Fluorescence Imaging of Actin Turnover Parses Early Stem Cell Lineage Divergence and Senescence

Prakhar Mishra, Daniel C. Martin, Ioannis Androulakis, Prabhas Moghe

Research output: Contribution to journalArticle

Abstract

This study describes a new approach to discern early divergence in stem cell lineage progression via temporal dynamics of the cytoskeletal protein, F-actin. The approach involves real-time labeling of human mesenchymal stem cells (MSCs) and longitudinal tracking of the turnover dynamics of a fluorogenic F-actin specific probe, SiR-actin (SA). Cells cultured in media with distinct lineage factors and labeled with SA showed lineage specific reduction in the actin turnover shortly after adipogenic (few minutes) and chondrogenic (3–4 hours) commitment in contrast to osteogenic and basal cultured conditions. Next, composite staining of SA along with the competing F-actin specific fluorescent conjugate, phalloidin, and high-content image analysis of the complementary labels showed clear phenotypic parsing of the sub-populations as early as 1-hour post-induction across all three lineages. Lastly, the potential of SA-based actin turnover analysis to distinguish cellular aging was explored. In-vitro aged cells were found to have reduced actin turnover within 1-hour of simultaneous analysis in comparison to cells of earlier passage. In summary, SiR-actin fluorescent reporter imaging offers a new platform to sensitively monitor emergent lineage phenotypes during differentiation and aging and resolve some of the earliest evident differences in actin turnover dynamics.

Original languageEnglish (US)
Article number10377
JournalScientific reports
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2019

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Cell Aging
Optical Imaging
Cell Lineage
Actins
Stem Cells
Cell Tracking
Phalloidine
Cytoskeletal Proteins
Mesenchymal Stromal Cells
Cultured Cells
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • General

Cite this

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title = "Fluorescence Imaging of Actin Turnover Parses Early Stem Cell Lineage Divergence and Senescence",
abstract = "This study describes a new approach to discern early divergence in stem cell lineage progression via temporal dynamics of the cytoskeletal protein, F-actin. The approach involves real-time labeling of human mesenchymal stem cells (MSCs) and longitudinal tracking of the turnover dynamics of a fluorogenic F-actin specific probe, SiR-actin (SA). Cells cultured in media with distinct lineage factors and labeled with SA showed lineage specific reduction in the actin turnover shortly after adipogenic (few minutes) and chondrogenic (3–4 hours) commitment in contrast to osteogenic and basal cultured conditions. Next, composite staining of SA along with the competing F-actin specific fluorescent conjugate, phalloidin, and high-content image analysis of the complementary labels showed clear phenotypic parsing of the sub-populations as early as 1-hour post-induction across all three lineages. Lastly, the potential of SA-based actin turnover analysis to distinguish cellular aging was explored. In-vitro aged cells were found to have reduced actin turnover within 1-hour of simultaneous analysis in comparison to cells of earlier passage. In summary, SiR-actin fluorescent reporter imaging offers a new platform to sensitively monitor emergent lineage phenotypes during differentiation and aging and resolve some of the earliest evident differences in actin turnover dynamics.",
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Fluorescence Imaging of Actin Turnover Parses Early Stem Cell Lineage Divergence and Senescence. / Mishra, Prakhar; Martin, Daniel C.; Androulakis, Ioannis; Moghe, Prabhas.

In: Scientific reports, Vol. 9, No. 1, 10377, 01.12.2019.

Research output: Contribution to journalArticle

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