Neural Progenitors for Sensory and Motor Repair

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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1365

Neural progenitors for sensory and motor repair

JAN HOEBER

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0058-0 UPPSALA urn:nbn:se:uu:diva-328590 2017 Dissertation presented at Uppsala University to be publicly examined in B/C8:305, Husargatan 3, Uppsala, Monday, 23 October 2017 at 10:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Professor Linda Greensmith (University College London, Institute of Neurology, Sobell Department of Motor Neuroscience and Movement Disorders).

Abstract Hoeber, J. 2017. Neural progenitors for sensory and motor repair. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1365. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0058-0.

Injury and neurodegenerative conditions of the spinal cord can lead to paralysis and loss of sensation. Cell therapeutic approaches can restore sensory innervation of the spinal cord following injury and protect spinal cord cells from degeneration. This thesis primarily focuses on the restoration of deaffarented sensory fibres following injury to the dorsal root and spinal cord. These injuries lead to the formation of a non-permissive glial scar that prevents sensory axons from reinnervating spinal cord targets. It takes advantage of a dorsal root injury model that closely mimics spinal root avulsion injuries occurring in humans. In the first part of the thesis, three different neural progenitor types from human or murine sources are tested for their regenerative properties following their transplantation to the site of dorsal root avulsion injury. In the second part, the ability of murine neural progenitors to protect spinal motor neurons from a neurodegenerative process is tested. In the first original research article, I show that human embryonic stem cell derived neural progenitors are able to restore sensorimotor functions, mediated by the formation of a tissue bridge that allows ingrowth of sensory axons into the spinal cord. In the second research article, I present that murine boundary cap neural crest stem cells, a special type of neural progenitor that governs the entry of sensory axons into the spinal cord during development, are unable to form a permissive tissue bridge. This is possibly caused by the contribution of transplant derived ingrowth non-permissive glial cells. In the third research article, I show that human neural progenitors derived from foetal sources are capable of stimulating sensory ingrowth and that they ameliorate the glial scar. When this approach is combined with the delivery of sensory outgrowth stimulating neurotrophic factors, these cells fail to form a permissive tissue bridge and fail to modify the glial scar. In the final research article, murine boundary cap neural crest stem cells are shown to protect motor neurons, which harbor an amyotrophic lateral sclerosis causing mutation, from oxidative stress. Oxidative stress is a pathological component of amyotrophic lateral sclerosis in human patients. Taken together, this thesis provides first evidence that sensory regeneration following a spinal root avulsion injury can be achieved by transplantation of human neural progenitors. In addition, it introduces murine boundary cap neural crest stem cells as interesting candidates for the cell therapeutic treatment of amyotrophic lateral sclerosis.

Keywords: Regenerative Neurobiology, Stem cells, Sensory regeneration, Spinal cord injury, Amyotrophic Lateral Sclerosis, Neurodegeneration, Oxidative Stress

Jan Hoeber, Department of Neuroscience, Regenerative neurobiology, Box 593, Uppsala University, SE-75124 Uppsala, Sweden.

ISSN 1651-6206 ISBN 978-91-513-0058-0 urn:nbn:se:uu:diva-328590 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-328590) "If the doors of perception were cleansed everything would appear to man as it is, inﬁnite." (William Blake, The Marriage of Heaven and Hell)

This work is dedicated to Joshua and Stefﬁ

List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Hoeber J, Trolle C*, König N*, Du Z, Gallo A, Hermans E, Aldskogius H, Shortland P, Zhang S-C, Deumens R, Kozlova EN (2015) Human Embryonic Stem Cell-Derived Progenitors Assist Functional Sensory Axon Regeneration after Dorsal Root Avulsion Injury. Scientiﬁc Reports, Volume 5, Article Number 10666.

II Hoeber J (2015) Sensory regeneration in dorsal root avulsion. Neural Regeneration Research, Volume 10, Issue 11.

III Trolle C, Ivert P, Hoeber J, Rocamonde-Lago I, Vasylovska S, Lukanidin L, Kozlova EN (2017) Boundary cap neural crest stem cell transplants contribute to the glial scar and do not assist regeneration of sensory axons. Regenerative Medicine, Volume 12, Issue 4.

IV Hoeber J*, König N*, Trolle C, Lekholm E, Zhou C, Pankratova S, Åkesson E, Fredriksson R, Aldskogius H, Kozlova EN (2017) A Combinatorial Approach to Induce Sensory Axon Regeneration into the Dorsal Root avulsed Spinal Cord. Stem Cells and Development, Volume 26 Issue 15.

V Aggarwal T*, Hoeber J*, Ivert P, Vasylovska S, Kozlova EN. (2017) Boundary Cap Neural Crest Stem Cells Promote Survival of Mutant SOD1 Motor Neurons. Neurotherapeutics, Volume 14, Issue 3. Co-Authored articles that are not part of the thesis

VI Ivert P, Otterbeck A, Panchenko M, Hoeber J, Vasylovska S, Zhou C, Garcia-Bennett A, Kozlova EN (2017) The Effect of Mesoporous Silica Particles on Stem Cell Differentiation. Journal of Stem Cell Research & Therapeutics Volume 2, Issue 3.

VII Schizas N, König N, Andersson B, Vasylovska S, Hoeber J, Kozlova EN, Hailer NP (2017) Neural Crest Stem Cells protect Spinal Cord Slice Cultures from Excitotoxic Neuronal Damage and Inhibit Glial Activation. submitted to Cell and Tissue Research

VIII Dmytriyeva O, Christiansen A, Hoeber J, Larsen KL, Køning A, Rasmussen KK, Soroka V, Klingelhofer J, Kozlova EN, Sangild T, Pankratova S (2017) VEGF-A versus VEGF-B: novel peptide mimetics derived from their receptor binding sites promote neurite outgrowth and neuronal survival of primary central and peripheral neurons in vitro. Manuscript

Reprints were made with permission from the publishers. Abbreviations

ALS amyotrophic lateral sclerosis BDA biotinylated dextran amine bNCSC boundary cap neural crest stem cell C cervical CAG chicken β-actin promoter coupled with the cytomegalovirus immediate-early enhancer CGRP calcitonin gene-related peptide CNS central nervous system CNTF ciliary neurotrophic factor CSPG chondroitin sulfate proteoglycan CTB cholera toxin subunit B DRA dorsal root avulsion DREZ dorsal root entry zone DRG dorsal root ganglion DRR dorsal root rhizotomy DRTZ dorsal root transitional zone EB embryoid body EGF epidermal growth factor FGF fibroblast growth factor FUS RNA-binding protein fused-in-sarcoma GDNF glial derived neurotrophic factor GFAP glial fibrillary acidic protein GLAST glutamate aspartate transporter GRP glial-restricted progenitor ESC embryonic stem cell hNP human spinal cord neural progenitor hscNSPC human spinal cord neural stem/progenitor cell IB4 isolectin B4 iPS induced pluripotent stem cell L lumbar MND motor neuron disease MNP motor neuron progenitor MSC mesenchymal stem cell NA numerical aperture NEAA non-essential amino acid NF200 neurofilament 200kD NG2 neural/glial antigen 2 NGF nerve growth factor Continued on next page NSC neural stem cell NT3 neurotrophin-3 OEC olfactory ensheating cell PBS phosphate buffered saline PFA paraformaldehyde PNS peripheral nervous system RA retinoic acid RNA ribonucleic acid ROS reactive oxygen species S sacral SOD1 Cu/Zn superoxide dismutase SHH sonic hedgehog T thoracic TDP-43 TAR DNA-binding protein 43 VAPB vesicle-associated membrane protein-associated protein B/C VEGF vascular endothelial growth factor VRTZ ventral root transitional zone Contents

1 Introduction 1 1.1 Development and anatomy of the spinal cord ...... 1 1.2 Restoration of sensory innervation of the spinal cord ...... 3 1.2.1 Sensory innervation of the spinal cord ...... 3 1.2.2 The PNS-CNS interface before and after injury . . . . . 5 1.2.3 Spinal root injury and the dorsal root avulsion model . . 7 1.2.4 Regeneration of sensory axons through the PNS-CNS interface ...... 9 1.2.5 Cell therapy for sensory regeneration in dorsal root injury 10 1.3 Protection of spinal motor control using neural progenitors . . . 12 1.3.1 The motor neuron disorder: Amyotrophic lateral sclerosis 12 1.3.2 Cell therapeutic spinal cord transplantation for ALS . . 13 1.3.3 Stem cell based in vitro disease modelling of ALS . . . 15

2 Aims of the study 17

3 Experimental procedures 18 3.1 Ethical permits and informed consent ...... 18 3.2 Cell culture ...... 18 3.2.1 Generation of transplantable human spinal cord neural progenitors derived from human embryonic stem cells [Paper I,III] ...... 18 3.2.2 In vitro differentiation of human spinal cord neural progenitors [Paper I] ...... 19 3.2.3 Generation of transplantable murine boundary cap neural crest stem cells [Paper III,V] ...... 19 3.2.4 Generation of transplantable human spinal cord neural stem/progenitors derived from human foetal spinal cord [Paper VI] ...... 19 3.2.5 In vitro differentiation of human spinal cord neural stem/ progenitor cells [Paper IV] ...... 20 3.2.6 Mesoporous silica particle loaded neurotrophic factor mimetics [Paper IV] ...... 20 3.2.7 Generation of motor neuron cultures from murine embryonic stem cells [Paper V] ...... 21 3.2.8 Magnetic activated cell sorting (MACS) [Paper V] . . . 21 3.2.9 In vitro analysis of motor neuron and boundary cap neural crest stem cell cultures [Paper V] ...... 21 3.3 Animal studies ...... 22 3.3.1 Dorsal root avulsion injury and cell transplantation [Paper I,III,IV] ...... 22 3.3.2 Cervical spinal cord cell injection [Paper V] ...... 22 3.3.3 Transganglionic tracing [Paper I,III,IV] ...... 23 3.3.4 Tissue and cell sphere collection and processing [Paper II,III-V] ...... 24 3.3.5 Immunohistochemistry [Paper II,III-V] ...... 24 3.3.6 Behavioural analyses [Paper I] ...... 25 3.4 Microscopy [Paper I,III-V] ...... 28 3.5 Image analysis [Paper I,III-V] ...... 28 3.6 Statistics [Paper I,III-V] ...... 29

4 Results and Discussion 30 4.1 Paper I ...... 30 4.2 Paper III ...... 33 4.3 Paper IV ...... 34 4.4 Paper V ...... 36

5 Conclusions 38

6 Future aims 39 6.1 Stimulation of sensory axon ingrowth by cell therapeutic means 39 6.2 Boundary cap neural crest stem cells for cell therapeutic transplantation in amyotrophic lateral sclerosis ...... 40

7 Sammanfattning på svenska 41

8 Acknowledgment 42

References 43 List of Figures

1.1 Development of a spinal cord segment ...... 2 1.2 Spinal cord segments and dermatomes ...... 3 1.3 Sensory innervation of the dorsal horn ...... 4 1.4 Transitional zones of the spinal cords afference and efference . 6 1.5 The dorsal root transitional zone after injury ...... 7 1.6 Experimental dorsal root avulsion injury ...... 9 1.7 Pathological features of stem cell based in vitro ALS models. . 16 3.1 Overview of conducted experiments ...... 22 3.2 Cell therapeutic approaches for dorsal root avulsion ...... 23 3.3 Stem cell injection into the cervical spinal cord ...... 24

