Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

Animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene functions.The field of genome modification in rabbits has progressed slowly.However,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic pathologies.These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice.This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

A booming field of large animal model research

Animal models are integral to the study of fundamental biological processes and the etiology of human diseases.Small animal models,especially those involving mice,have yielded abundant and significant insights,greatly enhancing our understanding of biological phenomena and disease mechanisms.

Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology.Meanwhile,pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases.Here,we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics,and the core concepts of computational data analysis.We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming,and long non-coding RNAs

Transposable elements(TEs) are mobile genomic sequences of DNA capable of autonomous and nonautonomous duplication. TEs have been highly successful,and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional,these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TEderived endogenous viral particles can even be detected. Asimilar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells(ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs(lnc RNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lnc RNAs, and how these links influence each other to modulate aspects of ESCs,embryogenesis, and somatic cell reprogramming.

The therapeutic prospects and challenges of human neural stem cells for the treatment of Alzheimer’s Disease

Alzheimer’s disease(AD)is a multifactorial neurodegenerative disorder associated with aging.Due to its insidious onset,protracted progression,and unclear pathogenesis,it is considered one of the most obscure and intractable brain disorders,and currently,there are no effective therapies for it.Convincing evidence indicates that the irreversible decline of cognitive abilities in patients coincides with the deterioration and degeneration of neurons and synapses in the AD brain.Human neural stem cells(NSCs)hold the potential to functionally replace lost neurons,reinforce impaired synaptic networks,and repair the damaged AD brain.They have therefore received extensive attention as a possible source of donor cells for cellular replacement therapies for AD.Here,we review the progress in NSC-based transplantation studies in animal models of AD and assess the therapeutic advantages and challenges of human NSCs as donor cells.We then formulate a promising transplantation approach for the treatment of human AD,which would help to explore the disease-modifying cellular therapeutic strategy for the treatment of human AD.

Base editors:development and applications in biomedicine

Base editor(BE)is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase,enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break(DSB)or requiring donor DNA templates in living cells.Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems,such as CRISPR/Cas9,as the DSB induced by Cas9 will cause severe damage to the genome.Thus,base editors have important applications in the field of biomedicine,including gene function investigation,directed protein evolution,genetic lineage tracing,disease modeling,and gene therapy.Since the development of the two main base editors,cytosine base editors(CBEs)and adenine base editors(ABEs),scientists have developed more than 100 optimized base editors with improved editing efficiency,precision,specificity,targeting scope,and capacity to be delivered in vivo,greatly enhancing their application potential in biomedicine.Here,we review the recent development of base editors,summarize their applications in the biomedical field,and discuss future perspectives and challenges for therapeutic applications.

A tunable, rapid, and precise drug control of protein expression by combining transcriptional and post-translational regulation systems

Rapid,precise,and tunable regulation of protein abundance would be significantly useful in a variety of biotechnologies and biomedical applications.Here,we describe a system that allows tunable and rapid drug control of gene expression for either gene activation or inactivation in mammalian cells.We construct the system by coupling Tet-on 3 G and small molecule-assisted shutoff systems,which can respectively induce transcriptional activation and protein degradation in the presence of corresponding small molecules.This dual-input drug inducer regulation system facilitates a bidirectional control of gene expression.The gene of interest can be precisely controlled by dual small molecules in a broad dynamic range of expression from overexpression to complete silence,allowing gene function study in a comprehensive expression profile.Our results reveal that the bidirectional control system enables sensitive dosage-and time-dependent regulation for either turn-on or shutoff of gene expression.We also apply this system for inducible genome editing and gene activation mediated by clustered regularly interspaced short palindromic repeats.The system provides an integrated platform for studying multiple biological processes by manipulating gene expression in a more flexible way.

Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process.Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline.Previous pig models with tetracycline regulatory elements were generated through random integration.This process often resulted in uncertain expression and unpredictable phenotypes,thus hindering their applications.Here,by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus,respectively,a double knock-in reporter pig model was generated.We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo.Two att P sites were arranged to flank the td Tomato to switch reporter gene.Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos.To display the flexible application of this system,we generated a pig strain with Dox-inducing h KRASexpression through phiC31 integrase-mediated cassette exchange.After eight months of Dox administration,squamous cell carcinoma developed in the nose,mouth,and scrotum,which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis.Overall,the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Generation of tooth-like structures from integration-free human urine induced pluripotent stem cells

Background:Tooth is vital not only for a good smile,but also good health.Yet,we lose tooth regularly due to accidents or diseases.An ideal solution to this problem is to regenerate tooth with patients’own cells.Here we describe the generation of tooth-like structures from integration-free human urine induced pluripotent stem cells(ifhU-iPSCs).Results:We first differentiated ifhU-iPSCs to epithelial sheets,which were then recombined with E14.5 mouse dental mesenchymes.Tooth-like structures were recovered from these recombinants in 3 weeks with success rate up to 30%for 8 different iPSC lines,comparable to H1 hESC.We further detected that ifhU-iPSC derived epithelial sheets differentiated into enamel-secreting ameloblasts in the tooth-like structures,possessing physical properties such as elastic modulus and hardness found in the regular human tooth.Conclusion:Our results demonstrate that ifhU-iPSCs can be used to regenerate patient specific dental tissues or even tooth for further drug screening or regenerative therapies.

Generation of rat blood vasculature and hematopoietic cells in rat-mouse chimeras by blastocyst complementation

Interspecies chimera through blastocyst complementation could be an alternative approach to create human organs in animals by using human pluripotent stem cells.A mismatch of the major histocompatibility complex of vascular endothelial cells between the human and host animal will cause graft rejection in the transplanted organs.Therefore,to achieve a transplantable organ in animals without rejection,creation of vascular endothelial cells derived from humans within the organ is necessary.In this study,to explore whether donor xeno-pluripotent stem cells can compensate for blood vasculature in host animals,we generated rat-mouse chimeras by injection of rat embryonic stem cells(rESCs)into mouse blastocysts with deficiency of Flk-1 protein,which is associated with endothelial and hematopoietic cell development.We found that rESCs could differentiate into vascular endothelial and hematopoietic cells in the rat-mouse chimeras.The whole yolk sac(YS)of Flk-1^EGFP/ECFP rat-mouse chimera was full of rat blood vasculature.Rat genes related to vascular endothelial cells,arteries,and veins,blood vessels formation process,as well as hematopoietic cells,were highly expressed in the YS.Our results suggested that rat vascular endothelial cells could undergo proliferation,migration,and self-assembly to form blood vasculature and that hematopoietic cells could differentiate into B cells,T cells,and myeloid cells in rat-mouse chimeras,which was able to rescue early embryonic lethality caused by Flk-1 deficiency in mouse.

SNX16 negatively regulates the migration and tumorigenesis of MCF-7 cells

Background:Sorting nexins are a large family of proteins that are associated with various components of the endosome system and they play many roles in processes such as endocytosis,intracellular protein trafficking and cell signaling.The subcellular distribution patterns of many of them remain controversial and their in vivo functions have not been characterized yet.Results:We investigated the subcellular distribution and function of SNX16 in this study.SNX16 is detected on Rab5-positive endosomes localized adjacent to focal adhesions at cell cortex.Inhibition of SNX23,polymerization of microtubule filaments as well as the PI3-kinase all disrupt the cell cortex distribution of SNX16.Ectopic expression of SNX16 reduces the migration and the tumor formation activity of MCF-7 cells.Conclusion:Our results indicate that,in addition to the PI3P,there is a SNX23-and microtubule-dependent cargo transport pathway required for the proper subcellular distribution of SNX16.SNX16 plays a negative regulatory role during cell migration and tumorigenesis.

3D chromatin architecture and epigenetic regulation in cancer stem cells

Dedifferentiation of cell identity to a progenitor-like or stem cell-like state with increased cellular plasticity is frequently observed in cancer formation.During this process,a subpopulation of cells in tumours acquires a stem cell-like state partially resembling to naturally occurring pluripotent stem cells that are temporarily present during early embryogenesis.Such characteristics allow these cancer stem cells(CSCs)to give rise to the whole tumour with its entire cellular heterogeneity and thereby support metastases formation while being resistant to current cancer therapeutics.Cancer development and progression are demarcated by transcriptional dysregulation.In this article,we explore the epigenetic mechanisms shaping gene expression during tumorigenesis and cancer stem cell formation,with an emphasis on 3D chromatin architecture.Comparing the pluripotent stem cell state and epigenetic reprogramming to dedifferentiation in cellular transformation provides intriguing insight to chromatin dynamics.We suggest that the 3D chromatin architecture could be used as a target for re-sensitizing cancer stem cells to therapeutics.

