LILI CUI SAFETY AND HOMING OF INTRACAROTIDLY DELIVERED MESENCHYMAL STEM CELLS FOR TREATING ISCHEMIC STROKE

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1 PUBLICATIONS OF THE UNIVERSITY OF EASTERN FINLAND Dissertations in Health Sciences LILI CUI SAFETY AND HOMING OF INTRACAROTIDLY DELIVERED MESENCHYMAL STEM CELLS FOR TREATING ISCHEMIC STROKE

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3 LILI CUI Safety and homing of intracarotidly delivered mesenchymal stem cells for treating ischemic stroke To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in CA102, Kuopio, on Friday, March 2 nd 2018, at 12 noon Publications of the University of Eastern Finland Dissertations in Health Sciences Number 450 Department of Neurology, Institute of Clinical Medicine School of Medicine, Faculty of Health Sciences University of Eastern Finland Kuopio 2018

4 Grano Oy Kuopio, 2018 Series Editors: Professor Tomi Laitinen, M.D., Ph.D. Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Associate Professor (Tenure Track) Tarja Malm, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI Kuopio, Finland ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L:

5 III Author s address: Supervisors: Department of Neurology, Institute of Clinical Medicine Faculty of Health Sciences, University of Eastern Finland KUOPIO FINLAND Docent Jukka Jolkkonen, Ph.D. Department of Neurology, Institute of Clinical Medicine Faculty of Health Sciences, University of Eastern Finland KUOPIO FINLAND Professor Pekka Jäkälä, M.D., Ph.D. Department of Neurology, Institute of Clinical Medicine Faculty of Health Sciences, University of Eastern Finland NeuroCenter, Kuopio University Hospital KUOPIO FINLAND Reviewers: Associate Professor Heli Skottman, PhD Faculty of Medicine and Life Sciences, BioMediTech Institute University of Tampere TAMPERE FINLAND Tomás Sobrino, PhD Department of Neurology, Hospital Clinico Universitário SANTIAGO DE COMPOSTELA SPAIN Opponent: Professor Mathias Hoehn, MD, PhD In-vivo-NMR Research Laboratory Max Planck Institute for Metabolism Research COLOGNE GERMANY

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7 Cui, Lili Safety and homing of intracarotidly delivered mesenchymal stem cells for treating ischemic stroke University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences p. V ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: ABSTRACT Stroke poses a huge burden on society as the long-term disability suffered by stroke patients demands special care and rehabilitation. At present, limited treatment options are available for ischemic stroke, and there is an urgent need for novel therapeutic approaches. Cell therapy is one of the most attractive strategies for enhancing post-stroke functional recovery, particularly mesenchymal stem cells (MSCs). Positive effects of transplantation of MSCs have been observed in animal models, but real functional improvements in patients are still to appear. The efficiency of MSC therapy in the clinic could be increased by facilitating MSCs homing towards the brain lesion, such as intra-arterial delivery and cell surface engineering. In this thesis, we first aimed to examine the factors associated with the safety of intracarotid rat bone marrow MSC transplantation into sham-operated rats monitored by laser Doppler flowmetry and MRI. Behavioral tests were used to evaluate the rats functional outcome after cell transplantation. Cerebral embolism could be evoked by the injected cells, and this was found to be related with cell dose and injection velocity. We then investigated several factors that might affect cell clumping and cell viability in vitro before transplantation using flow cytometry. A pulse-width assay was applied to quantify cell clumps in suspension. Immediate use of freshly-harvested cells stored in normal saline was found to create fewer cell clumps while achieving good cell viability. To further improve the safety and homing of intracarotidly delivered MSCs, MSCs were lentivirally transduced to overexpress integrin alpha-4 (ITGA4), a subunit of very late antigen-4 (VLA-4). Transendothelial cell migration ability was tested in vitro using a Boyden chamber assay, and in vivo using intravital microscopy and SPECT/CT imaging. The ITGA4- overexpressed MSCs revealed increased transendothelial migration in vitro, but not in vivo. They also reduced the cell-evoked cerebral embolism after intracarotid transplantation into stroke rats. In conclusion, the cell preparation and infusion procedure need to be carefully optimized to ensure safe and efficient intracarotid injection. ITGA4 overexpression of MSCs did not enhance their cerebral homing after intracarotid injection but increased their safety. National Library of Medicine Classification: QU 325, QU 375, WH 380, WL 356, WN 206, WG 104, WX 185 Medical Subject Headings: Brain Ischemia; Cerebral Infarction; Cell- and Tissue-Based Therapy; Cell Survival; Infusions, Intra-Arterial; Cell Transplantation; Mesenchymal Stromal Cells; Bone marrow; Transendothelial and Transepithelial Migration; Magnetic Resonance Imaging; Single Photon Emission Computed Tomography Computed Tomography; Laser-Doppler Flowmetry; Safety; Models, Animal; Rats

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9 Cui, Lili Valtimonsisäisen soluterapian turvallisuus ja solujen kulkeutuminen kokeellisessa aivoiskemiamallissa Itä-Suomen yliopisto, terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences s. VII ISBN (print): ISBN (pdf): ISSN (print): ISSN (pdf): ISSN-L: TIIVISTELMÄ Aivoverenkiertohäiriöt (AVH) ovat tärkein aikuisiän vammaisuuden syy aiheuttaen valtavan taakan yhteiskunnalle ja terveydenhuollolle. Uusia terapeuttisia hoitoja tarvitaan. Soluterapia on yksi lähestymistapa tehostaa toiminnallista AVH-kuntoutumista. Erityisesti mesenkymaalisia kantasoluja (MSC) on tutkittu paljon. Eläinmalleissa MSC-siirrolla on havaittu positiivisia vaikutuksia, mutta potilailla terapeuttinen teho ei ole vielä optimaalinen. Hoidon tehoa voidaan mahdollisesti lisätä tehostamalla solujen siirtymistä vaurioituneeseen aivokudokseen. Tässä väitöskirjatyössä selvitettiin valtimonsisäisen solusiirron turvallisuutta rotilla käyttäen laser-doppler virtausmittausta ja kuvantamista (MRI). Mahdollisia sivuvaikutuksia solusiirron jälkeen arvioitiin myös käyttäytymistesteillä. Tulokset osoittivat, että injisoidut solut aiheuttivat mikrotukoksia ja kudostuhoa, jotka selittyivät käytetyllä soluannoksella ja injektionopeudella. Seuraavaksi selvitettiin tekijöitä, jotka vaikuttavat in vitro solujen elinkelpoisuuteen ennen siirtoa. Solujen elinkelpoisuus ja mahdolliset solurykelmät määritettiin virtaussytometrialla. Normaaliin keittosuolaliuokseen kerättyjen tuoreiden solujen välitön käyttö lisäsi solujen elinkykyä. Lopuksi selvitettiin valtimonsisäisesti annettujen ITGA4:tä yliekspressoivien MCS-solujen turvallisuutta ja hakeutumista kohdekudokseen. Solujen kulkeutumista aivoverisuoniesteen läpi tutkittiin in vitro Boyden kammiossa ja in vivo käyttämällä intravitaalista mikroskopiaa ja SPECT/CT-kuvantamista. ITGA4:tä yliekspressoivat MSC:t läpäisivät solukalvon in vitro, mutta ei in vivo. Geenimuokkaus vähensi solujen aiheuttamia mikrotukoksia valtimonsisäisen annostelun jälkeen. Yhteenvetona voidaan todeta, että soluvalmiste ja annosteluprosessi ovat optimoitava huolellisesti turvallisen ja tehokkaan valtimonsisäisen annostelun varmistamiseksi. ITGA4- yliekspressio soluissa ei parantanut aivoon kulkeutumista valtimonsisäisen infuusion jälkeen, mutta lisäsi solujen turvallisuutta. Luokitus: QU 325, QU 375, WH 380, WL 356, WN 206, WG 104, WX 185 Yleinen Suomalainen asiasanasto: aivoverenkiertohäiriöt; kantasolut; kantasolujen siirto; koe-eläimet; koeeläinmallit; luuydin; annostelu; solusiirto; turvallisuus

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11 IX If we knew what it was we were doing, it would not be called research, would it? Albert Einstein

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13 XI Acknowledgements The thesis was carried out in the Department of Neurology, Institute of Clinical Medicine in the Faculty of Health Sciences, University of Eastern Finland during the years First of all, I would like to express my heartfelt gratitude to my main supervisor Adjunct Professor Jukka Jolkkonen, who gave me this opportunity to undertake my thesis work in the stroke recovery group and have been kind, patient and supportive all the time to guide me towards completion. I am also very grateful to my second supervisor Professor Pekka Jäkälä for his generous support whenever needed. Meanwhile, I need to thank my supervisor of my master thesis work, Professor Chuansheng Zhao, for his recommendation of me to study in Finland and also for his valuable advice and help when I encountered difficulties. I am also very thankful to the official reviewers of this thesis, Associate Professor Heli Skottman and Dr. Tomás Sobrino, for their in-depth review of this thesis. In addition, I would like to thank Dr. Ewen MacDonald for his timely linguistic help. I deeply thank all the coauthors of the articles included in this thesis. My special gratitude goes to Professor Johannes Boltze for his critical comments and valuable suggestions, and also to Dr. Franziska Nitzsche, Evgeny Pryazhnikov and Marina Tibeykina, Dr. Tuure Kinnunen, Dr. Johanna Nystedt and Erja Kerkelä, Jingwei Mu and Abdulhameed Bakreen for their significant contributions. I also would like to thank Ville Juutilainen, Kristina Kuptsova, Petra Gregorová and all the others who have provided technical assistance during my thesis work. I also want to express my gratitude to my previous colleague Bhimashankar Mitkari and and current colleague Shanshan Zhao, who helped me in many ways during my stay in our research group. Special thanks to Yawu Liu for his kind help, guidance and company during my stay in Finland, to Esa Koivisto for his positive energy and help with computer problems, and to Pasi Miettinen for his generous help in the histology lab. I sincerely thank my colleagues Maria Hytti, Eveliina Korhonen, Niina Piippo, Sofia Rantaaho and all the people in their group who shared the same corridor with me, for their company during lunch and coffee time and also for their kind help during my study. Great thanks also to Ulla, Sari and Tarja for their kindness and help. Moreover, I would like to thank Ossi Nerg, my host when I visited Kuopio University Hospital, for his guidance and arrangements, and also thank all the people I have met in the hospital for their demonstration. I would also like to thank all my Chinese friends (Wujun Xu, Ying Wang, Yanyan Gao, Xiaoyan Lan, Liqing Hao, Nanxiang Jin, etc) currently living or having lived in Kuopio for all their support and company during my stay in Finland. My life would be much less interesting without them. Moreover, I want to thank my flatemates and friends Thu, Thuy, Matlena and all others for their friendship and fond memories. Finally, greatest thanks to my fiance Fei Wang and my family for their unconditional love and support for so many years. The thesis was funded by CIMO Foundation, Academy of Finland, Faculty of Health Sciences in University of Eastern Finland, ORION Research Foundation, Finnish Cultural Foundation, Aarne and Aili Turusen Foundation, and Antti and Tyyne Soinisen Foundation Beijing, February 2018 Lili Cui

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15 XIII List of the original publications This dissertation is based on the following original publications: I. Cui LL, Kerkelä E, Bakreen A, Nitzsche F, Andrzejewska A, Nowakowski A, Janowski M, Walczak P, Boltze J, Lukomska B, Jolkkonen J. The cerebral embolism evoked by intra-arterial delivery of allogeneic bone marrow mesenchymal stem cells in rats is related to cell dose and injection velocity. Stem Cell Res Ther. 6: II. III. Cui LL, Kinnunen T, Boltze J, Nystedt J, Jolkkonen J. Clumping and viability of bone marrow derived mesenchymal stromal cells under different preparation procedures: a flow cytometry-based in vitro study. Stem Cells Int Cui LL, Nitzsche F, Pryazhnikov E, Tibeykina M, Tolppanen L, Rytkönen J, Huhtala T, Mu JW, Khiroug L, Boltze J, Jolkkonen J. Integrin α4 overexpression on rat mesenchymal stem cells enhances transmigration and reduces cerebral embolism after intracarotid injection. Stroke. 48: The publications were adapted with the permission of the copyright owners.

