The discovery of novel genes causative of rare inherited diseases often requires validation through functional experiments to probe the biological function of encoded proteins and understand how their function is disrupted by mutations. While in vivo animal models are not always available or physiologically accurate, the use of human cell lines may represent a valid alternative but, especially in neurogenetic disorders, this may present limitations related, for instance, to the differences with the complexity of the actual tissues affected by the disease. Joubert syndrome (JS) is a congenital cerebellar ataxia characterised by a specific cerebellar and brainstem malformation, the molar tooth sign (MTS). All known JS-linked genes encode proteins of the primary cilium, qualifying JS as a ciliopathy. Primary cilium is a subcellular organelle involved in organ development and generally functions as antenna-like signalling receptor on most cell types, including neural progenitors and neurons. Therefore, disruption of its structure or function leads to defects and developmental abnormalities that are associated with syndromic ciliopathies, like JS. Despite great strides in understanding cilia biology and disease, little is known about the biological process linking a potential ciliary dysfunction to the JS phenotype. This project set out to develop and optimise a cellular model of JS in order to produce cerebellar neuronal types that could be used to characterise the cellular effects of genetic mutations identified in JS patients. This is possible with induced pluripotent stem cells (iPSCs) that can be differentiated in any cell type while recapitulating the full genetic background of the patient. To this end, fibroblasts from JS patients with mutations in different genes were reprogrammed into iPSCs, and then characterised to confirm their pluripotency and stemness through immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for specific markers. Karyotype and short tandem repeat (STR) analyses were also performed to assess the stability and accuracy of the reprogramming process in the various cell lines produced. iPSCs from selected patients, with mutations in major JS genes (ciliogenesis and planar polarity effector 1 (CPLANE1) and retinitis pigmentosa GTPase regulator (RPGR) interacting protein 1 like (RPGRIP1L)), and control (Ctrl) iPSCs were then differentiated into neural stem cells (NSCs), and subsequently into cerebellar neurons using a two-dimensional (2D) cerebellar differentiation model. After up to 21 days of culture, heterogeneous populations of neurons were obtained, including granule cells and Purkinje cells (PCs), as showed by immunofluorescence and RT-qPCR for lineage-specific markers. In addition, ciliary length, number, and morphology were analysed through immunofluorescence of axoneme and basal body (BB) markers, and differences among patients and control lines were observed regarding the number of ciliated cells in culture and ciliary morphology. Finally, a three-dimensional (3D) cerebellar differentiation approach was trialled from selected iPSCs, in collaboration with the University La Sorbonne. Preliminary observations confirmed the formation of 3D organoid structures up to day 35, which represent a promising model for the future characterisation of JS-specific changes in mechanisms such as patterning, neuronal migration, or neuron-glia interaction. Results produced in this project confirm that iPSCs represent a useful in vitro model to analyse cerebellar development in humans and study the role of primary cilia and JS-related genes in disease mechanisms. This approach could also be beneficial for future high-throughput drug screening and testing of different therapeutic approaches for neurodevelopmental defects such as JS.
The discovery of novel genes causative of rare inherited diseases often requires validation through functional experiments to probe the biological function of encoded proteins and understand how their function is disrupted by mutations. While in vivo animal models are not always available or physiologically accurate, the use of human cell lines may represent a valid alternative but, especially in neurogenetic disorders, this may present limitations related, for instance, to the differences with the complexity of the actual tissues affected by the disease. Joubert syndrome (JS) is a congenital cerebellar ataxia characterised by a specific cerebellar and brainstem malformation, the molar tooth sign (MTS). All known JS-linked genes encode proteins of the primary cilium, qualifying JS as a ciliopathy. Primary cilium is a subcellular organelle involved in organ development and generally functions as antenna-like signalling receptor on most cell types, including neural progenitors and neurons. Therefore, disruption of its structure or function leads to defects and developmental abnormalities that are associated with syndromic ciliopathies, like JS. Despite great strides in understanding cilia biology and disease, little is known about the biological process linking a potential ciliary dysfunction to the JS phenotype. This project set out to develop and optimise a cellular model of JS in order to produce cerebellar neuronal types that could be used to characterise the cellular effects of genetic mutations identified in JS patients. This is possible with induced pluripotent stem cells (iPSCs) that can be differentiated in any cell type while recapitulating the full genetic background of the patient. To this end, fibroblasts from JS patients with mutations in different genes were reprogrammed into iPSCs, and then characterised to confirm their pluripotency and stemness through immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for specific markers. Karyotype and short tandem repeat (STR) analyses were also performed