Regeneration of immunocompetent B lymphopoiesis from pluripotent stem cells guided by transcription factors

Regeneration of functional B lymphopoiesis from pluripotent stem cells(PSCs)is challenging,and reliable methods have not been developed.Here,we unveiled the guiding role of three essential factors,Lhx2,Hoxa9,and Runx1,the simultaneous expression of which preferentially drives B lineage fate commitment and in vivo B lymphopoiesis using PSCs as a cell source.In the presence of Lhx2,Hoxa9,and Runx1 expression,PSC-derived induced hematopoietic progenitors(iHPCs)immediately gave rise to pro/pre-B cells in recipient bone marrow,which were able to further differentiate into entire B cell lineages,including innate B-1a,B-1b,and marginal zone B cells,as well as adaptive follicular B cells.In particular,the regenerative B cells produced adaptive humoral immune responses,sustained antigen-specific antibody production,and formed immune memory in response to antigen challenges.The regenerative B cells showed natural B cell development patterns of immunoglobulin chain switching and hypermutation via cross-talk with host T follicular helper cells,which eventually formed T cell-dependent humoral responses.This study exhibits de novo evidence that B lymphopoiesis can be regenerated from PSCs via an HSC-independent approach,which provides insights into treating B cell-related deficiencies using PSCs as an unlimited cell resource.

Proteins in DNA methylation and their role in neural stem cell proliferation and differentiation

Background:Epigenetic modifications,namely non-coding RNAs,DNA methylation,and histone modifications such as methylation,phosphorylation,acetylation,ubiquitylation,and sumoylation play a significant role in brain development.DNA methyltransferases,methyl-CpG binding proteins,and ten-eleven translocation proteins facilitate the maintenance,interpretation,and removal of DNA methylation,respectively.Different forms of methylation,including 5-methylcytosine,5-hydroxymethylcytosine,and other oxidized forms,have been detected by recently developed sequencing technologies.Emerging evidence suggests that the diversity of DNA methylation patterns in the brain plays a key role in fine-tuning and coordinating gene expression in the development,plasticity,and disorders of the mammalian central nervous system.Neural stem cells(NSCs),originating from the neuroepithelium,generate neurons and glial cells in the central nervous system and contribute to brain plasticity in the adult mammalian brain.Main body:Here,we summarized recent research in proteins responsible for the establishment,maintenance,interpretation,and removal of DNA methylation and those involved in the regulation of the proliferation and differentiation of NSCs.In addition,we discussed the interactions of chemicals with epigenetic pathways to regulate NSCs as well as the connections between proteins involved in DNA methylation and human diseases.Conclusion:Understanding the interplay between DNA methylation and NSCs in a broad biological context can facilitate the related studies and reduce potential misunderstanding.

CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells

The CCCTC-binding factor(CTCF)protein and its modified forms regulate gene expression and genome organization.However,information on CTCF acetylation and its biological function is still lacking.Here,we show that CTCF can be acetylated at lysine 20(CTCF-K20)by CREB-binding protein(CBP)and deacetylated by histone deacetylase 6(HDAC6).CTCF-K20 is required for the CTCF interaction with CBP.A CTCF point mutation at lysine 20 had no effect on self-renewal but blocked the mesoderm differentiation of mouse embryonic stem cells(mESCs).The CTCF-K20 mutation reduced CTCF binding to the promoters and enhancers of genes associated with early cardiac mesoderm differentia-tion,resulting in diminished chromatin accessibility and decreased enhancer-promoter interactions,impairing gene expression.In summary,this study reveals the important roles of CTCF-K20 in regulating CTCF genomic functions and mESC differentiation into mesoderm.

Deubiquitylating Nanog:novel role of USP21 in embryonic stem cell maintenance

Recently,three groups independently identified ubiquitin-specific peptidase 21(USP21)as an efficient deubiquitylase that reverses Nanog polyubiquitylation and stabilizes Nanog protein.In this preview,I have summarized the work of these three groups.

