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Fluorescence imaging based analysis of actin turnover: longitudinal profiling of stem cell phenotypes during differentiation

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TitleInfo
Title
Fluorescence imaging based analysis of actin turnover: longitudinal profiling of stem cell phenotypes during differentiation
Name (type = personal)
NamePart (type = family)
Mishra
NamePart (type = given)
Prakhar
NamePart (type = date)
1987-
DisplayForm
Prakhar Mishra
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Moghe
NamePart (type = given)
Prabhas
DisplayForm
Prabhas Moghe
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
School of Graduate Studies
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
Genre (authority = ExL-Esploro)
ETD doctoral
OriginInfo
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2020
DateOther (type = degree); (qualifier = exact); (encoding = w3cdtf)
2020-10
Language
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English
Abstract (type = abstract)
Stem cells derived from adults have become the focal point of cell-based regenerative therapies. The most commonly used stem cells, mesenchymal stem cells (abbreviated MSCs), have several traits such as the ability to migrate to injury site, immunomodulation, in vitro expansion and differentiation into a variety of cells. However, the widespread usage of MSCs is hampered by poor characterization, tissue culture induced senescence and innate heterogeneity that makes it challenging to predict their clinical efficacy. The traditional methods for evaluation of stem cells are primarily terminal assays that rely on discrete time-point data sets that offer limited, discontinuous information and often overlook the intrinsic phenotypic diversity of these cells. In this thesis, we address some of these issues by developing a novel platform for live cell imaging which can be used to monitor evolving MSCs by generating continuous data sets over the course of several hours or several days while sparing the cells for multiplexing with other traditional assays. Our imaging-based approach relies on a cell permeable fluorogenic probe, SiR-actin (SA), that becomes fluorescent only when it labels endogenous actin filaments (F-actin). The idea of harnessing actin as a sentinel stem cell reporter is supported from past research conducted by Moghe lab and numerous other research groups, as the cytoskeleton can modulate and display the phenotypic changes associated with lineage commitment. Unlike previous research that focused primarily on morphology of actin cytoskeleton, we focused on utilizing the shifting actin turnover rates in response to extracellular cues as the key reporter metric. After SA labelling, actin reorganization leads to removal of the SA probe from F-actin binding sites and subsequent decline in SA fluorescence. When cells are cultured in differentiation media, the rate of fluorescence loss of SA provided insights about the kinetics of lineage specific change in actin reorganization. We report that initiation of differentiation involves decline in actin turnover during adipogenesis and chondrogenesis within few hours. By combining SA with another F-actin probe, phalloidin, we were able to parse heterogenous single cells across the standard tri-lineages (adipogenic, osteogenic and chondrogenic) within 1 hour of stimulation. In addition, our approach enabled assessment of in vitro aging by demonstrating a slowdown in actin turnover within 1 hour of analysis. Next, to establish the link between actin turnover and stem cell differentiation, we employed immunolabeling to demonstrate co-occurrence of altered actin turnover with differentiation markers in MSCs as well induced pluripotent stem cells (iPSCs). We also propose that inherent actin turnover status of individual cells could be a determinant of their differentiation potential. To this end, we isolated cells based on differential SA expression and found that the actin turnover plays a role in determining the ability to differentiate towards osteogenic or adipogenic fate. In summary, we propose actin turnover as a novel dynamic marker as it provides immediate readouts of changing cell states that could be utilized to forecast stem cell behavior towards regenerative therapies.
Subject (authority = local)
Topic
Mesenchymal stem cells
Subject (authority = RUETD)
Topic
Cell and Developmental Biology
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
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ETD_11195
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application/pdf
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Extent
1 online resource (i, 171 pages) : illustrations
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
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TitleInfo
Title
School of Graduate Studies Electronic Theses and Dissertations
Identifier (type = local)
rucore10001600001
Location
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NjNbRU
Identifier (type = doi)
doi:10.7282/t3-tt7h-1f82
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RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Mishra
GivenName
Prakhar
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2020-09-24 09:45:03
AssociatedEntity
Name
Prakhar Mishra
Role
Copyright holder
Affiliation
Rutgers University. School of Graduate Studies
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License
Name
Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
Copyright
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Copyright protected
Availability
Status
Open
Reason
Permission or license
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DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2020-09-24T09:43:25
DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2020-09-24T09:43:25
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