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17 XV Contents 1 INTRODUCTION REVIEW OF THE LITERATURE STROKE Epidemiology and burdens Current treatment CELL THERAPY Different cell types Mesenchymal stem cells Neural stem cells Mononuclear cells Other cells Delivery route Intracerebral delivery Intravascular delivery Intraventricular delivery Others Therapeutic time window Cell dose Cell product manufacture MSC FOR ISCHEMIC STROKE Mechanisms of effects Cell replacement Immunomodulation Neuroprotection Angiogenesis, synaptogenesis and neurogenesis MSC homing into the brain The structure and function of BBB MSC homing cascade as compared with leukocytes Strategies to improve efficacy Genetic modification Cell preconditioning Combined therapy Others Stroke modeling in preclinical studies Behavioral tests in preclinical studies Clinical studies of MSCs for stroke RATIONALE 17 3 AIMS OF THE STUDY.19

18 XVI 4 MATERIAL AND METHODS PREPARATION AND CHARACTERIZATION OF BMMSCS (I-III) Culture and characterization of rat BMMSCs (I-III) ITGA4-gene modification of BMMSCs (III) MODIFIED BOYDEN CHAMBER ASSAY (III) FLOW CYTOMETRY (II, III) Sample preparation (II, III) Detection of cell clumps (II, III) Detection of cell viability (II) ANIMALS (I, III) Transient middle cerebral artery occlusion (I, III) Cell transplantation (I, III) CBF monitoring with laser Doppler flowmetry (I) Imaging (I, III) Magnetic resonance imaging (I, III) SPECT/CT imaging (III) Intravital microscopy (III) Behavioral tests (I, III) Limb placing test (I, III) Cylinder test (I) Open field test (I) Histology (I, III) Perfusion (I, III) Nissl staining (I, III) Modified Gallyas silver staining (I) IgG staining (I) Prussian blue staining (I) RECA-1 staining (III) VCAM-1 staining (III) STUDY DESIGNS STATISTICAL ANALYSIS.28 5 RESULTS EFFECTS OF CELL DOSE AND INFUSION VOLUME/VELOCITY ON SAFETY OF INTRACAROTID MSC DELIVERY Lower cell dose is safer for intracarotid delivery of BMMSCs Infusion velocity is related to the safety of intracarotid BMMSC delivery IN VITRO OPTIMIZATION OF CELL SUSPENSION Cell concentration, cell viability and clumping Storage solution and storage time, cell viability and clumping Freeze-thawing, cell viability and clumping...31

19 5.3 EFFECTS OF ITGA4 OVEREXPRESSION ON SAFETY AND HOMING OF INTRACAROTIDLY DELIVERED MSCS ITGA4 overexpression enhances in vitro transendothelial MSC migration ITGA4 overexpression does not increase in vivo transendothelial MSC migration ITGA4 overexpression decreases MSC aggregation and cerebral embolism DISCUSSION METHODOLOGICAL ISSUES SAFETY OF INTRACAROTID CELL DELIVERY (I) EFFECTS OF PREPARATION PROCEDURES ON CELL PRODUCT QUALITY (II) ITGA4 OVEREXPRESSION AND MSC HOMING AND SAFETY (III) FUTURE PERSPECTIVES CONCLUSIONS 41 REFERENCES...43 ORIGINAL PUBLICATIONS (I-III) XVII

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21 XIX Abbreviations Ang1 angiopoietin 1 EPC endothelial progenitor cell BBB blood-brain barrier ESC embryonic stem cell BDNF bfgf BI BM BMEC BMMSC BMMNC BMSC CBF CCA CFSE CNS CSF CXCR4 DALY DMSO DTI EC ECA enos brain-derived neurotrophic factor basic fibroblast growth factor Barthel Index bone marrow brain microvascular endothelial cell bone marrow derived- mesenchymal stem cell bone marrow derived- mononuclear cell bone marrow stromal cell cerebral blood flow common carotid artery carboxy fluoresceindiacetate succinimidyl ester central nervous system cerebrospinal fluid C-X-C chemokine receptor type 4 disability-adjusted life year dimethylsulfoxide diffusion tensor imaging endothelial cell external carotid artery endothelial nitric oxide ESS ET-1 FBS FMS FOV FGF-2 G-CSF GM-CSF HGF IA IBMIR ICA ICAM-1 ipsc ITGA4 IV LDF LFA-1 mbi Mac-1 European Stroke Scale endothelin-1 fetal bovine serum Fugl Meyer Scale field-of-view fibroblast growth factor-2 granulocyte colony stimulating factor granulocyte-macrophage colony-stimulating factor hepatocyte growth factor intra-arterial instant blood-mediated inflammatory reaction internal carotid artery intercellular adhesion molecule-1 induced pluripotent stem cell integrin α4 intravenous laser Doppler flowmetry leukocyte function-associated molecule-1 modified Barthel Index macrophage-1 antigen synthase 3

22 XX MAPC multipotent adult progenitor STEPS Stem Cell Therapies as an cells Emerging Paradigm in Stroke MCA middle cerebral artery SVZ subventricular zone MCP-1 monocyte chemoattractant TE echo time protein-1 TF tissue factors MMP matrix metalloproteinase T1DM type 1 diabetes mellitus MNC mononuclear cell tmcao transient middle cerebral mnss modified Neurological artery occlusion Severity Score TR repetition time mrs modified Rankin Score UCB umbilical cord blood MRI magnetic resonance imaging VCAM-1 vascular cell adhesion MSC mesenchymal stem cell molecule-1 MUSE multilineage-differentiating VEGF vascular endothelial growth stress-enduring factor MW molecular weight VLA-4 very late antigen-4 NIHSS National Institutes of Health Stroke Scale NS normal saline NSC neural stem cell NSPC neural stem/progenitor cell PBS phosphate-buffered saline PET positron emission computed tomography PIGF placental growth factor PT photothrombotic rtpa recombinant tissue plasminogen activator SDF-1 stromal cell-derived factor-1 SRRR Stroke Recovery and Rehabilitation Roundtable

23 1 1 Introduction Stroke is one of the leading global causes of disability and death. Despite acute care and spontaneous recovery, more than 50% of stroke patients are left with a functional impairment, imposing a huge burden on society. Around 80% of the stroke cases are ischemic. The current treatments of ischemic stroke are limited, such as intravenous thrombolysis and mechanical thrombectomy with a narrow time window of only a few hours after symptom onset, and rehabilitation in acute and post-acute period (1). Thus, there is a clear need for novel therapies to help stroke patients restore their neural function and reduce the extent of their disabilities. Recent progress in the field of cell therapy has introduced this approach as a promising option to enhance post-stroke recovery. In particular, mesenchymal stem cells (MSCs) have shown significant potential for clinical use due to their easy isolation, lack of ethical issues or significant immunogenicity (2). Positive effects of MSC transplantation after stroke have been seen in animal models; these are mediated via multiple mechanisms including immunomodulation, neuroprotection, and promotion of angiogenesis and neurogenesis (3). Recent clinical trials also have revealed the potential of MSC therapy (4, 5), but the functional outcome after administration of MSCs is still not optimal. Deeper understandings of the safety, bio-distribution, as well as the therapeutic mechanisms underpinning MSC therapy are still required before this technique can be successfully transferred to the clinic. Efficient homing of the infused cells to the ischemic brain without complications is crucial for their therapeutic potential in stroke. Intra-vascular cell delivery is the most commonly used method in both preclinical and clinical studies. However, most of the infused cells are found to be trapped in the peripheral organs (e.g., lungs, spleen, liver) after intravenous infusion (6, 7). Intra-arterial infusion is more efficient since it can increase the cell localization to the ischemic hemisphere by circumventing the pulmonary circulation (8-10), but then the risk of micro-occlusions needs to be addressed (11). Cell size, infusion velocity and technique have been suggested to be important factors governing safe intra-arterial delivery (12, 13). Moreover, MSCs still do not transmigrate efficiently into the brain parenchyma. Cell surface engineering may increase cell homing and reduce the potential risk of adverse events by decreasing the therapeutic cell dose. One of the most relevant cell adhesion pathways for MSC targeting is to exploit the very late antigen-4 (VLA-4)/ vascular cell adhesion molecule- 1 (VCAM-1) interaction (14). VLA-4 belongs to the integrin family of cell adhesion molecules and binds to the integrin receptor VCAM-1, which is overexpressed particularly on cerebral endothelial cells after ischemic stroke. Genetic cell engineering leading to VLA-4 overexpression might increase the cell homing capacity to the brain and thereby improve the functional outcome. Therefore, the current study has primarily focused on advancing safe and efficient intra-arterial MSC delivery for ischemic stroke.

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25 3 2 Review of the literature 2.1 STROKE Stroke is a cerebrovascular event that occurs after a vascular blockage (ischemic stroke) or bleeding (hemorrhagic stroke). Ischemic stroke interrupts the blood supply to the brain; it cuts off the supply of oxygen and nutrients and inflicts damage to the brain tissue. In acute stroke, the symptoms usually appear immediately after stroke onset, and last more than 24h. The most common symptoms include motor or sensory dysfunction, aphasia, hemianopia, headache, and dizziness. In severe cases, the patients can lose consciousness or even die. Common risk factors include hypertension, atrial fibrillation, dyslipidemia, diabetes, smoking, unhealthy diet, lack of exercise and advanced age (15, 16) Epidemiology and burdens Stroke affects 17 million people worldwide each year, and this number continues to rise due to the aging of societies coupled with population growth. Stroke has been the second greatest cause of death globally (>5 million deaths per year) during the last 15 years (17) and a leading cause of adult physical disability (18). Ischemic stroke accounts for approximately 80% of all cases. It is predicted that there will be a 34% increase in the total number of stroke cases from 2015 to 2035 in European Union, with a 32% increase in Disability-Adjusted Life Years (DALYs) lost (19). More than 50% of stroke survivors are left with some degree of motor or some other disability. The death rates from stroke have been declining over the last twenty years due to the improvements and faster acute treatment, meaning that more people survive and live with stroke s consequences (20). In 2015, the estimated total cost of stroke in Europe was 45 billion euros, with 22 billion euros being due to direct health care (72% for in-hospital care and 7% for drugs) and 15 billion euros attributable to indirect costs (19). In Finland, the total healthcare cost were million euros in 2015 (21, 22) Current treatment A healthy life style (e.g., quitting smoking, healthy diet, physical exercise, control of body weight) as well as interventions controlling the risk factors like hypertension, diabetes, and atrial fibrillation decrease an individual s chance of suffering a stroke (15, 23). With respect to acute ischemic stroke, intravenous thrombolysis by recombinant tissue plasminogen activator (rtpa) (24 26) up to 4.5h from symptom onset and mechanical thrombectomy up to 6h from symptom onset (27, 28) are the only effective treatments at present capable of restoring the blood flow to the brain, but since they have a narrow time window of only a few hours, they leave a large number of patients untreated. The thrombolysis rates vary from less than 1% of patients to 17% across Europe, and thrombectomy is currently unavailable to the majority of stroke patients (19). Although a stroke unit can improve the efficiency of acute care, only about 30% patients receive stroke unit care across Europe, with the values ranging from less than 10% to over 80% in different areas (19). For patients with disabilities after stroke, there is no other established treatment at present except for rehabilitation. However, the access to rehabilitation and long-term support is limited in many countries. Therefore, there is an urgent need to develop new effective therapies to help the patients restore their neural function after stroke and return to their premorbid societal and economical role.

