Human Intestinal Organoids as a Model of NGLY1 Deficiency in a Patient Population

人类肠道类器官作为患者群体 NGLY1 缺乏的模型

• 批准号: 9256798
• 负责人: Jessica M Donnelly
• 金额: $ 2.71万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-19 至 2017-12-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9256798
• 关键词: ASCL2 gene; Affect; Apoptosis; Autophagocytosis; BMI1 gene; Biological Assay; Biological Neural Networks; Biological Process; Biopsy; Categories; Cell Line; Cell Lineage; Cell physiology; Cells; Cellular Stress; Characteristics; Chronic; Clinical; Clinical Management; Complement; Congenital Disorders; Constipation; Cues; Data; Defect; Development; Developmental Delay Disorders; Disease; Disease model; Endoplasmic Reticulum; Enteral; Enteric Nervous System; Enteroendocrine Cell; Enzymes; Epithelial; Epithelium; Excision; Exhibits; Exons; Fibroblasts; Gastrointestinal Diseases; Gastrointestinal tract structure; Genetic study; Genotype; Goblet Cells; Hereditary Disease; Heterozygote; Homozygote; Human; Hyperplasia; Immunocompromised Host; Immunohistochemistry; Impairment; In Vitro; Individual; Inflammatory Bowel Diseases; Intestinal Motility; Intestines; Introns; Investigation; Isometric Exercise; LGR5 gene; Laboratories; Measurement; Mesenchymal; Methods; Modeling; Modification; Morphogenesis; Movement Disorders; Mus; Muscle; Mutation; Neural Crest; Neuronal Differentiation; Neurons; Neuropathy; OLFM4 gene; Organ; Organoids; Parents; Patients; Pattern; Peptide N-Glycosidase; Phenotype; Physiology; Pluripotent Stem Cells; Polysaccharides; Population; Process; Proliferation Marker; Protein Inhibition; Proteins; Quantitative Reverse Transcriptase PCR; Research Proposals; Role; Secretory Cell; Signal Pathway; Stem cells; Stress; Symptoms; System; Technology; Testing; Tissues; Translations; Transplantation; Western Blotting; Work; absorption; arm; base; biological adaptation to stress; cell motility; cell type; disabling symptom; exome sequencing; experience; falls; gastrointestinal; genotyped patients; glycosylation; human embryonic stem cell; in vitro Model; in vivo; induced pluripotent stem cell; intestinal epithelium; intestinal homeostasis; intestinal villi; liver injury; misfolded protein; monolayer; motility disorder; mutation carrier; myelination; nutrient absorption; patient population; progenitor; protein expression; protein function; response; stem cell population; targeted treatment

Project Summary NGLY1 deficiency is a rare, congenital disorder that is caused by mutations in the cytoplasmic peptide:N- glycanase, an enzyme associated with degradation of misfolded proteins. Symptoms of the disease include neuropathy with movement disorder, global developmental delay, liver damage, chronic constipation and the absence of tears. As NGLY1 deficiency has only recently been identified through whole exome sequencing of patients, no targeted therapies are available. Investigations into NGLY1 expression and function throughout the body are therefore imperative for developing better strategies for clinical management of this condition. The central hypothesis of this research proposal is loss of NGLY1 function elicits an ER stress response in the intestine that alters epithelial differentiation while abrogating enteric nervous system function, changes which together result in intestinal dysmotility. We have based this hypothesis on the key clinical features in patients and preliminary studies using human intestinal organoids (HIOs), or “mini-guts”, generated by differentiation of control (hESC) and patient-derived pluripotent stem cell (iPS) lines. HIOs generated from NGLY1-/- iPSCs, when transplanted into immunocompromised mice, form mature intestinal tissue which undergoes differentiation and morphogenesis to form intestinal villi, but with epithelial secretory cell hyperplasia (Goblet and enteroendocrine cells). Therefore, in Specific Aim 1a I will characterize the phenotype and function of control iPSC-derived HIOs (NGLY1+/+) compared to HIOs generated from iPSC lines established from the fibroblasts of NGLY1 deficient patients (NGLY1-/-) and their parents (NGLY1+/-). Intestinal cell lineages will be quantified through immunostaining and transplanted HIOs expanded ex vivo as human intestinal enteroid (HIE) monolayers to assess intestinal barrier function, secretion and absorption. Specific Aim 1b will complement these studies by quantifying ER stress responses within these HIOs, as NGLY1 associates with the endoplasmic reticulum associated degradation (ERAD) machinery. Collectively, we expect to confirm our initial finding of increases in secretory cell populations and identify that secretory cell function may be inefficient due to elevated ER stress and the inhibition of protein translation. As chronic constipation is a debilitating symptom of NGLY1 deficiency but the contribution of NGLY1 to intestinal motility is unknown, in Specific Aim 2 we will incorporate enteric neurons into HIOs, representing each NGLY1 genotype in each compartment (neuronal or epithelial), to assess their function and interaction with the muscle layer. To do this, we will work with collaborators who have demonstrated that a functional enteric neural network develops in xenografted HIOs when they are combined with neural crest progenitors before transplantation (HIOs + ENS). Immunostaining will be used to assess neuronal differentiation with analysis of contractility using isometric force assays in organ chambers. At the completion of these studies we expect to have elucidated how mutations in NGLY1 impact intestinal differentiation and function.

