ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1365

Neural progenitors for sensoryand motor repair

JAN HOEBER

ISSN 1651-6206ISBN 978-91-513-0058-0urn:nbn:se:uu:diva-328590

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 ofPhilosophy (Faculty of Medicine). The examination will be conducted in English. Facultyexaminer: Professor Linda Greensmith (University College London, Institute of Neurology,Sobell Department of Motor Neuroscience and Movement Disorders).

AbstractHoeber, J. 2017. Neural progenitors for sensory and motor repair. Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 1365. 67 pp. Uppsala: ActaUniversitatis Upsaliensis. ISBN 978-91-513-0058-0.

Injury and neurodegenerative conditions of the spinal cord can lead to paralysis and lossof sensation. Cell therapeutic approaches can restore sensory innervation of the spinal cordfollowing injury and protect spinal cord cells from degeneration. This thesis primarily focuseson the restoration of deaffarented sensory fibres following injury to the dorsal root and spinalcord. These injuries lead to the formation of a non-permissive glial scar that prevents sensoryaxons from reinnervating spinal cord targets. It takes advantage of a dorsal root injury modelthat closely mimics spinal root avulsion injuries occurring in humans. In the first part of thethesis, three different neural progenitor types from human or murine sources are tested for theirregenerative 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 froma neurodegenerative process is tested.

In the first original research article, I show that human embryonic stem cell derived neuralprogenitors are able to restore sensorimotor functions, mediated by the formation of a tissuebridge 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 progenitorthat governs the entry of sensory axons into the spinal cord during development, are unableto form a permissive tissue bridge. This is possibly caused by the contribution of transplantderived ingrowth non-permissive glial cells. In the third research article, I show that humanneural progenitors derived from foetal sources are capable of stimulating sensory ingrowthand that they ameliorate the glial scar. When this approach is combined with the delivery ofsensory outgrowth stimulating neurotrophic factors, these cells fail to form a permissive tissuebridge and fail to modify the glial scar. In the final research article, murine boundary capneural crest stem cells are shown to protect motor neurons, which harbor an amyotrophic lateralsclerosis causing mutation, from oxidative stress. Oxidative stress is a pathological componentof amyotrophic lateral sclerosis in human patients.

Taken together, this thesis provides first evidence that sensory regeneration following a spinalroot 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 celltherapeutic 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, UppsalaUniversity, SE-75124 Uppsala, Sweden.

© Jan Hoeber 2017

ISSN 1651-6206ISBN 978-91-513-0058-0urn:nbn:se:uu:diva-328590 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-328590)

"If the doors of perception were cleansedeverything 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 textby 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 AssistFunctional Sensory Axon Regeneration after Dorsal Root AvulsionInjury. Scientific Reports, Volume 5, Article Number 10666.

II Hoeber J (2015) Sensory regeneration in dorsal root avulsion. NeuralRegeneration 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 celltransplants contribute to the glial scar and do not assist regeneration ofsensory 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) ACombinatorial Approach to Induce Sensory Axon Regeneration intothe 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 MutantSOD1 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 MesoporousSilica Particles on Stem Cell Differentiation. Journal of Stem CellResearch & Therapeutics Volume 2, Issue 3.

VII Schizas N, König N, Andersson B, Vasylovska S, Hoeber J, KozlovaEN, Hailer NP (2017) Neural Crest Stem Cells protect Spinal CordSlice Cultures from Excitotoxic Neuronal Damage and Inhibit GlialActivation. 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 mimeticsderived from their receptor binding sites promote neurite outgrowthand neuronal survival of primary central and peripheral neurons invitro. Manuscript

Reprints were made with permission from the publishers.

Abbreviations

ALS amyotrophic lateral sclerosisBDA biotinylated dextran aminebNCSC boundary cap neural crest stem cellC cervicalCAG chicken β -actin promoter coupled with the cytomegalovirus immediate-early

enhancerCGRP calcitonin gene-related peptideCNS central nervous systemCNTF ciliary neurotrophic factorCSPG chondroitin sulfate proteoglycanCTB cholera toxin subunit BDRA dorsal root avulsionDREZ dorsal root entry zoneDRG dorsal root ganglionDRR dorsal root rhizotomyDRTZ dorsal root transitional zoneEB embryoid bodyEGF epidermal growth factorFGF fibroblast growth factorFUS RNA-binding protein fused-in-sarcomaGDNF glial derived neurotrophic factorGFAP glial fibrillary acidic proteinGLAST glutamate aspartate transporterGRP glial-restricted progenitorESC embryonic stem cellhNP human spinal cord neural progenitorhscNSPC human spinal cord neural stem/progenitor cellIB4 isolectin B4iPS induced pluripotent stem cellL lumbarMND motor neuron diseaseMNP motor neuron progenitorMSC mesenchymal stem cellNA numerical apertureNEAA non-essential amino acidNF200 neurofilament 200kDNG2 neural/glial antigen 2NGF nerve growth factor