26 4 2.2 CELL THERAPY Cell-based therapy is one of the most promising innovative paradigms for the treatment of ischemic stroke. Positive effects of cell transplantation after stroke have been shown in numerous animal studies (29, 30) Different cell types Most of the cells used are stem cells, which can self-renew and differentiate into several different cell types. The stem cells can be categorized as either pluripotent or multipotent depending on their differentiation potential. Pluripotent stem cells, like embryonic stem cells (ESCs) and induced pluripotent stem cells (ipscs), have the potential to differentiate into almost any cell type. Multipotent stem cells can differentiate into certain cell types, and can be found in both fetal and adult tissues. The most commonly used cells in preclinical studies are listed below (figure 1). Figure 1. Number of preclinical studies using different cell therapies for treating ischemic stroke that report structural (infarct size) or functional outcomes between 2000 and 2017 (Cui et al., unpublished data) Mesenchymal stem cells Mesenchymal stem cells (MSCs) are multipotent cells that can give rise to a few unique, differentiated mesenchymal cell types. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed a set of standards to define human MSCs: 1) they must adhere to plastic tissue culture plates; 2) be positive for CD105, CD73, CD90 and negative for CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR; and 3) be able to differentiate into osteoblasts, adipocytes and chondroblasts under standard in vitro differentiating conditions (31). MSCs are considered to have significant clinical utility potential due to their ease of isolation, lack of significant immunogenicity allowing allogenic transplantation without immunosuppression (32, 33), and lack of ethical controversies. Therefore, MSCs have been the focus of the emerging therapeutics for various diseases such as myocardial infarction, cartilage and bone injury, Crohn s disease, graft-versus-host disease, spinal cord injury, multiple sclerosis, as well as ischemic stroke (34). MSCs can be isolated from multiple tissues by a density gradient centrifugation method. The most frequently used source is bone marrow, followed by umbilical cord blood (UCB), adipose tissue or placenta. However, phenotypic differences may exist in the MSCs due to the different source tissue microenvironments, and this may confer distinct functional properties on the cells (35). For example, one study showed that adipose tissue-derived MSCs displayed distinct immunophenotypes on surface expression of CD34 +, PODXL, CD36, CD49f, CD106 and CD146, and more pronounced adipogenic differentiation capability relative to BMMSCs (36). Another study revealed that placenta-derived MSCs possess better immunoregulatory properties compared to their cord-derived counterparts (37). Differences in cell donors may also affect cell characterization. For instance, expression of the gene for interleukin-1α for

27 5 MSCs isolated from aged rats have been shown to be 8-fold lower than cells from the young ones (38) Neural stem cells There is a small amount of endogenous neural stem cells (NSCs) located in the subgranular zone of dental gyrus, the subventricular zone (SVZ) and the subependymal zone of the spinal cord in the adults (39). After ischemic stroke, the endogenous stem cells become activated, migrate towards the lesion area and differentiate, but it remains unclear whether or not they can functionally integrate into the local network (40). Therefore, the endogenous neurogenesis may be insufficient to repair the tissue of injured brain (41), and could be impeded due to the microglia activation post-stroke (42). A number of animal studies have shown improved brain repair and functional recovery after the transplantation of NSCs in ischemic stroke. Those exogenous NSCs may elicit endogenous stem cell production at the site of injury (43, 44), reduce neuronal apoptosis, improve the microenvironment around the ischemic areas and promote functional recovery of damaged neurons (45, 46). A phase 1, first-in-man study using intracerebral transplantation of up to 20 million human NSCs in 11 patients 6-60 months after ischemic stroke reported improved neurological function without any adverse events (47). However, there are still some limitations associated with the utility of NSCs in the clinic, such as the large-scale production of cells (48) and safety concerns about tumorigenesis (49) Mononuclear cells A mononuclear cell (MNC) is any cell that has a single round nucleus. MNCs can be easily obtained from various tissues using density gradient centrifugation technique (50 53). Bone marrow-derived MNCs (BMMNCs) are the most commonly used (49); they consist of various proportions of differentially matured B-cells, T-cells, monocytes, and a smaller proportion of progenitor cells such as hematopoietic stem cells, MSCs, endothelial progenitor cells (EPCs), and very few embryonic-like cells. Although MNCs may contain MSCs, MNCs are preferred in some cases because they are easier to acquire and there is no need to grow cells. In addition, the cell size is much smaller compared to MSCs. There is a growing evidence for the beneficial effect of MNC transplantation in experimental stroke (54 58). MNCs have been demonstrated to have the ability to cross the blood-brain barrier (BBB) (59), exert neuro-protective effects (60), promote angiogenesis and neurogenesis (61) and attenuate the post-stroke inflammatory response. A few clinical studies of MNCs transplantation for ischemic stroke have also been conducted (62 64). Selected cell populations from MNCs have been investigated to search for the most effective types. For instance, compared to BMMNCs negative for the C-X-C chemokine receptor type 4 (CXCR4), the CXCR4 positive counterparts exhibit a greater migratory capacity and are more effective in improving neovascularization, releasing trophic factors and facilitating brain repair after acute ischemia (65). Moreover, among CXCR4 + BMMNCs, the CXCR4 + CD45 - subpopulation has been found to be superior to CXCR4 + CD45 - cells or the unfractionated cells in ameliorating cerebral damage (66). The CD34 + and CD34 - subpopulations have also been compared (67, 68), but no clear superiority in the overall effect could be attributed to the CD34 + cells. Another study reported monocytes as being the essential cellular component of MNCs obtainable from UCB in evoking neuroprotective effects following stroke in rats (69) Other cells The other cells such as ESCs, ipscs and EPCs have also been tested for stroke treatment (70 73). ESCs are pluripotent stem cells derived from the inner cell mass of a blastocyst, an earlystage pre-implantation embryo which is formed 4-5 days post-fertilization consisting of cells. The mouse ESCs were firstly isolated by Martin Evans (74), and human ESCs were later successfully collected by Thomson and his group (75). There has been substantial

28 6 interest in using ESCs to differentiate different cell types for regenerative medicine, drug discovery and immunotherapy, but the ethical issues surrounding their isolation from human blastocysts limit their wider use. Moreover, Erdö and coworkers reported the generation of aggressively growing neoplastic formations resembling primitive neural structures after intracerebral transplantation of allogeneic ESCs into the mouse brain (76). ipscs were initially derived from adult mouse fibroblasts by retroviral transduction with certain crucial genes (e.g., Oct-¾, Sox2, c-myc and Klf4) by Yamanaka s group (77). The development of this technique provides the possibility of generating ipscs on an individual basis, overcomes the ethical issue and may accelerate the implementation of stem cells for clinical treatment of various diseases. For example, transplantation of undifferentiated (71) or neuronal lineage-differentiated ipscs (78, 79) into animals with ischemic stroke have been shown to be able to migrate into the injured brain area, differentiate into neuron-like cells, reduce the inflammatory response and improve the endogenous neurogenesis and functional recovery. However, the risk of tumorigenesis and genetic modifications of ipscs should be aware. Kawai et al. reported tridermal teratoma after implantation of ipscs in the ischemic brain at 28 days after cell transplantation (80). EPCs are cells expressing both human stem cell marker CD34 or CD133, and endothelial cell marker CD31, vascular endothelial growth factor (VEGF) receptor 2, von Willebrand factor, VE-cadherin or Tie2; they are thought to act as a cellular reservoir to maintain the vascular homeostasis by monitoring and repairing dysfunctional endothelium (81, 82). They can be isolated from bone marrow, peripheral blood or UCB. EPCs have been implicated in playing an important role in cerebral neovascularization after focal cerebral ischemia (83), and exogenous administration of EPCs attenuated ischemic brain injury and improved functional recovery in rats (73, 84). Cells from other sources are also being investigated. For instance, multilineagedifferentiating stress-enduring (MUSE) cells, a stress-tolerant, unique multipotent stem cell population identified in mesenchymal tissues, have been shown to replenish new neuronal cells and significantly improve neurological function in a rodent stroke model (85, 86). The dental pulp stem cells, neural crest-derived stem cells residing within the perivascular niche of the dental pulp, also exerted therapeutic effects after cerebral ischemic injury (87) Delivery route The cells can be transplanted via intravascular (intravenous or intra-arterial), intraparenchymal, intraventricular and intranasal routes (figure 2). Each delivery route has its own pros and cons. Although positive effects have been reported after cell delivery via different routes, the optimal route with maximal efficacy and safety after ischemic stroke remains to be elucidated. A few studies have compared therapeutic effects of different delivery routes, but the results are somewhat conflicting (88 90). Figure 2. Number of preclinical studies using different cell delivery routes for treating ischemic stroke that report structural (infarct size) or functional outcomes between 2000 and 2017 (Cui et al., unpublished data)..

29 Intracerebral delivery Intracerebral cell delivery, via stereotaxic transplantation with craniectomy, was used for stroke treatment to examine whether the implanted cells could replace the lost neurons and rebuild neural circuits. In 2000, Li et al. transplanted bone marrow non-hematopoietic cells into the ischemic brain of mice and found improved functional recovery (91). Those transplanted cells concentrated mainly around the infusion site, some also transmigrated into multiple areas of the brain including the corpus callosum and cortex. Later on, positive effects have been seen after intracerebral cell transplantation into stroke animals in many preclinical studies (33, 92, 93). A few clinical studies have also reported that intracerebral transplantation of allogeneic neuronal cells or NSCs is safe and confers some benefits for stroke patients (47, 94, 95). Although intracerebral cell delivery allows for spatially precise cell deposition within or next to a lesion, the required stereotaxic infusion procedure makes this delivery invasive and limits its clinical use. For example, hemorrhage was reported in about 3.1% of patients with Parkinson s disease, who received electrode deposition to obtain deep brain stimulation, with 1.4% being symptomatic (96). Temporal worsening of motor deficits and seizures has also been reported in one study using fetal porcine cell transplantation in 5 patients with basal ganglia infarcts, although it is unclear whether this is due to transplanted cells per se (97). Other complications such as headache, somnolence, and subdural hematoma have also been observed in clinical studies (95, 98) Intravascular delivery Intravascular cell delivery is the most commonly used approach in both preclinical and clinical studies due to the minimal invasiveness and straightforward accessibility, particularly with intravenous delivery. In 2001, Chen and colleagues reported that intravenously administered human UCB cells could enter the brain, survive, migrate and improve functional recovery after an ischemic stroke in rats (99). Similarly, they found the intravenously infused bone marrow stromal cells (BMSCs) in the ipsilateral ischemic hemisphere and other organs, a few cells expressing neural markers, with significant somatosensory and motor function recovery (100). However, no reduction of infarct size was observed in either study and the amount of infused cells in the brain was rather small (~10%). Subsequently, the number of studies using IV cell delivery has increased. Some of these later studies have explored the in vivo bio-distribution of cells after IV delivery; they confirmed that the majority of cells accumulated in internal organs like spleen, liver and kidneys after the pulmonary first-pass effect, with very few cells being detected in the brain (9, ). Although the distribution pattern is similar regardless of cell type or infusion time (9), pulmonary passage of BMMNCs could be 30-fold while that of NSCs and multipotent adult progenitor cells was double that of MSCs (103). Intra-arterial (IA) cell delivery is considered as more efficient compared to IV delivery because the cells can bypass the pulmonary circulation, thus more cells are available to enter into the brain (9, 104). Moreover, along with the development of the endovascular treatment technique after stroke, IA delivery also has the potential advantage of selectively targeting cells to the brain requiring a smaller cell dose at the same time while the patient is closely monitored for safety. The first study of IA cell delivery after stroke was published in 2001 by Li et al., who injected allogeneic BMSCs into rats from the internal carotid artery (ICA) 1d after ischemia (105). The injected cells were localized and directed to the territory of middle cerebral artery, promoting a functional improvement after cerebral ischemia. Later on, there have also been some other preclinical studies reporting positive therapeutic effects after IA cell delivery. Although intravascular cell delivery is generally considered as safe, there are potential risks of complications such as vascular embolism, particularly with larger cells like MSCs (figure 3). Pulmonary embolism has been reported after IV delivery of allogeneic MSCs in animal studies (103, 106). It has also been reported in patients after multiple IV infusions of autologous human adipose tissue-derived stem cells (107). The cell surface structures

