Development of a standardised methodology for the isolation and culture of murine mesenchymal stromal cells from different tissues and comparison of their migratory, leukocyte recruitment and immunomodulatory potential in vivo

Cuesta Gomez, Nerea (2020) Development of a standardised methodology for the isolation and culture of murine mesenchymal stromal cells from different tissues and comparison of their migratory, leukocyte recruitment and immunomodulatory potential in vivo. PhD thesis, University of Glasgow.

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Abstract

Since the discovery of mesenchymal stromal cells (MSCs) ability to repair craniofacial defects and immunomodulatory properties, they became focus of research due to their cellular therapeutic potential. The Advanced Therapeutics Department within the Scottish National Blood Transfusion Service (SNBTS) has developed standardised methodologies for the isolation of MSCs from pancreatic islets, visceral adipose tissue, liposuction aspirate, bone marrow and umbilical cord. However, studying and comparing their in vivo function and the immunomodulatory potential is essential prior to their use within a clinical setting. The immunomodulatory abilities of human MSCs have been studied using mouse models that lack a control for mismatched major histocompatibility complex molecule expression. For this reason, this study aimed to objectively compare the phenotype and potential immunomodulatory functions in vivo of murine MSCs isolated from the bone marrow (BM MSCs), islets of Langerhans (Is MSCs) and adipose tissue (Ad MSCs) in a stringent, standardised manner, without any species or gender mismatch that could lead to both cell-mediated and humoral immune responses.

First, for use within a clinical settings MSCs need to be infused and home and engraft into the target tissue. However, most cells get entrapped in the lung and only a small percentage home and remain in the target tissue. Size could be essential to avoid lung entrapment and this study has described that murine BM MSCs are slightly smaller than Is and Ad MSCs, suggesting that they could be a better source of MSC if being delivered intravenously. Moreover, chemokine receptor expression targets immune cells into specific tissues. Comparison of chemokine receptor transcription showed that Ad MSCs have a greater transcription of CXCR4 combined with a very low transcription of other chemokine receptors, suggesting that they will more likely suffer from lung entrapment compared to BM and Is MSCs. BM MSCs have the highest transcription of CCR7 and CXCR6, and therefore, we hypothesise that BM MSCs will be more successful to reach lymphoid organs. Is MSCs have a greater potential to migrate towards the kidneys due to higher transcription of CCR1 and CXCR3. Ad MSCs, on the contrary, transcribed statistically significantly more CCR3 than Is and BM MSCs and therefore, they have a greater potential to migrate towards the skin if they avoid entrapment within lungs.

Under resting conditions, MSCs from the three tissue sources were able to secrete chemokines at similar levels; however, this secretion did not produce a recruitment of leukocytes above control levels. Inflammatory stimulation led to increased secretion of chemokines where Is MSCs secreted the highest levels of CCL2, CCL5, CXCL1 and CXCL10, while CXCL12 was secreted at higher levels by BM MSCs. CCL2, CCL5 and CXCL1 are strong chemoattractans, but despite the higher secretion by Is MSCs under inflammatory conditions, Ad MSCs were able to recruit significantly more leukocytes in vivo than BM and Is MSCs. More importantly, Ad MSCs were the only MSCs able to produce the recruitment of T cells. Recipient cytotoxic cells are considered detrimental in clinical settings, but they are essential to initiate MSC-mediated immunosuppression; thus, we could hypothesise that Ad MSCs have a greater immunosuppression potential than BM and Is MSCs.

Chemokines not only have leukocyte recruitment properties, as examples, CXCL1, CXCL2 and CXCL12 have angiogenic properties while CXCL10 has angiostatic potential. Chemokines are not the only molecules secreted by MSCs with the potential to regulate angiogenesis. Ad MSCs secreted the most IL-6, which can promote VEGF secretion, but Is MSCs secreted the most VEGF under resting and stimulatory conditions, which combined with the increased secretion of CXCL1, CXCL2 and CXCL12, we hypothesise that Is MSCs could have greater re-vascularisation potential.

Altogether, this study highlights that MSCs from different sources differ in their ability to recruit and immunomodulate surrounding immune cells in vivo. These differences have the potential to influence their clinical performance.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Chemokines, Mesenchymal stromal cells, Cell therapy
Subjects: Q Science > QR Microbiology > QR180 Immunology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation > Immunology
Funder's Name: Scottish National Blood Transfusion Service (SNBTS)
Supervisor's Name: Graham, Prof Gerard and John, Prof Campbell
Date of Award: 2020
Depositing User: Nerea Cuesta Gomez
Unique ID: glathesis:2020-81738
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 20 Oct 2020 11:18
Last Modified: 21 Oct 2020 09:15
URI: http://theses.gla.ac.uk/id/eprint/81738

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