Continued on next page

NSC neural stem cellNT3 neurotrophin-3OEC olfactory ensheating cellPBS phosphate buffered salinePFA paraformaldehydePNS peripheral nervous systemRA retinoic acidRNA ribonucleic acidROS reactive oxygen speciesS sacralSOD1 Cu/Zn superoxide dismutaseSHH sonic hedgehogT thoracicTDP-43 TAR DNA-binding protein 43VAPB vesicle-associated membrane protein-associated protein B/CVEGF vascular endothelial growth factorVRTZ ventral root transitional zone

Contents

1 Introduction 11.1 Development and anatomy of the spinal cord . . . . . . . . . . 11.2 Restoration of sensory innervation of the spinal cord . . . . . . 3

1.2.1 Sensory innervation of the spinal cord . . . . . . . . . . 31.2.2 The PNS-CNS interface before and after injury . . . . . 51.2.3 Spinal root injury and the dorsal root avulsion model . . 71.2.4 Regeneration of sensory axons through the PNS-CNS

interface . . . . . . . . . . . . . . . . . . . . . . . . . 91.2.5 Cell therapy for sensory regeneration in dorsal root injury 10

1.3 Protection of spinal motor control using neural progenitors . . . 121.3.1 The motor neuron disorder: Amyotrophic lateral sclerosis 121.3.2 Cell therapeutic spinal cord transplantation for ALS . . 131.3.3 Stem cell based in vitro disease modelling of ALS . . . 15

2 Aims of the study 17

3 Experimental procedures 183.1 Ethical permits and informed consent . . . . . . . . . . . . . . 183.2 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.2.1 Generation of transplantable human spinal cord neuralprogenitors derived from human embryonic stem cells[Paper I,III] . . . . . . . . . . . . . . . . . . . . . . . . 18

3.2.2 In vitro differentiation of human spinal cord neural pro-genitors [Paper I] . . . . . . . . . . . . . . . . . . . . . 19

3.2.3 Generation of transplantable murine boundary cap neuralcrest stem cells [Paper III,V] . . . . . . . . . . . . . . . 19

3.2.4 Generation of transplantable human spinal cord neuralstem/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 factormimetics [Paper IV] . . . . . . . . . . . . . . . . . . . 20

3.2.7 Generation of motor neuron cultures from murine em-bryonic 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 neuralcrest stem cell cultures [Paper V] . . . . . . . . . . . . 21

3.3 Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . 223.3.1 Dorsal root avulsion injury and cell transplantation [Paper

I,III,IV] . . . . . . . . . . . . . . . . . . . . . . . . . . 223.3.2 Cervical spinal cord cell injection [Paper V] . . . . . . 223.3.3 Transganglionic tracing [Paper I,III,IV] . . . . . . . . . 233.3.4 Tissue and cell sphere collection and processing [Paper

II,III-V] . . . . . . . . . . . . . . . . . . . . . . . . . . 243.3.5 Immunohistochemistry [Paper II,III-V] . . . . . . . . . 243.3.6 Behavioural analyses [Paper I] . . . . . . . . . . . . . . 25

3.4 Microscopy [Paper I,III-V] . . . . . . . . . . . . . . . . . . . 283.5 Image analysis [Paper I,III-V] . . . . . . . . . . . . . . . . . . 283.6 Statistics [Paper I,III-V] . . . . . . . . . . . . . . . . . . . . . 29

4 Results and Discussion 304.1 Paper I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304.2 Paper III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.3 Paper IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.4 Paper V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

5 Conclusions 38

6 Future aims 396.1 Stimulation of sensory axon ingrowth by cell therapeutic means 396.2 Boundary cap neural crest stem cells for cell therapeutic trans-

plantation 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 . . . . . . . . . . . . . 21.2 Spinal cord segments and dermatomes . . . . . . . . . . . . . 31.3 Sensory innervation of the dorsal horn . . . . . . . . . . . . . 41.4 Transitional zones of the spinal cords afference and efference . 61.5 The dorsal root transitional zone after injury . . . . . . . . . . 71.6 Experimental dorsal root avulsion injury . . . . . . . . . . . . 91.7 Pathological features of stem cell based in vitro ALS models. . 163.1 Overview of conducted experiments . . . . . . . . . . . . . . . 223.2 Cell therapeutic approaches for dorsal root avulsion . . . . . . 233.3 Stem cell injection into the cervical spinal cord . . . . . . . . . 24