1. Introduction

1.1 Development and anatomy of the spinal cord During neurulation, the edges of the neural plate lift upwards and form the neural groove. When the edges converge, the neural groove is converted into the closed neural tube and the enclosed neural canal. The neural canal later forms the central canal of the spinal cord and the ventricles of the brain. The neural tube divides itself into the four subdivisions of the central nervous system (CNS). At the level of the prenatal spinal cord, a small group of cells migrate away from the closing neural tube and form the neural crest that later gives rise to the dorsal root ganglia (DRG). In later stages, the neural tube separates into the dorsal grey column that differentiates into neurons that mediate sensory information, the ventral gray column that gives rise to spinal motor neurons and interneurons, and the surrounding marginal layer that forms the white matter of the spinal cord (top of Figure 1.1)1. In the mature spinal cord, the peripheral white matter is comprised of glial cells and myelinated axons, and the central gray matter that is comprised of the soma, dendrites and synapses of most spinal neurons. In transverse orientation, the white matter separates into the dorsal, lateral and ventral column, and likewise the gray matter into dorsal, intermediate and ventral horn (bottom of Figure 1.1)2. The cyto-architecture of the gray matter can be classiﬁed by the modality of information that is conveyed to neurons of distinct areas, classically referred to as Rexed’s Laminae. The dorsal horn is comprised of Laminae I to IV that receive sensory information from the body’s surface and Laminae V and VI that receive sensory information from inside the body. The intermediate horn is roughly equivalent to Lamina VII, and relays information between spinal cord areas. Lamina X surrounds the central canal and encompasses axons that cross from one spinal cord side into the other. The ventral horn is comprised of the Laminae VIII and IX and contains the soma of all spinal motor neurons and interneurons that modulate motor control (see also Figure 1.3)3,4. When oriented on the osseous vertebral column the spinal cord resides in, the spinal cord can be separated into ﬁve levels that are named after their longitudinal localization along the body: the cervical (C), thoracic (T), lumbar (L), sacral (S) and coccygeal spinal cord. Each level is further divided into individual spinal cord segments that receive afferent information from sensory neurons of the peripheral nervous system (PNS) residing in the DRG. These sensory neurons extend axons into the spinal cord along the dorsal root and

1 Figure 1.1. Development of a spinal cord segment. terminate on spinal neurons that mediate reﬂex actions or convey information to the brain. Efferent information originating from the brain is conveyed towards appropriate segments and to spinal motor neurons residing inside the ventral horn. Spinal motor neurons project axons along the ventral root and innervate skeletal muscles and glands. Dorsal and ventral roots join together at the DRG and form the spinal nerves. Due to the topographic sensory innervation of the skin, individual parts of the body’s surface can be classiﬁed as belonging to individual spinal cord segments and are generally referred to as dermatomes (Figure 1.2)5.

2 Figure 1.2. The spinal cord segments and dermatomes. Modiﬁed from Spinal- research.org (October 29, 2015).

1.2 Restoration of sensory innervation of the spinal cord 1.2.1 Sensory innervation of the spinal cord The spinal cord receives sensory innervation by pseudo-unipolar sensory neurons that reside in the dorsal root ganglia (DRG) and from which a bifurcated axon is extended. The distal process of this axon projects into the periphery and receives information from the trunk and limbs. Its proximal process con- veys this information through the dorsal root. The majority of proximal processes enter the spinal cord through the Lissauer tract situated directly above Lamina I6. DRG can be classified as either visceral DRG that innervate the internal organs and non-visceral DRG that covey information from the skin, muscles and joints7. With the exception of muscle spindle sensory axons (Ia fibres) that primarily innervate spinal motor neurons of the same segment8, sensory neurons generally transmit information to defined areas of the spinal cord, brain stem and thalamus9. The modality of sensory information transmitted

3 by sensory neurons is determined by the type of sensory receptors that are associated with the terminal branch of the sensory axon.

Figure 1.3. Rexed’s laminae and the sensory innervation of the dorsal horn. Modiﬁed from Lallemend and Ernfors 2012 10.

Sensory information that originates from the skin or from inside the body is referred to as somatic sensory information. It encompasses four different sensory modalities, proprioception, mechanoreception, nociception and ther- moception4, and these can be attributed to sensory neurons classified by distinct electrical conduction velocities and expression of specific receptors and signalling molecules (Figure 1.3). Aα and some Aβ fibres are thickly myelinated and show the highest conduction velocities. They primarily transmit proprioceptive information and encompass the Ia, II and Ib muscle afferents. These afferents contribute to the spinocerebellar proprioceptive pathway and convey information from muscle spindles and golgi organs to spinal motor neurons in the ventral horn. Aβ and Aδ fibres are thinly myelinated and have intermediate conduction velocities, together with some C fibres they mainly transmit mechanoreceptive information of cutaneous and subcutaneous origin and primarily terminate in the deeper dorsal horn laminae. C-fibres are unmyelinated and exhibit the lowest conduction velocities, together with some Aδ fibres they convey nociceptive and thermoceptive information from the skin to superficial dorsal horn laminae10,11.

4 Characterization of sensory neurons by the expression of distinct molecules is generally more complicated due to the multitude and non-exclusiveness of expressed marker proteins. Therefore, only non-visceral sensory neurons will be regarded in the following classification. Sensory neurons use glutamate as their primary neurotransmitter but they utilize distinct sets of glutamate trans- porters. Proprioceptive and mechanoreceptive fibres often show high expression of the vesicular glutamate transporter 1 (VGluT1). In contrast, nociceptive fibres express primarily the vesicular glutamate transporter 2 (VGluT2)12. Myelinated large and medium sized sensory neurons express neurofilament 200kD (NF200), small and medium sized sensory neurons express calcitonin gene-related peptide (CGRP) and substance P, and small and medium sized sensory neurons express the glial derived neurotrophic factor (GDNF) receptor and show binding of Griffonia simplicifolia isolectin B4 (IB4)13. In addition, there are smaller populations of sensory neurons that show expression of CGRP and bind IB4, and some that express tyrosine hydroxylase14–16. More recent single-cell transcriptomic efforts confirm classical markers but also suggest a far more complex landscape of sensory neurons subtypes than previously believed17,18.

1.2.2 The PNS-CNS interface before and after injury The dorsal root branches out into smaller dorsal rootlets as it approaches the spinal cord surface. The dorsal rootlets terminate on a sheet of densely packed astrocytic processes that build up the glia limitans that covers the whole surface of the spinal cord19. Where axons enter the spinal cord, the glia limitans extends into the rootlet, while the rootlet remains wrapped in a sheet of Schwann cells that form its peripheral border. This anatomical structure de- marcates the transitional zone between the PNS and the CNS and is referred to as either the dorsal root transitional zone (DRTZ) or dorsal root entry zone (DREZ) (Figure 1.4)19. Axons originating from neuronal soma residing in the spinal cord exit the spinal cord through the ventral root transitional zone (VRTZ) that is organized in a similar way. Myelinated axons that traverse through the glia limitans at the DRTZ and VRTZ have a transitional node at this site. Their myelin sheet is formed by oligodendrocytes inside the spinal cord and by transitional Schwann cells in the periphery20,21. The pronounced central tissue projection into the dorsal root is absent from the ventral root (Figure 1.4). During development both the DRTZ and VRTZ are formed by a subset of late migrating neural crest cells, that are referred to as boundary cap neural crest stem cells (bNCSC)22,23. At later developmental stages, these highly migratory cells also give rise to satellite cells and a small population of nociceptive neurons in the DRG, Schwann cells of the spinal nerve root and migrate as far as the skin where they differentiate into terminal glia24,25.

5 Figure 1.4. Transitional zones (TZ) of the spinal cords afference (dorsal root, DRTZ) and efference (ventral root, VRTZ). Note the characteristic central tissue projection (CTP) at the DRTZ. Modiﬁed from Fraher 2000 19.

Injury to the spinal nerve leads to axonal outgrowth of sensory neurons as part of a strong regenerative response and is indicated by the up-regulation of regeneration associated genes including growth associated protein (GAP)-43, activating transcription factors (ATFs) and c-Jun26,27. In contrast, injury to the dorsal root results in a very limited outgrowth of sensory axons and less pronounced activation of regeneration associated genes and extracellular matrix proteins associated with axonal regeneration28–32. In addition, the central tissue projection of the DRTZ undergoes a strong astrogliotic response indicated by proliferation and swelling of astrocytes, their tight organization and an increase in the expression of glial ﬁbrillary acidic protein (GFAP). The resulting glial scar renders the DRTZ impenetrable for regenerating ﬁbres that extend beyond the site of injury33,33,34. Already Santiago Ramón y Cajal described in his original works that sensory axons that approach the glial scar either collapse, turn around or become immobile (Figure 1.5)35. The collapse and repellence of regenerating axons is generally attributed to factors that are prominent also in the glial scar following spinal cord injury. Astrocytes that react to injury release high levels of the chondroitin sulfate proteoglycans (CSPG): aggrecan, brevican, vesi- can, phosphacan and neural/glial antigen 2 (NG2)36,37. Oligodendrocytes and degenerating sensory axons deposit myelin associated axon growth in- hibitors38. The three best studied myelin associated axon growth inhibitors are the myelin-associated glycoprotein (MAG), Nogo proteins and the oligodendrocyte myelin glycoprotein that all bind the Nogo receptor and inhibit axonal outgrowth by activation of the Rho-ROCK pathway. Stimulation of this pathway leads to the collapse and retraction of the growth cone of outgrow- ing axons39,40. At the injured DRTZ, a phagocytic microglia response slowly clears the myelin debris but ultimately fails to allow sensory regeneration41. Immobile axons are of great interest to regeneration approaches as they might contain the potential to regrow after removal of the outgrowth inhibiting

6 agents. Originally these axons were believed to terminate on reactive astrocytes where they form synapse-like structures42,43. Recently these cells have been characterized as oligodendrocyte progenitor cells positive for the proteoglycan NG2 and represent a class of glial cells distinct from astrocytes39,44–46.

Figure 1.5. The dorsal root transitional zone after injury to the dorsal root. Modiﬁed from Smith et al 2012 47.

1.2.3 Spinal root injury and the dorsal root avulsion model Spinal root avulsion injuries are the result of traction forces that pull the spinal root nerve sheets away from the spinal cord until both the dorsal and ventral root are ripped from the cord48. In contrast to the rupture of the spinal root that appears post-ganglionic and thereby is not effecting the spinal cord directly, spinal root avulsion injury is always a pre-ganglionic injury resulting in the complete destruction of the DRTZ and/or VRTZ, and a longitudinal spinal cord injury. The complexity of the injury leads to the degeneration of affected roots, an inflammatory response and the subsequent formation of a glial scar at the site of injury49. In adults, spinal root avulsion most often occurs during high kinetic traffic or sport accidents, whereas in newborn children it is most often caused by dif- ficulties during delivery48,50. In about 70% of all cases the brachial plexus is affected and one or several roots of the C5-T1 segments are avulsed. The often young patients suffer from partial to complete paralysis of the affected extremity, loss of sensibility and in some cases severe untreatable chronic pain51,52. Nerve and muscle grafts show poor recovery but are usually benefi-

7 cial51. Reimplantation of the ventral root is routinely used in patients, it results in regrowth of axons along the ventral root and improves motor deﬁcits. In contrast, reimplantation of the dorsal root fails to induce ingrowth of sensory axons into the spinal cord53. Several rodent models to study sensory regeneration through the scarred DRTZ have been established so far. The two most commonly used models are the dorsal root rhizotomy or cut (DRR) and the dorsal root crush model. In DRR, the dorsal root is cleanly cut peripheral to the DRTZ using micro- scissors and is often followed by rejoining the cut surfaces using tissue sealants or sutures19. In dorsal root crush, the root is squeezed using a blunt forceps until sensory axons are completely disrupted. The endoneurial tube remains intact and no further procedure is necessary19. Sensory axons might to a very limited extend grow back into Laminae II and dorsal column following dorsal root crush54. However, regrowth is practically absent following DRR or dorsal root reimplantation55. The more recently developed dorsal root avulsion (DRA) model is characterized by the pulling of individual roots away from the spinal cord until their complete avulsion from the spinal cord is achieved, while leaving the ventral root undisturbed (Figure 1.6). Compared to the DRR and dorsal root crush model, DRA does not only result in injury to the dorsal root but also directly damages the DRTZ and the dorsal white and gray matter of segments that are connected to avulsed roots. On the cellular level, avulsion of several dorsal roots leads to rapid invasion of neutrophils into the ipsi-lateral dorsal horn and the loss of dorsal horn neurons. Inside the spinal cord, DRA results in bilateral astrogliosis, inﬂammation, chronic reduction of vascularization, immediate neurodegeneration and a second wave of neurodegeneration two weeks after injury56,57. For the study of pain, DRA has become an interesting model as well. DRA of selected thoracic and lumbar levels leads to the development of chronic pain below the level of avulsed segments. Consistent with observations in rodent models of true brachial plexus injury where both dorsal and ventral roots are avulsed at the cervical level58–60, the pain can be relieved by administration of glial activation inhibitors but becomes elevated in the presence of opioids61–63. These injuries strongly affect spinal neurons indicated by upregulation of the stimulus inducible gene c-Fos64,65. Interestingly, avulsion of the dorsal and ventral root of a single lumbar level (segmental spinal root avulsion) also results in chronic pain, but it manifests in the form of hypersensitivity to mechanical and thermal stimuli at dermatomes that correspond to the affected spinal cord segment66.

