Convergence of human pluripotent stem cell,organoid, and genome editing technologies

The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.     

Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation

Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patientspecific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.Key words: induced pluripotent stem cells (iPSCs), hepatic differentiation, liver ngraftment, disease modeling, drug testing, alpha-1 antitrypsin, liver cirrhosis, hepatocellular carcinoma, cell therapy     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.Key words: induced pluripotent stem cells (iPSCs), blood, B lymphocytes, hematopoietic differentiation, hepatic differentiation, disease modeling, drug testing     

Liver engraftment potential of hepatic cells derived from patient-specific induced pluripotent stem cells

Human induced pluripotent stem cells (iPSCs) are potential renewable sources of hepatocytes for drug development and cell therapy. Differentiation of human iPSCs into different developmental stages of hepatic cells has been achieved and improved during the last several years. We have recently demonstrated the liver engraftment and regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo. Here we describe the in vitro and in vivo activities of hepatic cells derived from patient specific iPSCs, including multiple lines established from either inherited or acquired liver diseases, and discuss basic and clinical applications of these cells for disease modeling, drug screening and discovery, gene therapy and cell replacement therapy.     

Hematopoietic cells as sources for patient-specific iPSCs and disease modeling

In addition to being an attractive source for cell replacement therapy, human induced pluripotent stem cells (iPSCs) also have great potential for disease modeling and drug development. During the recent several years, cell reprogramming technologies have evolved to generate virus-free and integration-free human iPSCs from easily accessible sources such as patient skin fibroblasts and peripheral blood samples. Hematopoietic cells from umbilical cord blood banks and Epstein Barr virus (EBV) immortalized B lymphocyte repositories represent alternative sources for human genetic materials of diverse backgrounds. Ability to reprogram these banked blood cells to pluripotency and differentiate them into a variety of specialized and functional cell types provides valuable tools for studying underlying mechanisms of a broad range of diseases including rare inherited disorders. Here we describe the recent advances in generating disease specific human iPSCs from these different types of hematopoietic cells and their potential applications in disease modeling and regenerative medicine.     

Plant genome editing: ever more precise and wide reaching

Genome-editing technologies consisting of targeted mutagenesis and gene targeting enable us to modify genes of interest rapidly and precisely. The discovery in 2012 of CRISPR/Cas9 systems and their development as sequence-specific nucleases has brought about a paradigm shift in biology. Initially, CRISPR/Cas9 was applied in targeted mutagenesis to knock out a target gene. Thereafter, advances in genome-editing technologies using CRISPR/Cas9 developed rapidly, with base editing systems for transition substitution using a combination of Cas9 nickase and either cytidine or adenosine deaminase being reported in 2016 and 2017, respectively, and later in 2021 bringing reports of transversion substitution using Cas9 nickase, cytidine deaminase and uracil DNA glycosylase. Moreover, technologies for gene targeting and prime editing systems using DNA or RNA as donors have also been developed in recent years. Besides these precise genome-editing strategies, reports of successful chromosome engineering using CRISPR/Cas9 have been published recently. The application of genome editing to crop breeding has advanced in parallel with the development of these technologies. Genome-editing enzymes can be introduced into plant cells, and there are now many examples of crop breeding using genome-editing technologies. At present, it is no exaggeration to say that we are now in a position to be able to modify a gene precisely and rearrange genomes and chromosomes in a predicted way. In this review, we introduce and discuss recent highlights in the field of precise gene editing, chromosome engineering and genome engineering technology in plants.     

Derivation of male germ cells from induced pluripotent stem cells by inducers: A review

Induced pluripotent stem cells (iPSCs) refer to stem cells that are artificially produced using a new technology known as cellular reprogramming, which can use gene transduction in somatic cells. There are numerous potential applications for iPSCs in the field of stem cell biology becauase they are able to give rise to several different cell features of lineages such as three-germ layers. Primordial germ cells, generated via in vitro differentiation of iPSCs, have been demonstrated to produce functional gametes. Therefore, in this review we discussed past and recent advances in the in vitro differentiation of germ cells using pluripotent stem cells with an emphasis on iPSCs. Although this domain of research is still in its infancy, exploring development mechanisms of germ cells is promising, especially in humans, to promote future reproductive and developmental engineering technologies. While few studies have evaluated the ability and efficiency of iPSCs to differentiate toward male germ cells in vitro by different inducers, the given effect was investigated in this review.     

Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells

Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.     