1. Introduction

1.1 Development and anatomy of the spinal cordDuring neurulation, the edges of the neural plate lift upwards and form theneural groove. When the edges converge, the neural groove is converted intothe closed neural tube and the enclosed neural canal. The neural canal laterforms the central canal of the spinal cord and the ventricles of the brain. Theneural tube divides itself into the four subdivisions of the central nervous sys-tem (CNS). At the level of the prenatal spinal cord, a small group of cellsmigrate away from the closing neural tube and form the neural crest that latergives rise to the dorsal root ganglia (DRG). In later stages, the neural tube sep-arates into the dorsal grey column that differentiates into neurons that mediatesensory information, the ventral gray column that gives rise to spinal motorneurons and interneurons, and the surrounding marginal layer that forms thewhite matter of the spinal cord (top of Figure 1.1)1.

In the mature spinal cord, the peripheral white matter is comprised of glialcells and myelinated axons, and the central gray matter that is comprised ofthe soma, dendrites and synapses of most spinal neurons. In transverse orien-tation, the white matter separates into the dorsal, lateral and ventral column,and likewise the gray matter into dorsal, intermediate and ventral horn (bottomof Figure 1.1)2.

The cyto-architecture of the gray matter can be classified by the modality ofinformation that is conveyed to neurons of distinct areas, classically referredto as Rexed’s Laminae. The dorsal horn is comprised of Laminae I to IV thatreceive sensory information from the body’s surface and Laminae V and VIthat receive sensory information from inside the body. The intermediate hornis roughly equivalent to Lamina VII, and relays information between spinalcord areas. Lamina X surrounds the central canal and encompasses axons thatcross from one spinal cord side into the other. The ventral horn is comprisedof the Laminae VIII and IX and contains the soma of all spinal motor neuronsand 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 lon-gitudinal localization along the body: the cervical (C), thoracic (T), lumbar(L), sacral (S) and coccygeal spinal cord. Each level is further divided intoindividual spinal cord segments that receive afferent information from sensoryneurons of the peripheral nervous system (PNS) residing in the DRG. Thesesensory neurons extend axons into the spinal cord along the dorsal root and

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Figure 1.1. Development of a spinal cord segment.

terminate on spinal neurons that mediate reflex actions or convey informationto the brain. Efferent information originating from the brain is conveyed to-wards appropriate segments and to spinal motor neurons residing inside theventral horn. Spinal motor neurons project axons along the ventral root andinnervate skeletal muscles and glands. Dorsal and ventral roots join togetherat the DRG and form the spinal nerves. Due to the topographic sensory inner-vation of the skin, individual parts of the body’s surface can be classified asbelonging to individual spinal cord segments and are generally referred to asdermatomes (Figure 1.2)5.

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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 cord1.2.1 Sensory innervation of the spinal cordThe spinal cord receives sensory innervation by pseudo-unipolar sensory neu-rons that reside in the dorsal root ganglia (DRG) and from which a bifurcatedaxon is extended. The distal process of this axon projects into the peripheryand receives information from the trunk and limbs. Its proximal process con-veys this information through the dorsal root. The majority of proximal pro-cesses enter the spinal cord through the Lissauer tract situated directly aboveLamina I6.

DRG can be classified as either visceral DRG that innervate the internalorgans and non-visceral DRG that covey information from the skin, musclesand joints7. With the exception of muscle spindle sensory axons (Ia fibres)that primarily innervate spinal motor neurons of the same segment8, sensoryneurons generally transmit information to defined areas of the spinal cord,brain stem and thalamus9. The modality of sensory information transmitted

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by sensory neurons is determined by the type of sensory receptors that areassociated with the terminal branch of the sensory axon.

Figure 1.3. Rexed’s laminae and the sensory innervation of the dorsal horn. Modifiedfrom Lallemend and Ernfors 201210.