8 Figure 1.6. Experimental dorsal root avulsion injury. Reprinted by permission from Neural Regeneration Research (Hoeber, 2015), copyright (2015).

1.2.4 Regeneration of sensory axons through the PNS-CNS interface The scarred PNS-CNS interface also serves as a model to study sensory regeneration following spinal cord injury. It is often favoured due to its limited effect on the well-being of the studied species and controlled impact on the sensory system67. Sensory regeneration in the spinal cord can be approached from multiple angles. The two most prominent approaches focus on direct manipulation of the growth non-permissive environment of the scar and/or the stimulation of sensory axonal outgrowth pathways47. In one of the earliest studies that aim to render the glial scar permissive, the spinal cord of neonatal animals was irradiated. This resulted in the replacement of astrocytes and oligodendrocytes, that are normally localised at the DRTZ, by Schwann cells. In these animals, injury did not induce a typical glial scar and thus sensory axons were able to grow back into the spinal cord68,69. Less invasive is the enzymatic digestion of outgrowth inhibiting CSPGs using the bacterial chondroitinase ABC70. Interestingly, at the DRTZ chondroitinase ABC treatment only succeeds when combined with the stimulation of axonal outgrowth of sensory neurons and ideally stimulation of the mTor pathway71,72. Axonal ingrowth of sensory axons can also be achieved by inhibition of the Nogo receptor, the common downstream target of myelin associated axon growth inhibitors40,73. Stimulation of axonal outgrowth pathways of sensory neurons can be eas- ily achieved by injury to the peripheral part of the sensory axon as this results in axonal elongation of not only the peripheral but also the central part of the bifurcated axon74. This process relies on dual leucine zipper kinase (DLK) for effective signalling of an injury signal to the sensory soma and involves the hypoxia-inducible factor 1α before regeneration pathways are ac- tivated75,76. The "conditional lesion" is sufﬁcient for sensory axons to grow beyond a cut and rejoined dorsal root, into implanted nerve grafts and even results in ingrowth of sensory axons into the spinal cord following dorsal root crush77–79. Regrowth can be further promoted by the local delivery of neu-

9 rotrophic factors77. However, "conditional lesion" only promotes the regeneration of thickly myelinated sensory axons and not peptidergic and IB4 reactive sensory axons78,80,81. Interestingly, induction of an inflammatory response at the DRG alone leads to a comparable increase in axonal outgrowth71,82. A less invasive approach to induce sensory regeneration focuses on the delivery of neurotrophic factors that are known to control axonal outgrowth. These factors can be delivered using either injection of recombinant viruses that induce expression of neurotrophic factors by host cells or direct delivery of neurotrophic factors by implanted delivery devices such as infusion pumps. Myelinated sensory axons can be stimulated to grow back into the spinal cord using viral expression of neurotrophin-3 (NT3), and peptidergic sensory axons can be stimulated using viral delivery of fibroblast growth factor 2 (FGF 2) and neurotrophic growth factor (NGF)83. But ingrowing fibres do not show topographic specificity inside the spinal cord83. As a side effect, NGF overexpression induces sprouting of uninjured nociceptive axons leading to chronic pain in both injured and healthy animals83,84. To achieve topographic specificity of regenerating axons, NGF overexpression can be restricted to the dorsal horn and can be combined with virally induced expression of the axonal outgrowth repelling Semaphorin 3A in the ventral horn. This approach succeeds in two ways, it reduces the NGF induced chronic pain and achieves projection of ingrowing sensory axons to appropriate laminae85,86. Direct infusion of the injured dorsal root with NGF, NT3 or GDNF results in the regeneration of specific sensory axon types. NGF results in ingrowth of peptidergic axons, NT3 in ingrowth of NF200 expressing axons and GDNF in regrowth of NF200 positive, peptidergic and IB4 binding axons87. Acute delivery of NT3 induces outgrowth of myelinated sensory axons and leads to the recovery of proprioception88. However, it fails when treatment starts one week after the injury89. Thus, for this approach to succeed the time point of treatment is an important factor. In addition, NT3 induced outgrowth can be enhanced by viral induction of the mTor pathway90. Recently, systemic administration of the GDNF-family member artemin has been reported to induce regeneration of all three fibre types to appropriate laminae and to restore sensorimotor and nociceptive functions91,92. The mechanism behind this remains elusive and more recent evidence suggests that the effect of artemin might be limited to nociceptive axons93,94.

1.2.5 Cell therapy for sensory regeneration in dorsal root injury Apart from the previously presented approaches to induce sensory regeneration, also cell and tissue transplantation has provided promising results. It is generally believed that cell and tissue transplants that originate from early stages of development combine multiple beneﬁcial effects. Early approaches aimed to circumvent the scarred DRTZ completely by transplanting foetal

10 spinal cord tissue between the cut dorsal root and spinal cord. These approaches succeeded in stimulating ingrowth and synaptic integration of all types of sensory axons and could be combined with the local delivery of NGF to further improve axonal outgrowth95–100. Peripheral nerve grafts and ar- tificial nerve conduits succeed in a similar way when combined with local delivery of NGF85,101. In one of the earliest approach to overcome and not circumvent the reactive DRTZ, embryonic astrocytes were mounted on a cell carrying matrix and transplanted to the site of dorsal root crush, this allowed ingrowth of sensory axons by modifying the local glial environment at the DRTZ and provided trophic support to sensory axons102. For DRR, tissue adhesive alone or when combined with mono-nuclear cells results in limited sensory ingrowth103. Al- ternatively, injured sensory neurons can be completely replaced by DRG cells of embryonic origin. These cells differentiate into neurons that extend axonal projections into the periphery and re-innervate spinal cord neurons104–109. More recent studies focus on the identification of immature cell types that can be gained from the host and used for regenerative purposes. The ideal candidate would already support ingrowth of PNS originating axons into the mature CNS in its tissue of origin. During development, olfactory ensheathing cells (OEC), a type of radial glia, provide a growth supportive substrate for PNS originating olfactory axons to grow into the olfactory bulb that belongs to the CNS. Injections of OEC either into the dorsal horn or DRTZ allow sensory regeneration after DRR110,111. When OEC are applied to the cut surface of the dorsal root and the spinal cord before the root is reattached using a tissue adhesive, they interact with Schwann cells and astrocytes to form a permissive tissue bridge that allows ingrowth of sensory axons and reinnervation of dorsal horn neurons112,113. Special care has to be taken that OEC are collected from the olfactory bulb alone and are not injected as single cells, as otherwise they fail to exert their regenerative effect114,115. OEC can be obtained directly form the adult host. This paved the way for the currently ongoing clinical trials that test their regenerative potential in spinal cord injury patients116,117. All the aforementioned studies have used either models of DRR or dorsal root crush. DRA presents an additional challenge as it not only involves deaf- ferentiation and a more pronounced glial response, it also leads to the complete destruction of the DRTZ, neuronal loss and chronic pain. In analogy to the effect of OEC, boundary cap neural crest stem cells (bNCSC) could exert similar beneficial effects as they also contribute to the establishment of the PNS-CNS interface during development. For this reason, they were chosen as our first cell type of interest and their effect on DRA is presented in Paper III. Embryonic stem cell (ESC) derived spinal cord neural progenitors are another good candidate as they provide functional recovery in spinal cord injury and can be directed to an exclusive spinal cord lineage in vitro, prior to trans- plantation118–120. Directed differentiation into neural progenitors is another benefit of this approach as it minimizes the ESC inherent tumorigenic poten-

11 tial and chance to induce chronic pain in the host121,122. For these reasons ESC derived spinal cord neural progenitors were chosen as the second cell type of interest and tested in Paper I. An alternative source of spinal cord neural progenitors is foetal tissue. These cells form neurospheres in vitro and can be cultured over extend periods of time with only small changes in their cellular composition. In addition, they improve the functional outcome of spinal cord injury and do not cause chronic pain123–125. Thus, spinal cord neural progenitors from foetal tissue have been selected as our third cell type of interest and are tested in combination with neurotrophic factor delivery in Paper IV.

1.3 Protection of spinal motor control using neural progenitors 1.3.1 The motor neuron disorder: Amyotrophic lateral sclerosis Motor neuron diseases (MND) are a set of lethal progressive neurodegenerative disorders that are characterized by the degeneration of motor neurons situated in the cerebral cortex, brain stem and spinal cord. Their symptoms most often include muscle spasticity and atrophy of bulbar muscles and/or muscles of the limbs. Amyotrophic lateral sclerosis (ALS) is the most common form of MND but with a prevalence of 2-6 cases per 100.000 people still considered a rare disease126. In classical ALS, death by respiratory failure sets-in once motor neurons that control the diaphragm degenerate approximately 3-5 years after the initial diagnosis. So far, apart from the anti-glutamatergic drug Riluzole that results in a modest increase of survival of patients, no effective treatment exists127,128. Most cases of ALS share no family history and are considered sporadic (sALS). Around 10% of all ALS cases are based on the inheritance of spe- ciﬁc gene mutations and are summarized under the term familiar ALS (fALS). Nevertheless most cases share a similar pathophysiology while their origin might differ. 13 types of fALS can be distinguished, the most studied mutated protein is Cu/Zn superoxide dismutase (SOD1) that accounts for 20% of all fALS cases, followed by C9orf72 that accounts for 40%, TAR DNA- binding protein 43 (TDP-43) that accounts for 1-4% and the ribonucleic acid (RNA)-binding protein fused-in-sarcoma (FUS) that accounts for 4% of all fALS cases128. Several other rare protein mutations have been identiﬁed as well, for example in the vesicle-associated membrane protein-associated protein B/C (VAPB)128. The complex genetic background of fALS poses a great challenge as alone for the SOD1 protein over 150 different mutations are reported to be involved in ALS of which the most common are point mutations resulting in amino acid substitutions at D90A, A4V, H46R and G93A129. Mu- tations in SOD1 are believed to compromise the proteins function to inhibit reactive oxygen species in the cytosol and mitochondria, implicating their in-

12 volvement in the pronounced oxidative stress and mitochondrial dysfunction observed in fALS130,131. Apart from oxidative stress, multiple processes contribute to the degeneration of motor neurons in ALS. Neuroinﬂammation and an altered immuno- logical response132,133, hyper-excitability and glutamate excitotoxicity134,135, mitochondrial dysfunction131, protein aggregation136, altered axonal transport and dysregulated RNA processing have all been linked to this outcome137,138. In recent years, it became clear that microglia, astrocytes and oligodendrocytes are involved in some of these processes and generate a toxic spinal cord micro-environment that makes motor neurons less stable and reduces their survival135,139,140. This renders ALS a multi-factorial disease and suggests that treatment would have to provide support to motor neurons and render the spinal cord micro-environment less harmful to their survival.

