The underlying mechanisms of mesenchymal stem cell migration in ischemic stroke patients

Anastassia Kostenko

U1145373

Supervisor: Dr. Mohammed Meah

Biomedical Science

School of Health, Sport and Bioscience

Contents

Chapters 1. Aknowledgemens..............................................................	Page number 2
2. Abstract .........................................................................	3
3. Chapter 1 - Introduction ................................................	4
4. Chapter 2 - Methods ......................................................	5
5. Chapter 3 - What is stroke ..............................................	6-10
6. Available treatments for stroke .......................................	10-12
7. Chapter 4 - Stem Cells ...................................................	13-14
8. Embryonic stem cells ......................................................	13
9. Adult stem cells ..............................................................	13-14
10. Induced pluripotent stem cells ......................................	14
11. Mesenchymal stem cells ...............................................	15
12. MSCs for treatment of stroke ........................................	16-17
13. Chapter 5 - Migratory mechanisms of MSCs....................	18-21
14. Chapter 6 - Discussion .................................................	22-24
15. Conclusion .................................................................	25
16. References ....................................................................	26

Abstract

Stroke is a major cause of death and disability worldwide. Currently available treatments are aimed at minimising damage after stroke and prevention of recurring stroke episodes, rather than neuroregeneration. Many studies have suggested that mesenchymal stem cells (MSC) have the ability to differentiate into a variety of tissue types and promote functional recovery in stroke patients via immunomodulatory and trophic factor production.

The mechanisms that induce MSC transendothelial migration towards inflamed and damaged regions are not fully understood. This research project aimed to elucidate underlying migratory mechanisms of MSCs.

Currently known mechanisms are passive or active homing. Passive homing results in MSC entrapment in narrow blood vessels. Active homing shows similarity to leukocyte migratory mechanisms. It suggests that MSCs travel within the blood vessels following a chemokine gradient produced by damaged tissues. MSC chemoattractants include: SDF-1, CXCL3-16, XCL1-2, CX3CL1, CCL1-5. The following cytokines and growth factors are involved in MSC trafficking: PDGF-BB, PDGF-AB, TGF-β1, TNF-α, VEGF. Integrins (VLA-4, α6β1, α8β1, α9β1), adhesions (VCAM-1, ICAM-1, CD44, PECAM, CD105) and selectins (P-selectin, E-selectin) are involved in MSC rolling and transendothelial migration. Tissue invasion is mediated by matrix metalloproteinases (MMP-1 and 2). Receptors expressed by MSCs include: CXCR1-6, CX3CR1, CCR1-9, TLR3, 4, 9.

This research shows that there is a vast heterogeneity of MSC phenotypes, as different MSCs express a variety of receptors. Studies show that priming and retroviral transduction in vitro can increase MSC homing. The lack of universally available MSC characteristics requires more in depth research into molecular signalling pathways underlying MSC transfer within the blood vessels, penetration through the vascular endothelium and tissue invasion.

Chapter 1 – Introduction

Every year, 15 million people worldwide suffer a stroke. Nearly six million die and another five million are left permanently disabled (World Heart Federation., 2014).

Stroke results from a rapid cessation in adequate amount of blood supply reaching sections of the brain. Strokes can be ischaemic (hypoxia induced neuronal death due to vascular obstruction) or haemorrhagic (caused by intracerebral bleeds). Presently available therapies are: anti-coagulants (prevent blood clotting), thrombolysis (removes blood clots by enzymatic digestion) and thrombectomy (surgical removal of the blood clot). However, due to the associated risk of increased intracranial bleeding and the narrow time window, the efficacy of available treatments is limited.

Therefore, alternative novel stem cell therapies are heavily investigated. There are three major types of stem cells, which include: embryonic stem cells, adult stem cells and induced pluripotent stem cells. The following chapters (3 and 4) elaborate more on stroke and stem cells.

Mesenchymal stem cells (MSC) fall into the adult stem cell category and are investigated in particular. This is because they can be obtained fairly effortlessly from multiple sites, such as: bone marrow, adipose tissue, umbilical cord, and umbilical cord blood. MSCs are easy to culture in vitro up to significant amounts, without ethical concerns. Also, due to the lack of alloreactive T lymphocyte production response MSCs are suitable for transplantation between HLA incompatible individuals (Le Blanc et al., 2003).

First clinical trials have confirmed improved functional recovery in stroke patients after MSC delivery (Bang et al., 2005). Numerous animal studies, such as those done by: Tsai et al., (2014) and Lindvall et al., (2004) support this statement.

Benefits of MSCs in post-ischaemic brain are mediated by trophic, immunomodulatory and antimicrobial molecules produced by MSCs in response to the cytokines and chemokines secreted by the damaged tissues (Table 4). Table 5 further elaborates on exact beneficial neuroprotective and neuroregenerative effects of MSCs in the brain of patients who suffered a stroke. The subchapter: MSCs for treatment of stroke provides more information on this topic (Chapter 4).

Although many molecules mediating MSC migration have been identified (Table 6), unfortunately, the exact mechanisms of MSC migration through the blood vessels and stromal tissues towards the site of injury still remain unclear (Doeppner et al., 2010). It is essential to understand the molecular mechanisms underlying MSC transfer from the infusion site to the damaged region in order to create functional MSC treatment for stroke.

Ruster et al., (2006) provide support for active mechanism of MSC migration. They state that MSCs adhere to P-selectins on endothelial cells. This adhesion is mediated by very late antigen-4 (VLA-4) on MSC surface and vascular cell adhesion molecule-1 (VCAM-1), expressed on endothelial cell surface interaction. P-selectins and VCAM-1 mediate MSC rolling and therefore transmigration. Stromal derived factor-1 (SDF-1) is also essential for MSC migration (Liu et al., 2011). Presence of the cognate receptor for SDF-1, CXCR4 on the cell surface of MSCs is still not fully confirmed, due to the vast heterogeneity of MSC phenotypes from different sources. However, Tsai et al., (2011), Shi et al., (2007) and Bhakta et al., (2006) have confirmed that modification of MSCs in culture can lead to increased extracellular CXCR4 expression (Chapter 5). These findings open broad possibilities in future treatment production, as desired receptors increasing MSC migration and homing can be expressed using in vitro priming strategies and retroviral transduction.

The aim of this project is to identify the known migratory mechanisms of MSCs towards ischaemically damaged regions of the brain after infusion, show identified MSC phenotypes and possible in vitro MSC modifications that improve tropism and homing to the damaged regions.