Sensory information that originates from the skin or from inside the bodyis referred to as somatic sensory information. It encompasses four differentsensory modalities, proprioception, mechanoreception, nociception and ther-moception4, and these can be attributed to sensory neurons classified by dis-tinct electrical conduction velocities and expression of specific receptors andsignalling molecules (Figure 1.3). Aα and some Aβ fibres are thickly myeli-nated and show the highest conduction velocities. They primarily transmitproprioceptive information and encompass the Ia, II and Ib muscle afferents.These afferents contribute to the spinocerebellar proprioceptive pathway andconvey information from muscle spindles and golgi organs to spinal motorneurons in the ventral horn. Aβ and Aδ fibres are thinly myelinated and haveintermediate conduction velocities, together with some C fibres they mainlytransmit mechanoreceptive information of cutaneous and subcutaneous originand primarily terminate in the deeper dorsal horn laminae. C-fibres are un-myelinated and exhibit the lowest conduction velocities, together with someAδ fibres they convey nociceptive and thermoceptive information from theskin to superficial dorsal horn laminae10,11.

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Characterization of sensory neurons by the expression of distinct moleculesis generally more complicated due to the multitude and non-exclusiveness ofexpressed marker proteins. Therefore, only non-visceral sensory neurons willbe regarded in the following classification. Sensory neurons use glutamate astheir primary neurotransmitter but they utilize distinct sets of glutamate trans-porters. Proprioceptive and mechanoreceptive fibres often show high expres-sion of the vesicular glutamate transporter 1 (VGluT1). In contrast, nocicep-tive fibres express primarily the vesicular glutamate transporter 2 (VGluT2)12.

Myelinated large and medium sized sensory neurons express neurofilament200kD (NF200), small and medium sized sensory neurons express calcitoningene-related peptide (CGRP) and substance P, and small and medium sizedsensory neurons express the glial derived neurotrophic factor (GDNF) recep-tor and show binding of Griffonia simplicifolia isolectin B4 (IB4)13. In addi-tion, there are smaller populations of sensory neurons that show expressionof CGRP and bind IB4, and some that express tyrosine hydroxylase14–16.More recent single-cell transcriptomic efforts confirm classical markers butalso suggest a far more complex landscape of sensory neurons subtypes thanpreviously believed17,18.

1.2.2 The PNS-CNS interface before and after injuryThe dorsal root branches out into smaller dorsal rootlets as it approaches thespinal cord surface. The dorsal rootlets terminate on a sheet of densely packedastrocytic processes that build up the glia limitans that covers the whole sur-face of the spinal cord19. Where axons enter the spinal cord, the glia limi-tans extends into the rootlet, while the rootlet remains wrapped in a sheet ofSchwann cells that form its peripheral border. This anatomical structure de-marcates the transitional zone between the PNS and the CNS and is referredto 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 thespinal cord through the ventral root transitional zone (VRTZ) that is organizedin a similar way. Myelinated axons that traverse through the glia limitansat the DRTZ and VRTZ have a transitional node at this site. Their myelinsheet is formed by oligodendrocytes inside the spinal cord and by transitionalSchwann cells in the periphery20,21. The pronounced central tissue projectioninto the dorsal root is absent from the ventral root (Figure 1.4).

During development both the DRTZ and VRTZ are formed by a subset oflate migrating neural crest cells, that are referred to as boundary cap neuralcrest stem cells (bNCSC)22,23. At later developmental stages, these highlymigratory cells also give rise to satellite cells and a small population of no-ciceptive neurons in the DRG, Schwann cells of the spinal nerve root andmigrate as far as the skin where they differentiate into terminal glia24,25.

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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 200019.

Injury to the spinal nerve leads to axonal outgrowth of sensory neurons aspart of a strong regenerative response and is indicated by the up-regulation ofregeneration associated genes including growth associated protein (GAP)-43,activating transcription factors (ATFs) and c-Jun26,27. In contrast, injury to thedorsal root results in a very limited outgrowth of sensory axons and less pro-nounced activation of regeneration associated genes and extracellular matrixproteins associated with axonal regeneration28–32. In addition, the central tis-sue projection of the DRTZ undergoes a strong astrogliotic response indicatedby proliferation and swelling of astrocytes, their tight organization and an in-crease in the expression of glial fibrillary acidic protein (GFAP). The resultingglial scar renders the DRTZ impenetrable for regenerating fibres that extendbeyond the site of injury33,33,34.