1.3.2 Cell therapeutic spinal cord transplantation for ALS Stem cell based cell therapeutic approaches are believed to modulate multiple systems at once as transplanted cells show a high degree of plasticity141. Ideally transplanted cells would have the ability to alter the immune- and inflammatory response of the host, provide a protective environment for ALS affected spinal motor neurons and even replace dying cells142. Most current cell therapeutic approaches achieve one or two of these goals, whereas cell replacement is the most elusive so far. Strategies that aim to replace dying spinal motor neurons focus on the transplantation of motor neuron progenitors (MNP), neural stem cells (NSC) derived from human (hESC) or mouse embryonic stem cells (mESC), or induced pluripotent stem cells (iPS). Early studies utilizing murine MNP failed to show long-term benefits143, more recently human MNP transplantation achieved neuroprotection possibly by the secretion of the neurotrophic factors NT-3 and 4, NGF and vascular endothelial growth factor (VEGF) by transplanted cells144. In contrast to MNP transplants, NSC transplants from the human foetal nervous system are extensively studied due to their beneficial paracrine effects145,146. Intra-spinal injection of NSC result in the successful integration of NSC derived neurons into the spinal cord circuitry, synaptic innervation of motor neurons and improve survival of ALS mice147,148. This effect requires grafted NSC to be in close proximity to hosts motor neurons and the outcome can be improved by performing multiple injections along the spinal cord149,150. In addition, transplanted NSC can be genetically modified to over-express VEGF. VEGF increases motor neuron survival and motor functions151 and might stimulate endogenous NSC populations152. NSC derived from iPS show robust differentiation once inside the host spinal cord and improve several aspects of the ALS pathology by modulation of the inflammatory response and neurotrophic factor secretion153,154. However, ultimately

13 these approaches fail to replace dying neurons. In conclusion, MNP and NSC generally improve survival time and motor functions most likely due to their neurotrophic and immunomodulatory properties155. With more and more evidence pointing towards a special role of glial cells in the ALS pathology156, increasing efforts are made to test the effect of glial progenitor transplantation on ALS animals. In an initial study, embryonic glial-restricted progenitors (GRP) differentiated primarily into astrocytes when injected into the spinal cord of ALS rats and improved motor neuron loss, motor functions and survival. The transplanted cells effect was attributed to the amelioration of glutamate excitotoxicity by increasing the expression of the astrocytic glutamate transporter glutamate transporter-1 (GLT-1)157. Both GLT-1 and the astrocytic glutamate aspartate transporter (GLAST) are major regulators of glutamatergic excitotoxicity158. Human GRP from foetal sources fail to exhibit the same properties possibly due to their longer maturation time to become fully functional astrocytes159,160. Umbilical cord blood cells transplanted to the spinal cord of ALS animals exhibit properties of astrocytes, in addition they secrete anti-inflammatory cytokines. This approach prolongs survival time and ameliorates ALS pathology161,162. Another type of glial progenitor, the OEC receives a lot of attention in clinical trials due to its beneficial properties in spinal cord injury163. These cells remyelinate neurons, are neuroprotective and improve survival of ALS animals164. However, they seem to have no effect on human ALS patients165,166. Apart from the cell therapeutic approaches mentioned above, mesenchymal stem cells (MSC) are good candidates due to their well documented neurotrophic and immunomodulatory properties. They are most often adminis- tered systemically or injected into the cerebrospinal fluid and are generally beneficial167. When injected into the cerebrospinal fluid they can migrate into the spinal cord grey matter where they give rise to cells that exhibit properties of astrocytes. They protect motor neurons, reduce inflammation and extend the lifespan of ALS mice168. Similarly when human bone marrow derived MSC are transplanted directly into the spinal cord, they decrease inflammation, improve motor function and extend survival169. One major limitation of all the pre-clinical studies presented here is the exclusive use of SOD1G93A mice and rats. Despite these shortcomings, NSC and MSC transplants are the most promising approaches for cell therapeutic strategies in ALS and this is reflected in the multitude of ongoing clinical studies centred around their use141,167. Very little is known about the potentially beneficial effects of other neuroprotective stem cell types. bNCSC transplanted into the spinal cord show cell protective and angiogenic properties and differentiate into both neurons and astrocytes170–174. These features render them interesting candidates for the cell therapeutic treatment of ALS. Their effect on ALS motor neurons is presented in Paper V.

14 1.3.3 Stem cell based in vitro disease modelling of ALS Since the advent of stem cell science and the development of efficient directed differentiation protocols, in vitro modelling of many neurological diseases has been greatly improved. Not only is it now possible to gain defined neuronal and glial cell population from human stem cells and tissue allowing the fo- cused study of their contribution to complex disease phenotypes, they can also be genetically modified or directly collected from patients exhibiting genetic abnormalities. The development of highly efficient protocols to gain motor neurons and astrocytes from embryonic and induced pluripotent stem cells allowed for several ALS in vitro models to emerge175. Initial steps on this path were made using the well characterized SOD1G93A mice lines. From these, mESC were collected and the resulting SOD1G93A mESC lines characterized. mESC derived SOD1G93A motor neuron cultures show an increased loss of motor neurons176,177, aberrant SOD1 accumula- tion176,177, a neurotoxic astrocyte phenotype176 and abnormal morphology177. Further, these cells were successfully used in a high-throughput screen of small molecules that revealed Kenpaullone, a GSK-3α/β and HGK inhibitor involved in neuronal apoptosis, that greatly improves motor neuron survival177. Most of the hallmarks identified in mESC derived SOD1G93A motor neurons are also present in primary and iPS derived murine SOD1G93A motor neu- rons176,178 and have been corroborated in SOD1 mutated motor neurons derived from human embryonic and induced pluripotent stem cells177,179–181. In addition to the previously present cellular abnormalities, human iPS derived SOD1G93A motor neurons revealed neurofilament dysregulation that could contribute to the degeneration of neurites and observed hyper-excitability of SOD1 G93A motor neurons180,182. Following the insights gained from the initial SOD1G93A mESC lines, the effect of other common fALS mutations on motor neurons was elucidated. Motor neurons derived from iPS harbouring the TDP-43 mutation show reduced neurite length, an abnormal protein level of TDP-43 and protein aggre- gation183,184. In addition, the cell line allowed the identification of anacardic acid in a high-throughput screen of small molecules. Anacardic acid is a his- tone acetyltransferase inhibitor, that ameliorates the reduced neurite length and abnormal levels of TDP-43184. Motor neurons derived from iPS harbouring the C9orf72 mutation confirm glutamate excitotoxicity and hyper-excitability as a cause of neuronal death and revealed RNA toxicity and deficient nucle- oplasmatic transport to contribute to the C9orf72 pathology182,185–188. Less well understood are the cell pathological features of motor neurons derived from iPS harbouring VAPB or FUS mutations, as so far only decreased levels of VAPB and hyper-excitability of FUS motor neurons are reported in these lines182,189,190. Apart from the motor neuron pathology, also the neurotoxic effect of astrocytes in ALS has been corroborated for SOD1 and C9orf72 using a combination of stem cell derived motor neurons and fALS astrocytes from

15 primary and stem cell sources191–196. In contrast, TDP-43 mutated astrocytes show typical aggregation of TDP-43 but are not toxic to motor neurons197. Taken together, the most common observation in in vitro stem cell derived fALS afflicted motor neurons is protein aggregation, reduced survival of neurons, shorter neurites and hyper-excitability and for fALS afflicted astrocytes, their neurotoxic effect on motor neurons (Figure 1.7). In all these models sur- prisingly little is known about their susceptibility to oxidative stress, a feature that is commonly linked to mitochondrial dysfunction in ALS. This is even more important as in a recent gene profiling study that analysed motor neurons derived from iPS cells from sALS patients, mitochondrial dysfunction was linked to the degeneration of motor neurons in most cases198.

Figure 1.7. Pathological features of stem cell based in vitro ALS models.

16 2. Aims of the study

The general aim of this thesis was to analyse the potential of neural progenitors for regenerative purposes in the spinal cord. Its major focus lies on neural progenitor transplantation for the recovery of sensory sensation and to a lesser extend on the potential beneﬁts of neural progenitor transplantation for the treatment of amyotrophic lateral sclerosis.

Specific hypothesis were tested: • The first aim of this thesis was to analyse the potential benefits of neural progenitor transplantation from human and murine sources to the site of dorsal root avulsion in mice. (Paper I, reviewed in Paper II, Paper III) • The second aim of this thesis was to analyse whether the observed human neural progenitor induced sensory regeneration could be translated to stem cells from alternative human sources and whether their beneficial effect could be improved by the co-administration of neurotrophic factors. (Paper IV) • The third aim of this thesis was to analyse the effect of murine neural progenitors on the survival of SOD1G93A motor neurons. (Paper V)

17 3. Experimental procedures

3.1 Ethical permits and informed consent All animal experiments were approved by the Uppsala Regional Ethical Com- mittee for Animal Experiments under the permits C298/11 and C178/14 in accordance with European Ethical directives. Human abortion tissue was collected from elective routine abortions, used for the extraction of foetal spinal cord tissue and established as spinal cord neurosphere cultures in accordance to the ethical permit # 2008/158-33/3, 2011/1101-32 approved by the Regional Human Ethics Committee Stockholm and with the informed consent of the pregnant woman. For the generation of CAG::hrGFP hESC, the commercially available H9 hESC line was used199.

3.2 Cell culture 3.2.1 Generation of transplantable human spinal cord neural progenitors derived from human embryonic stem cells [Paper I,III] Stable expression of humanized recombinant green fluorescent protein (hrGFP) under the control of the CAG promoter (chicken β-actin promoter coupled with the cytomegalovirus immediate-early enhancer) was introduced to hESC using the transcription activator-like effector nuclease (TALEN) genome editing technique180,200,201. The resulting CAG::hrGFP hESC line was patterned to a neuroepithelial fate according to the previously published protocol180. Neuroepithelia were sustained in cell culture media containing DMEM/F12, Neurobasal, GlutaMAX, neuronal cell culture supplements B27 and N2, and the defined small molecule mixture of GSK3 inhibitor CHIR99021, TGF/ Ac- tivin/ Nodal receptor inhibitor SB435142, bone morphogenic protein receptor ALK2 inhibitor DMH1, and the caudalizing morphogen retinoic acid (RA) together with the sonic hedgehog (SHH) agonist purmorphamine. Neuroepithe- lial cells were differentiated towards a spinal cord neural progenitor lineage in suspension culture. To induce directed differentiation, the defined small molecule mixture was replaced by higher concentrated RA and the SHH agonist Ag1.3. After seven days, spherical cell conglomerates had formed that expressed markers of ventral spinal cord neural progenitors. From here on, these conglomerates are referred to as human spinal cord neural progenitor (hNP) spheres.

18 3.2.2 In vitro differentiation of human spinal cord neural progenitors [Paper I] hNP spheres were dissociated and seeded onto poly-l-ornithin and laminin precoated glass cover-slips in differentiation medium containing DMEM/F12, Neurobasal, GlutaMAX, with neuronal cell culture supplements B27 and N2. To promote neuronal survival, neurotrophic factors glial derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) were added to the medium. 10 and 21 days after initiation of terminal differentiation, cells were ﬁxed using 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS). Cell populations present in hNP spheres and terminally differentiating human spinal cord neural progenitors were analysed using immunocytochemistry.

3.2.3 Generation of transplantable murine boundary cap neural crest stem cells [Paper III,V] For the generation of boundary cap neural crest stem cell (bNCSC) cultures, embryonic day 11 dorsal root ganglia from transgenic mice expressing red fluorescent protein variant DsRed.MST under the control of the CAG promoter (CAG::DsRed.MST, Jackson Laboratory,202) and from CAG::eGFP transgenic mice (Jackson Laboratory) were collected from the isolated cord and mechano- enzymatically dissociated. Single cells were cultivated in N2-medium containing DMEM/F12 supplemented with N2, B27, epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) until free floating neurospheres had formed (for a detailed description of the establishment of these cell lines see Aldskogius et al. 203 and for their characterization Hjerling-Leffler et al. 170). bNCSC neurospheres were used for cell transplantation (Paper III), in vitro differentiation on poly-D-lysine and laminin precoated glass cover-slips in DMEM/F12 and Neurobasal supplemented with non-essential amino acids (NEAA), B27, N2 (Paper III) and co-culture with mESC derived motor neurons (Paper V).

