The Use of Stem Cells to Study Autism Spectrum Disorder

Autism spectrum disorder (ASD) affects as many as 1 in 68 children and is said to be the fastest-growing serious developmental disability in the United States. There is currently no medical cure or diagnostic test for ASD. Furthermore, the U.S. Food and Drug Administration has yet to approve a single drug for the treatment of autism’s core symptoms. Despite numerous genome studies and the identification of hundreds of genes that may cause or predispose children to ASD, the pathways underlying the pathogenesis of idiopathic ASD still remain elusive. Post-mortem brain samples, apart from being difficult to obtain, offer little insight into a disorder that arises through the course of development. Furthermore, ASD is a disorder of highly complex, human-specific behaviors, making it difficult to model in animals. Stem cell models of ASD can be generated by performing skin biopsies of ASD patients and then dedifferentiating these fibroblasts into human-induced pluripotent stem cells (hiPSCs). iPSCs closely resemble embryonic stem cells and retain the unique genetic signature of the ASD patient from whom they were originally derived. Differentiation of these iPSCs into neurons essentially recapitulates the ASD patient’s neuronal development in a dish, allowing for a patient-specific model of ASD. Here we review our current understanding of the underlying neurobiology of ASD and how the use of stem cells can advance this understanding, possibly leading to new therapeutic avenues.     

人诱导多能干细胞来源功能性胰岛β细胞的体外定向分化方法的建立

1型糖尿病是由胰岛β细胞功能受损、胰岛素分泌不足所致,目前,主要通过外源性胰岛素补充来治疗,但外源性胰岛素无法精准调控血糖,严重低血糖可危及生命。胰岛移植是一种替代疗法,但面临器官供体不足和异种来源胰岛β细胞存在人畜共患病交叉感染风险的问题。因此,获得足量且安全的胰岛β细胞是1型糖尿病细胞治疗面临的难题。本研究旨在通过人诱导多能干细胞(human induced pluripotent stem cells, hiPSCs)在体外向胰岛β细胞分化,提供一种潜在的1型糖尿病治疗新策略。为实现这一目标,我们采用了结合2D和3D培养系统的分化策略,模拟胰岛β细胞的体内发育环境,并使用多种生长因子调节在胰腺发育和β细胞分化中发挥重要作用的关键信号包括Notch信号通路(Notch signaling pathway)、Wnt信号通路(Wnt signaling pathway)、TGF-β/Smad信号通路(TGF-β/Smad signaling pathway)等,在体外将hiPSC定向诱导分化至胰岛β细胞。结果显示,在2D、3D结合的培养条件下,分化过程中定型内胚层细胞,胰腺祖细胞,胰腺...     

雷帕霉素介导的caspase 9同源二聚化调控人类多能干细胞自杀

人诱导多能干细胞（human induced pluripotent stem cells,hiPSCs）在再生医学领域具有广阔的应用前景。然而,多能干细胞（pluripotent stem cells,PSCs）具有肿瘤化风险,成为其临床应用最主要的安全性问题。雷帕霉素是一种安全和广泛使用的免疫抑制药物,通过诱导FKBP12与FRB片段的异源二聚起作用。为了保障hiPSCs治疗的安全性,本研究将雷帕霉素诱导的caspase 9（riC9）基因插入到AAVS1安全位点,构建了含有EF1α启动子、FRB-FKBP-Caspase9（CARD结构域）融合蛋白和嘌呤霉素抗性基因的供体,并与sgRNA/Cas9载体共转染hiPSCs。用嘌呤霉素筛选2周后,收集单个克隆进行基因和表型分析。最后,用雷帕霉素诱导caspase9同源二聚化,激活工程细胞的凋亡。通过对筛选获得的5个hiPSCs克隆鉴定,表明供体DNA准确敲入内源性AAVS1位点。hiPSCs保持正常的多潜能状态和增殖能力。雷帕霉素可诱导caspase 9同源二聚化,并激活细胞凋亡程序。本研究通过药物精确调控caspase 9的同源二聚化来启动细胞自杀,实现了可控的hiPSCs存活,这为保证hiPSCs治疗的安全性提供了新的策略。     

Prospective,Blinded Exploratory Evaluation of the PlayWisely Program in Children with Autism Spectrum Disorder

The purpose of the study was to explore a low-cost intervention that targets an increasingly common developmental disorder. The study was a blinded, exploratory evaluation of the PlayWisely program on autism symptoms and essential learning foundation skills (attention, recognition, and memory skills) in children with a diagnosis of autism, autism spectrum disorder (ASD), pervasive developmental disorder – not otherwise specified (PDD-NOS), and Asperger syndrome (AS). Eighteen children, 1 to 10 years of age, were evaluated using the Childhood Autism Rating Scale, Second Edition (CARS2); the PlayWisely Interactive Test of Attention, Recognition, and Memory Skills; Autism Treatment Evaluation Checklist (ATEC), and the Modified Checklist for Autism in Toddlers (M-CHAT). There were significant treatment effects for the PlayWisely measure on the Yellow Sets that examine recognition; Purple Sets that examine brain region agility and early memory skills; Blue Sets that examine phonemic awareness and recognition; and for the Total Sets, with a similar trend toward improvement in the Green Sets that examine perception and Red Sets that examine attention. No other measures reached statistical significance. The results suggest that PlayWisely can improve recognition, brain region agility, phonemic awareness, letter recognition, and early memory skills in ASD. It was observed by the parents, coaches, and study investigators that the children who were less than 3 years of age showed improvements in autism symptoms; however, the group was too small to reach statistical significance. Future studies are needed to see if this intervention can mitigate autism symptoms in very young children with ASD.     

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。     

诱导性多能干细胞与肝样细胞分化

肝脏疾病正逐渐成为全球棘手的医疗问题。肝细胞是肝脏生理活动的主要承担者,在肝脏疾病的研究以及药物的研发和测试方面有着举足轻重的作用。然而,体外分离培养的原代肝细胞面临在体外不能无限增殖和稳定表达肝脏特异基因等问题。有强大的自我更新能力和三胚层分化潜能的诱导性多能肝细胞(iPSCs)能被诱导因子、外源基因和小分子化合物等定向诱导分化为功能性肝细胞。同时,还避免了伦理、宗教以及免疫排斥等诸多问题。本文简要综述了从不同策略诱导iPSCs成为功能性肝细胞的研究方法和成果,并对该领域进行小结和展望。     

分化细胞经特定因子诱导重编程为多能干细胞

胚胎干细胞在再生医学领域有着十分诱人的应用前景。但是现有胚胎干细胞建系技术不能避开对卵细胞的操作, 成为ES细胞临床应用的障碍。通过反转录病毒载体系统, 在小鼠和人类高度分化细胞中表达干细胞因子Oct4, Sox2, Klf4和/或c-Myc等基因, 再经过干细胞标志因子Nanog或Oct4筛选, 可以获得与ES细胞特性十分近似的诱导多能干细胞系。这种不依赖于卵细胞的多能干细胞建系方法无疑是干细胞实验技术的重大进展, 也是对现有重编程理论假设的突破。综述了诱导多能干细胞系建系实验结果, 并对诱导重编程的机制和诱导多能干细胞系的临床应用前景进行了讨论。