Already Santiago Ramón y Cajal described in his original works that sen-sory axons that approach the glial scar either collapse, turn around or becomeimmobile (Figure 1.5)35. The collapse and repellence of regenerating axonsis generally attributed to factors that are prominent also in the glial scar fol-lowing spinal cord injury. Astrocytes that react to injury release high levelsof the chondroitin sulfate proteoglycans (CSPG): aggrecan, brevican, vesi-can, phosphacan and neural/glial antigen 2 (NG2)36,37. Oligodendrocytesand degenerating sensory axons deposit myelin associated axon growth in-hibitors38. The three best studied myelin associated axon growth inhibitorsare the myelin-associated glycoprotein (MAG), Nogo proteins and the oligo-dendrocyte myelin glycoprotein that all bind the Nogo receptor and inhibit ax-onal outgrowth by activation of the Rho-ROCK pathway. Stimulation of thispathway leads to the collapse and retraction of the growth cone of outgrow-ing axons39,40. At the injured DRTZ, a phagocytic microglia response slowlyclears the myelin debris but ultimately fails to allow sensory regeneration41.

Immobile axons are of great interest to regeneration approaches as theymight contain the potential to regrow after removal of the outgrowth inhibiting

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agents. Originally these axons were believed to terminate on reactive astro-cytes where they form synapse-like structures42,43. Recently these cells havebeen characterized as oligodendrocyte progenitor cells positive for the proteo-glycan 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. Modifiedfrom Smith et al 201247.

1.2.3 Spinal root injury and the dorsal root avulsion modelSpinal root avulsion injuries are the result of traction forces that pull the spinalroot nerve sheets away from the spinal cord until both the dorsal and ventralroot are ripped from the cord48. In contrast to the rupture of the spinal root thatappears post-ganglionic and thereby is not effecting the spinal cord directly,spinal root avulsion injury is always a pre-ganglionic injury resulting in thecomplete destruction of the DRTZ and/or VRTZ, and a longitudinal spinalcord injury. The complexity of the injury leads to the degeneration of affectedroots, an inflammatory response and the subsequent formation of a glial scarat the site of injury49.

In adults, spinal root avulsion most often occurs during high kinetic trafficor 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 isaffected and one or several roots of the C5-T1 segments are avulsed. The of-ten young patients suffer from partial to complete paralysis of the affectedextremity, loss of sensibility and in some cases severe untreatable chronicpain51,52. Nerve and muscle grafts show poor recovery but are usually benefi-

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cial51. Reimplantation of the ventral root is routinely used in patients, it resultsin regrowth of axons along the ventral root and improves motor deficits. Incontrast, reimplantation of the dorsal root fails to induce ingrowth of sensoryaxons into the spinal cord53.

Several rodent models to study sensory regeneration through the scarredDRTZ have been established so far. The two most commonly used modelsare 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 sealantsor sutures19. In dorsal root crush, the root is squeezed using a blunt forcepsuntil sensory axons are completely disrupted. The endoneurial tube remainsintact and no further procedure is necessary19. Sensory axons might to a verylimited extend grow back into Laminae II and dorsal column following dorsalroot crush54. However, regrowth is practically absent following DRR or dorsalroot reimplantation55.

The more recently developed dorsal root avulsion (DRA) model is char-acterized by the pulling of individual roots away from the spinal cord untiltheir complete avulsion from the spinal cord is achieved, while leaving theventral root undisturbed (Figure 1.6). Compared to the DRR and dorsal rootcrush model, DRA does not only result in injury to the dorsal root but alsodirectly damages the DRTZ and the dorsal white and gray matter of segmentsthat are connected to avulsed roots. On the cellular level, avulsion of severaldorsal roots leads to rapid invasion of neutrophils into the ipsi-lateral dorsalhorn and the loss of dorsal horn neurons. Inside the spinal cord, DRA resultsin bilateral astrogliosis, inflammation, chronic reduction of vascularization,immediate neurodegeneration and a second wave of neurodegeneration twoweeks after injury56,57.

For the study of pain, DRA has become an interesting model as well. DRAof selected thoracic and lumbar levels leads to the development of chronicpain below the level of avulsed segments. Consistent with observations in ro-dent models of true brachial plexus injury where both dorsal and ventral rootsare avulsed at the cervical level58–60, the pain can be relieved by adminis-tration of glial activation inhibitors but becomes elevated in the presence ofopioids61–63. These injuries strongly affect spinal neurons indicated by up-regulation of the stimulus inducible gene c-Fos64,65. Interestingly, avulsionof the dorsal and ventral root of a single lumbar level (segmental spinal rootavulsion) also results in chronic pain, but it manifests in the form of hypersen-sitivity to mechanical and thermal stimuli at dermatomes that correspond tothe affected spinal cord segment66.