3.2.4 Generation of transplantable human spinal cord neural stem/progenitors derived from human foetal spinal cord [Paper VI] Human foetal spinal cord cells were isolated from human first trimester spinal cord tissue (6-9 weeks of gestation) and maintained as free floating human spinal cord neural stem/progenitor (hscNSPC) neurospheres. Cells were cultured in DMEM/F12 supplemented with glucose, Hepes, Heparin and N2 together with neurotrophic factors EGF, fibroblast growth factor 2 (FGF2) and CNTF125.

19 3.2.5 In vitro differentiation of human spinal cord neural stem/ progenitor cells [Paper IV] hscNSPC neurospheres were dissociated and seeded onto poly-l-ornithin and laminin precoated glass cover-slips in DMEM/F12 and Neurobasal supplemented with NEAA, N2, B27 and neurotrophic factors GNDF and CNTF or neurotrophic factor peptide mimetics for GDNF (Gliafin204) and CNTF (Cin- trofin205) delivered by mesoporous silica particles206. 5 and 8 days after initiation of terminal differentiation, hscNSPC were collected, ribonucleic acid (RNA) was isolated and RNA-to-cDNA synthesis was performed following the manufacturers protocol. Migration of differentiating hscNSPC was analysed using wide-field microscopy, and an expression profile for multiple differentiation associated marker genes was established using qRT-PCR analysis. For used primers please see Table 3.1.

Table 3.1. Primers used for qRT-PCR Target Forward Primer Reverse Primer Genes of interest Nestin 5’-aaggagaatcaagaactaatg-3’ 5’-ctttgtcagaggtctcag-3’ MAP2 5’-accgaggaagcattgattg-3’ 5’-aagttcgttgtgtcgtgtt-3’ β-3-tubulin 5’-aagttctgggaagtcatc-3’ 5’-ttgtagtagacgctgatc-3’ GFAP 5’-ccgtctggatctggagag-3’ 5’-tcctcctcgtggatcttc-3’ KI67 5’-gagacgcctggttactat-3’ 5’-gcagagcatttatcagatg-3’ OLIG2 5’-atccaatctcaatatctg-3’ 5’-tctactctgaatgtctat-3’ SOX2 5’-agagagaaagaaagggagagaa-3’ 5’-gccgccgatgattgttat-3’ GDNF 5’-agagactgctgtgtattg-3’ 5’-tcctcatcttccattctg-3’ CNTF 5’-ttctcttctaatggaatatagc-3’ 5’-caggctaaacttgtatgc-3’

Housekeeping Genes ACTB 5’-cagatcatgtttgagaccttc-3’ 5’-agaggcgtacagggatag-3’ GAPDH 5’-cctcaagatcatcagcaat-3’ 5’-ttccacgataccaaagtt-3’ B2M 5’-gactggtctttctatctct-3’ 5’-cttcaaacctccatgatg-3’

3.2.6 Mesoporous silica particle loaded neurotrophic factor mimetics [Paper IV] Mesoporous silica with an average particle size of 12 µm and average pore size of 20 nm were characterized before206,207. Particles were loaded with GDNF and CNTF peptide mimetics via impregnation in water and successive evaporation. Their release kinetics in simulated body ﬂuid was char- acterized206. For the loading of particles, the GDNF peptide mimetic Gli- aﬁn (153- ETMYDKILKNLSRSR-167; UniProtKB entry no. Q07731), that interacts with neural cell adhesion molecule (NCAM) and has neuritogenic

20 effects similar to GDNF204, and the CNTF peptide mimetic Cintroﬁn (148- DGGLFEKKLWGLKV-161; UniProtKB entry no. P26441), that interacts with other members of the CNTF receptor complex and has neuritogenic and neuroprotective effects205, were used.

3.2.7 Generation of motor neuron cultures from murine embryonic stem cells [Paper V] HB9::GFP mESC lines harbouring either the human SOD1G93A, human wild type SOD1176 or expressing endogenous murine SOD1208 were established on a monolayer of mouse embryonic ﬁbroblast cells in serum free propagation medium containing KoDMEM, KoSerum-Replacement, GlutaMAX, NEAA, β-mercaptoethanol and penicillin streptomycin. Following a slightly adapted version of the original protocol for motor neuron differentiation208 (for details please see Paper V), mESC lines were transferred to suspension culture dishes where they formed embryoid bodies (EB) in neural differentiation medium containing DMEM/F12, Neurobasal, GlutaMAX, neuronal cell culture supplements B27 and N2, β-mercaptoethanol and penicillin-streptomycin. Motor neuron differentiation of EBs was induced by the addition of RA and Ag1.3 to EB medium two days after the start of the suspension culture. EBs were dissociated after 7 days of culture and in some cases depleted of GLAST+ glial cells using magnetic activated cell sorting (MACS).

3.2.8 Magnetic activated cell sorting (MACS) [Paper V] In some cultures, mESC derived motor neurons were depleted of GLAST+ cells using magnetic activated cell sorting (MACS) against the extracellular epitope of GLAST the astrocyte-speciﬁc L-glutamate/L-aspartate trans- porter209, following the previously published protocol Machado et al. 210. Us- ing the same principle, bNCSC cultures were enriched for GLAST+ cells in some experiments.

3.2.9 In vitro analysis of motor neuron and boundary cap neural crest stem cell cultures [Paper V] GLAST-depleted motor neuron suspensions were seeded together with GLAST -enriched bNCSC onto poly-l-ornithin and laminin precoated glass cover-slips in DMEM/F12 and Neurobasal supplemented with GlutaMAX, N2, B27 and neurotrophic factors GNDF and CNTF. Under these conditions, mESC-derived motor neurons mature over the course of one week indicated by the expression of motor neuron speciﬁc markers208. Over the same time course, progressive loss of motor neurons occurs208. The percentage of surviving motor neurons

21 alone or in the presence of bNCSC was calculated for multiple time-points after initiation of differentiation. In additional experiments, cultures were treated with increasing levels of H2O2 and motor neuron and bNCSC survival rates were calculated.

Figure 3.1. Overview of conducted experiments.

3.3 Animal studies 3.3.1 Dorsal root avulsion injury and cell transplantation [Paper I,III,IV] Adult male nu/nu NMRI mice underwent dorsal root avulsion (DRA) and human spinal cord neural progenitor spheres or murine bNCSC neurosphere transplantation following previously established protocols57,172,173. Mice were kept under 2.5% isoflurane gas-anaesthesia while laminectomies were performed along the T13+L1 vertebrae of the left side. The dura was opened and the exposed L3-L5 dorsal roots were pulled one after another away from the spinal cord with steady force until they ruptured at the PNS-CNS interface. This led to a longitudinal dorsal spinal cord injury and destroyed the dorsal rootlets. Avulsed dorsal roots were repositioned to the site of avulsion and human and murine cell spheres were placed at the site of injury. Two groups in Paper IV received 20 µg of mesoporous silica particles loaded with neurotrophic factor mimetics Gliafin and Cintrofin locally applied to the site of injury (Figure 3.2).

3.3.2 Cervical spinal cord cell injection [Paper V] Adult male nu/nu NMRI mice received injections into the left cervical spinal cord. Mice were kept under 2.5% isoﬂurane gas-anaesthesia while partial

22 Figure 3.2. Overview of dorsal root avulsion cell therapeutic approaches presented in this thesis.

laminectomy along the C3-C5 vertebrae of the left side were performed. The dura was opened and GLAST-enriched bNCSC and SOD1G93A MN cell suspensions were injected into the spinal cord tissue and the wound was closed (Figure 3.3). The procedure was based on a published protocol from Lepore 211, for a detailed description please see Paper V.

3.3.3 Transganglionic tracing [Paper I,III,IV] Regeneration of sensory fibres was analysed by transganglionic tracing with either cholera toxin subunit B (CTB) or lectin Griffonia Simplicifolia Agglu- tinin isolectin B4 (IB4)212,213. In naive and sham animals, CTB exclusively labels myelinated and IB4 exclusively labels unmyelinated sensory axons214. For tracer injections, animals were kept under 2.5% isoflurane gas-anaesthesia while the left sciatic nerve was exposed and 1% (wt/vol) tracer solution was injected through the epineurium of the nerve using a fine glass pipette, following the previously published procedure215. Spinal cord tissue was collected three days after injection of the tracer.

23 Figure 3.3. Stem cell injection into the cervical spinal cord. Modiﬁed from Lepore 211 .

3.3.4 Tissue and cell sphere collection and processing [Paper II,III-V] Mice received an overdose of anaesthetics and were then transcardially per- fused with saline followed by 4%PFA/14% picric acid solution. Cervical or lumbar spinal cord tissue was collected, immersed in fixation solution for four hours and transferred to cryo-protection in 15% sucrose in PBS at 4 ◦C overnight. In vitro grown cell spheres were collected from the culture dish and fixed in 4%PFA in PBS for seven minutes followed by five minutes incuba- tion in 15% sucrose in PBS. Fixed tissue and spheres were embedded in OCT compound and flash frozen in a stream of CO2 gas or embedded in pulverized dry ice and cryo-sections were prepared using a cryostat set to -22 ◦C. Spinal cord pieces were cut in a coronal orientation.

3.3.5 Immunohistochemistry [Paper II,III-V] Tissue cryo-sections were thawed at room temperature and incubated in blocking solution containing bovine serum albumin, Triton X-100 and sodium azide (NaN3) in PBS. Mixed primary antibodies in blocking solution were applied in empirically determined concentrations and either incubated for four hours at room temperature or at 4 ◦C overnight. Following PBS washing of tissue sections, mixed secondary ﬂuorophore-conjugated antibodies targeting the host species of the primary antibodies were diluted to working concentration in

24 blocking solution and applied to sections for one hour at room temperature. After three PBS washes, tissue sections were covered with mounting solution containing glycerol and propyl-gallate in PBS. Immunocytochemistry was performed likewise after ﬁxation of cell cultures in 4%PFA in PBS for seven minutes at room temperature. To visualize the nucleus of cells, tissue sections or cell cultures were incubated with 1:10000 Hoechst 33342 in PBS for ﬁve minutes prior to embedding in mounting medium. For a complete list of all used primary and secondary antibodies please see Table 3.2.

3.3.6 Behavioural analyses [Paper I] Animals underwent behavioural testing for several parameters of sensorimotor skills and development of hypersensitivity to a repetitive stimulus (allodynia). In all cases, both the ipsi- and contra-lateral hind paw was analysed but effects of DRA and hNP transplantation were conﬁned to the ipsi-lateral side. Five months after DRA surgery, animals receiving hNP transplants underwent a second surgery that resulted in the complete transection of the reattached dorsal roots close to their respective dorsal root ganglion (DRG) and animals were analysed for two additional weeks.

Gait analysis Animals were tested with the CatWalk gait system that allows recording of several aspects of gait by automated illumination, detection and recording of the paw contact points of animals running in a straight line on a glass surface. Six parameters were analysed (base-of-support, paw print intensity, paw print area, stride length, stance phase and swing phase) that are commonly affected in models of neurotraumata217,218.

Van Frey filaments Van Frey hairs often also referred to as filaments are designed to apply a constant Newton force when pressed against a rigid surface and can be used to test the responsiveness of animals to defined mechanical stimulation when applied to specific parts of the skin. Here, the responsiveness of the animal’s glabrous mid-plantar hind paw was analysed by repeatedly applying a van Frey filament of a supra-threshold force (5.5g) and recording the paw withdrawal frequency. To detect hypersensitivity to a mechanical stimulus induced by DRA or cell transplantation, the paw withdrawal threshold was recorded using filaments ranging from 0.04 to 2g applied in an up-down fashion219.

Grip strength Grip strength as a combination of sensory perception and muscle coordination was recorded by placing the animal close to a metal bar connected to a Newton meter and let one hind paw voluntarily grip the bar. The animal was then

25 slowly pulled back and the force at the time of release of the paw from the bar was recorded.