30 8 could influence the lung clearance rate of intravenously delivered MSCs. For example, Lee and colleagues reported that PODXL (hi) /CD49f (hi) human MSCs were less prone to produce lethal pulmonary emboli compared to the PODXL (lo) /CD49f (lo) ones after IV infusion into mice (108). In another study, a higher expression level of CD49d and CD49f on human MSCs was found to induce a higher lung clearance rate in mice (109). Vascular embolism has also been reported after IA delivery of MSCs in preclinical studies (10, 11, 110, 111) as well as in patients (112). The vascular blockage by MSCs can occur already at the precapillary level due to their large size, resulting in an immediate reduction of blood flow in the arteriole and downstream capillaries (113). The local hypoxic-ischemic environment caused by microembolism not only can cause a loss of the majority of transplanted cells, but also evoke additional hypoxic-ischemic stress to the surrounding tissues. In the case of intracarotid MSC infusion, disturbances of cerebral blood flow (CBF) and cerebral embolism could occur (11). Janowski et al. demonstrated that intracarotid infusion of smaller cells such as glial-restricted precursor cells led to significantly less decrease in CBF compared to MSCs, and a slower infusion velocity of 0.2 ml/minute seemed to be safe while a velocity 1 ml/minute could cause stroke with even infusion of vehicle (12). The 3D spheroid-cultured MSCs were reported to have a smaller size compared to their conventional 2D-cultured counterparts and thus reduce the risk of cerebral embolism after IA infusion (114, 115). Intracarotid infusion using a microneedle with preserved common carotid artery (CCA) flow could also decrease the risk of microembolism (13). In addition to the mechanical vascular obstruction, culture-expanded MSCs can also elicit an innate immune attack - instant blood-mediated inflammatory reaction (IBMIR) after exposure to blood due to the surface expression of prothrombotic tissue/stromal factors, particularly tissue factors (TF) (106, 116). The intensity of IBMIR increases with higher cell dose and prolonged ex vivo expansion, and also varies between different MSC donors (116). However, the occurrence of thrombotic events was rather limited at the commonly applied dose of 1-3 million cells/kg. Anticoagulation treatment via heparin (400 U/kg IV) has been reported to prevent BMSC-induced coagulation, reducing the mortality, preventing weight loss and improving the therapeutic effects in a mice model of experimental colitis (117). For stroke patients, combined use of rtpa and MSCs may hold more potential due to clinical feasibility, e.g., systemic infusion of MSCs has been reported to inhibit intracranial hemorrhage after rtpa therapy for stroke rats (118). Post-stroke epilepsy occurs in 3-30% of patients after stroke (119). Interestingly, a recent study reported that two patients after IA and five patients after IV administration of autologous BMMNCs developed seizures (7). It is difficult to establish a causal relationship between increased seizures and cell therapy due to the small number of patients in these early phase trial studies, but this symptom may be due to altered perilesional excitability, cerebral microembolism or systemic immune response. Another potential concern for systemic cell delivery is their immunosuppressive effect. Although cell-induced immunosuppression is one important mechanism for their efficacy, it could be also harmful after stroke by amplifying the post-stroke immune deficiency syndrome and thus increase the risk for infections (120) Intraventricular delivery The ventricular system is the place where cerebrospinal fluid (CSF) is produced. It consists of the lateral ventricles, the 3 rd and 4 th ventricles which connect with the central canal of the spinal cord allowing the CSF to circulate. As an alternative to intracerebral delivery, cells can also be transplanted by a single trajectory targeting the lateral ventricles or by intrathecal infusion. One study examined the effect of intracisternally transplanted SVZ cells into stroke rats, and found that the transplanted cells selectively migrated towards the ischemic parenchyma with significant improvement of neurological function (121), but these positive results were not confirmed in another report (122). Intrathecally delivered cells through lumbar puncture into stroke rats have also been reported to survive, migrate to the ischemic

31 9 area and differentiate into neurons and astrocytes with improved functional outcome (123, 124). Intracerebroventricular or intrathecal cell delivery has also been reported in patients (49, 125, 126). Headache is a frequently reported complication after intraventricular cell delivery, mainly related with the operation procedure (126). Another potential risk is hydrocephalus since the injected cells might adhere to the ventricular wall and cause a dysfunction of CSF circulation. Others symptoms such as lumbosacral radiculopathies, which can cause pain and neurological deficits, have also been reported after intrathecal cell delivery (127). Ectopic survival and proliferation of engrafted cells could also occur since the administered cells can widely distribute along the ventricular wall. There is one clinical case report of a multifocal donor-derived glioneuronal neoplasm after intracerebellar and intrathecal infusion of human fetal NSCs in a commercial stem cell transplantation center (49). Thus it is crucial to determine the biodistribution of the infused cells after intraventricular delivery Others Intranasal administration has been a recently explored noninvasive delivery route that bypasses the BBB and directly guides therapeutics to the central nervous system (CNS). A few studies have indicated that intranasally administered BMSCs can migrate to different brain regions along olfactory and trigeminal nerves innervating the nasal passage (128, 129). One study delivered BMSCs intranasally at 24h after the stroke and found that the cells reached the ischemic cortex and were deposited outside of the vasculatures as early as 1.5h after administration (130). Intraperitoneal delivery has been examined in rat models of neonatal hypoxia-ischemia (131, 132). There is also one study comparing the intraperitoneal and IV infusion of BMMSCs into adult stroke rats; it was found that the infused cells had reached the brain after both types of delivery, but there was a better functional improvement after IV delivery (133). Ectopic cell engraftment has also been described after intranasal cell delivery. Lee et al. reported that after successful intranasal delivery of immortalized HB1.F3 human NSCs with firefly luciferase gene (F3-effluc) into BALB/c nude mice, the cell signal migrated toward the brain area at 4h and gradually decreased over 2 days (134). In mice in which there had been an initial lung signal, the lung signal disappeared over 5 days but reappeared 2 weeks later. Ex vivo imaging and histology confirmed that the lung signal originated from the tumors in the lungs formed by those F3-effluc cells (134). However, this type of complication seems unlikely in immunocompetent recipients given the immunological barrier function of the airway mucous tissue as well as the graft infusion and immune response in most tissues Therapeutic time window The optimal therapeutic time window for cell treatment of stroke remains unclear. Cells delivered after either the acute or chronic phase after stroke have shown therapeutic effects in experimental studies (54, 135, 136). However, approximately two thirds of the preclinical cell therapy studies for stroke have delivered the cells within 24h after stroke (29). Neuroprotection is considered as the main effect of cell transplantation at such early time points (32, 33, 135). The opening of BBB and increased expression of various chemotactic signals could facilitate the migration of cells towards the ischemic zone and penetrate into the brain parenchyma (14, ), but the hostile environment may also endanger the longterm cell survival. For cell delivery at later time points, the infused cells are thought to exert more of a neurorestorative effect by inducing remodeling of the injured brain via angiogenesis, neurogenesis, and axonal and dendritic sprouting (140).

32 10 Figure 3. Complications after cell therapies for stroke (with permission from 141) Cell dose Most of the preclinical studies used a cell dose of <5 million/kg ( ), but higher cell doses even as many as >100 million/kg have been used for MNCs (145, 146). A meta-analysis involving 60 preclinical studies described a dose-response association between treatment effect and the number of injected cells (147). Nevertheless, another meta-analysis of MSC therapy for stroke revealed that a lower MSC dose was related with a greater behavioral recovery (148). This may be because a higher cell dose also introduces the risk of adverse events such as vascular embolism (142). Multiple infusions of lower cell dose may be one alternative to reduce the risk of complications while maintaining the efficacy. However, one study compared a single high dose (3 million) of human MSCs with multiple lower doses (1 million) after experimental stroke, and found that better therapeutic effects were achieved with the single high dose (149). Another study did not find multiple infusions to be superior to a single infusion (150). It is even more challenging to estimate the ideal cell dose for clinical studies. The Stem Cell Therapies as an Emerging Paradigm in Stroke (STEPS) group suggested determining the maximal tolerated dose from the literature and a dose-response curve (151) Cell product manufacture The clinical application of therapeutic cells requires cell production under the conditions of Good Manufacturing Practice. Therapeutic cell products are also subjected to the advanced therapy medicinal cell product legal framework of the European Medicines Agency or similar regulations, such as those issued by Office of Cellular, Tissue and Gene Therapies of

33 11 the Food and Drug Administration in the United States. The strict quality control of cell products excludes the presence of viral contaminants, mycoplasma, endotoxins or xenogeneic supplements. This reduces the adverse events related to the manufacturing process and enhances the safety of novel cell products for patients. However, there have still been some adverse events reported after cell therapy that are related to improper cell product manufacturing. For example, dimethylsulfoxide (DMSO), a cryoprotectant frequently used in the cryopreservation of cells, has been reported to cause allergic reactions (152) or aseptic meningitis after intrathecal administration of a DMSO-containing cell suspension (126). More severe complications like transient encephalopathy, stroke and seizures could also occur after systemic infusion of DMSO-containing cell suspensions (153, 154). 2.3 MSC FOR ISCHEMIC STROKE MSC transplantation holds great potential for treatment of ischemic stroke, and has been investigated both in preclinical and clinical studies. Positive effects of MSC therapy have been demonstrated in numerous preclinical studies. The suggested mechanisms of their effects on stroke include direct repair and replacement of damaged brain tissue, as well as the indirect support for endogenous repair process due to the secretion of cytokines, growth factors and other molecules Mechanisms of effects Cell replacement In vitro studies have shown that MSCs are capable of differentiating into cells of multiple lineages, including neuronal, glial and endothelial cells, leading to the hope that the transdifferentiation of transplanted MSCs may play a role in improving outcome after stroke ( ). Some studies have described the expression of neuronal, glial and endothelial markers of infused MSCs in the ischemic brain in rodent models (91, 144, ). Nevertheless, there is still no convincing evidence that MSCs can directly differentiate into cells that replace the injured ischemic tissue and functionally integrate into the local network (157, 163, 164). MSCs may exert a more indirect role in promoting the changes leading to a functional recovery Immunomodulation A cascade of inflammatory responses occurs after ischemic stroke starting from the early damaging events caused by arterial occlusion up to the late regenerative processes (165). MSCs are known to be immunomodulatory in attenuating both innate and adaptive immune responses. MSCs can regulate T-cell mediated processes by inhibiting T-cell proliferation, promoting a T-regulatory cell phenotype, and by the non-specific suppression of CD4 + and CD8 + T cells ( ). They can also modulate the function of B cells and antigen-presenting cells (167, 170). In vitro experiments also revealed a significant reduction in leukocyte proliferation and changes in differentiation when co-cultured with MSCs (171, 172). Many of the effects that MSCs exert on the immune system are directly related with the neural repair processes after stroke. For example, MSCs can promote the resolution of post-stroke inflammatory milieu that can impede repair, and change the activated macrophages so that they take on a repair-regulatory phenotype (173). The immunomodulatory effect of MSCs can also be remote. MSCs entrapped in the spleen or lungs after systemic administration can have immunomodulatory effects on remote organs including the brain ( ) Neuroprotection MSCs have been found to secrete numerous factors that can promote the brain repair and recovery like small molecular factories. An ischemic stroke evokes abundant necrosis and apoptosis of neurons, glial cells, and endothelial cells. Investigators have consistently found that the delivery of MSCs in the acute stage after stroke can reduce apoptosis and increase

