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. 2016 Jan 5;113(1):170-5.
doi: 10.1073/pnas.1522297113. Epub 2015 Dec 22.

Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI

Dong-ki Kim  1 , Hidetaka Nishida  1 , Su Yeon An  1 , Ashok K Shetty  1 , Thomas J Bartosh  1 , Darwin J Prockop  2
Affiliations

Chromatographically isolated CD63+CD81+ extracellular vesicles from mesenchymal stromal cells rescue cognitive impairments after TBI

Dong-ki Kim et al. Proc Natl Acad Sci U S A. .

Abstract

Extracellular vesicles (EVs) secreted by cells present an attractive strategy for developing new therapies, but progress in the field is limited by several issues: The quality of the EVs varies with the type and physiological status of the producer cells; protocols used to isolate the EVs are difficult to scale up; and assays for efficacy are difficult to develop. In the present report, we have addressed these issues by using human mesenchymal stem/stromal cells (MSCs) that produce EVs when incubated in a protein-free medium, preselecting the preparations of MSCs with a biomarker for their potency in modulating inflammation, incubating the cells in a chemically defined protein-free medium that provided a stable environment, isolating the EVs with a scalable chromatographic procedure, and developing an in vivo assay for efficacy of the cells in suppressing neuroinflammation after traumatic brain injury (TBI) in mice. In addition, we demonstrate that i.v. infusion of the isolated EVs shortly after induction of TBI rescued pattern separation and spatial learning impairments 1 mo later.