不同打靶位点影响人诱导多能干细胞MYO7A杂合突变位点的基因校正中CRISPR/Cas9介导的同源重组效率

应用CRISPR-Cas9系统对人诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）进行基因编辑,为疾病模型的建立、致病机制研究、药物筛选及基因校正治疗疾病提供了更广阔的平台。相对于CRISPR-Cas9介导的基因敲除,应用该系统介导的同源重组实现基因点突变或突变校正效率要低、且难度偏大。为了实现对MYO7A杂合点突变（c.4118C>T）的人iPSCs的点突变校正,本文构建了表达maxGFP的pX330质粒。针对需校正的突变位点,设计5组识别序列并连接到maxGFP-pX330中构建靶向质粒。将5组打靶质粒分别转染HEK 293FT细胞48 h,细胞表达GFP;测序结果显示,MYO7A基因相应位点出现杂峰,表明打靶质粒具有打断活性。将同源模版单链寡核苷酸链（single-stranded DNA oligonucleotides, ssODN）与打靶质粒共同电转入人iPSCs后48 h,经流式分选出(5.8±2.2)%的细胞表达GFP。分选后细胞行单克隆扩增并测序。结果显示,打靶质粒1和ssODN组合对点突变校正未成功;打靶质粒2、3、4、5与ssODN组合均获得了校正后的细胞株。本研究表明,打断位点是影响同源重组校正效率的关键因素。当应用CRISPR/Cas9（或其它核酸酶）介导的同源重组进行基因编辑操作时,可以同时选择多个打靶位点造成基因组不同位置上的双链打断（double-stranded break, DSB）位点,以获得目的单克隆细胞株。本研究为应用CRISPR-Cas9系统对人诱导多能干细胞进行基因编辑提供了有力参考。     

CRISPR/Cas9 system: a powerful technology for in vivo and ex vivo gene therapy

CRISPR/Cas9 is a versatile genome-editing tool which is widely used for modifying the genome of both prokaryotic and eukaryotic organisms for basic research and applications. An increasing number of reports have demonstrated that CRISPR/Cas9-mediated genome editing is a powerful technology for gene therapy. Here, we review the recent advances in CRISPR/Cas9-mediated gene therapy in animal models via different strategies and discuss the challenges as well as future prospects.     

基因组编辑脱靶研究进展

近年各种基因组编辑技术的成功研发为人类疾病的治疗与预防谱写了新的篇章,这些技术对应的基因组编辑工具主要包括锌指核酸酶(ZFNs)、转录激活子样效应因子核酸酶(TALENs)和最近发现的规律成簇间隔短回文重复(CRISPR)/Cas系统。这些工具相应的脱靶问题目前是制约基因组编辑技术介导人类疾病治疗的重要瓶颈。本文将分别从基因组编辑工具的介绍、脱靶的现状、解决优化的方案和检测方法进行总结与探讨,通过比较,进一步了解基因组编辑工具的优缺点及相关脱靶检测方法的适用性。     

Optimized TAL effector nucleases (TALENs) for use in treatment of sickle cell disease

TAL effector nucleases (TALENs) represent a new class of artificial nucleases capable of cleaving long, specific target DNA sequences in vivo and are powerful tools for genome editing with potential therapeutic applications. Here we report a pair of custom-designed TALENs for targeted genetic correction of the sickle cell disease mutation in human cells, which represents an example of engineered TALENs capable of recognizing and cleaving a human disease-associated gene. By using a yeast reporter system, a systematic study was carried out to optimize TALEN architecture for maximal in vivo cleavage efficiency. In contrast to the previous reports, the engineered TALENs were capable of recognizing and cleaving target binding sites preceded by A, C or G. More importantly, the optimized TALENs efficiently cleaved a target sequence within the human β-globin (HBB) gene associated with sickle cell disease and increased the efficiency of targeted gene repair by >1000-fold in human cells. In addition, these TALENs showed no detectable cytotoxicity. These results demonstrate the potential of optimized TALENs as a powerful genome editing tool for therapeutic applications.     

人工核酸酶介导的基因组编辑技术

传统的基因组编辑技术是基于胚胎干细胞和同源重组实现生物基因组定向改造,但是该技术打靶效率低,严重制约了生命科学以及医学的研究.因此,研究新的基因组编辑技术十分重要.人工核酸酶介导的基因组编辑技术是通过特异性识别靶位点造成DNA双链断裂,引起细胞内源性的修复机制实现靶基因的修饰.与传统的基因组编辑技术相比,人工核酸酶技术打靶效率高,这对于基因功能的研究、构建人类疾病动物模型以及探索新型疾病治疗方案有着重要的意义.人工核酸酶技术有3种类型：锌指核酸酶(ZFN)、类转录激活因子核酸酶(TALEN)及规律成簇的间隔短回文重复序列(CRISPR).本文将对以上3种人工核酸酶技术的原理以及在生命科学和医学研究的应用进行综述.     

Targeted genome editing in pluripotent stem cells using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are designer nucleases capable of cleaving a prespecified target DNA within complex genomes. ZFNs consist of a non-specific endonuclease domain fused to an engineered DNA-binding domain that tethers the nuclease activity to the chosen chromosomal site. The endonuclease-induced DNA double strand break triggers a cellular DNA damage response, resulting in double strand break repair by either accurate homologous recombination (HR) or error-prone non-homologous end-joining (NHEJ). Thus, ZFNs are powerful tools for targeted genome engineering in a variety of mammalian cell types, including embryonic (ESCs) and induced pluripotent stem cells (iPSCs). As a paradigm for genome editing in pluripotent stem cells, we describe the use of ZFNs in murine ESCs for generating knockout alleles by NHEJ without selection or by HR employing different selection schemes.