Graduate Seminar Series: Cell and Tissue Stream
Graduate Seminar Series for the Institute of Biomedical Engineering (BME). This day is for cell and tissue stream presenters.
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Presentation Title: Mechanical Control of Exosome Formation by Mesenchymal Stromal Cells
Rationale: Mesenchymal stromal cells (MSC) have vast potential as cell therapies to repair
hypertrophic scars due to their availability from donors, their capacity to regenerate damaged
tissues, and immune toleration by the host. However, expansion on conventionally stiff cell
culture surfaces mechanically activates MSCs into scar-forming myofibroblasts. Myofibroblast
activation also associates with loss of regenerative potential, reducing the effectiveness of MSC
in therapies. We found that growing human umbilical cord perivascular MSC on ‘skin-soft’ (E
modulus=5 kPa) silicone substrates suppresses myofibroblast activation, compared to MSC
grown on ‘scar-stiff’ (100 kPa) substrates. Transplanted onto hypertrophic rat skin wounds,
‘skin-soft’-grown MSC suppressed whereas ‘scar-stiff’-grown MSC fostered scarring after 9 d.
Because grafted human MSC were not detectable in rat wounds 4 d post-wounding, we set out to
study how mechanical environment controls MSC secretion of soluble factors, with a focus on
extracellular vesicles (EV).
Objective: To identify EV mediators produced of soft-grown MSC that suppress scarring.
Hypothesis: Mechanical environment of MSCs controls the biogenesis and content of exosomes.
Methods: Human umbilical cord MSC were directly isolated on ‘skin-soft’ and ‘scar-stiff’
substrates and expanded for 2 passages (P2). At P3, conditioned media were collected and
fractionated into soluble cytokines and EV, using phase separation assays and
ultracentrifugation. Differential expression of soluble cytokines was quantified using a Luminex
cytokine assay. EV fractions (microvesicles and exosomes) were analyzed for surface markers
(e.g., CD9, CD63, CD81, CD40) and size distributions using nano-particle resolution flow
cytometry, Western blotting, sub-diffraction microscopy, transmission electron microscopy, and
atomic force microscopy. MicroRNAs were quantified using nano-string analysis as signal-
carrying entities in EV. All data were normalized to the numbers of MSC producing the
respective factors and subjected to subsequent signalling pathway analysis.
Results: Flow cytometry, Western blotting and protein content quantification reveal overall
higher production of both, cytokines and exosomes by soft-grown versus stiff-grown MSC. The
ratios between exosomes and microvesicles in the EV fractions are affected by the mechanical
environment of the producing MSC. Expression patterns of soluble cytokines and proteins
contained in the EV fractions substantially differed between stiff- and soft-grown MSC.
Likewise, EV produced by either soft-or stiff-grown MSC contained microRNA sets
characteristic for the respective mechanical condition. In general, factors that are unique or
highly enriched in the secretomes obtained from soft- versus stiff-grown MSC are indicative of
inflammation- and scar-suppression.
Conclusion: Transplantation of MSC expanded in a tissue-soft mechanical environment is a
promising approach to improve the healing of large area wounds such as burns. However, for
specific therapeutic applications, delivery of their active trophic factors identified in our study
may prove beneficial and more effective.
Supervisor Name: Boris Hinz
Year of Study: 1
Program of Study: MASc
Zoom link: https://us02web.zoom.us/j/89610372821?pwd=azd4SCtYVWtreVovaGNPV1c2NGY2Zz09
Meeting ID: 896 1037 2821
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