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

1.2.4 Regeneration of sensory axons through the PNS-CNSinterface

The scarred PNS-CNS interface also serves as a model to study sensory re-generation following spinal cord injury. It is often favoured due to its limitedeffect on the well-being of the studied species and controlled impact on thesensory system67. Sensory regeneration in the spinal cord can be approachedfrom multiple angles. The two most prominent approaches focus on directmanipulation of the growth non-permissive environment of the scar and/or thestimulation 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 re-placement of astrocytes and oligodendrocytes, that are normally localised atthe DRTZ, by Schwann cells. In these animals, injury did not induce a typ-ical glial scar and thus sensory axons were able to grow back into the spinalcord68,69. Less invasive is the enzymatic digestion of outgrowth inhibitingCSPGs using the bacterial chondroitinase ABC70. Interestingly, at the DRTZchondroitinase ABC treatment only succeeds when combined with the stimu-lation of axonal outgrowth of sensory neurons and ideally stimulation of themTor pathway71,72. Axonal ingrowth of sensory axons can also be achievedby inhibition of the Nogo receptor, the common downstream target of myelinassociated 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 re-sults in axonal elongation of not only the peripheral but also the central partof the bifurcated axon74. This process relies on dual leucine zipper kinase(DLK) for effective signalling of an injury signal to the sensory soma and in-volves the hypoxia-inducible factor 1α before regeneration pathways are ac-tivated75,76. The "conditional lesion" is sufficient for sensory axons to growbeyond a cut and rejoined dorsal root, into implanted nerve grafts and evenresults in ingrowth of sensory axons into the spinal cord following dorsal rootcrush77–79. Regrowth can be further promoted by the local delivery of neu-

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rotrophic factors77. However, "conditional lesion" only promotes the regener-ation of thickly myelinated sensory axons and not peptidergic and IB4 reactivesensory axons78,80,81. Interestingly, induction of an inflammatory response atthe DRG alone leads to a comparable increase in axonal outgrowth71,82.

A less invasive approach to induce sensory regeneration focuses on the de-livery of neurotrophic factors that are known to control axonal outgrowth.These factors can be delivered using either injection of recombinant virusesthat induce expression of neurotrophic factors by host cells or direct deliveryof neurotrophic factors by implanted delivery devices such as infusion pumps.Myelinated sensory axons can be stimulated to grow back into the spinal cordusing viral expression of neurotrophin-3 (NT3), and peptidergic sensory axonscan be stimulated using viral delivery of fibroblast growth factor 2 (FGF 2) andneurotrophic growth factor (NGF)83. But ingrowing fibres do not show topo-graphic specificity inside the spinal cord83. As a side effect, NGF overexpres-sion induces sprouting of uninjured nociceptive axons leading to chronic painin both injured and healthy animals83,84. To achieve topographic specificity ofregenerating axons, NGF overexpression can be restricted to the dorsal hornand can be combined with virally induced expression of the axonal outgrowthrepelling Semaphorin 3A in the ventral horn. This approach succeeds in twoways, it reduces the NGF induced chronic pain and achieves projection ofingrowing sensory axons to appropriate laminae85,86.

Direct infusion of the injured dorsal root with NGF, NT3 or GDNF resultsin the regeneration of specific sensory axon types. NGF results in ingrowthof peptidergic axons, NT3 in ingrowth of NF200 expressing axons and GDNFin regrowth of NF200 positive, peptidergic and IB4 binding axons87. Acutedelivery of NT3 induces outgrowth of myelinated sensory axons and leads tothe recovery of proprioception88. However, it fails when treatment starts oneweek after the injury89. Thus, for this approach to succeed the time point oftreatment is an important factor. In addition, NT3 induced outgrowth can beenhanced by viral induction of the mTor pathway90. Recently, systemic ad-ministration of the GDNF-family member artemin has been reported to induceregeneration of all three fibre types to appropriate laminae and to restore sen-sorimotor and nociceptive functions91,92. The mechanism behind this remainselusive and more recent evidence suggests that the effect of artemin might belimited to nociceptive axons93,94.

1.2.5 Cell therapy for sensory regeneration in dorsal root injuryApart from the previously presented approaches to induce sensory regener-ation, also cell and tissue transplantation has provided promising results. Itis generally believed that cell and tissue transplants that originate from earlystages of development combine multiple beneficial effects. Early approachesaimed to circumvent the scarred DRTZ completely by transplanting foetal

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spinal cord tissue between the cut dorsal root and spinal cord. These ap-proaches succeeded in stimulating ingrowth and synaptic integration of alltypes of sensory axons and could be combined with the local delivery of NGFto further improve axonal outgrowth95–100. Peripheral nerve grafts and ar-tificial nerve conduits succeed in a similar way when combined with localdelivery of NGF85,101.

