Comparison of Exosomes Secreted by Culture‐Expanded Annulus Fibrosus, Nucleus Pulposus, and Bone Marrow Stromal Cells for Intervertebral Disc Repair

Abstract

Exosomes have emerged as an alternative to cell therapies in many fields. Bone marrow stromal cells (MSCs) have been almost exclusively used to generate exosomes as progenitor cells are known to rapidly generate exosomes with a high therapeutic potential. Progenitor cells can be obtained from different connective tissues, and it has been shown that the therapeutic potential of exosomal cargo differs among tissues. We postulate the most efficacious exosomes are produced by progenitor cells derived from the tissue that is to be treated. Our long‐term goal is to develop exosome therapies for treatment of intervertebral disc degeneration. The intervertebral disc is made up of two regions that can experience degradation, the outer annulus fibrosus and inner nucleus pulposus. Therefore, the objective of this study was to compare exosomes from MSCs to exosomes from annulus fibrosus and nucleus pulposus cells that adopt progenitor cell properties with culture‐expansion. Bone marrow MSCs, annulus fibrosus, and nucleus pulposus cells were isolated from sheep and culture expanded. Over 20 days of expansion, all cell types grew in a steady exponential fashion for around 28 population doublings and expressed progenitor cell CD markers. Secreted exosomes were purified by ultracentrifugation and identified by transmission electron microscopy and western blot for exosomal proteins. The size and number of exosomes produced was analyzed by nanoparticle tracking analysis. All cell types secreted exosomes that were not significantly different in size or number. MSCs, annulus fibrosus and nucleus pulposus cells all took up exosomes from each cell type. This data confirms that manufacturing of functional exosomes is comparable among these cell types. Exosomal content is currently being analyzed to identify differences among cell types and includes total proteome by protein mass spectrometry, as well as RNA and mi‐RNA content through RNA sequencing.Support or Funding InformationColorado State University Orthopaedic Bioengineering Research Laboratory