Real-time tracking of stem cell viability, proliferation, and differentiation with autonomous bioluminescence imaging.
Autobioluminescence
Bacterial luciferase
Bioimaging
Luciferase
Luciferin
Lux
MSC
Stem cells
iPSC
Journal
BMC biology
ISSN: 1741-7007
Titre abrégé: BMC Biol
Pays: England
ID NLM: 101190720
Informations de publication
Date de publication:
03 07 2020
03 07 2020
Historique:
received:
11
01
2020
accepted:
18
06
2020
entrez:
5
7
2020
pubmed:
6
7
2020
medline:
5
6
2021
Statut:
epublish
Résumé
Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20-30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.
Sections du résumé
BACKGROUND
Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous.
RESULTS
We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20-30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system.
CONCLUSIONS
Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.
Identifiants
pubmed: 32620121
doi: 10.1186/s12915-020-00815-2
pii: 10.1186/s12915-020-00815-2
pmc: PMC7333384
doi:
Substances chimiques
Luminescent Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
79Subventions
Organisme : NIEHS NIH HHS
ID : R44 ES026269
Pays : United States
Organisme : National Science Foundation
ID : 1530953
Pays : International
Organisme : NIGMS NIH HHS
ID : R42 GM116622
Pays : United States
Références
Stem Cells Transl Med. 2014 Jul;3(7):821-35
pubmed: 24833591
Mol Ther Methods Clin Dev. 2014 Nov 12;1:14053
pubmed: 26052521
Gene. 2009 Jan 1;428(1-2):20-4
pubmed: 18976699
Sci Transl Med. 2017 Feb 15;9(377):
pubmed: 28202772
Science. 1995 Jun 23;268(5218):1766-9
pubmed: 7792603
EMBO Rep. 2009 Nov;10(11):1259-64
pubmed: 19798101
Bioeng Bugs. 2011 Jan-Feb;2(1):8-16
pubmed: 21636983
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5672-6
pubmed: 8516317
Nat Methods. 2015 Jul;12(7):595-6
pubmed: 26125590
PLoS One. 2014 Oct 01;9(10):e107885
pubmed: 25271765
Eur J Nucl Med Mol Imaging. 2009 May;36(5):771-9
pubmed: 19096841
PLoS Genet. 2011 May;7(5):e1002085
pubmed: 21637780
Genesis. 2010 Jan;48(1):63-72
pubmed: 20014345
PLoS One. 2014 May 02;9(5):e96347
pubmed: 24788811
FEBS Lett. 2001 Oct 5;506(2):140-2
pubmed: 11591388
J Biomed Opt. 2011 Apr;16(4):047003
pubmed: 21529093
Microbiol Rev. 1991 Mar;55(1):123-42
pubmed: 2030669
Angew Chem Int Ed Engl. 2013 Jan 21;52(4):1175-9
pubmed: 23212783
Cell Stem Cell. 2008 Aug 7;3(2):196-206
pubmed: 18682241
J Biol Chem. 1990 Sep 25;265(27):16581-7
pubmed: 2204626
Infect Immun. 2000 Jun;68(6):3594-600
pubmed: 10816517
J Am Chem Soc. 2014 Sep 24;136(38):13277-82
pubmed: 25208457
Nat Protoc. 2013 Jan;8(1):162-75
pubmed: 23257984
Int J Stem Cells. 2008 Nov;1(1):1-7
pubmed: 24855503
PLoS One. 2010 Aug 27;5(8):e12441
pubmed: 20805991
Nucl Med Commun. 2003 Sep;24(9):1003-9
pubmed: 12960600
Proc Natl Acad Sci U S A. 2019 Dec 2;:
pubmed: 31792180
Proc Natl Acad Sci U S A. 2018 Jan 30;115(5):962-967
pubmed: 29339494
Proc Natl Acad Sci U S A. 2012 Jul 3;109(27):E1848-57
pubmed: 22645348
Proc Natl Acad Sci U S A. 2018 Dec 11;115(50):12728-12732
pubmed: 30478037