In one of the earliest approach to overcome and not circumvent the reac-tive DRTZ, embryonic astrocytes were mounted on a cell carrying matrix andtransplanted to the site of dorsal root crush, this allowed ingrowth of sensoryaxons by modifying the local glial environment at the DRTZ and providedtrophic support to sensory axons102. For DRR, tissue adhesive alone or whencombined with mono-nuclear cells results in limited sensory ingrowth103. Al-ternatively, injured sensory neurons can be completely replaced by DRG cellsof embryonic origin. These cells differentiate into neurons that extend axonalprojections into the periphery and re-innervate spinal cord neurons104–109.

More recent studies focus on the identification of immature cell types thatcan be gained from the host and used for regenerative purposes. The idealcandidate would already support ingrowth of PNS originating axons into themature CNS in its tissue of origin. During development, olfactory ensheathingcells (OEC), a type of radial glia, provide a growth supportive substrate forPNS originating olfactory axons to grow into the olfactory bulb that belongs tothe CNS. Injections of OEC either into the dorsal horn or DRTZ allow sensoryregeneration after DRR110,111. When OEC are applied to the cut surface ofthe dorsal root and the spinal cord before the root is reattached using a tissueadhesive, they interact with Schwann cells and astrocytes to form a permissivetissue bridge that allows ingrowth of sensory axons and reinnervation of dorsalhorn neurons112,113. Special care has to be taken that OEC are collected fromthe olfactory bulb alone and are not injected as single cells, as otherwise theyfail to exert their regenerative effect114,115. OEC can be obtained directly formthe adult host. This paved the way for the currently ongoing clinical trials thattest their regenerative potential in spinal cord injury patients116,117.

All the aforementioned studies have used either models of DRR or dorsalroot crush. DRA presents an additional challenge as it not only involves deaf-ferentiation and a more pronounced glial response, it also leads to the completedestruction of the DRTZ, neuronal loss and chronic pain. In analogy to the ef-fect of OEC, boundary cap neural crest stem cells (bNCSC) could exert similarbeneficial effects as they also contribute to the establishment of the PNS-CNSinterface during development. For this reason, they were chosen as our firstcell type of interest and their effect on DRA is presented in Paper III.

Embryonic stem cell (ESC) derived spinal cord neural progenitors are an-other good candidate as they provide functional recovery in spinal cord injuryand can be directed to an exclusive spinal cord lineage in vitro, prior to trans-plantation118–120. Directed differentiation into neural progenitors is anotherbenefit of this approach as it minimizes the ESC inherent tumorigenic poten-

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tial and chance to induce chronic pain in the host121,122. For these reasons ESCderived spinal cord neural progenitors were chosen as the second cell type ofinterest and tested in Paper I. An alternative source of spinal cord neural pro-genitors is foetal tissue. These cells form neurospheres in vitro and can becultured over extend periods of time with only small changes in their cellularcomposition. In addition, they improve the functional outcome of spinal cordinjury and do not cause chronic pain123–125. Thus, spinal cord neural progeni-tors from foetal tissue have been selected as our third cell type of interest andare tested in combination with neurotrophic factor delivery in Paper IV.

1.3 Protection of spinal motor control using neuralprogenitors

1.3.1 The motor neuron disorder: Amyotrophic lateral sclerosisMotor neuron diseases (MND) are a set of lethal progressive neurodegenera-tive disorders that are characterized by the degeneration of motor neurons sit-uated in the cerebral cortex, brain stem and spinal cord. Their symptoms mostoften include muscle spasticity and atrophy of bulbar muscles and/or musclesof the limbs. Amyotrophic lateral sclerosis (ALS) is the most common formof MND but with a prevalence of 2-6 cases per 100.000 people still consid-ered a rare disease126. In classical ALS, death by respiratory failure sets-inonce motor neurons that control the diaphragm degenerate approximately 3-5years after the initial diagnosis. So far, apart from the anti-glutamatergic drugRiluzole that results in a modest increase of survival of patients, no effectivetreatment 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 originmight differ. 13 types of fALS can be distinguished, the most studied mu-tated 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 allfALS cases128. Several other rare protein mutations have been identified aswell, for example in the vesicle-associated membrane protein-associated pro-tein B/C (VAPB)128. The complex genetic background of fALS poses a greatchallenge as alone for the SOD1 protein over 150 different mutations are re-ported to be involved in ALS of which the most common are point mutationsresulting in amino acid substitutions at D90A, A4V, H46R and G93A129. Mu-tations in SOD1 are believed to compromise the proteins function to inhibitreactive oxygen species in the cytosol and mitochondria, implicating their in-

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volvement in the pronounced oxidative stress and mitochondrial dysfunctionobserved in fALS130,131.