Keywords: MSCs; efficacy assay; exosomes; neuroinflammation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Defining conditions for production of EVs. Cultures of human MSCs (donor 6015) at 70–80% confluent were transferred to the media indicated and incubated for 6–48 h. (A) Expression of mRNA for CD63 was increased by culture for 48 h in CDPF medium compared with culture in CCM with standard concentration of FBS (16.6%) or reduced FBS (2%). Assay by RT-PCR. (B) Expression of mRNA for CD63 was increased with time of incubation in CDPF. (C) Secretion of CD63+ was increased with time in CDPF. Medium was assayed by ELISA for vesicle-bound protein (Fig. S1). (D) RT-PCR assays in MSCs incubated in CDPF indicated that the proinflammatory cytokine IL-1β was expressed for up to 6 h and that expression of the inflammation-modulating protein TSG-6 increased between 2 and 24 h. Expression of the antiapoptotic/calcium-phosphate metabolic protein STC-1 peaked at about 6 h. (E) Expression of mRNA for TSG-6 increased with time of incubation in CDPF. (F) Secretion of TSG-6 increased with time in CDPF. Assay by ELISA. (G) Schematic for protocol developed for production of EVs by MSCs.
Fig. 2.
Fig. 2.
Survival of the MSCs under the culture conditions, comparisons of four donors, and demonstration that most of the secreted proteins and EVs are anionic. (A) Survival of MSCs in CDPF. MSCs were expanded to about 70% confluence and then incubated an additional 48 h in CCM, CDPF, or PBS. (Top) Cultures labeled with Hoechst, Calcein AM, and propidium iodide (PI) demonstrate viable cells in CDPF but not PBS. (Bottom) Assays of the same cultures by flow cytometry after labeling with PI and Annexin V demonstrated survival in CCM or CDPF medium but not PBS. (B) Comparisons of four different preparations of MSCs (donors 235, 6015, 7052 and 7074) after incubation as in Fig. 1G. (i) CD63+ in medium assayed by ELISA. (ii) TSG-6 in medium assayed by ELISA. (C) Small-scale assays in SDS-electrophoretic gels demonstrated that most of the medium proteins bound to and were eluted from an anionic resin and not a cationic resin. Gels were stained with silver.
Fig. S1.
Fig. S1.
ELISA for CD63+ proteins on EVs. (A) Schematic of the assay. (B) Standard curve prepared with varying amounts of protein from pooled fractions of column (Fig. 3 A, ii). Assays by nanoparticle diffusion analysis indicated 1 µg = 0.51 × 109 EVs.
Fig. 3.
Fig. 3.
Chromatographic isolation and characterization of EVs from the medium. (A) Preparation and characterization of CD63+ EVs from medium of MSCs incubated as in Fig. 1G. (i) Assay by SDS-electrophoretic gel of medium eluted from anion exchange column with 0.5 M NaCl. Gel was silver-stained. (ii) Assays of eluted fractions for protein and CD63. (B) Recovery of CD63+ protein from the column was slightly greater than recovery by centrifuging the same samples at 100,000 × g for 12 h. (C) Assays of eluted fractions by nanoparticle diffusion analysis demonstrated that the mean size of the vesicles ranged from 209 ± 1.8 nm (SEM) to 231 ± 3.2 nm. The three peaks at the lower concentration (ii) were 85, 165, and 236 nm. (Insets) Photos of nanoparticles in the instrument.
Fig. S2.
Fig. S2.
Assays of epitopes on the isolated EVs. The EVs were trapped on a magnetic bead covalently linked to anti-CD63 and then assayed for additional epitopes by flow cytometry (35). Data were gated for single beads. (Left) Detection of CD63 as a function of the concentration of protein added. In the remaining panels, EVs were positive for CD63 and CD81 but not for CD9.
Fig. 4.
Fig. 4.
Dose–response data for suppression of neuroinflammation by EVs after TBI. (A) Immunochemistry of brain sections demonstrated that TBI increased IL-1β in GFAP+ astrocytes. Sections from brain recovered 12 h after TBI and sections from region indicated were stained for DAPI, IL-1β, and GFAP. (B) Dose-dependent decrease in IL-1β after i.v. administration of PBS or EVs. Amounts varied from 3.5 to 30 µg of protein or 1.8–15.3 × 109 EVs. PBS or EVs were administered 1 h after TBI, and assays were by ELISA on homogenates of ipsilateral brain isolated 12 h after TBI. The i.v. administration of 1 million MSCs cultured in CCM had little effect, apparently because they were not fully activated in 12 h to express TSG-6 by embolization of the lung (21).
Fig. S3.
Fig. S3.
Plasma EVs after i.v. injection of EVs or MSCs from two donors into C57BL/6J mice. (A) Schematic of experiment. (B) ELISAs for CD63+ EVs in plasma. ELISAs were performed as in Fig. S1. n = 3 mice for each condition.
Fig. S4.
Fig. S4.
ELISAs for cytokines after TBI. (A) Brain levels of IL-1β and IL-6 in ipsilateral brain 6 h to 7 d after TBI. n = 4 mice for each time point. (B) Schematic for the experiment in C and D. (C) IL-6 in brain 12 h after TBI and i.v. PBS or 30 µg of protein from pooled fraction from Fig. 3 A, ii. (D) Plasma levels of IL-10.
Fig. 5.
Fig. 5.
Improved cognitive function after TBI and i.v. EVs. About 1 h after TBI, each mouse received i.v. PBS or 30 µg of protein (about 15.3 × 109 EVs) from the pooled peak from the anion exchange column (pooled CM-Q) (Fig. 3 A, ii). Behavior in the water maze was tested 28–33 d after TBI. The pattern separation test was performed 35 d after TBI. (A) In the water maze, treated mice with TBI learned to locate the hidden platform with the same latency as controls after four trials and better than TBI mice that received PBS. There was no significant effect of the therapy on the number of entries to platform zone (B) or time spent in the platform zone (C) in the probe test. (D) The treated mice performed better than TBI mice that received PBS in the pattern separation test.
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References

    1. Maas AI, Stocchetti N, Bullock R. Moderate and severe traumatic brain injury in adults. Lancet Neurol. 2008;7(8):728–741. - PubMed
    1. Heneka MT, Kummer MP, Latz E. Innate immune activation in neurodegenerative disease. Nat Rev Immunol. 2014;14(7):463–477. - PubMed
    1. Lozano D, et al. Neuroinflammatory responses to traumatic brain injury: Etiology, clinical consequences, and therapeutic opportunities. Neuropsychiatr Dis Treat. 2015;11:97–106. - PMC - PubMed
    1. Reis C, et al. What’s new in traumatic brain injury: Update on tracking, monitoring and treatment. Int J Mol Sci. 2015;16(6):11903–11965. - PMC - PubMed
    1. Loane DJ, Stoica BA, Faden AI. Neuroprotection for traumatic brain injury. Handb Clin Neurol. 2015;127:343–366. - PMC - PubMed

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