项目概要 NGLY1 缺陷是一种罕见的先天性疾病，由细胞质肽 N- 突变引起 聚糖酶，一种与错误折叠蛋白质降解相关的酶。该疾病的症状包括 神经病伴有运动障碍、整体发育迟缓、肝损伤、慢性便秘和 没有眼泪。由于 NGLY1 缺陷最近才通过全外显子组测序发现 对于患者来说，没有针对性的治疗方法。对 NGLY1 表达和功能的研究 因此，身体有必要制定更好的策略来临床管理这种疾病。 本研究提案的中心假设是 NGLY1 功能的丧失会引起内质网应激反应 肠改变上皮分化，同时消除肠神经系统功能，改变 共同导致肠道蠕动障碍。我们的这一假设基于患者的关键临床特征 以及使用通过分化产生的人类肠道类器官（HIO）或“迷你肠道”进行的初步研究 对照（hESC）和患者来源的多能干细胞（iPS）系。从 NGLY1-/- iPSC 生成的 HIO， 当移植到免疫功能低下的小鼠体内时，形成成熟的肠道组织，并经历 分化和形态发生形成肠绒毛，但伴有上皮分泌细胞增生（杯状 和肠内分泌细胞）。因此，在具体目标 1a 中，我将描述以下物质的表型和功能： 对照 iPSC 衍生的 HIO (NGLY1+/+) 与从建立的 iPSC 系生成的 HIO 进行比较 NGLY1 缺陷患者 (NGLY1-/-) 及其父母 (NGLY1+/-) 的成纤维细胞。肠细胞谱系将是 通过免疫染色进行量化，移植的 HIO 离体扩增为人肠肠样 (HIE) 单层细胞评估肠道屏障功能、分泌和吸收。具体目标 1b 将 通过量化这些 HIO 内的 ER 应激反应来补充这些研究，因为 NGLY1 与 内质网相关降解（ERAD）机制。总的来说，我们希望确认我们的 初步发现分泌细胞群增加，并确定分泌细胞功能可能是 由于内质网应激升高和蛋白质翻译受到抑制而效率低下。由于慢性便秘是 NGLY1 缺乏的衰弱症状，但 NGLY1 对肠道蠕动的贡献尚不清楚， 具体目标 2 我们将把肠神经元纳入 HIO，代表每个 NGLY1 基因型 隔室（神经元或上皮细胞），以评估其功能以及与肌肉层的相互作用。为此， 我们将与合作者合作，他们已经证明了功能性肠神经网络的发展 异种移植的 HIO 在移植前与神经嵴祖细胞结合（HIO + ENS）。 免疫染色将用于评估神经元分化，并使用等距分析收缩性 器官室中的力测定。在完成这些研究后，我们希望能够阐明突变是如何发生的 NGLY1 影响肠道分化和功能。