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How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols

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 c...

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Autores principales: Kalia, Anil Kumar, Rösseler, Corinna, Granja-Vazquez, Rafael, Ahmad, Ayesha, Pancrazio, Joseph J., Neureiter, Anika, Zhang, Mei, Sauter, Daniel, Vetter, Irina, Andersson, Asa, Dussor, Gregory, Price, Theodore J., Kolber, Benedict J., Truong, Vincent, Walsh, Patrick, Lampert, Angelika
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Journal Experts 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635298/
https://www.ncbi.nlm.nih.gov/pubmed/37961300
http://dx.doi.org/10.21203/rs.3.rs-3127017/v1
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author Kalia, Anil Kumar
Rösseler, Corinna
Granja-Vazquez, Rafael
Ahmad, Ayesha
Pancrazio, Joseph J.
Neureiter, Anika
Zhang, Mei
Sauter, Daniel
Vetter, Irina
Andersson, Asa
Dussor, Gregory
Price, Theodore J.
Kolber, Benedict J.
Truong, Vincent
Walsh, Patrick
Lampert, Angelika
author_facet Kalia, Anil Kumar
Rösseler, Corinna
Granja-Vazquez, Rafael
Ahmad, Ayesha
Pancrazio, Joseph J.
Neureiter, Anika
Zhang, Mei
Sauter, Daniel
Vetter, Irina
Andersson, Asa
Dussor, Gregory
Price, Theodore J.
Kolber, Benedict J.
Truong, Vincent
Walsh, Patrick
Lampert, Angelika
author_sort Kalia, Anil Kumar
collection PubMed
description 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.
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spelling pubmed-106352982023-11-13 How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols Kalia, Anil Kumar Rösseler, Corinna Granja-Vazquez, Rafael Ahmad, Ayesha Pancrazio, Joseph J. Neureiter, Anika Zhang, Mei Sauter, Daniel Vetter, Irina Andersson, Asa Dussor, Gregory Price, Theodore J. Kolber, Benedict J. Truong, Vincent Walsh, Patrick Lampert, Angelika Res Sq Article 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. American Journal Experts 2023-10-28 /pmc/articles/PMC10635298/ /pubmed/37961300 http://dx.doi.org/10.21203/rs.3.rs-3127017/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
Kalia, Anil Kumar
Rösseler, Corinna
Granja-Vazquez, Rafael
Ahmad, Ayesha
Pancrazio, Joseph J.
Neureiter, Anika
Zhang, Mei
Sauter, Daniel
Vetter, Irina
Andersson, Asa
Dussor, Gregory
Price, Theodore J.
Kolber, Benedict J.
Truong, Vincent
Walsh, Patrick
Lampert, Angelika
How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title_full How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title_fullStr How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title_full_unstemmed How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title_short How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
title_sort how to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635298/
https://www.ncbi.nlm.nih.gov/pubmed/37961300
http://dx.doi.org/10.21203/rs.3.rs-3127017/v1
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