26 Table 3.2. Antibodies used for immunohisto- and immunocytochemistry Primary Antigen Host Cat no. Source Dilution ACSA-1(GLAST) Mouse 130-095-822 Miltenyi Biotec 1:100 β-3-tubulin Mouse 32-2600 Invitrogen 1:500 Calbindin Rabbit CB-38a Swant 1:2000 CGRP Goat ab36001 Abcam 1:200 ChAT Goat AB144P Millipore 1:100 Chx10 Sheep AB9016 Millipore 1:400 CTB Goat 703 List Biochemical Laboratories 1:1000 DCX Rabbit ab18723 Abcam 1:1000 GFAP Mouse MAB3402 Millipore 1:200 GFAP Rabbit 2016-04 DAKO 1:500 GFP Mouse A11120 Molecular Probes 1:100 GFP-FITC Goat ab6662 Abcam 1:250 huSOD1 misfolded Mouse MM-0070-2 Medimabs 1:100 Hsp27 Rabbit SPA-803 Nordic Biosite 1:300 HuC/D Mouse A21271 Novex (Life Technologies) 1:400 HuNu Mouse MAB1281 Chemicon 1:50 huKi67 Rabbit ab92742 Abcam 1:50 Iba1 Rabbit 019-19741 Wako 1:500 IB4 Goat AS-2104 List Biochemical Laboratories 1:100 Islet1 Mouse 40.2D6 DSHB 1:200 Laminin Rabbit L9393 Sigma 1:50 Map2 Chicken ab5392 Abcam 1:1500 Msx1+2 Mouse AB 531788 DSHB 1:50 Mts1 (S100A4) Rabbit see publication Ambartsumian et al. 216 1:400 NG2 Rabbit ab5320 Abcam 1:200 Nkx6.1 Mouse F55A10 DSHB 1:20 Olig2 Rabbit ab18723 Millipore 1:500 p75NTR Goat AF1157 RD Systems 1:500 Pax7 Mouse Product PAX7 DSHB 1:50 RT97 (NF200kD) Mouse 1178709 Mannheim boehringer 1:50 SOX2 Goat Sc-17320 Santa Cruz 1:200 Stem123 (huGFAP) Mouse AB-123-U-050 StemCells 1:1000 TH Rabbit P40101 Pel-Freez 1:100 VGluT1 Rabbit 135 303 Synaptic Systems 1:1000 VGluT2 Guinea pig AB2251 Millipore 1:5000 VIAAT Rabbit 131 002 Synaptic Systems 1:100

Secondary AMCA 350 Rabbit 711-155-152 Jackson 1:100 Alexa 350 Mouse A10035 Invitrogen 1:500 Alexa 488 Mouse A21202 Invitrogen 1:1000 Alexa 488 Rabbit A11008 Invitrogen 1:1000 Alexa 546 Chicken A11040 Invitrogen 1:1000 Alexa 555 Goat A21432 Invitrogen 1:1000 Alexa 555 Rabbit A31572 Invitrogen 1:1000 Cy3 Guinea pig 706-165-148 Jackson 1:1000 Cy3 Mouse 715-165-151 Jackson 1:1000 Alexa 647 Mouse A31571 Invitrogen 1:1000 Alexa 647 Rabbit A31573 Invitrogen 1:500

27 3.4 Microscopy [Paper I,III-V] In vitro differentiated stem cells were imaged using either a Plan-Apochromat 10x objective (numerical aperture (NA) 0.25) or a Plan-Apochromat 20x objective (NA 0.75). Images for CTB quantiﬁcation were taken using a Plan- Apochromat 10x objective (NA 0.45) attached to an epiﬂuorescence micro- scope equipped with a CCD camera. For Papers I-IV, tissue sections and cell spheres were analysed using Zeiss LSM700, LSM710 and LSM780 confocal laser scanning microscopes. Images that contributed data to the same quantitative analysis were never collected with different microscopes. Single images were captured using a Plan-Apochromat 20x objective (NA 0.8), a LD LCI Plan-Apochromat 25x objective (NA 0.8) and a LD C-Apochromat 40x W Corr (NA 1.1) objective. For orthogonal projections, images were taken with a Plan-Apochromat 40x objective (NA 1.3) or a Plan-Apochromat 63x objective (NA 1.4). Z-stacks were taken with an optical slice thickness of 1 µm at an interval of 1 µm or 0.5 µm at an interval of 0.5 µm. For Paper III and V, tile- scanned images were captured using a Plan-Apochromat 20x objective (NA 0.8).

3.5 Image analysis [Paper I,III-V] For Paper I-IV, images of every 10th tissue section containing transplanted cells spanning along the L3-L5 segments of the spinal cord were processed using Fiji ImageJ2220. For Paper V, every 5th tissue section along the C3- C5 segments was analysed. Double and triple labelled cells were counted in a semi-sterological manner in confocal z-stacks spanning the whole 14 µm tissue section (Paper I) or in single confocal images (Paper III and IV) using the Fiji ImageJ2 cell-counter plugin. High magniﬁcation z-stacks for double labelled tissue sections were deconvolved using Huygens Essential software (Scientiﬁc Volume Imaging). Immunoreactive area was analysed using self- written ImageJ macros and implemented Fiji ImageJ2 functions for image pre- processing steps and subsequent data collection as reported in Papers I+IV. In general, images were background subtracted using the ImageJ implemented rolling ball algorithm and contrast enhanced to a saturation of 0.35, followed by automatic thresholding of the immuno-positive area using either Otsu’s or Tsai’s method221,222. In some analysis no suitable automatic thresholding technique could be found and therefore a manual threshold had to be set. In all cases, the site of dorsal root avulsion injury was set as the region of interest (ROI).

28 3.6 Statistics [Paper I,III-V] Data was described using mean values and the standard error of the mean. Bio- logical replicates were tested for normality using the Kolmogorov-Smirnov test and for homoscedasticity using scatter plots of the raw data. When these assumptions were violated, either the Mann-Whitney U test or for multiple comparisons the Kruskal-Wallis analysis of variance followed by Dunn’s Mul- tiple Comparison test was used. When normality and homoscedasticity could be assumed and only two conditions were tested a two-sample Student’s t-test was used, in cases where more then two conditions were analysed one-way ANOVA followed by Dunn’s Multiple Comparison test or Tukey’s test was used. For data sets that consisted of two independent categorical variables, two-way ANOVA was used followed by Sidak’s multiple comparison. The confidence interval was stated at the 95% confidence level, placing statistical significance at p < 0.05. Plotting of data and statistical analyses were performed in GraphPad Prism 6.

29 4. Results and Discussion

4.1 Paper I Human spinal cord neural progenitor (hNP) spheres expressed typical markers of ventral spinal cord neural progenitors of the neural lineage. Long-term in vitro differentiation primarily gave rise to cholinergic and inhibitory neurons and astrocytes. When transplanted to the site of dorsal root avulsion (DRA), hNP cells nearly exclusively differentiated into inhibitory neurons and astrocytes. Up to ﬁve month after transplantation, engrafted hNP continued to show expression of doublecortin, a marker typical for immature neurons. This corroborates the previously observed long maturation time of human neural progenitors from human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPS) after transplantation to the rodent central nervous system (CNS)223,224. It raises the question whether similarly long maturation times are expected when human neural progenitors would be transplanted to the humans CNS. Doublecortin is a 40-kDa microtubule-associated protein expressed by neural progenitors and post-mitotic neuronal precursors. It gets down-regulated with the appearance of markers of mature neurons and disappears shortly after birth in mice and pigs225–227. In humans, doublecortin is prominent in the sub-ventricular zone up to 18 months after birth and from there on it is nearly absent from the brain228. In the adult murine CNS, doublecortin is associated with neurogenesis, while newly born neurons cease to express doublecortin at the latest one month after their generation229. Taking the doublecortin expression in the young human brain and adult murine CNS into consideration, the presented maturation time here might lie within a reasonable time-frame. The month long maturation time of hNP might have implications on cell replacement strategies, as these aim to provide quick replacement of dying neurons in neurodegenerative diseases and after injury to the CNS230. It suggests that transplanting even further differentiated cells could increase the immediate effect of neuronal cell therapy and in parallel would reduce the risk of teratoma formation by stem cell derived cell types231. hNP intermingled with the dorsal root stump and interfered with the dorsal astrocytic scar and basal lamina. This resulted in the formation of openings in the otherwise continuous spinal cord surface and were referred to as "gates" in Paper I. The formation of "gates" shares many properties with the previously described growth permissive "tissue bridges" formed by transplanted olfactory ensheathing cells (OEC) at the PNS-CNS interface112. Like hNP transplants,

30 OEC directly contact endogenous astrocytes and show no migration into the spinal cord, but allow biotinylated dextran amine (BDA) labelled sensory axons to transverse the OEC transplant and grow into the spinal cord112. BDA is neuroanatomical tracer that is taken up by injured and uninjured axons and distributes by passive diffusion232,233. Thus, it does not allow to draw conclusions about the type of ingrowing sensory axons. Here, we performed transganglionic tracer injections of cholera toxin subunit B (CTB) or isolectin B4 (IB4) into the sciatic nerve resulting in the active transport of tracer to the neuronal soma in the dorsal root ganglion, and to myelinated and unmyelinated axonal projections of sensory neurons in the spinal cord, respec- tively212–214,234. Special care needs to be taken when CTB and IB4 are used after axotomy of the sciatic nerve, as CTB is transported by both myelinated and unmyelinated fibres and IB4 might fail to be transported beyond the dorsal root ganglion (DRG)235,236. This effect renders it difficult to analyse the type of regenerating sensory fibres by transganglionic tracing alone. So far, no report states whether sensory axons undergo similar changes in their ability to transport these tracers after dorsal root injury. Here, we could observe both tracers inside the DRG and adjacent nerve fibres, whereas only CTB tracer was found inside the ipsi-lateral spinal cord dorsal horn. Further, CTB positive profiles were found in both the superficial and deep dorsal horn, overlapping at least partially with dorsal horn areas that are innervated by proprioceptive and mechanoreceptive input in uninjured animals10,237. In addition to transganglionic tracing, both neurofilament 200kD (NF200) and calcitonin gene-related peptide (CGRP) expressing sensory fibres were found in the transplant area. Of these, only NF200 expressing fibres were able to cross the PNS-CNS interface through "gates" in the spinal cord surface. These were accompanied by fibres originating from the human transplant that superficially grew into the dorsal spinal cord. The selective ingrowth of NF200 positive fibres and the finding that only CTB traceable sensory axons were found in the dorsal horn, leads to the conclusion that primarily myelinated, and not unmyelinated or peptidergic fibres regenerated across the PNS-CNS interface. The neuronal population of engrafted cells could function as synaptic relays to restore the sensory circuitry after DRA. Foetal spinal cord tissue grafted into the injured adult spinal cord is able to functionally reunite separated segments of the spinal cord238. This approach proves to be equally efficient for the restoration of sensory connectivity99,100. The observed functional reconnec- tion is attributed to host sensory axons passing through the foetal graft and the formation of a spinal cord-transplant relay99,100,239. So far, a similar relay function of transplanted stem cell derived neurons to restore the sensory connectivity to the spinal cord has not been reported. Foetal neural stem cells transplanted to the murine spinal cord are able to establish synapses and integrate structurally into the host’s motor circuitry147,148. In addition, murine embryonic stem cell derived motor neurons transplanted into

