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1.
Stem Cell Res Ther ; 15(1): 99, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38581069

RESUMO

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.


Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Reprodutibilidade dos Testes , Células Receptoras Sensoriais/metabolismo , Dor/metabolismo , Diferenciação Celular/fisiologia
2.
Neuropharmacology ; 253: 109967, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38657946

RESUMO

Botulinum neurotoxin type A BoNT/A is used off-label as a third line therapy for neuropathic pain. However, the mechanism of action remains unclear. In recent years, the role of voltage-gated sodium channels (Nav) in neuropathic pain became evident and it was suggested that block of sodium channels by BoNT/A would contribute to its analgesic effect. We assessed sodium channel function in the presence of BoNT/A in heterologously expressed Nav1.7, Nav1.3, and the neuronal cell line ND7/23 by high throughput automated and manual patch-clamp. We used both the full protein and the isolated catalytic light chain LC/A for acute or long-term extracellular or intracellular exposure. To assess the toxin's effect in a human cellular system, we differentiated induced pluripotent stem cells (iPSC) into sensory neurons from a healthy control and a patient suffering from a hereditary neuropathic pain syndrome (inherited erythromelalgia) carrying the Nav1.7/p.Q875E-mutation and carried out multielectrode-array measurements. Both BoNT/A and the isolated catalytic light chain LC/A showed limited effects in heterologous expression systems and the neuronal cell line ND7/23. Spontaneous activity in iPSC derived sensory neurons remained unaltered upon BoNT/A exposure both in neurons from the healthy control and the mutation carrying patient. BoNT/A may not specifically be beneficial in pain syndromes linked to sodium channel variants. The favorable effects of BoNT/A in neuropathic pain are likely based on mechanisms other than sodium channel blockage and new approaches to understand BoNT/A's therapeutic effects are necessary.


Assuntos
Toxinas Botulínicas Tipo A , Células-Tronco Pluripotentes Induzidas , Canal de Sódio Disparado por Voltagem NAV1.7 , Neuralgia , Humanos , Neuralgia/tratamento farmacológico , Toxinas Botulínicas Tipo A/farmacologia , Toxinas Botulínicas Tipo A/uso terapêutico , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Analgésicos/farmacologia , Animais , Canal de Sódio Disparado por Voltagem NAV1.3/genética , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células HEK293 , Linhagem Celular
3.
Pflugers Arch ; 476(6): 975-992, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38538988

RESUMO

Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel NaV1.7, which is a validated pain target. NaV1.7 deficiency leads to pain insensitivity, whereas NaV1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag NaV1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface NaV1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust NaV1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes NaV1.7 surface expression. A clinically effective NaV1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Canal de Sódio Disparado por Voltagem NAV1.7 , Células Receptoras Sensoriais , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação , Sistemas CRISPR-Cas
4.
Res Sq ; 2023 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-37961300

RESUMO

Background: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic symptoms. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs for disease modelling remain key challenges to study human nociception in vitro. Here, we report a detailed characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Methods: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Expression profiling of sensory neurons was performed with Immunocytochemistry and in situ hybridization techniques. Manual patch clamp and high throughput cellular screening systems (Fluorescence imaging plate reader, automated patch clamp and multi-well microelectrode arrays recordings) were applied to functionally characterize the generated sensory neurons. Results: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. High throughput systems confirmed functional expression of Na+ and K+ ion channels. Multi-well microelectrode recordings display spontaneously active neurons with sensitivity to increased temperature indicating expression of heat sensitive ion channels. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. Conclusions: We validated the efficiency of two differentiation protocols and their potential application for understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

5.
Methods Mol Biol ; 2429: 175-188, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35507161

RESUMO

Induced pluripotent stem cells (iPS-cells) have significantly expanded our experimental possibilities, by creating new strategies for the molecular study of human disease and drug development. Treatment of pain has not seen much improvement over the past decade, likely due to species differences in preclinical models. Thus, iPS-cell derived sensory neurons offer a highly welcome translational approach for research and drug development. Although central neuronal differentiation is relatively straightforward, the successful and reliable generation of peripheral neurons requires more complex measures. Here, we describe a small molecule-based protocol for the differentiation of human sensory neurons from iPS-cells which renders functional nociceptor-like cells within several weeks.


Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Humanos , Células Receptoras Sensoriais
6.
Neurobiol Pain ; 8: 100055, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33364527

RESUMO

In this concise Mini-Review we will summarize ongoing developments of new techniques to study physiology and pathophysiology of the peripheral sensory nervous system in human stem cell derived models. We will focus on recent developments of reprogramming somatic cells into induced pluripotent stem cells, neural differentiation towards neuronal progenitors and human sensory neurons. We will sum up the high potential of this new technique for disease modelling of human neuropathies with a focus on genetic pain syndromes, such as gain- and loss-of-function mutations in voltage-gated sodium channels. The stem cell derived human sensory neurons are used for drug testing and we will summarize their usefulness for individualized treatment identification in patients with neuropathic pain. The review will give an outlook on potential application of this technique as companion diagnostics and for personalized medicine.

7.
Cell Mol Life Sci ; 77(17): 3401-3422, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31712992

RESUMO

Cell-based therapies using adult stem cells are promising options for the treatment of a number of diseases including autoimmune and cardiovascular disorders. Among these, vascular wall-derived mesenchymal stem cells (VW-MSCs) might be particularly well suited for the protection and curative treatment of vascular damage because of their tissue-specific action. Here we report a novel method for the direct conversion of human skin fibroblasts towards MSCs using a VW-MSC-specific gene code (HOXB7, HOXC6 and HOXC8) that directs cell fate conversion bypassing pluripotency. This direct programming approach using either a self-inactivating (SIN) lentiviral vector expressing the VW-MSC-specific HOX-code or a tetracycline-controlled Tet-On system for doxycycline-inducible gene expressions of HOXB7, HOXC6 and HOXC8 successfully mediated the generation of VW-typical MSCs with classical MSC characteristics in vitro and in vivo. The induced VW-MSCs (iVW-MSCs) fulfilled all criteria of MSCs as defined by the International Society for Cellular Therapy (ISCT). In terms of multipotency and clonogenicity, which are important specific properties to discriminate MSCs from fibroblasts, iVW-MSCs behaved like primary ex vivo isolated VW-MSCs and shared similar molecular and DNA methylation signatures. With respect to their therapeutic potential, these cells suppressed lymphocyte proliferation in vitro, and protected mice against vascular damage in a mouse model of radiation-induced pneumopathy in vivo, as well as ex vivo cultured human lung tissue. The feasibility to obtain patient-specific VW-MSCs from fibroblasts in large amounts by a direct conversion into induced VW-MSCs could potentially open avenues towards novel, MSC-based therapies.


Assuntos
Fibroblastos/citologia , Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Reprogramação Celular , Metilação de DNA , Modelos Animais de Doenças , Fibroblastos/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Pulmão/citologia , Pulmão/patologia , Linfócitos/citologia , Linfócitos/metabolismo , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Comunicação Parácrina , Pneumonia/patologia , Pneumonia/terapia
8.
Stem Cell Res ; 33: 20-24, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30296670

RESUMO

Angelman syndrome (AS) is a neurodevelopmental disorder with leading symptoms of happy demeanor, intellectual disability, ataxia and seizures. AS can be caused by genetic and epigenetic aberrations, resulting in the absence of functional UBE3A protein in the brain. UBE3A is an imprinted gene, which is, in neurons of the brain, expressed exclusively from maternal chromosome 15. The generated iPSC line was derived from skin fibroblasts of a patient with AS, who, due to an imprinting defect, lacked DNA methylation at the chromosome 15 imprinting center, which controls maternal-specific expression of UBE3A. Resource table.


Assuntos
Síndrome de Angelman/genética , Impressão Genômica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Criança , Feminino , Humanos
10.
Sci Rep ; 6: 30792, 2016 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-27484051

RESUMO

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders, such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome, we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs, both parental alleles are present, distinguishable by the mutation, and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation, in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein.


Assuntos
Síndrome de Angelman/genética , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/metabolismo , Domínio Catalítico , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Metilação de DNA , Derme/citologia , Derme/metabolismo , Epigênese Genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Ligação Proteica , Deleção de Sequência , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo
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