Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids

Adhya, Dwaipayan ; Chennell, George ; Crowe, James A LU orcid ; Valencia-Alarcón, Eva P ; Seyforth, James ; Hosny, Neveen A ; Yasvoina, Marina V ; Forster, Robert ; Baron-Cohen, Simon and Vernon, Anthony C , et al. (2021) In Molecular Autism 12.
Abstract

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked... (More)

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.

RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.

CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; ; ; and , et al. (More)
; ; ; ; ; ; ; ; ; and (Less)
publishing date
type
Contribution to journal
publication status
published
keywords
Cell Culture Techniques, Cerebral Cortex/cytology, Fluorescent Antibody Technique, Humans, Induced Pluripotent Stem Cells/cytology, Microscopy/methods, Organoids, Spheroids, Cellular
in
Molecular Autism
volume
12
article number
4
publisher
BioMed Central (BMC)
external identifiers
  • scopus:85099923117
  • pmid:33482917
ISSN
2040-2392
DOI
10.1186/s13229-021-00413-1
language
English
LU publication?
no
id
43f6b82b-b0e5-45dc-badd-db6e649a7462
date added to LUP
2021-12-14 13:52:11
date last changed
2024-11-03 13:06:02
@article{43f6b82b-b0e5-45dc-badd-db6e649a7462,
  abstract     = {{<p>BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points.</p><p>RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes.</p><p>CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro.</p>}},
  author       = {{Adhya, Dwaipayan and Chennell, George and Crowe, James A and Valencia-Alarcón, Eva P and Seyforth, James and Hosny, Neveen A and Yasvoina, Marina V and Forster, Robert and Baron-Cohen, Simon and Vernon, Anthony C and Srivastava, Deepak P}},
  issn         = {{2040-2392}},
  keywords     = {{Cell Culture Techniques; Cerebral Cortex/cytology; Fluorescent Antibody Technique; Humans; Induced Pluripotent Stem Cells/cytology; Microscopy/methods; Organoids; Spheroids, Cellular}},
  language     = {{eng}},
  month        = {{01}},
  publisher    = {{BioMed Central (BMC)}},
  series       = {{Molecular Autism}},
  title        = {{Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids}},
  url          = {{http://dx.doi.org/10.1186/s13229-021-00413-1}},
  doi          = {{10.1186/s13229-021-00413-1}},
  volume       = {{12}},
  year         = {{2021}},
}