to assess the stability and accuracy of the reprogramming process in the various cell lines produced. iPSCs from selected patients, with mutations in major JS genes (ciliogenesis and planar polarity effector 1 (CPLANE1) and retinitis pigmentosa GTPase regulator (RPGR) interacting protein 1 like (RPGRIP1L)), and control (Ctrl) iPSCs were then differentiated into neural stem cells (NSCs), and subsequently into cerebellar neurons using a two-dimensional (2D) cerebellar differentiation model. After up to 21 days of culture, heterogeneous populations of neurons were obtained, including granule cells and Purkinje cells (PCs), as showed by immunofluorescence and RT-qPCR for lineage-specific markers. In addition, ciliary length, number, and morphology were analysed through immunofluorescence of axoneme and basal body (BB) markers, and differences among patients and control lines were observed regarding the number of ciliated cells in culture and ciliary morphology. Finally, a three-dimensional (3D) cerebellar differentiation approach was trialled from selected iPSCs, in collaboration with the University La Sorbonne. Preliminary observations confirmed the formation of 3D organoid structures up to day 35, which represent a promising model for the future characterisation of JS-specific changes in mechanisms such as patterning, neuronal migration, or neuron-glia interaction. Results produced in this project confirm that iPSCs represent a useful in vitro model to analyse cerebellar development in humans and study the role of primary cilia and JS-related genes in disease mechanisms. This approach could also be beneficial for future high-throughput drug screening and testing of different therapeutic approaches for neurodevelopmental defects such as JS.
In vitro modelling of Joubert syndrome: development and characterisation of patient-derived induced pluripotent stem cells-based models
POLLARA, LIDIA
2023-07-31
Abstract
The discovery of novel genes causative of rare inherited diseases often requires validation through functional experiments to probe the biological function of encoded proteins and understand how their function is disrupted by mutations. While in vivo animal models are not always available or physiologically accurate, the use of human cell lines may represent a valid alternative but, especially in neurogenetic disorders, this may present limitations related, for instance, to the differences with the complexity of the actual tissues affected by the disease. Joubert syndrome (JS) is a congenital cerebellar ataxia characterised by a specific cerebellar and brainstem malformation, the molar tooth sign (MTS). All known JS-linked genes encode proteins of the primary cilium, qualifying JS as a ciliopathy. Primary cilium is a subcellular organelle involved in organ development and generally functions as antenna-like signalling receptor on most cell types, including neural progenitors and neurons. Therefore, disruption of its structure or function leads to defects and developmental abnormalities that are associated with syndromic ciliopathies, like JS. Despite great strides in understanding cilia biology and disease, little is known about the biological process linking a potential ciliary dysfunction to the JS phenotype. This project set out to develop and optimise a cellular model of JS in order to produce cerebellar neuronal types that could be used to characterise the cellular effects of genetic mutations identified in JS patients. This is possible with induced pluripotent stem cells (iPSCs) that can be differentiated in any cell type while recapitulating the full genetic background of the patient. To this end, fibroblasts from JS patients with mutations in different genes were reprogrammed into iPSCs, and then characterised to confirm their pluripotency and stemness through immunofluorescence and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for specific markers. Karyotype and short tandem repeat (STR) analyses were also performed to assess the stability and accuracy of the reprogramming process in the various cell lines produced. iPSCs from selected patients, with mutations in major JS genes (ciliogenesis and planar polarity effector 1 (CPLANE1) and retinitis pigmentosa GTPase regulator (RPGR) interacting protein 1 like (RPGRIP1L)), and control (Ctrl) iPSCs were then differentiated into neural stem cells (NSCs), and subsequently into cerebellar neurons using a two-dimensional (2D) cerebellar differentiation model. After up to 21 days of culture, heterogeneous populations of neurons were obtained, including granule cells and Purkinje cells (PCs), as showed by immunofluorescence and RT-qPCR for lineage-specific markers. In addition, ciliary length, number, and morphology were analysed through immunofluorescence of axoneme and basal body (BB) markers, and differences among patients and control lines were observed regarding the number of ciliated cells in culture and ciliary morphology. Finally, a three-dimensional (3D) cerebellar differentiation approach was trialled from selected iPSCs, in collaboration with the University La Sorbonne. Preliminary observations confirmed the formation of 3D organoid structures up to day 35, which represent a promising model for the future characterisation of JS-specific changes in mechanisms such as patterning, neuronal migration, or neuron-glia interaction. Results produced in this project confirm that iPSCs represent a useful in vitro model to analyse cerebellar development in humans and study the role of primary cilia and JS-related genes in disease mechanisms. This approach could also be beneficial for future high-throughput drug screening and testing of different therapeutic approaches for neurodevelopmental defects such as JS.| File | Dimensione | Formato | |
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