31 the spinal cord ventral horn are able to grow into the ventral root and form neu- romuscular junctions on host skeletal muscles240. These studies indicate that stem cell derived neurons are able to integrate into the spinal cord circuitry. The complete absence of VGluT1 and VGluT2 positive terminals from areas of hNP engraftment suggests the absence of synaptic connectivity between sensory fibres and the transplant, as primary sensory neurons are typically glutamatergic12. Together with the limited ingrowth of hNP fibres into the spinal cord, this renders it unlikely that human neurons functioned as relays for sensory axons and contributed to the observed sensorimotor improvement. The 3rd, 4th and 5th lumbar DRG encompass nearly all the contribution of sensory fibres from the sciatic nerve in mice and rats241,242. Following L3-L5 DRA, animals showed a severe reduction of mechanoreceptive and sensorimotor abilities of the ipsi-lateral hind paw but their gait remained unaffected. De- spite DRA of all lumbar segments that contribute to the hind paw dermatomes in rodents243,244, animals showed only about 50% reduction in their ability to react to a mechanical stimulus applied to this hind paw. The strength of the used van Frey filament (5.5g) could provide an explanation for the lack of complete desensitisation. Filaments of sufficient strength not only stimulate the cutaneous hind paw but also minimally displace the leg, leading to a substantial change in the nature of the stimulus219. DRA of T13+L1 results in the development of bilateral allodynia of the hind paws61,62. In contrast, avulsion of dorsal and ventral roots (spinal root avulsion) of L5 results in allodynia occurring only on the ipsi-lateral side66. Even transplantation of neural stem cells alone can lead to the development of allodynia122,245. These findings made it substantial to test for the development of allodynia in our DRA model, but we could not observe any development of hypersensitivity to noxious mechanical stimuli in either DRA alone or animals receiving hNP transplants. The absence of allodynia in animals undergoing L3-L5 DRA alone might be explained by the avulsion of multiple adjacent roots, as this results in a continuous longitudinal injury of the dorsal horn along these segments. This outcome might resemble the more controlled micro-surgical DREZotomy that relies on selective ablation of the dorsal root transitional zone and reduces chronic pain after brachial plexus injury in pa- tients246,247. Animals receiving hNP transplants showed improved mechanical nociceptive sensitivity from seven weeks after transplantation up to five months after transplantation. In addition, animals regained the majority of their strength to grip with the ipsi-lateral hind paw. Both tests record a motor response of the hind paw, this suggests that sensory fibres were conveying meaningful sensory information into the spinal cord and were able to elicit an appropriate motor response. Five months after hNP transplantation, dorsal roots that were previously avulsed were dissected close to the DRG during a second surgery. This resulted in the complete loss of observed sensorimotor improvements, indicat- ing that the behavioural improvements were caused by regenerating sensory

32 fibres entering the spinal cord along previously avulsed dorsal roots and not by sprouting from neighbouring segments or trophic effects of the transplanted cells. Taken together, the improvement of mechanical nociceptive sensitivity and sensorimotor coordination, the expression of NF200 by ingrowing fibres and their uptake and transport of CTB as well as their projection pattern in the spinal cord suggests the regeneration of mechanoreceptive and/or proprioceptive sensory axons innervating dorsal horn neurons. After the findings presented in Paper I, a recent study showed that also OEC are able to form a tissue bridge after DRA avulsion248. In contrast to hNP transplants that primarily rescue NF200 positive axons and proprioception, OEC transplants stimulate primarily peptidergic axons and ameliorate the DRA induced chronic pain. In theory, these two cell types would complement one another in their regenerative potential and it would be most interesting to see the effect of their co-transplantation to a model of DRA.

4.2 Paper III During development, boundary cap neural crest stem cells (bNCSC) form the interface between the spinal roots and cord22,23. This makes them particularly interesting for transplantation approaches to induce sensory ingrowth after DRA. It allows a close match between the targeted tissue and the origin of transplantation material, a clear advantage over the human spinal cord neural progenitors used in Paper I and IV that were either differentiated towards the ventral spinal cord lineage in vitro (Paper I) or collected from primary tissue of the foetal spinal cord (Paper IV). Another possible benefit of bNCSC transplantation is the use of allogenic instead of xenogenic cells. Nevertheless, all transplantation approaches tested here are expected to induce an immune response. hESC show reduced immunogenicity by differential expression of major histology complexes and immunomodulatory properties but it is unclear whether this transcends to hESC derived progenitors249. Therefore, both hNP spheres and hscNSPC neurospheres are expected to be rejected by the host. Allogenic transplantation of bNCSC faces the same problem. For these reasons, immunodeficient mice were used in all studies and represent a major limitation of the studies presented in this thesis. To overcome the problem of allogenic and xenogenic transplantation, future studies will have to focus on induced pluripotent cells that can be patterned to both bNCSC250–252 and spinal cord neural progen- itors253. The use of patient specific iPS and in vitro differentiation of the therapeutic cell type of interest results in histocompatibility that is believed to overcome the need for immunosupression or -modulation254,255. More recent studies challenge this view and so far it remains unclear whether iPS transplantation does not face the same challenges as transplanting hESC and their progeny256.

33 Similar to hNP spheres, bNCSC transplanted to the site of DRA showed good survival, integrated into the tissue and generated a gap in the glial scar of the injured spinal cord. Few transplanted cells even migrated into the spinal cord tissue. This suggested the formation of a tissue bridge similar to the one formed by hNP spheres. Tracing of injured sensory fibres revealed sensory axon outgrowth into the injured dorsal root and their termination at the interface between dorsal root and bNCSC transplant, corroborating the previous observation that bNCSC attract neurofilament 200kD positive sensory fibres173. These fibres never crossed over into the spinal cord tissue. Findings from Paper I and lessons from OEC induced sensory regeneration suggest glial progenitors to play a crucial role in the establishment of a growth permissive tissue bridge. Here, we found one major difference between hNP sphere formed tissue bridges that allowed ingrowth of sensory fibres and those that were formed by bNCSC and did not allow ingrowth. bNCSC at the interface between injured dorsal root and bNCSC transplant extensively co-expressed the glial marker GFAP and Mts1/S100A4. In contrast, hNP and hscNSPC transplants showed no detectable Mts1/S100A4 expression (Paper III and Paper IV[data not shown]). In mice, Mts1/S100A4 was expressed by white matter astrocytes and became up-regulated in response to injury. This finding is in line with previous studies in rats that describe intracellular Mts1/S100A4 as a marker of sensory outgrowth inhibiting reactive white matter astrocytes257. Intracellular Mts1/S100A4 reduces injury-induced migration and might contribute to the stabilization of the glial scar258. Surpris- ingly expression of Mts1/S100A4 could already be found in undifferentiated bNCSC neurospheres, in contrast to hNP spheres that showed no detectable expression. This suggests that for future transplantation approaches, special emphasis should be placed on glial progenitors and their interaction with the local environment at the scarred spinal cord/spinal root interface.

4.3 Paper IV hscNSPC grafted to the site of DRA remained immature up to two months after transplantation, indicated by expression of Sox2 and Olig2 and showed continuous proliferation. In contrast to previous ﬁndings that neurotrophic factor mimetics for glial derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) lead to increased differentiation of transplanted stem cells206, co-transplantation of hscNSPC and neurotrophic factor mimetics had no effect on the observed differentiation pattern or proliferation of hscNSPC. The overall immaturity of hscNSPC and their unaltered differentiation proﬁles after co-transplantation with neurotrophic factor mimetics renders it unlikely that immaturity is required for hscNSPC to promote sensory axon regeneration.

34 hscNSPC transplants of both conditions differed only in one aspect, their degree of integration into the injured spinal cord tissue. hscNSPC are transplanted to the surface of the injured spinal cord, thus human cells found inside the spinal cord have to originate from migratory hscNSPC. Extensive migration was observed when hscNSPC were transplanted alone. Here, Olig2 positive human cells were found in the ipsi-lateral and contra-lateral gray matter and migrated deep into the ventral horn. In contrast, Olig2 positive human cells in the group treated with neurotrophic factor mimetics were predomi- nantly confined to the ipsi-lateral dorsal horn. During spinal cord development, Olig2 positive cells in the spinal cord give primarily rise to motor neurons and oligodendrocytes259. hscNSPC transplanted to the site of DRA may have the potential to serve as a replacement for lost oligodendrocytes and spinal motor neurons in the ipsi-lateral ventral horn as well as for the avulsion-induced loss of dorsal horn neurons48,56,66. In contrast, hscNSPC injected into the injured spinal cord do not give rise to oligodendrocytes125. This suggests that either the way of delivery or more likely differences in the initial local environment have an effect on the survival of oligodendrocyte progenitors or the differentiation of hscNSPC. Due to the lack of studies that systematically compare ways of human neural progenitor delivery to the spinal cord, this question needs to remain unanswered. Following injection into healthy spinal cord grey matter, human spinal cord neural progenitors are able to migrate far away from their initial site and spread both into gray and white matter areas260. When degenerative CNS tissue is present, neural stem cells show a remarkable ability to migrate towards the lesion site261–265, an effect that is attributed to cues provided by the lesion environment261,262,266,267. The local administration of neurotrophic factor mimetics might disturb this environment enough to no longer stimulate directed migration of transplanted cells and could explain the reduced integration of hscNSPC transplants into the spinal cord tissue. hscNSPC transplantation allowed CTB traceable sensory fibres to grow into the injured dorsal horn. Interestingly, administration of neurotrophic factor mimetics alone led to limited ingrowth of CTB traceable sensory fibres. Local application of the neurotrophic factor GDNF enhances the regenerative drive in damaged sensory neurons and ameliorates the inhibitory effect of reactive astrocytes87,268. Thus, the local application of the GDNF peptide mimetic to the site of DRA could explain this finding. The combination of neurotrophic factor mimetics and hscNSPC grafts did not promote regeneration. Analysis of the spinal cord revealed strong GFAP expression at the site of DRA. When hscNSPC were transplanted alone, the site of injury showed reduced expression of murine GFAP but increased presence of human GFAP. In contrast, when cells were transplanted together with neurotrophic factor mimetics this effect was diminished and the majority of the injured dorsal horn showed strong murine GFAP expression. Further, DRA led to a 2.5 fold increase in neural/glial antigen 2 (NG2) immunoreactivity com-

35 pared to naive animals that was not present in animals receiving hscNSPC alone. Co-transplantation with neurotrophic factor mimetics negated this effect and NG2 levels at the site of DRA remained at injury typical levels. In the context of spinal cord injury, NG2 is regarded as an axonal growth inhibiting chondroitin sulfate proteoglycans (CSPG)37, but its role as an inhibitory proteoglycan is disputable. Knock-out of NG2 fails to ameliorate sensory axonal ingrowth and NG2 positive cells are permissive to neurite outgrowth of DRG neurons in vitro32,269. More recent evidence suggests a role for NG2 positive cells in the immobilization of regenerating sensory axons after dorsal root injury44. Taken together, the negative synergistic effect on sensory regeneration observed when hscNSPC were combined with neurotrophic factor mimetics suggests that successful regeneration of CTB traceable sensory ﬁbres by human spinal cord neural progenitors is dependent on transplant integration into the spinal cord tissue. Further, hscNSPC provide a growth promoting environment for sensory ﬁbres by reducing the presence of growth inhibitory components typical for the spinal cord glial scar.