Lower genomic stability of induced pluripotent stem cells reflects increased non-homologous end joining

Background:Induced pluripotent stem cells(iPSCs)and embryonic stem cells(ESCs)share many common features,including similar morphology,gene expression and in vitro differentiation profiles.However,genomic stability is much lower in iPSCs than in ESCs.In the current study,we examined whether changes in DNA damage repair in iPSCs are responsible for their greater tendency towards mutagenesis.Methods:Mouse iPSCs,ESCs and embryonic fibroblasts were exposed to ionizing radiation(4 Gy)to introduce dou-ble-strand DNA breaks.At 4 h later,fidelity of DNA damage repair was assessed using whole-genome re-sequencing.We also analyzed genomic stability in mice derived from iPSCs versus ESCs.Results:In comparison to ESCs and embryonic fibroblasts,iPSCs had lower DNA damage repair capacity,more somatic mutations and short indels after irradiation.iPSCs showed greater non-homologous end joining DNA repair and less homologous recombination DNA repair.Mice derived from iPSCs had lower DNA damage repair capacity than ESC-derived mice as well as C57 control mice.Conclusions:The relatively low genomic stability of iPSCs and their high rate of tumorigenesis in vivo appear to be due,at least in part,to low fidelity of DNA damage repair.

Two-step protocol for regeneration of immunocompetent T cells from mouse pluripotent stem cells

Numerous efforts have been attempted to regenerate T cells in culture dish from pluripotent stem cells(PSCs).However,in vitro generated T cells exhibited extremely low activity and compromised immunocompetency in vivo.Here,we describe a two-step protocol for regenerating functional T cells using an inducible Runx1-Hoxa9-PSC(iR9-PSCs)line.The procedure mainly includes generation of induced hematopoietic progenitor cells(iHPCs)in vitro,transplantation,and development of functional induced T cells(iT)in vivo via transplantation.The entire induction process in vitro requires 21 days before iHPCs transplantation.The development of mature T cells in vivo takes 4 to 6 weeks post-transplantation.We provide a simple and reproducible approach for functional T cell regeneration from iR9-PSCs for research purpose.

Transitions between epithelial and mesenchymal states during cell fate conversions

Cell fate conversion is considered as the changing of one type of cells to another type including somatic cell reprogramming （de-differentiation）, differentiation, and trans-differentiation, Epithelial and mesenchymal cells are two major types of cells and the transitions between these two cell states as epithelial-mesenchymal transi- tion （EMT） and mesenchymal-epithelial transition （MET） have been observed during multiple cell fate conversions including embryonic development, tumor progression and somatic cell reprogramming. In addition, MET and sequential EMT-MET during the generation of induced pluripotent stem cells （iPSC） from fibroblasts have been reported recently. Such observation is consistent with multiple rounds of sequential EMT-MET during embryonic development which could be considered as a reversed process of reprogramming at least partially. Therefore in current review, we briefly discussed the potential roles played by EMT, MET, or even sequential EMT-MET during different kinds of cell fate conversions. We also provided some preliminary hypotheses on the mechanisms that connect cell state transitions and cell fate conversions based on results collected from cell cycle, epigenetic regulation, and sternness acquisition.

Neural progenitor cells from human induced pluripotent stem cells generated less autogenous immune response

The breakthrough development of induced pluripotent stem cells(iPSCs)raises the prospect of patient-specific treatment for many diseases through the replacement of affected cells.However,whether iPSC-derived functional cell lineages generate a deleterious immune response upon auto-transplantation remains unclear.In this study,we differentiated five human iPSC lines from skin fibroblasts and urine cells into neural progenitor cells(NPCs)and analyzed their immunogenicity.Through co-culture with autogenous peripheral blood mononuclear cells(PBMCs),we showed that both somatic cells and iPSC-derived NPCs do not stimulate significant autogenous PBMC proliferation.However,a significant immune reaction was detected when these cells were co-cultured with allogenous PBMCs.Furthermore,no significant expression of perforin or granzyme B was detected following stimulation of autogenous immune effector cells(CD3+CD8 T cells,CD3+CD8+T cells or CD3 CD56+NK cells)by NPCs in both PBMC and T cell co-culture systems.These results suggest that human iPSC-derived NPCs may not initiate an immune response in autogenous transplants,and thus set a base for further preclinical evaluation of human iPSCs.