We are currently investigating the use of human mesenchymal stromal cells (MSCs) for tissue repair by injecting MSCs into the damaged organ. Recent discoveries indicate that many of the therapeutic benefits of MSCs can be attributed to secretion of various biomolecules which can be secreted via exosomes, small membrane vesicles of endocytic origin. Most cell types secrete exosomes which contain proteins, DNA, mRNA, and microRNA, all of which which are thought to play a role in cell-cell communication. While previous research efforts largely focused on the characterization of proteins found in exosomes, our current research focuses on exosomal RNAs. Increasing evidence suggests that exosome formation and release are regulated by the autophagy pathway, a homeostatic quality control pathway that recycles proteins and organelles via recognition, sequestration, and lysosomal degradation. Conditions that stimulate autophagy pathway can inhibit exosome release, but at the same time pharmacological inhibitors of autophagy enhance the release of exosomes. For our study, we propose utilizing a subtype of MSCs called ?marrow-isolated adult multilineage inducible? (MIAMI) cells due to their ease of isolation from bone marrow, differentiation capacity, their immunomodulatory and tissue repair capacities, and ability to secrete various chemokines/growth factors. We hypothesize that autophagy mediates release of exosomes from MIAMI cells, regulates their RNA content and their immunomodulatory capacity. To stimulate MIAMI cells, we will expose them to inflammatory response stimulator, IFN?, while simultaneously applying pharmacologic stimulators or inhibitors of autophagy. Subsequently, we will isolate and characterize MIAMI cell-derived exosomes by using NanoSight, electron microscopy and immunoblotting to characterize and compare exosomes size distributions, exosome yield and markers (CD9, CD63 and CD81) (Aim 1). We will then determine how modulation of autophagy regulates exosomal RNA content by identifying and validating long (more than 200 nucleotides) and short (less than 200 nucleotides) RNAs in MIAMI cells-derived exosomes (Aim 2). Lastly, we will evaluate immunoregulatory properties of MIAMI cells upon modulation of autophagy using MIAMI cell-T cell co-cultures and subsequent flow cytometry analysis to assess T cell markers of proliferation and cytokine secretion (Aim 3). Results of this mechanistic study will increase our understanding of the role that autophagy plays in regulating RNA content in exosomes and it will also reveal whether targeting autophagy could be used to manipulate RNA content and subsequently immunomodulation. Ultimately, such knowledge is anticipated to foster further development of cell therapies for tissue regeneration. 1

Public Health Relevance

The development of stem cells-based therapies for tissue regeneration is an important goal that seeks to improve tissue function using innovative approaches. This project will explore the role of autophagy, a cell self-digestion pathway, in regulating therapeutic biomolecules secreted by stem cells. Altogether, the proposed project will contribute to understanding how autophagy affects therapeutic properties of stem cells with an ultimate goal of improving their therapeutic properties.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM128189-01
Application #
9516549
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Maas, Stefan
Project Start
2018-07-01
Project End
2021-06-30
Budget Start
2018-07-01
Budget End
2021-06-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Nova Southeastern University
Department
Other Health Professions
Type
Sch Allied Health Professions
DUNS #
002971240
City
Fort Lauderdale
State
FL
Country
United States
Zip Code
33314