Üç boyutlu organoid kültür temelli çip-üstü-rett sendromu modeli geliştirilmesi
Küçük Resim Yok
Tarih
2024
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Son yıllarda, kalıtsal, enfeksiyöz, nörodejeneratif, nörogelişimsel bozukluklar ve kanser başta olmak üzere çeşitli hastalıkların modellenmesi ve çözümüne yönelik geliştirilen, potansiyel yeni ilaçların etkinlik ve güvenlik profillerinin pre-klinik olarak değerlendirilmesi için in vivo fizyolojiyi ve doku mikroçevresini daha iyi taklit eden in vitro üç boyutlu (3B) hücre kültürü çalışmaları önem kazanmıştır. Bu kapsamda, tez çalışmasında, günümüzde birçok araştırmanın ilgi odağı olan biyomühendislik ürünü 3B beyin organoidleri ile çip-üstü-organ sistemleri temel alınmış, dünyada çığır açan biyoteknolojik gelişmeler olan CRISPR/Cas9 genom düzenleme sistemi, indüklenmiş pluripotent kök hücreler (iPKH) ve DNA sekanslama işlemleri ise araç olarak kullanılmıştır. Tez çalışmasının amacı; iPKH'larda CRISPR/Cas9 teknolojisinin kullanılmasıyla, henüz tedavisi olmayıp pre-klinik çalışmalarının geliştirilmesine devam edilen nadir genetik nörogelişimsel hastalıklar (NGH) grubundaki Rett Sendromuna (RTT) yönelik MECP2 geninde fonksiyon kaybı mutasyonunu oluşturmak, sağlıklı/mutant iPKH'lardan farklılaştırılan nöral/glial hücrelerle 2B-3B statik-dinamik modeller kurmak, ve en nihayetinde moleküler/fonksiyonel açıdan yeterli matürasyona ulaşmış serebral organoidler ile mikro-biyomühendislik ürünü organoid zekası çalışmaları için çip-üstü-RTT modelini geliştirmek ve akabinde hastalık modelini en gerçeğe yakın koşullarda potansiyel ilaç denemesiyle çalışmak olmuştur. Paralelinde, RTT patogenezinde önemli bir hücre olan merkezi sinir sistemi yerleşik immün hücresi mikroglia popülasyonu açısından farklı serebral organoid gelişimi metotlarını kıyaslamak, farklı dinamik etkilere sahip mikroakışkan çip platformlarında organoid matürasyonlarını incelemek ve RTT dahil NGH'ların bölge spesifik modellenebilmesi amacıyla orta beyin organoidi çalışmalarını yürütmek de hedef alınmıştır. Bu amaçla ilk olarak, MECP2 mutant iPKH'lerin CRISPR/Cas9 teknoloji ile oluşturulması için, yeni dizayn gRNA'lar kullanılarak knock-out (KO) mutasyonları oluşturulmuş, western blot (WB), sanger DNA dizileme, yeni nesil dizileme, qRT-PCR ve immünofloresan (IF) boyama teknikleri ile mutant hücrelerin detaylı karakterizasyonları yapılmıştır. Ardından, sağlıklı-mutant iPKH'lerden farklılaştırılan nöral/glial hücreler ile 2B-3B statik-dinamik sistemlerde birlikte kültür çalışmaları gerçekleştirilmiş, hastalık fenotipi ve genotipinin gösterilmesine yönelik karakterizasyon çalışmaları yürütülmüştür. En nihayetinde, hücresel içerik ve moleküler/fonksiyonel matürasyonu açısından en ideal serebral organoid matürasyon sisteminin belirlenmesi için dört farklı dinamik sistemde (orbital çalkalamalı kültür, spinner flask, RCCS-mikrogravite ve yeni tasarım µ-platform sistemi) kıyaslamalı çalışmalar yürütülmüş, detaylı karakterizasyonlar ile ideal sistem tanımı yapılmıştır. Akabinde, sağlıklı kontrol dahilinde, iki biyomühendislik ürünü ve bir hasta kaynaklı MECP2 mutant iPKH hatlarından, RTT organoidleri geliştirilmiş ve morfolojik gözlemler, vDISCO toku temizleme sonrası tüm yapı konfokal görüntülemesi, IF, qRT-PCR, glutamat analizi ve ELISA testleri ile detaylı karakterizasyonları yapılmıştır. Son olarak, organoid temelli çip-üstü-RTT modeli kurulmuş ve potansiyel ilaç molekülü olan JQ1 denemesi sonrası yenilikçi hastalık modelinde RTT patogenezi araştırılmıştır. Paralel çalışmalar kapsamında ise, mikroglia zenginliği, yapısal morfogenez ve ileri matürasyon bakımından, Ormel ve Lancester serebral organoid yöntemleri, orta beyin organoidi geliştirme yöntemleri ve tubing-rocking mikroplatform sistemleri, FUnGI doku temizleme yöntemi sonrası tüm yapı konfokal görüntülemesi ile kıyaslanmıştır.