34 12 endogenous neurogenesis and synaptogenesis ( ). This process probably occurs through the secretion of prosurvival and antiapoptotic factors, as well as indirect stimulation of CNS parenchymal cells to secrete various neuroprotective, neurotrophic, and prooligodendrogenic factors like brain-derived neurotrophic factor (BDNF), epidermal growth factor, and stem cell factor (93, 177, ). MSCs can also increase tpa expression and decrease the expression of plasminogen activator inhibitor 1 in astrocytes via the sonic hedgehog signaling pathway after stroke in vitro (186). Increased tpa activity in astrocytes has also been seen after MSC delivery into mice at 24h post-stroke, which promotes neuroprotection as well as neurite growth (179) Angiogenesis, synaptogenesis and neurogenesis Angiogenesis in the perilesional area plays an important role in mediating survival and regeneration of neurons after stroke. MSCs have been shown to be capable of secreting proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bfgf), insulin-like growth factor 1, nerve growth factor, and placental growth factor (PIGF) (159, ). Many of these angiogenic factors possess additional neuroprotective roles. For instance, VEGF not only can promote angiogenesis, but it also has potent anti-inflammatory effects as well as promoting the recruitment and differentiation of endogenous neuronal precursors ( ). Moreover, MSCs share some similarities with pericytes that may allow the infused MSCs to have an additional neural repair effect after stroke such as being mediated by the maintenance of vascular supply (113). MSCs could also support normal interactions of pericytes with astrocytes and endothelia to sustain the BBB integrity (196). MSC therapy could also increase brain plasticity such as synaptogenesis and neurogenesis after stroke (3). Synaptogenesis is the process of formation of a new synapse. It could be enhanced by angiogenesis with sufficient O2 and glucose supply to the tissue from the blood vessels (138). Increased expression of synaptophysin, an indicator of synaptogenesis has been revealed after MSC transplantation into stroke rats (179). MSC delivery after stroke has also been found to enhance survival, proliferation and differentiation of endogenous neuronal precursors in the subgranular zone of hippocampus and in the SVZ (177, 178) MSC homing into the brain MSC homing is defined as the arrest of MSCs within the tissue vasculature followed by transmigration across the vascular endothelium. It has been hypothesized that increased engraftment of MSCs into the site of injury can improve the therapeutic effect by amplifying their local trophic or paracrine activity. However, the cerebral homing after systemic MSC delivery is rather limited, due to the lack of several key homing molecules as well as the existence of BBB The structure and function of BBB The BBB is formed by cellular interactions between brain microvascular endothelial cells (BMECs), astrocytes, pericytes and neurons. The BMECs have tight junctions that restrict the diffusion of ions and polar molecules and regulate the paracellular permeability of endothelial cells ( ). They also contain a small number of endocytotic caveolae that are involved in the receptor-dependent and -independent transcytosis (200). In addition, BMECs express a large number of active or passive transporters that regulate the passage of nutrients (e.g., glucose, amino acids) from the blood to the brain (199, 201). The BBB plays a crucial role in brain homeostasis by restricting the passage of leukocytes and molecules into and out of the brain. However, after brain inflammation or injury, the BBB becomes compromised with significantly upregulated cellular trafficking through the BBB.

35 MSC homing cascade as compared with leukocytes The transmigration of leukocyte across BBB has been well characterized as a multistep adhesion/migration cascade including tethering and rolling, firm adhesion, crawling and transendothelial migration (14, 202). During inflammation, a number of cell surface adhesion molecules (e.g., P- and E-selectins, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and chemoattractants (e.g., stromal cell-derived factor-1 (SDF-1) and monocyte chemoattractant protein-1 (MCP-1) are upregulated on BMECs. The leukocytes initiate a transient selectin-mediated tethering and rolling on the endothelium. This subsequently triggers the activation of integrin on leukocytes such as leukocyte function-associated molecule-1 (LFA-1, ligand for ICAM-1), macrophage-1 antigen (Mac-1, ligand for ICAM-1) and very late antigen-4 (VLA-4, a heterodimer of integrin α4β1, ligand for VCAM-1). The leukocytes then slow down and become arrested on the BMECs via integrin-mediated firm adhesion. The arrested leukocytes undergo polarization and lateral crawling on the luminal surface, searching for the optimal spot for transmigration. Eventually, leukocytes migrate across the endothelial barrier preferably by the paracellular route. The endothelial adhesion of leukocyte induces clustering of endothelial cell surface adhesion molecules (i.e., ICAM-1 and VCAM-1), and triggers downstream signaling pathways that disrupt the tight junctions and promote paracellular migration. Transcellular migration may play a major role in leukocyte trafficking where endothelial cells (ECs) have very tight junctions (203). After passing through the endothelial barrier, leukocytes can penetrate the endothelial basement membrane and pericytes facilitated by extracellular matrix degradation which is mediated by matrix metalloproteinases (MMPs) (figure 4). MSCs are considered to share certain similarities with leukocytes during the homing process, but the extent and mechanisms for their transmigration across BBB remain to be clarified. MSCs express some homing molecules such as chemokine receptors (e.g., CXCR4, CCR2) and cell adhesion molecules (e.g., CD44, CD99, integrin β1), but lack certain key homing molecules like P-selectin glycoprotein ligand 1, LFA-1, Mac-1 and integrin α4 (ITGA4, a subunit of VLA-4), which may limit their homing ability. CD44 and galectin-1 have been suggested as alternative selectin ligands (204, 205). Moreover, passive entrapment, instead of active endothelial interaction, may also play a role in the firm adhesion and subsequent transmigration of MSCs. The VLA-4/VCAM-1 interaction has been shown to be important in MSC firm adhesion and transmigration to BMECs ( ). For example, blocking the integrin β1 subunit of VLA-4 substantially decreases their homing capabilities (209). In contrast to leukocytes, MSCs show little lateral migration along the endothelium during crawling. During the early stage of transmigration, MSCs formed bleb-like protrusions, particularly at sites having close contact with the endothelium, instead of lamellipodia and invadosomes for leukocytes. MMPs such as MMP-2 have been shown to help MSCs overcome the endothelial basement barrier, but MMP-9 seems to play a minor role ( ). The MSC transmigration occurs in the time scale of hours, whereas for leukocytes, it usually happens within minutes (213).

36 14 Figure 4. Homing cascade of leukocytes (modified from 202) Strategies to improve efficiency Genetic modification Various ways of genetic engineering of MSCs has been explored to enhance their therapeutic effects after stroke (table 1). For example, Kang et al. used adenovirus transfection to induce the overexpression of BDNF on human adipose tissue stromal cells. The transplantation of BDNF-overexpressed cells significantly improved the functional recovery in stroke rats as compared to those animals receiving non-modified cells (214). There are also similar studies using human amniotic membrane- and bone marrow-derived MSCs ( ). Induced overexpression of other proteins such as VEGF, Noggin, basic fibroblast growth factor-2, neurogenin 1 or 2 has also been reported to enhance the BMSC-induced functional outcome after stroke in rats ( ). MSCs have also been engineered with the CXCR4 gene to promote their migration (223, 224). Survivin and hypoxia-inducible factor 1α have also been explored as ways to increase cell survival after stroke ( ). However, most of these studies used viral vectors, which carry a potential safety concern and thus may limit their clinical use Cell preconditioning One of the problems after cell therapy for ischemic stroke is the massive death of the delivered cells due to the local hypoxia/ischemia environment. A sublethal hypoxic preconditioning (0.5% O2) of MSCs made the cells more resistant to necrotic and apoptotic insults, promoted the neuronal differentiation of MSCs and conferred additional structural and functional benefits after the transplantation of the cells into stroke animals (130, 144, 229, 230). Preconditioning of MSCs with drugs, such as valproate and lithium, melatonin, has also been found to enhance the migration of MSCs by increasing CXCR4 or MMP-9 expression, or increasing the survival of MSCs, respectively, which further reduced brain infarction and improved neurobehavioral outcomes ( ). Induction of neuronal differentiation of MSCs using conditioned medium before transplantation has also been examined in attempts to enhance their efficacy (93, 235). However, in one study, the pretreated and non-treated MSCs achieved similar therapeutic outcomes (93), while another study showed better therapeutic effect after transplantation of pre-treated MSCs (235).

37 15 Table 1. Methods used to improve MSC therapy in experimental stroke. Techniques Description Reference Genetic modification BDNF, NGF promote neuronal survival, neurogenesis and synaptogenesis ( , 236, 237) VEGF, Ang1 promote angiogenesis, neuroprotection (218, 219, 238, 239) Noggin promote neurogenesis and oligodendrogenesis (236, 237, 240) HGF anti-apoptosis, neuroprotection (220) Survivin, HIF-1 increase MSC survival and secretion of protective cytokines ( ) Neurogenin 1/2 promote neural differentiation of MSCs (221, 222) CXCR4 promote MSC migration, neuroprotection and angiogenesis (223, 224) GM-CSF anti-apoptosis, promote angiogenesis and neurogenesis (241) FGF-2 neuroprotection and vasodilation (242) PIGF neuroprotection and angiogenesis (188) Cell pretreatment G-CSF increase MSC proliferation and differentiation, neurogenesis (243) Valproate, Lithium, or both increase MSC migration (231, 232) Hypoxia increase MSC survival (130, 144) Isoflurane short exposures to low isoflurane concentrations promotes MSC survival and (244) migration Melatonin increase MSC survival (234) Pronase transiently modify cell surface adhesion proteins (8) Culture in neural-conditioned media or coculture with neurons increase neural differentiation of MSCs (93, 245) Combined with drugs Simvastatin a hydroxyl methylglutaryl-coenzyme A reductase inhibitor (246, 247) DETA/NONOate a nitric oxide donor (248) MCI-186 a free radical scavenger (249) Niaspan a vitamin which can increase high density lipoprotein level, improve endothelial function, (250) reduce inflammation, increase plaque stability Adrenomedullin a potent vasodilator peptide (251) FK506 (Tacrolimus) an immunosuppressant (252) Erythropoietin a glycoprotein that controls erythropoiesis (253) Oxiracetam a new ring gamma-aminobutyric acid derivative (254) G-CSF a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream (255) Ginsenoside Rg1, Quercetin, chemicals extracted from Chinese herbal Sodium Ferulate medicines ( ) Others Combined with rehabilitation treadmill (259, 260) Use of biomaterials thermoreversible gelation polymerhydrogel (261) MSC: mesenchymal stem cell, BDNF: brain-derived neurotrophic factor, NGF: nerve growth factor, VEGF: vascular endothelial growth factor, Ang1: angiopoietin 1, HGF: hepatocyte growth factor, HIF-1: hypoxiainducible factor-1, CXCR4: C-X-C chemokine receptor type 4, GM-CSF: granulocyte-macrophage colonystimulating factor, FGF-2: fibroblast growth factor-2, PIGF: placental growth factor, G-CSF: granulocytecolony stimulating factor.

