Enforced mesenchymal stem cell tissue colonization counteracts immunopathology

Mesenchymal stem/stromal cells (MSCs) are distributed within all tissues of the body. Though best known for generating connective tissue and bone, these cells also display immunoregulatory properties. A greater understanding of MSC cell biology is urgently needed because culture-expanded MSCs are increasingly being used in treatment of inflammatory conditions, especially life-threatening immune diseases. While studies in vitro provide abundant evidence of their immunomodulatory capacity, it is unknown whether tissue colonization of MSCs is critical to their ability to dampen/counteract evolving immunopathology in vivo. To address this question, we employed a murine model of fulminant immune-mediated inflammation, acute graft-versus-host disease (aGvHD), provoked by donor splenocyte-enriched full MHC-mismatched hematopoietic stem cell transplant. aGvHD induced the expression of E-selectin within lesional endothelial beds, and tissue-specific recruitment of systemically administered host-derived MSCs was achieved by enforced expression of HCELL, a CD44 glycoform that is a potent E-selectin ligand. Compared to mice receiving HCELL− MSCs, recipients of HCELL+ MSCs had increased MSC intercalation within aGvHD-affected site(s), decreased leukocyte infiltrates, lower systemic inflammatory cytokine levels, superior tissue preservation, and markedly improved survival. Mechanistic studies reveal that ligation of HCELL/CD44 on the MSC surface markedly potentiates MSC immunomodulatory activity by inducing MSC secretion of a variety of potent immunoregulatory molecules, including IL-10. These findings indicate that MSCs counteract immunopathology in situ, and highlight a role for CD44 engagement in unleashing MSC immunobiologic properties that maintain/establish tissue immunohomeostasis.


Statistics
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Sample size
To calculate sample size in survival analysis (log-rank test) we define a type I error (alpha)= 0.05, a type error (beta)= 0.20, an hypothesized survival rate in group 1 (i.e FucmAdMSCs-treated-group)=0.8, an hypothesized survival rate in group 2 (i.e UmAdMSCs-treated group)=0.4, and a ratio of sample sizes in group 1/group 2= 1 (equal sample size in both groups), obtaining a total of n=27 animals/group. With only n=16 animals/group, a similar number of animals used in other previously reported studies using the same GvHD model, we obtain a p=0.0198 when compared survival of both treatment groups (FucAdMSCs vs UmAdMSCs-treated groups). Number of animals was not finally augmented in order to safeguard the animal welfare (3R principles). For all other experiments we used a minimum of n=3 replicates.
Data exclusions No data were excluded from the analyses.

Replication
We confirmed the reproducibility of data by independently repeating all of the experiments at least three times. All attempts at replication were successful.
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Blinding
Histopathological analyses were performed by a single pathologist blinded to the treatment groups using coded slices. Survival and GvHD clinical scores were also monitored by researchers in a blind fashion.
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Validation
Antibodies used for flow cytometry experiments were previously validated on murine AdMSCs, and the obtained percentages corresponded well to the expected values. Immunohistochemistry antibodies were also previously validated on appropriate fixed paraffin-embedded mouse tissue sections.

nature research | reporting summary
October 2018 Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used.

Animals and other organisms
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This study did not involve wild animals.

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Ethics oversight
All animal procedures were approved by the Institutional Animal Care and Use Committee at University of Murcia and performed according to the guidelines of our institution (approved protocol A13150201). The Institutional Review Board of the University Hospital Virgen de la Arrixaca (Murcia, Spain) approved the protocols used to obtain and process all human samples. As needed, written informed consent was obtained from donors as per Helsinki Declaration guidelines.
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Flow Cytometry
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