In vitro three-dimensional (3D) cell culture studies developed in recent years have gained great importance for recapitulation of various diseases, especially hereditary, infectious, neurodegenerative, neurodevelopmental disorders and cancer. As such, in vitro 3D cell culture better mimics in vivo physiology and tissue microenvironment for preclinical evaluation in regards to the efficacy and safety of potential new drugs. In this context, bioengineered 3D brain organoids and organ-on-a-chip systems, which are the focus of attention for many researchers, form the basis of this thesis. Some of the groundbreaking biotechnological developments such as the CRISPR/Cas9 genome editing system, induced pluripotent stem cells (iPSCs) and DNA sequencing processes have also been used as tools. The aim of this thesis has been to create a loss-of-function mutation in the MECP2 gene by using CRISPR/Cas9 technology in iPSCs for recapitulation of Rett Syndrome (RTT), one of the rare genetic neurodevelopmental disease (NDD) for which preclinical studies are still being developed. Additionally, 2D-3D static-dynamic models with neural/glial cells differentiated in healthy/mutant iPSCs have been formulated. Finally, the development of a micro-bioengineered RTT-on-a-chip model for organoid intelligence studies included highly mature cerebral organoids in terms of molecular/functional features, and to study this model under the most realistic conditions through potential drug trials has been undertaken. In parallel, comparison of different cerebral organoid development methods comparing the abundance of the central nervous system resident immune cell microglia population which is an important cell in the pathogenesis of RTT, examining organoid maturations on microfluidic platforms with different dynamic effects, and also midbrain organoid studies were also targeted in order to model NDDs including RTT via region-specifically. For this purpose, knock-out (KO) mutations on MECP2 gene mutant iPSCs were created initially using newly designed gRNAs with CRISPR/Cas9 technology. Subsequently, detailed characterization of mutant cells were performed by western blot (WB), sanger DNA sequencing, next generation sequencing, qRT-PCR and immunofluorescence (IF) staining techniques. Afterwards, co-culture studies were carried out with neural/glial cells differentiated from healthy/mutant iPSCs in 2D-3D static/dynamic systems, and characterization studies were carried out to ascertain the disease phenotype and genotype. Finally, comparative studies were carried out in four different dynamic systems, namely orbital shaking culture, spinner flask, RCCS-microgravity and newly designed µ-platform, to determine the most ideal cerebral organoid maturation system in terms of cellular content and molecular/functional maturation, and the ideal system was defined with detailed characterizations. Subsequently, RTT organoids were developed from two bioengineered and one patient-derived MECP2 mutant iPSC lines within the healthy control and detailed characterizations were made with morphological observations, whole structure confocal imaging after vDISCO tissue clearing, IF, qRT-PCR, glutamate analysis and ELISA tests. Finally, RTT organoid-on-chip model was established and the pathogenesis of RTT was investigated in this innovative disease model after the trial of JQ1, a potential drug molecule. In parallel studies, Ormel and Lancester cerebral organoid methods, midbrain organoid generation methods and tubing-rocking microplatform systems were compared with whole structure confocal imaging after the FUnGI tissue clearing, in terms of microglia richness, structural morphogenesis and advanced maturation.
In vitro three-dimensional (3D) cell culture studies developed in recent years have gained great importance for recapitulation of various diseases, especially hereditary, infectious, neurodegenerative, neurodevelopmental disorders and cancer. As such, in vitro 3D cell culture better mimics in vivo physiology and tissue microenvironment for preclinical evaluation in regards to the efficacy and safety of potential new drugs. In this context, bioengineered 3D brain organoids and organ-on-a-chip systems, which are the focus of attention for many researchers, form the basis of this thesis. Some of the groundbreaking biotechnological developments such as the CRISPR/Cas9 genome editing system, induced pluripotent stem cells (iPSCs) and DNA sequencing processes have also been used as tools. The aim of this thesis has been to create a loss-of-function mutation in the MECP2 gene by using CRISPR/Cas9 technology in iPSCs for recapitulation of Rett Syndrome (RTT), one of the rare genetic neurodevelopmental disease (NDD) for which preclinical studies are still being developed. Additionally, 2D-3D static-dynamic models with neural/glial cells differentiated in healthy/mutant iPSCs have been formulated. Finally, the development of a micro-bioengineered RTT-on-a-chip model for organoid intelligence studies included highly mature cerebral organoids in terms of molecular/functional features, and to study this model under the most realistic conditions through potential drug trials has been undertaken. In parallel, comparison of different cerebral organoid development methods comparing the abundance of the central nervous system resident immune cell microglia population which is an important cell in the pathogenesis of RTT, examining organoid maturations on microfluidic platforms with different dynamic effects, and also midbrain organoid studies were also targeted in order to model NDDs including RTT via region-specifically. For this purpose, knock-out (KO) mutations on MECP2 gene mutant iPSCs were created initially using newly designed gRNAs with CRISPR/Cas9 technology. Subsequently, detailed characterization of mutant cells were performed by western blot (WB), sanger DNA sequencing, next generation sequencing, qRT-PCR and immunofluorescence (IF) staining techniques. Afterwards, co-culture studies were carried out with neural/glial cells differentiated from healthy/mutant iPSCs in 2D-3D static/dynamic systems, and characterization studies were carried out to ascertain the disease phenotype and genotype. Finally, comparative studies were carried out in four different dynamic systems, namely orbital shaking culture, spinner flask, RCCS-microgravity and newly designed µ-platform, to determine the most ideal cerebral organoid maturation system in terms of cellular content and molecular/functional maturation, and the ideal system was defined with detailed characterizations. Subsequently, RTT organoids were developed from two bioengineered and one patient-derived MECP2 mutant iPSC lines within the healthy control and detailed characterizations were made with morphological observations, whole structure confocal imaging after vDISCO tissue clearing, IF, qRT-PCR, glutamate analysis and ELISA tests. Finally, RTT organoid-on-chip model was established and the pathogenesis of RTT was investigated in this innovative disease model after the trial of JQ1, a potential drug molecule. In parallel studies, Ormel and Lancester cerebral organoid methods, midbrain organoid generation methods and tubing-rocking microplatform systems were compared with whole structure confocal imaging after the FUnGI tissue clearing, in terms of microglia richness, structural morphogenesis and advanced maturation.
Açıklama
Anahtar Kelimeler
Biyomühendislik, Bioengineering
