inhibits viral replication.Together,our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection,mediated by cytoplasmic chromatin from the infected cells.Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19.In addition,these findings extend our knowledge in host defense against viral infection by showing that host cells’self-nucleic acids can be employed as a“danger signal”to alarm the immune system.

PhaseFIT:live-organoid phase-fluorescent image transformation via generative AI

Organoid models have provided a powerful platform for mechanistic investigations into fundamental biological processes involved in the development and function of organs.Despite the potential for image-based phenotypic quantification of organoids,their complex 3D structure,and the time-consuming and labor-intensive nature of immunofluorescent staining present significant challenges.In this work,we developed a virtual painting system,PhaseFIT(phase-fluorescent image transformation)utilizing customized and morphologically rich 2.5D intestinal organoids,which generate virtual fluorescent images for phenotypic quantification via accessible and low-cost organoid phase images.This system is driven by a novel segmentation-informed deep generative model that specializes in segmenting overlap and proximity between objects.The model enables an annotation-free digital transformation from phase-contrast to multi-channel fluorescent images.The virtual painting results of nuclei,secretory cell markers,and stem cells demonstrate that PhaseFIT outperforms the existing deep learning-based stain transformation models by generating fine-grained visual content.We further validated the efficiency and accuracy of PhaseFIT to quantify the impacts of three compounds on crypt formation,cell population,and cell stemness.PhaseFIT is the first deep learning-enabled virtual painting system focused on live organoids,enabling large-scale,informative,and efficient organoid phenotypic quantification.PhaseFIT would enable the use of organoids in high-throughput drug screening applications.

A shared role of the myocardin-family transcriptional coactivators in cardiomyocyte maturation

Dear Editor,In development,after cells make a commitment to their fates,they undergo a continuous and adaptive maturation process to eventually reach their terminal states.Cell maturity often decays during aging and pathogenesis.The immature phenotypes of stem cell-differentiated cells deposit a major bottleneck in regenerative medicine.