Skip to main content

Lund University Publications

LUND UNIVERSITY LIBRARIES

NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities

Tolomeo, Anna Maria ; Laterza, Cecilia LU ; Grespan, Eleonora ; Michielin, Federica ; Canals, Isaac LU ; Kokaia, Zaal LU orcid ; Muraca, Maurizio ; Gagliano, Onelia and Elvassore, Nicola (2021) In Frontiers in Cellular Neuroscience 15.
Abstract

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based... (More)

Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 βIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 βIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.

(Less)
Please use this url to cite or link to this publication:
author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
induced neurons, microfluidics, mmRNA, NGN2, transcriptional programming
in
Frontiers in Cellular Neuroscience
volume
15
article number
602888
publisher
Frontiers Media S. A.
external identifiers
  • scopus:85101878038
  • pmid:33679325
ISSN
1662-5102
DOI
10.3389/fncel.2021.602888
language
English
LU publication?
yes
id
983e4d61-91cb-4ab5-9a28-ba6c034e797d
date added to LUP
2022-03-21 17:15:23
date last changed
2024-06-19 09:16:58
@article{983e4d61-91cb-4ab5-9a28-ba6c034e797d,
  abstract     = {{<p>Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 βIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 βIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.</p>}},
  author       = {{Tolomeo, Anna Maria and Laterza, Cecilia and Grespan, Eleonora and Michielin, Federica and Canals, Isaac and Kokaia, Zaal and Muraca, Maurizio and Gagliano, Onelia and Elvassore, Nicola}},
  issn         = {{1662-5102}},
  keywords     = {{induced neurons; microfluidics; mmRNA; NGN2; transcriptional programming}},
  language     = {{eng}},
  month        = {{02}},
  publisher    = {{Frontiers Media S. A.}},
  series       = {{Frontiers in Cellular Neuroscience}},
  title        = {{NGN2 mmRNA-Based Transcriptional Programming in Microfluidic Guides hiPSCs Toward Neural Fate With Multiple Identities}},
  url          = {{http://dx.doi.org/10.3389/fncel.2021.602888}},
  doi          = {{10.3389/fncel.2021.602888}},
  volume       = {{15}},
  year         = {{2021}},
}