38 Combined therapy Several studies have combined multiple types of drugs together with MSC delivery to test whether this could potentiate the therapeutic effects of infused cells. For instance, simvastatin, a hydroxyl methyl glutaryl-coenzyme A reductase inhibitor which can lower the cholesterol level and is commonly prescribed to stroke patients, can also upregulate the expression of BDNF, VEGF and endothelial nitrix oxide synthase, increase tpa activities, and eventually augment CBF, promote angiogenesis and improve the functional outcome after stroke ( ). A combined treatment of simvastatin with BMSCs was found to exert the best therapeutic effects compared to simvastatin or MSC monotherapy, perhaps because simvastatin significantly increased SDF-1 expression and CXCR4 in BMSCs and thus increased the migration of the BMSCs towards the lesion (246, 247). Other drugs such as MCI- 186, oxiracetam and adrenomedullin have also been demonstrated to have synergistic effects (248, 249, 251, 253). Combined treatment of some chemicals extracted from Chinese herbal medicines like sodium ferulate and Ginsenoside Rg1 with MSCs also revealed enhanced efficacy ( ). Furthermore, the combined use of Niaspan has been reported to attenuate the adverse effects such as cerebral hemorrhage and arteriosclerosis of BMSCs treatment in type 1 diabetes mellitus (T1DM) rats subjected to an ischemic stroke (250). However, the combined therapy does not always work. Balseanu et al. showed that the combination of granulocyte colony-stimulating factor (G-CSF, 50 µg/kg daily for 28 days) with BMMSCs (single dose of 1 million cells/kg, iv.) starting at 6h post-stroke did not achieve a superior effect compared to G-CSF alone after experimental stroke in aged rats (255). A similar result has also been seen in another study using the combination of G-CSF and BMMNCs after stroke in senescent rats (266). One study conducted in hypertensive rats reported that the delivery of BMMNCs at 48h after stroke even abolished the efficacy of G- CSF, perhaps because the transplanted cells in the spleen had hindered the clearance of granulocytes that were increased by G-CSF (267) Others As an important part for stroke treatment, the effect of rehabilitation on cell therapy is one aspect to be considered. Sasaki et al. showed that those rats that received daily rehabilitation with treadmill running exercise after an IV infusion of MSCs showed enhanced therapeutic effects compared to those receiving either MSC infusion or rehabilitation alone (260). An enriched environment has also been claimed to exert additional benefits when combined with a form of cell therapy (268, 269). The exploitation of biomaterials such as thermoreversible gelation polymer hydrogel has been attracting increasing interest as a way to improve the survival and migration of transplanted cells (261) Stroke modeling in preclinical studies A valid experimental stroke model is essential for preclinical studies. The filament induced tmcao in rodents is currently the most widely used, followed by distal MCAO, photothrombotic stroke and endothelin-1 (ET-1) (270). Each stroke model has its own pros and cons. The filament model can produce a stroke allowing for reperfusion (271, 272), but the damage is highly variable and often extensive, with significant morbidity and mortality. Distal MCAO produces a cortical stroke. It is relatively easy to perform with high survival rates in the animals, but somatosensory properties rather than motor function are more severely altered (273). ET-1 is a vasoconstrictor which evokes stroke after local infusion into the brain parenchyma or adjacent to MCA. It is highly targetable and generates a cortical stroke with excellent behavioral readouts, but it is limited by the lack of reproducible effect of ET-1 in mice. Photothrombotic (PT) stroke is highly reproducible, relatively non-invasive and technically simple to perform. It is ideal for in vivo optical studies and for lesioning a specific cortical region. However, it is not possible to target the subcortical region without fiber optic implantation (274, 275).

39 Behavioral tests in preclinical studies Most of the behavioral tests are used to assess sensorimotor function. Hicks et al. summarized that the three most frequently used behavioral tests in for functional outcome assessment after cell-based therapy were the modified Neurological Severity Score (mnss), adhesive removal test and rotarod test (29). This situation seems to have remained the same from 2009 until today (276). The minimal workload and simplicity may be the reasons for selection of these particular behavioral tests. However, these frequently used tests do not have adequate sensitivity aside from identifying the initial deficits after stroke. Other commonly used behavioral tests with countless variations include cylinder test, grid walking test, tapered beam-walking test and treadmill test (29, 276). Use of behavioral tests that could provide quantitative data, show long term impairment, and not be affected by repeated testing, is recommended (e.g., Montoya staircase test). Water-maze (277, 278), elevated plusmaze (161), open-field (279), and forced swimming tests (280) have also been used to evaluate cognition, spontaneous activity or psychological status changes like anxiety or depression after stroke Clinical studies of MSCs for stroke In 2005, Bang et al. reported a clinical study of IV infusion of autologous bone marrowderived MSCs twice 4 weeks after stroke onset in five patients. During the one year followup, no adverse events were observed related with the cell delivery. Moreover, there was a significant improvement in performance in daily living using Barthel s Index and a tendency of improvement in degree of disabilities evaluated by modified Rankin Score (mrs) in the MSC-treated patients (281). Five years later they reported again with 16 treated patients. An increased survival and decreased mrs were observed as compared with control, although not statistically significant (282). A few other clinical studies also demonstrated the therapeutic potential of MSC therapy in stroke patients (table 2), but the effect cannot be considered definitive due to the small sample sizes without proper control. A recent multicenter clinical trial reported no beneficial effects compared to placebo control after the intravenous infusion of multipotent adult progenitor cells (MAPCs) into 67 patients 24-48h after the onset of ischemic stroke. However, post-hoc analysis revealed benefits of cell transplantation in earlier time points of 24-36h (5). No severe adverse events except for headache, transient fever, or nausea have been reported. More multi-centered, randomized, placebo controlled, double-blinded trials with larger sample sizes are urgently needed to investigate the effects of MSCs on stroke patients. 2.4 RATIONALE Intravascular MSC delivery holds significant potential for ischemic stroke, but the efficacy is far from optimal, perhaps due to limited homing of the delivered cells. IA delivery is more efficient than intravenous administration, but cell preparation and infusion protocol should be carefully optimized to avoid micro-embolism as well as ensuring efficacy. The VLA- 4/VCAM-1 interaction is essential for firm adhesion and transendothelial migration (283). MSCs typically express integrin β1, a subunit of VLA-4, but only a minimal level of integrin α4 (ITGA4) (284, 285). This may limit their homing capabilities into the ischemic tissue. It has been hypothesized that ITGA4 overexpression on MSCs by genetic engineering may enhance their homing into the injured brain after ischemic stroke and thereby improve functional outcome.

40 18 Table 2. Clinical studies of MSC therapy for patients with ischemic stroke. Year Number of patients Cells Treatment Follow-up Results Adverse events Reference treated, 25 controls 16 treated, 36 controls 6 treated, 6 controls treated autologous BMMSCs autologous BMMSCs autologous BMMSCs autologous BMMSCs IV delivery of first at 4-5 weeks and then at 7-9 weeks after symptom onset IV delivery of first at 4-5 weeks and then at 7-9 weeks after symptom onset IV delivery of at 7-12 months post-stroke, followed by 8 weeks of physiotherapy IV delivery of cells at days poststroke 12 months 5 years 24 weeks 12 months Significantly improved BI and a trend in mrs consistently during the follow-up Tendency of increased survival rate and decreased mrs score in MSCtreated patients compared to control No significant improvement in FMS and mbi results, increased number of cluster activation of Brodmann areas BA4 and BA6 at 24 weeks Improved functional outcome as measured by NIHSS and mrs, and reduced lesion volume by MRI No (281) Transient fever (282) No (286) Transient fever, nausea, slight itching, slight appetite loss, local pain (189) treated UCMSCs IA delivery of cells at days post-stroke 6 months 2 patients demonstrated improved muscle strength and improved mrs No (287) treated UCMSCs alone or with NSPCs IV delivery of /kg or followed by 3 infusions of /patient and NSPCs at /patient through the cerebellomedullary cistern from less than 1 week to 2 years after stroke onset 2 years treated UCMSCs Intrathecal delivery >1 year treated treated, 62 controls allogeneic SB623 cells MAPCs Intracerebral delivery of cells 6-60 months after stroke Intravenous delivery of cells 24-48h poststroke 12 months 90 days A trend of improvement in NIHSS, mrs and BI 5 patients had an improved outcome by HAI rating scale Significant improvement in ESS, NIHSS and FMS No significant improvement in global stroke recovery compared to control Fever, dizziness, recurrent stroke in one patient 10 months after receiving 4 intravenous infusions of MSCs Headache, low-grade fever, low back pain, lower limb pain Procedural headache, muscle spasticity, gait disturbance (288) (289) (290) No (5) MSC: mesenchymal stem cell, BMMSC: bone marrow-derived mesenchymal stem cell, UCMSC: umbilical cord-derived mesenchymal stem cell, NSPC: neural stem/progenitor cell, SB623 cell: Notch-1 transfected BMMSC, ESS: European Stroke Scale, NIHSS: National Institutes of Health Stroke Scale, mrs:modified Ranking Scale, FMS: Fugl Meyer Scale, BI: Barthel index, mbi: modified Barthel Index, MAPC: multipotent adult progenitor cell.

41 19 3 Aims of the study The aim of the study was to advance the development of safe and efficient intracarotid MSC transplantation for stroke. Our hypotheses were as follows: 1. omplications of intra-arterial cell delivery would be dependent on cell dose, infusion volume and infusion velocity (I). 2. The in vitro cell clumping and viability before transplantation could be related with cell concentration, storage solution, storage time and the freeze-thawing procedure (II). 3. The lentiviral-transduced VLA-4 overexpression on MSCs could improve the cerebral homing and reduce the risk of cerebral embolism after intracarotid cell delivery (III).

42 20

43 21 4 Materials and methods 4.1 PREPARATION AND CHARACTERIZATION OF BMMSCS (I-III) Culture and characterization of rat BMMSCs (I-III) Oricell TM Wistar rat BMMSCs (Cyagen Bioscience Inc., Neu-Isenburg, Germany, Cat. No. RAWMX-01001) were used. In study I and II, the cells were cultured in OriCell MSC growth medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine and 1% penicillinstreptomycin (all reagents from Cyagen Biosciences Inc., Cat. No. GUXMX-90011) at 37 C under 5 % CO2. The medium was changed twice a week. The cells were passaged after reaching 80-90% confluency. Rat BMMSCs at passage 5 were cryopreserved in the proteinfree Oricell TM NCR cryopreservation medium (Cyagen Biosciences Inc., Cat. No. NCPF-10001) for further use. In study III, BMMSCs were seeded in growth medium (KnockOutTM DMEM/F-12, Life Technologies) supplemented with 1% GlutaMAX (Life Technologies), 7.5% FBS and 1% penicillin/streptomycin (PAA Laboratories GmbH, Pasching, Austria) at 37 C under 5% O2 and 5% CO2, and cryopreserved in FBS with 10% DMSO for further use ITGA4-gene modification of BMMSCs (III) Lentiviral transfer vectors carrying the cdna sequence (NM_ ) for rat ITGA4 in front of a tdtomato sequence were transferred to the MSC monolayer. After 6h of incubation, the supernatant was replaced with growth medium. After culturing for 2 more passages, ITGA4-positive clones were sorted based on tdtomato overexpression using flow cytometry (BD FACS-ARIA), and expanded thereafter. TdTomato overexpressing control MSCs (CTRL- MSCs) were also prepared accordingly. To confirm the ITGA4 overexpression after lentiviral transduction, quantitative RT-PCR was performed using TaqMan Fast Advanced MasterMix (Thermo Fisher Scientific), TaqMan Gene Expression Assay probes for ITGA4 (Rn _m1, Thermo Fisher Scientific) and ITGB1 (Rn _m1, Thermo Fisher Scientific). For immunocytometry, MSCs grown on the glass cover slides were fixed and triton-permeabilized, blocked with PBS/10% normal goat serum, and then incubated with rabbit-anti-itga4 antibody (1:250, Cell Signaling Technology, Leiden, The Netherlands) at 4 C overnight. After washing, the MSCs were incubated with IgG-anti-rabbit AF488 (1:250) for 1.5h. The cover slides were then washed and mounted with fluorescent mounting medium (Dako) containing DAPI. Images were acquired using a fluorescent microscope (Eclipse Ti-E, Nikon GmbH, Düsseldorf, Germany). The rat MSC identification kit (R&D Systems, Minneapolis, USA) was used to verify the mesenchymal identity of tri-lineage differentiation. A growth curve was used to determine their proliferative potential. 4.2 MODIFIED BOYDEN CHAMBER ASSAY (III) A modified Boyden chamber setup (figure 5) was used to assess the transendothelial migration capacity of MSCs by FluoroBlok or the standard transwell assay (8µm pore size, Corning) (291). The rat cerebral endothelial cell line GPNT was grown to a confluent monolayer onto a collagen IV coated trans-well migration insert and then stimulated with TNFα (100ng/mL, Peprotech, Rocky Hill, USA) overnight. MCP-1 (Peprotech; 10ng, 50ng or 100ng in 0.6mL medium) was then added to the bottom of the chamber. A total of 25,000 MSCs, which were pre-labeled with carboxy fluoresceindiacetate succinimidyl ester (CFSE)

44 22 or PKH26 were seeded onto the GPNT monolayer and allowed to transmigrate overnight. For FluoroBlok assay, migrated MSCs were visualized from the lower side of the transwell mesh using an inverted fluorescence microscope (Eclipse Ti-E, Nikon). For standard transwell assay, multilayered Z-stack images were acquired using confocal laser scanning microscopy (Zeiss LSM710, ZEN software). The number of MSCs was quantified with ImageJ. Figure 5. Modified Boyden chamber. 4.3 FLOW CYTOMETRY (II, III) The samples were run on FACS Canto II flow cytometer (BD Biosciences, San Jose, USA) using FACSDiva software. The data was analyzed using Flowjo Sample preparation (II, III) Cultured rat BMMSCs at passage 5 (study II) or passage 8 (study III) were harvested before FACS analysis. Cryopreserved BMMSCs were thawed in a water bath at 37 C before being decanted into the complete growth medium (study II). In study II, after centrifugation, the cells were resuspended in phosphate-buffered saline (PBS) without calcium or magnesium, normal saline (NS; 0.9% NaCl) or complete medium to examine the effect of the storage solution, and then they were maintained at 37 C for 3 h, 6 h or 9 h at a concentration of /ml before measurement to investigate the effect of storage time. Fresh cells were also resuspended in PBS at a concentration of /ml, /ml, /ml and /ml, respectively, to determine the effect of cell concentration. In study III, freshly-harvested MSCs were resuspended in PBS at a concentration of /ml and analyzed immediately Detection of cell clumps (II, III) The flow cytometry-based pulse-width assay was used to quantify cell clumps. FSC-W was plotted against FSC-A by adjusting the photomultiplier tube voltage and area scaling factor (292). After calibration of the FSC-W axis using standardized polystyrene microspheres (Poly-sciences Inc, Cat. No ), the clumps or large cells >30 µm were gated (figure 6). Figure 6. Quantification of cell clumps in suspension using flow cytometry-based pulse-width assay.

