(176h) Proteomic Analysis Reveals the Key Role of Integrated Stress Response in Restoring the Stemness of Culture Expanded Mesenchymal Stem Cells in 3D Aggregates
AIChE Annual Meeting
2019
2019 AIChE Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Poster Session: Engineering Fundamentals in Life Science
Monday, November 11, 2019 - 3:30pm to 5:00pm
Mesenchymal
stem cells (MSCs)
are the most commonly tested cells in cell therapy in a wide range of diseases
but culture-induced reduction in therapeutic potency is a major barrier in MSCs
translation. Aggregation of MSCs has been found to improve tissue repair and regeneration capability due to enhanced cell survival, improved cell adhesion and retention, and
increased secretory functions. Although
studies have suggested that actin-mediated contractility regulates properties
of MSCs aggregates, the underlying mechanism remains to be fully understood. In the current study, a proteomic approach is utilized to
investigate the differential expression of proteins to identify key pathways
that are altered during 3D aggregation. Based on label-free proteomics analysis, a
total of 246, 197, 195 differentially expressed proteins (DEPs) (fold change
≥ 2, p ≤ 0.05) were identified in bone marrow derived MSCs (BMSCs),
adipose derived MSCs (ASCs), and dermal fibroblasts (FB), respectively, when
analyzed between planar (2D) versus aggregates (3D). Gene oncology (GO) annotation of the DEPs in
all three cell types was found to be principally
associated with metabolic process, cellular component organization or
biogenesis, and biological regulation in biological process items. Meanwhile, these DEPs were mainly connected
to binding, catalytic activity, and structural molecule activity in the
molecular function categories. In
cellular component subsets, the DEPs were primarily related to cell, organelle,
and protein-containing complexes. While
there were many similarities in the GO analysis there were also distinct
differences such as: MSCs and FB contained proteins belonging to molecular
transducer and activity in the molecular function. Besides, multi-organism
process and synapse were enriched items in MSCs and FB, but not in ASCs,
indicating cell type-dependent mechanism. Canonical pathway investigation by Ingenuity Pathway Analysis
(IPA) revealed that EIF2 signaling, regulation of eIF4 and p70S6k signaling,
and mTOR signaling were among the top pathways significantly altered during
aggregation, suggesting aggregation-induced integrated stress response
(ISR). To further analyze the role of
ISR during aggregation, Western blot was carried out and showed that eIF2ꭤ
phosphorylation expression increased in aggregates of MSCs and ASCs but not in
FB, suggesting ISR’s differential activity in MSCs and ASCs versus FB. This is further confirmed by the increased
expression of stem cell genes SOX2 and NANOG and increased autophagy in MSC and
ASC aggregates in contrast to FB. The results
demonstrate that 3D aggregation induced acute ISR restores the stemness of MSCs
and ASCs through eIF2ꭤ phosphorylation and the absence of such regulatory
mechanism in FB. Together, the results
reveal a novel mechanism and the key regulatory role of EIF2 in maintaining
MSCs/ASCs homeostasis and stemness.
stem cells (MSCs)
are the most commonly tested cells in cell therapy in a wide range of diseases
but culture-induced reduction in therapeutic potency is a major barrier in MSCs
translation. Aggregation of MSCs has been found to improve tissue repair and regeneration capability due to enhanced cell survival, improved cell adhesion and retention, and
increased secretory functions. Although
studies have suggested that actin-mediated contractility regulates properties
of MSCs aggregates, the underlying mechanism remains to be fully understood. In the current study, a proteomic approach is utilized to
investigate the differential expression of proteins to identify key pathways
that are altered during 3D aggregation. Based on label-free proteomics analysis, a
total of 246, 197, 195 differentially expressed proteins (DEPs) (fold change
≥ 2, p ≤ 0.05) were identified in bone marrow derived MSCs (BMSCs),
adipose derived MSCs (ASCs), and dermal fibroblasts (FB), respectively, when
analyzed between planar (2D) versus aggregates (3D). Gene oncology (GO) annotation of the DEPs in
all three cell types was found to be principally
associated with metabolic process, cellular component organization or
biogenesis, and biological regulation in biological process items. Meanwhile, these DEPs were mainly connected
to binding, catalytic activity, and structural molecule activity in the
molecular function categories. In
cellular component subsets, the DEPs were primarily related to cell, organelle,
and protein-containing complexes. While
there were many similarities in the GO analysis there were also distinct
differences such as: MSCs and FB contained proteins belonging to molecular
transducer and activity in the molecular function. Besides, multi-organism
process and synapse were enriched items in MSCs and FB, but not in ASCs,
indicating cell type-dependent mechanism. Canonical pathway investigation by Ingenuity Pathway Analysis
(IPA) revealed that EIF2 signaling, regulation of eIF4 and p70S6k signaling,
and mTOR signaling were among the top pathways significantly altered during
aggregation, suggesting aggregation-induced integrated stress response
(ISR). To further analyze the role of
ISR during aggregation, Western blot was carried out and showed that eIF2ꭤ
phosphorylation expression increased in aggregates of MSCs and ASCs but not in
FB, suggesting ISR’s differential activity in MSCs and ASCs versus FB. This is further confirmed by the increased
expression of stem cell genes SOX2 and NANOG and increased autophagy in MSC and
ASC aggregates in contrast to FB. The results
demonstrate that 3D aggregation induced acute ISR restores the stemness of MSCs
and ASCs through eIF2ꭤ phosphorylation and the absence of such regulatory
mechanism in FB. Together, the results
reveal a novel mechanism and the key regulatory role of EIF2 in maintaining
MSCs/ASCs homeostasis and stemness.