4.4 Paper V The primary goal of this study was to test the effect of bNCSC on the survival of SOD1G93A motor neurons with the future goal to use bNCSC in cell therapeutic transplantation experiments. SOD1G93A motor neurons show reduced survival in vitro176. This ﬁnding could be corroborated here. In line with the well established neurotoxic role of SOD1G93A astrocytes176,191,193,270, removal of SOD1G93A astrocytes increased SOD1G93A motor neuron survival by approximately 75%. SOD1G93A motor neuron survival can be improved by the addition of healthy astrocytes alone176. Depending on the used culture conditions, bNCSC can differentiate into astrocytes and neurons (171 and Paper VI) or peripheral neurons and Schwann cells170,271. This made it necessary to separate bNCSC into neurons and astrocytes for successive experiments. bNCSC astrocytes when cultured together with astrocyte depleted SOD1G93A motor neurons improved survival even further. In contrast, embryonic stem cell derived astrocytes, puriﬁed in the same way as bNCSC astrocytes, did not increase the survival of SOD1G93A motor neurons. Oxidative stress is a common pathological component of both sALS and fALS and appears at least in spinal cord and brain stem areas272,273. It remains unclear whether oxidative stress is a major mechanism behind ALS or symptomatic for ALS, but it is tightly linked to excitotoxicity, mitochondrial and cytoskeletal dysfunction and protein aggregation, all of which are neurodegenerative processes that are involved in ALS273,274. Oxidative stress is induced by reactive oxygen species (ROS) that are a constant side product

36 of aerobic metabolic reactions. Under normal conditions, the cellular anti- oxidant defence system can keep ROS levels balanced275. SOD1G93A astrocytes are a major source of ROS in ALS156,276. To simu- late this pathological environment, we induced strong oxidative stress in astrocyte deprived wild type motor neuron cultures and SOD1G93A motor neuron cultures. Interestingly, SOD1G93A motor neurons were more vulnerable to oxidative stress than wild type motor neurons. So far, no clear explanation exists why motor neurons are more burdened by oxidative stress than other cell types277. The addition of bNCSC astrocytes to SOD1G93A motor neurons resulted in an increase of motor neuron survival. It remains unclear whether bNCSC astrocytes exert this function by paracrine effects, as bNCSC are known to secret a multitude of trophic factors and/or by cell-cell con- tact174,278, or whether they are able to buffer ROS as the induced oxidative stress had no effect on bNCSC survival. Co-transplantation of bNCSC astrocytes and SOD1G93A motor neurons to the spinal cord of mice resulted in a higher number of surviving SOD1G93A motor neurons than the transplantation of SOD1G93A motor neurons alone. This indicates that bNCSC astrocytes are able to protect SOD1G93A motor neurons from injection induced stress and/or from the neuroinﬂammatory environment caused by the transplantation induced insult of the spinal cord. Apart from the aforementioned properties of bNCSC, unpublished results from Pa- per VII suggest that bNCSC are able to protect spinal neurons from excitotoxic damage, whereas the underlying mechanism remains unknown. This is particularly interesting as ALS is characterised by hyper-excitability of spinal neurons and glutamate excitotoxicity both of which contribute to the increased vulnerability of ALS motor neurons134,182. In sALS, expression of the primary astrocytic glutamate transporter GLT- 1, a major regulator of the glutamate clearance cycle, is reduced by over 90%279,280. Reduced expression of GLT-1 might have a negative impact on the spinal neurons ability to cope with increased extracellular levels of gluta- mate281. Transplanted embryonic glial-restricted progenitors are able to ameliorate glutamate excitotoxicity in ALS mice by increasing the expression of GLT-1157. In addition to their proposed cell-protective properties, transplanted bNCSC astrocytes might be able to act in a similar way. Transplantation experiments of puriﬁed bNCSC astrocytes into the spinal cord of ALS animals will hopefully shed light onto these questions.

37 5. Conclusions

• Human spinal cord neural progenitor transplantation partially restores sensorimotor functions of dorsal root avulsed mice (Paper I). • Transplantation of human spinal cord neural progenitors from either human embryonic stem cells or foetal abortion tissue promotes regeneration of sensory axons by providing a growth permissive environment at the site of dorsal root injury (Paper I+IV). • Engrafted human spinal cord neural progenitors from both human embryonic stem cell and foetal sources show robust survival and differentiation towards glial cells and neurons but distinctively different migration patterns and levels of maturity (Paper I+IV). • Murine boundary cap neural crest stem cells are not able to promote sensory recovery, possibly due to their contribution to the axonal growth inhibitory glial scar (Paper III). • Peptide mimetics of the neurotrophic factors GDNF and CNTF applied to the site of dorsal root avulsion lead to ingrowth of sensory axons. In contrast, combinatorial therapy of human spinal cord neural progenitors and peptide mimetics results in synergistic effects that prevents ingrowth (Paper IV). • Murine boundary cap neural crest stem cells promote survival of amyotrophic lateral sclerosis effected motor neurons and protect them from H2O2 induced oxidative stress (Paper V).

38 6. Future aims

6.1 Stimulation of sensory axon ingrowth by cell therapeutic means Before the treatment of brachial plexus injuries using human spinal cord neural progenitor transplantation can be envisioned, the cell type(s) responsible for the observed ingrowth of CTB positive fibres needs to be determined. Both hNP and hscNSPC transplants give rise to a multitude of different neural cell types of varying stages of maturity and it remains unclear what combination of factors is crucial for the observed improvements. The results from papers I, III and IV suggest that modification of the glial scar and the direct contact between transplanted cells and the scarred spinal cord is required for the formation of a growth permissive tissue bridge. The tissue bridge has to reach through the scar to allow sensory axons to grow into the spinal cord gray matter. A sub-population of transplanted cells that later gives rise to astrocytes plays a major role in the formation of the tissue bridge. It remains unclear whether the co-transplanted neuronal cell populations are functional or even necessary for the observed effect. For these reasons, future studies should focus on the isolation of astrocytic progenitor populations. As presented in Paper III, the presence of un- favourable astrocytic progenitor types already at the stage of neurosphere culture could be most crucial for the pre-evaluation of ideal cell therapeutic candidates. To achieve this goal, currently available cell separation techniques like immunopanning or magnetic activated cell sorting (MACS) to purify astrocytic progenitor populations followed by single cell transcriptomal profiling could help to distinguish between cell populations282. In addition, this would support the identification of specific markers that allow the formulation of better differentiation protocols or purification attempts before transplantation . For the presented cell therapeutic approach to be considered in a clinical setting, the culture and pre-differentiation of stem cells needs to comply with good manufacturing practice (GMP) standards. Cell therapeutic candidates should be gathered from patient derived iPS cells to minimise the required immunosupression after transplantation. First steps into this direction have already been taken283,284, for example the stem cell differentiation protocol used in Paper I can be directly used for the differentiation of iPS cells180, and a modified xenogenic-free culture system of human iPS derived spinal cord neural progenitors has been established285. If the collection of an astrocytic progenitor population of the spinal lineage is the main goal, differentiation

39 protocols that focus on high yields and reproducible cell compositions need to be established ﬁrst and adapted to follow GMP standards286–288.

6.2 Boundary cap neural crest stem cells for cell therapeutic transplantation in amyotrophic lateral sclerosis The experiments in Paper V have shown that the beneficial effects of boundary cap neural crest stem cells (bNCSC) are not limited to pancreatic islet cells174,289, and while they do not promote sensory regeneration as presented in Paper III, they are safe to be used for spinal cord transplantation172,173. In line with their better studied effect on β-cells278, the initial results suggest that transplanted bNCSC have to be in proximity to their target, similar to the beneficial effect of neuronal stem cells transplanted to ALS animals149,150. This increases the challenge of cell delivery as treatment will most likely require multiple intra-spinal injections. It remains unclear whether in a transplantation setting bNCSC would act in a similar way as reported in Paper V. To corroborate their beneficial effect, bNCSC need to be injected into the spinal cord of ALS mice. A pilot study is currently under way and initial results are promising but not yet conclusive. So far, it remains unknown how bNCSC improve ALS motor neuron survival in vitro. Future studies, should focus on elucidating the mechanism behind the improvement of SOD1G93A motor neuron survival in vitro and on additional aspects of the cytopathology of ALS. A multitude of different stem cell based in vitro models can be used for this purpose (see section 1.3.3). More thorough analyses have to be carried out to characterize the paracrine effects of bNCSC in co-culture and after transplantation. Taken the extensive amount of cell therapeutic ALS studies into consideration141,167, the secre- tory profile of bNCSC could be of major interest. First attempts to elucidate whether direct contact between bNCSC and SOD1G93A motor neurons is necessary, suggest that direct contact is required. So far, these assays lack conclusive proof, but if the initial observation holds true, special emphasis should be placed on cell-cell adhesion molecules and their effect on SOD1G93A motor neuron survival. Interestingly, multiple members of the cadherin and collage gene families are dysregulated in mice carrying a fALS-associated FUS muta- tion290. These could be interesting candidates for a directed analysis as both gene families are involved in synapse formation, axonal guidance and motor neuron differentiation291.

40 7. Sammanfattning på svenska

Skador och neurodegenerativa sjukdomar i ryggmärgen kan leda till förlamn- ing och förlust av känsel. Stamcellstransplantation kan återställa förlorade förbindelser som förmedlar sensorisk information och skydda ryggmärgsceller från degeneration. I allmänhet kan nerver återväxa efter skada men de kan inte växa in i den ärrade ryggmärgen. Tre olika typer av stamceller, som ger upphov till ryggmärgsceller, undersöktes med avseende på deras förmåga att övervinna denna växthämmande ärrvävnad. Stamcellerna transplanterades till det skadade området och växt av skadade sensoriska nerver analyserades. Av de tre typer av spinala stamceller som undersöktet, möjliggjorde de celler med ursprung i humana embryonala stamceller eller human fetal vävnad att nervfi- brer som förmedlar sensorisk information kunde växa igenom det ärrade om- rådet och nå oskadade delar av ryggmärgen. Humana ryggmärgsceller från embryonala stamceller förbättrade känseln markant hos ryggmärgsskadade djur. I syfte att ytterligare förbättra den observerade nervväxten kombinerades behandling med humana ryggmärgsceller från fetal vävnad med tillförsel av faktorer vilka stimulerar återväxt av sensoriska fibrer. I kontrast mot förvänt- ningarna förlorade de transplanterade humana ryggmärgscellerna sin förmåga att understödja sensoriska fibrer och att tillåta inväxt genom ryggmärgsärret. Den sista typen av ryggmärgsstamcell som prövades erhölls från platsen för de sensoriska fibrernas inväxt hos friska embryonala möss. Dessa celler, vilka kallas "boundary cap neural crest stem cells" (bNCSC), kunde inte stimulera inväxt av sensoriska fibrer utan bidrog med återväxthämmande celler till ryg- gmärgsärret. I den sista delen av avhandlingen upptäcktes att bNCSC skyd- dar de ryggmärgsceller som ansvarar för kontrollen av muskler mot oxidativ stress. Oxidativ stress är en degenerativ process, vilken är typisk för motorneu- ronsjukdomen amyotrofisk lateral skleros. Denna avhandling erbjuder första beviset för att transplantation av humana ryggmärgsceller kan stödja sensoriska nervers återväxt in i den skadade ryg- gmärgen. Avhandlingen lyfter också fram bNCSC som intressanta kandidater för stamcellsbaserad behandling av amyotrofisk lateral skleros.

41 8. Acknowledgment

I am grateful to my supervisor Elena Kozlova for inspiring discussions on- and off-topic and to allow me to pursue this work in her laboratory. I thank Håkan Aldskogious for providing the small nudges that are sometimes necessary to go "beyond" and his profound knowledge. Thanks to my co-supervisor Åsa Mackenzie to always have an ear for a PhD-student’s troubles. Special thanks to Robert Fredriksson, you might not have been an official co-supervisor but I surely see you as one. I am indebted to Niclas König and Carl Trolle, not only for their surgical expertise that made Paper I, III and IV possible but also for the great scientific and non-scientific discourses we had over the years and the fantastic teamwork. I thank Svitlana Vasylovska for the great company and all the shared experience. Ninnie Abrahamsson, I thank you for your invaluable technical assistance, resourceful introduction to stem cell culture and to always keep me updated on the intricate social network of our working environment. Thank you Tanya Aggarwal for our constructive discussions and your caring that goes beyond the time spent in the lab. Special thanks go to Emilia Lekholm for a great collaboration and for enduring my mundane complains about PhD life. Thank you Patrik Ivert for lending us your surgical skills and to Ronald Deumens for the great collaboration, your good spirit and shared insight to behavioural studies. I also have to thank my past students: Markus Petermann, our project might not have turned out as we wished but I am looking forward to our future collaborations; Iris Rocamonde-Lago, with- out you there would be no Paper III, I wish you all the best for your ongoing education; Ali Inan El-Naggar, it was a pleasure teaching you stem cell culture and discussing scientific theory. Last but not least, I thank my parents Elke and Heiner for giving me all the tools to get this far and my brother Cedric for keeping me on my "mental" toes. Finally, I thank the two main pillars of my life, my wife Steffi and my son Joshua. To you: "As I leave the second most important job I could ever hold, I cherish even more the first - as a husband and father." (Gordon Brown, 1951)

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67 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1365 Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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