45 Detection of cell viability (II) The CellEvent TM Caspase-3/7 Green Flow Cytometry Assay Kit (Thermo Fisher Scientific, Cat. No. C10427) was used to assess cell apoptosis and death following the manufacturer s instructions. Briefly, CellEvent TM Caspase-3/7 Green Detection Reagent was added to the cell suspension and then incubated in the dark at 37 C for 30 minutes to stain the apoptotic cells. SYTOX AADvanced TM dead cell stain solution was then added to the suspension during the final 5 minutes of staining. Then the stained cell samples were run by flow cytometry. 4.4 ANIMALS A total of 104 male RccHan Wistar rats weighing g were from the Laboratory Animal Center, Kuopio, Finland (study I; n=42) and Envigo, The Netherlands (study III, n=62). The animals were housed under a temperature controlled environment (21 ± 1 C, humidity 50-60%, light period 07:00-19:00) with food and fresh water available ad libitum throughout the experiment. All animal studies were approved by the Animal Ethics Committee (Hämeenlinna, Finland) and performed in accordance with the European Community Council Directives 86/609/EEC guidelines. All efforts were made to minimize the number of animals used and to ensure their welfare throughout the studies Transient middle cerebral artery occlusion (tmcao, I, III) Transient focal cerebral ischemia was induced by the intraluminal filament technique as described previously (293) (III). Anesthesia induction of rats was achieved by 5% isoflurane for 1-2 minutes and then maintained at % with an O2/N2O mixture (30 %/70 %) at a surgical depth of anesthesia using a nose mask throughout the operation. The body temperatures of rats were maintained at 37 C using a heating pad connected to a rectal probe (Harvard Homeothermic Blanket Control Unit). After a midline cervical incision, the right CCA, the external carotid artery (ECA) and the ICA were exposed. After cutting the ECA, a heparinized nylon filament (diameter 0.28 mm) with blunt tip was inserted via the ECA stump and advanced into the ICA until resistance was felt ( cm). After 1-2 h of middle cerebral artery (MCA) occlusion, the filament was gently removed and the ECA was carefully closed by electrocoagulation or suturing, leaving a long ECA stump for cell infusion. The sham-operated rats were treated in a similar manner, but without the filament insertion (I, III). Locally applied lidocaine was placed on the wound (I, III) and buprenorphine (0.03 mg/kg, RB Pharmaceuticals Ltd.) was administered subcutaneously to relieve any postoperative pain (I). Moreover, NS was supplemented intraperitoneally and softened food pellets were given after surgery to prevent weight loss (I, III). MRI or limb placing test was performed on the next day to assess the success of the tmcao operation Cell transplantation (I, III) The rat BMMSCs were transplanted through the re-exposed ECA stump while the blood flow in ICA was maintained (figure 7). After cell transplantation, the ECA stump was carefully closed by electrocoagulation.

46 24 Figure 7. Scheme of cell transplantation. The cells were infused from the ECA stump after sham or tmcao surgery. In study I, cells in PBS were injected into rats at two days after the sham operation, and the group assignments were as shown in table 3. Table 3. Group assignment in study I. Infusion Cell dose Infusion volume Number of Stroke model time (x10 6 ) (ml) rats (min) 0.00 Sham Sham Sham Sham (iron-labeled) Sham Sham Sham Sham In study III, control or ITGA4-overexpressed cells in 0.5 ml PBS were infused within 3 minutes at one day after sham or tmcao surgery. The group assignments are as shown in table 4. Table 4. Group assignment in study III. Stroke Cell dose Number of Infused cells model (x10 6 ) rats Sham DiD-labeled CTRL-MSCs Intravital microscopy Sham DiD-labeled ITGA4-MSCs with 24h follow-up tmcao DiD-labeled CTRL-MSCs tmcao DiD-labeled ITGA4-MSCs Intravital microscopy tmcao DiD-labeled CTRL-MSCs with 3d follow-up tmcao DiD-labeled ITGA4-MSCs SPECT/CT imaging tmcao 111 In-oxine-labeled CTRL-MSCs tmcao 111 In-oxine-labeled ITGA4-MSCs Stroke lesion confirmation tmcao PBS 0 2 tmcao: transient middle cerebral artery occlusion, CTRL-MSCs: control mesenchymal stem cell, ITGA4- MSCs: integrin alpha4-overexpressed mesenchymal stem cells, PBS: phosphate buffered saline.

47 CBF monitoring with laser Doppler flowmetry (I) The PeriFlux System 4000 (Perimed, Järfälla, Sweden) was used to monitor the local CBF. After an incision to expose the skull, a probe was placed above the sensorimotor cortex (1 mm posterior, 3 mm lateral to bregma). The CBF signal was then recorded from 5 min before cell infusion and subsequently for 30 min afterwards. The mean values of signal before cell infusion were used as baseline. The mean values of signal were also measured during cell infusion, and at 5 min periods during the 30 min follow-up after cell infusion. The changes of signal were expressed as percentage of baseline. Area under curve was calculated to reveal the overall CBF changes. The laser Doppler signal was recorded and analyzed using PeriSoft for Windows Imaging (I, III) Magnetic resonance imaging (I, III) In study I, MRI was performed at 24 h after cell transplantation using a Bruker 9.4T horizontal scanner to detect micro-occlusion or hemorrhage. In order to track the transplanted cells, cells were incubated overnight with 25 µg/ml Molday ION Rhodamine B (BioPAL, Worcester, USA, CL-50Q02-6A-50), a super paramagnetic iron oxide formulation, before transplantation. The rats were anesthetized with 2-2.5% isoflurane (5% for induction) in a gas mixture of 30% O2/70% N2 delivered via a nose mask. T2 weighted images were acquired using the following parameters: repetition time (TR) = 3.0 s, echo time (TE) = 32 ms, average=16, matrix size of , field-of-view (FOV) = 30 mm 30 mm, 15 slices with 1 mm slice thickness. For T2* weighted images, TR=750 ms, TE=3.0 ms, TE2=6.0 ms, average=6, FOV=25.6 mm 25.6 mm, 15 slices with 1 mm slice thickness. Lesion volumes were measured by Mat lab R2012a, Aedes 1.0. A semi-quantitative scale was also used to evaluate lesions on MRI images: 0 point, no lesions; 1 point, 1-5 focal lesions ( 2 mm); 2 points, 5-10 focal lesions; 3 points, more than 10 focal lesions or confluent lesions. In study III, MRI was performed with a 7T horizontal scanner (Bruker) at 1 day after MCAO, before cell transplantation and SPECT/CT imaging in Charles River. T2-weighted scans were acquired from isoflurane-anesthetized animals using a multi-slice multi-echo sequence with TR of 2.5s, FOV of mm, 18 slices with 1 mm thickness SPECT/CT imaging (III) SPECT imaging was performed at 1h, 24h, and 48h after cell transplantation. MSCs were labeled with 111 In-oxine (37MBq/ml in Tris-buffer for 30min at 37 C, Nycomed Amersham, Little Chalfont, UK) before being transplanted. Whole body 3D images combined with CT were acquired as the reference. Helical SPECT imaging was then performed at the same coordinates with 45s/frame. High resolution multi-pinhole apertures (NSP-105-R15-WB) were used to enhance resolution. After SPECT imaging, helical CT was subsequently performed with 180 projections at 55kVp. SPECT image reconstruction was conducted using HiSPECT and image processing was done using InVivoScope software. Radioactivity was calculated after normalization to the volume (% ID/cm 3 ). After the last scanning, the animals were euthanized and blood, heart, lung, liver, spleen and brain were collected. Radioactivity was then measured by gamma counting normalized to the organ weight (% ID/g) Intravital microscopy (III) Immediately before initial imaging, a 3 3mm round cranial window (294) was made above the right sensorimotor cortex of the rats. The FV1000MPE two-photon microscope (Olympus; OLYMPUS FLUOVIEW Version 4.2 software) with the 25 water immersion high NA objective was used. Before imaging, FITC-conjugated dextran (MW = 2000 kda, Sigma) was injected in order to visualize the cerebral vasculature. MSCs were labeled with DiD (5µL/mL for 25min at 37 C in darkness, Life Technologies) before infusion to increase their visibility under the microscope. A Mai Tai Broad Band DeepSee laser was used for excitation at 800nm. Emitted light was collected using bandpass filters ( nm for FITC-conjugated dextran

48 26 and nm for DiD). Arterioles and venules were discriminated by longitudinal line scans (295). Z-stack images or time-lapse recordings were collected with zoom factor 1 at pixels or pixels, respectively. The CBF of at least one pair of arteriole/venules was recorded before and after cell transplantation. Imaging was performed during and up to 72h after cell transplantation. At least 5 fields with a size of µm were chosen for each imaging. The 3D images were obtained using Imaris CBF measurement and quantification of the transplanted MSCs were done with ImageJ Behavioral tests (I, III) Limb placing test (I, III) The limb placing test was used to assess the sensorimotor integration of fore- and hindlimb responses to tactile and proprioceptive stimulation (296). This test has seven tasks in total. In study III, only four tasks were performed. The tasks are scored as follows: 2 points, the rat performs normally; 1 point, the rat performs with a delay (>2 s) and/or incompletely, and 0 point, the rat does not perform normally Cylinder test (I) The cylinder test was used to assess imbalance between the spontaneous use of impaired and non-impaired forelimbs (297). The rat was placed in a transparent cylinder (ø 20 cm) and videotaped during the light period of the light: dark cycle through a mirror placed at 45 o angle beneath the cylinder. Exploratory activity for 1 to 3 min was recorded and later analyzed using a program with slow motion capabilities. The numbers of contacts by left, right or both forelimbs were counted. The imbalance in rats forelimb use was calculated as: [(use of left or right forelimb+0.5 both forelimb uses)/ (total contacts)] x 100% Open field test (I) The open field test was used to measure spontaneous locomotor activity (298). The test was run under dimmed conditions to avoid any inhibitory effect of light. The apparatus is a black circular arena with a diameter of 116 cm surrounded by a 40 cm high wall. The location of the experimental animal was recorded using an infrared-sensitive video camera-computer linkup. The field was divided into six preprogrammed zones, and the test lasted 10 min in total. EthoVision XT 7.1 software was used to record and analyze the behavioral data Histology (I, III) Perfusion (I, III) In study I, the rats were perfused at 48 h post-transplantation, while in study III, the rats were perfused at either 24 h or 72 h post-transplantation for histology. All the rats were perfused transcardially with NS followed by 4% paraformaldehyde in PBS. The brains were carefully removed from the skulls, post-fixed and cryoprotected. A sliding microtome was used to cut frozen sections (35 µm) for staining. Images were obtained using a Zeiss Axio Imager M2 microscope or confocal laser scanning microscope (Zeiss LSM 800 Airyscan) and processed in Zen software Nissl staining (I, III) Nissl staining was used to reveal the cerebral infarct (I, III). Infarct volume (mm 3 ) was measured from Nissl-stained slices after correction for edema using an image analysis system MCID (299) (III) Modified Gallyas silver staining (I) Modified Gallyas silver staining was used to visualize degenerating terminals and cell bodies (300). Briefly, it contains the following steps: 1) alkaline pretreatment (ph 13); 2) silver impregnation; 3) washing; 4) development at ph monitored by an indicator and 5)

49 27 washing in acetic acid. Finally, the sections were mounted with Depex mounting medium and coverslipped IgG staining (I) IgG staining was used to assess the BBB integrity (301). After washing in PBS, the slices were incubated in 1% H2O2 for 15 minutes, blocked in 2% normal goat serum for 2 h followed by incubation with biotinylated sheep anti-rat IgG (1:200, AbD Serotec, Oxford, UK) for 48 h at 4 C. Then the sections were rinsed with Tris-buffered saline with 0.5% Triton X-100 (TBS-T) and incubated with streptavidin-horseradish peroxidase conjugate (1:1000, GE Healthcare, UK) for 1 h, and developed with diaminobenzidine for 4-6 minutes Prussian blue staining (I) Prussian blue staining was used to visualize the iron-labeled MSCs in the brain (302). The sections were incubated with 2% potassium ferrocyanide in 2% HCl for 5 minutes. Neutral red was used for counterstaining. The sections were then mounted with Depex mounting medium and coverslipped RECA-1 staining (III) Blood vessels were stained using RECA-1 (rat endothelial cell antibody). The free-floating sections were incubated with mouse anti-rat RECA-1 antibody (1:2000, AbDSerotec) overnight at room temperature and then washed with TBS-T, followed by incubation of Alexa Fluor 488 goat anti-mouse IgG (1:400, Life Technologies) for 2h. After repeated washing, the slices were mounted on slides with mounting medium containing DAPI and coverslipped VCAM-1 staining (III) The free-floating sections were incubated with rabbit anti-rat VCAM-1 antibody (1:500, Abcam) overnight at room temperature and then washed with TBS-T, followed by incubation of Alexa Fluor 405 goat anti-rabbit IgG antibody (1:500, Abcam) for 2h. After repeated washing, the slices were mounted on slides with mounting medium and coverslipped. 4.5 STUDY DESIGNS Figure 8 shows an overall summary of the study designs. Cells were infused on postoperative day 2 in study I and post-operative day 1 in study III.

50 28 Figure 8. Study designs. 4.6 STATISTICAL ANALYSIS The statistical analysis was performed using SPSS software Comparisons between two groups were done with two-tailed Student s t-test or Mann-Whitney test. Other comparisons were done using repeated measures ANOVA or two-way ANOVA followed by Bonferroni post hoc correction for multiple comparisons. The level of significance was P < Data are presented as mean ± standard deviation.

51 29 5 Results 5.1 EFFECTS OF CELL DOSE AND INFUSION VOLUME/VELOCITY ON SAFETY OF INTRACAROTID MSC DELIVERY Lower cell dose is safer for intracarotid delivery of BMMSCs For rats infused with different cell doses but with the same velocity (0.5ml/3min), there was a cell dose related reduction in the CBF in rats after cell transplantation as recorded by LDF. In contrast, CBF seemed to slightly increase in the control group (figure 9A). Cell-induced lesions were found mainly in the cortex and subcortical white matter, but some were also detected in the striatum and even in the brain stem (figure 9B). No signs of hemorrhage were observed. There was a cell dose-related increase in lesion size (r = 0.81, P < 0.001) and MRI score (r = 0.85, P < 0.001). The T2* images of rats received iron-labeled MSCs showed hypointense signal primarily in the ipsilateral hemisphere and mainly close to the lesions (data not shown). The T2* signal was rarely seen in rats without embolisms. Prussian blue staining revealed cells being trapped in the micro-vessels. Nissl and silver staining revealed focal brain damage in the same region as observed with MRI. IgG staining showed BBB leakage in the lesion core (data not shown). In the limb placing test, the mean score for left limbs in group (11.2 ± 5.0) was significantly lower than the corresponding value in the other groups (14) (P < 0.01). No significant difference was found between groups in the cylinder test. In the open field test, although no significant difference was observed between groups after cell transplantation, velocity, total distance moved and moving duration were found to negatively correlate with cell dose (r = -0.42, -0.42, and -0.49, respectively, P < 0.05) (figure 9C). Figure 9. Cell injection with different doses. There was a dose-related reduction of CBF signal by LDF (A), increase of lesion size on MRI (B, red arrow), and (C) reduction of moving velocity and total distance and duration by open field test. * P<0.05 vs. PBS group.

52 Infusion velocity is related to the safety of intracarotid BMMSC delivery For rats infused at different velocities but with the same dose ( ), CBF decreased extensively in 0.5 ml/6 min group. No significant difference was found between the other groups (figure 10A). The MRI score was the highest and the lesion size was the largest in the 0.5 ml/6 min group, but the differences with other groups were not statistically significant (figure 10B). In the limb placing test, the mean score of left limbs in 0.5 ml/6 min group (10.8 ± 4.0) was significantly lower than that in both the 0.5 ml/3 min (max score 14) and the 1.0 ml/3 min group (max score 14) (P < 0.05), but not in the 1.0 ml/6 min group (11.3 ± 5.5). No significant difference was found between groups in the animals performance in the cylinder or open field test (figure 10C). Figure 10. Cell injection with different velocities. CBF decreases the most in 0.5ml/6min group as recorded by LDF (A). Lesion size is the largest in 0.5ml/6min group on MRI (red arrow) (B). No significant difference was found in the open field test (C). *P<0.05 vs. 0.5ml/3min group. 5.2 IN VITRO OPTIMIZATION OF CELL SUSPENSION Cell concentration, cell viability and clumping Since we observed a cell dose-related increase in cerebral embolism after IA MSC delivery, clumps of MSC suspensions in PBS at different concentrations were quantified to exclude the possibility of increased cell clumping in vitro. For the fresh rat BMMSCs in PBS with different cell concentrations, cell viability was found to correlate positively (r=0.94, P < 0.001) with cell concentrations, and viability in the /ml group was significantly higher than that in the /ml group (P < 0.001) (figure 11A). No increase in the extent of cell clumping was observed with escalating concentrations (figure 11B). No significant correlation was found between the percentage of cell clumps and cell concentration.

53 31 Figure 11. Cell viability (A) and percentage of cell aggregates (>30 µm) (B) for cells with different concentrations. ***P< Storage solution and storage time, cell viability and clumping After harvesting from culture plates (0 h), cell viability in PBS (>65%) was significantly reduced (P < 0.001), with a similar number of cell clumps to that in the medium (figure 12). The cell viability was comparable in NS and medium (>90%), but significantly fewer cell clumps were present in NS (P < 0.01 compared to those in PBS or growth medium). Along with increasing storage time, there was a time-dependent reduction in the viability in all three solutions. The reduction was the most pronounced in NS (r = -0.96, P < 0.001) and least evident in medium (r = -0.73, P < 0.001). Cell clumping also increased in medium (r=0.64, P < 0.001), but not in NS or PBS (figure 12). Figure 12. Cell viability (A) and percentage of cell aggregates (>30 µm) (B) for cells in different storage solutions for different durations. * P<0.05, ** P<0.01, *** P<0.001: PBS vs. growth medium group; ## P<0.01, ### P<0.001: normal saline vs. growth medium group Freeze-thawing, cell viability and clumping There was a significant overall viability reduction for the thawed cells preserved in growth medium and NS (P < 0.01), and a trend for those preserved in PBS compared with fresh cells (figure 13). In comparison with the fresh cells, the number of cell clumps significantly increased after thawing for the cells stored in NS (P < 0.05), whereas a decrease was observed for cells in PBS (P < 0.05). The thawed cells stored in medium displayed the highest viability (P < 0.05 vs. NS, P < vs. PBS) although no significant difference was observed in the percentage of cell clumps.

54 32 Figure 13. Cell viability (A) and percentage of cell aggregates (>30 µm) (B) for freshlyharvested and freeze-thawed cells in different storage solutions. *, # P<0.05, ** P<0.01, ***, ### P< EFFECTS OF ITGA4 OVEREXPRESSION ON SAFETY AND HOMING OF INTRACAROTIDLY DELIVERED MSCS ITGA4 overexpression enhances in vitro transendothelial MSC migration Lentiviral transduction of MSCs and subsequent sorting resulted in a significantly higher mrna level of ITGA4 (P < 0.05 vs. CTRL-MSCs, figure 14A). ITGA4 protein was also found to be expressed throughout the ITGA4-modified MSCs (ITGA4-MSCs) according to immunocytochemistry. The lentiviral transduction and sorting procedure did not affect MSC growth or differentiation. The Boyden chamber assay revealed significantly increased migratory activity of ITGA4-MSCs in the presence of 50 ng (P < 0.01) and 100 ng MCP1 (P < 0.001) compared to CTRL-MSCs (figure 14B,C). Figure 14. Lentiviral transduction resulted in higher mrna level of ITGA4 (A). FluoroBlok assay showed increased transmigration of ITGA4-MSCs in the presence of 50ng and 100ng MCP1 (B and C). *P<0.05, ** P<0.01, ***P< ITGA4 overexpression does not increase in vivo transendothelial MSC migration The diameter of suspended DiD-labeled MSCs was µm as measured by fluorescence microscopy, with an average diameter of around 18.0 µm for both ITGA4- and CTRL-MSCs. After infusion, most of the MSCs disappeared from the field of view under the two-photon microscope within 5 minutes. The arrested MSCs were found entrapped inside vessels

55 33 smaller than their cell size (diameter 5-10 µm) (figure 15A). No statistical difference in the number of MSCs in the brain within 2h after transplantation was revealed between CTRLand ITGA4-MSCs (figure 15B). The number of observed MSCs declined at 24h after transplantation, and the remaining MSCs were still entrapped within the cerebral vasculature at 24h or 72h post-transplantation. No transendothelial migration was observed. Consistently, the majority of the MSCs were entrapped in the blood vessels at both 24h and 72h after transplantation by histology. The whole body biodistribution of MSCs by SPECT/CT imaging also showed initial, transient cell localization in the ipsilateral cerebrum but without any significant increase in the ITGA4-MSCs signal (data not shown). Figure 15. Intra-vital microscopy revealed DiDlabeled MSC (red) entrapped in microvessels (green) (A). No significant difference in the number of MSCs in the brain as observed by intravital microscopy between groups (B) ITGA4 overexpression decreases MSC aggregation and cerebral embolism Cell aggregates (diameter >30 µm) were observed in 2 of 6 sham rats and 3 of 5 stroke rats after CTRL-MSCs administration, but in only 1 of 5 sham rats and in none of stroke rats after ITGA4-MSCs administration under the two-photon microscope. Those cell aggregates blocked the arteries, resulting in a CBF reduction and local ischemia (figure 16A). Transplantation of CTRL-MSCs into stroke rats caused a significant decrease of CBF (P < 0.05) as well as an increase in lesion volume (P < 0.05) compared to the situation with ITGA4-MSCs (figure 16B). In total, 4 out of 14 rats died following CTRL-MSC transplantation, while 1 out of 13 rats died after ITGA4-MSC transplantation. There were significantly more cell aggregates with diameter >30 µm in the CTRL-MSC suspension as measured by flow cytometry (P < 0.01). Figure 16. Vascular embolism caused by large cell aggregates (red) after injection (A). Change of arterial blood flow and lesion volume in different groups (B). *P<0.05, **P<0.01.