Apart from oxidative stress, multiple processes contribute to the degener-ation 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 transportand dysregulated RNA processing have all been linked to this outcome137,138.In recent years, it became clear that microglia, astrocytes and oligodendro-cytes are involved in some of these processes and generate a toxic spinal cordmicro-environment that makes motor neurons less stable and reduces theirsurvival135,139,140. This renders ALS a multi-factorial disease and suggeststhat treatment would have to provide support to motor neurons and render thespinal cord micro-environment less harmful to their survival.

1.3.2 Cell therapeutic spinal cord transplantation for ALSStem cell based cell therapeutic approaches are believed to modulate multi-ple systems at once as transplanted cells show a high degree of plasticity141.Ideally transplanted cells would have the ability to alter the immune- and in-flammatory response of the host, provide a protective environment for ALSaffected spinal motor neurons and even replace dying cells142. Most currentcell therapeutic approaches achieve one or two of these goals, whereas cellreplacement is the most elusive so far.

Strategies that aim to replace dying spinal motor neurons focus on the trans-plantation of motor neuron progenitors (MNP), neural stem cells (NSC) de-rived from human (hESC) or mouse embryonic stem cells (mESC), or inducedpluripotent stem cells (iPS). Early studies utilizing murine MNP failed to showlong-term benefits143, more recently human MNP transplantation achievedneuroprotection possibly by the secretion of the neurotrophic factors NT-3and 4, NGF and vascular endothelial growth factor (VEGF) by transplantedcells144. In contrast to MNP transplants, NSC transplants from the humanfoetal nervous system are extensively studied due to their beneficial paracrineeffects145,146. Intra-spinal injection of NSC result in the successful integra-tion of NSC derived neurons into the spinal cord circuitry, synaptic innerva-tion of motor neurons and improve survival of ALS mice147,148. This effectrequires grafted NSC to be in close proximity to hosts motor neurons andthe outcome can be improved by performing multiple injections along thespinal cord149,150. In addition, transplanted NSC can be genetically modi-fied to over-express VEGF. VEGF increases motor neuron survival and motorfunctions151 and might stimulate endogenous NSC populations152. NSC de-rived from iPS show robust differentiation once inside the host spinal cord andimprove several aspects of the ALS pathology by modulation of the inflam-matory response and neurotrophic factor secretion153,154. However, ultimately

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these approaches fail to replace dying neurons. In conclusion, MNP and NSCgenerally improve survival time and motor functions most likely due to theirneurotrophic and immunomodulatory properties155.

With more and more evidence pointing towards a special role of glial cellsin the ALS pathology156, increasing efforts are made to test the effect ofglial progenitor transplantation on ALS animals. In an initial study, embry-onic glial-restricted progenitors (GRP) differentiated primarily into astrocyteswhen injected into the spinal cord of ALS rats and improved motor neuronloss, motor functions and survival. The transplanted cells effect was attributedto the amelioration of glutamate excitotoxicity by increasing the expression ofthe astrocytic glutamate transporter glutamate transporter-1 (GLT-1)157. BothGLT-1 and the astrocytic glutamate aspartate transporter (GLAST) are ma-jor regulators of glutamatergic excitotoxicity158. Human GRP from foetalsources fail to exhibit the same properties possibly due to their longer matu-ration time to become fully functional astrocytes159,160. Umbilical cord bloodcells transplanted to the spinal cord of ALS animals exhibit properties of as-trocytes, in addition they secrete anti-inflammatory cytokines. This approachprolongs survival time and ameliorates ALS pathology161,162. Another type ofglial progenitor, the OEC receives a lot of attention in clinical trials due to itsbeneficial properties in spinal cord injury163. These cells remyelinate neurons,are neuroprotective and improve survival of ALS animals164. However, theyseem to have no effect on human ALS patients165,166.

Apart from the cell therapeutic approaches mentioned above, mesenchy-mal stem cells (MSC) are good candidates due to their well documented neu-rotrophic and immunomodulatory properties. They are most often adminis-tered systemically or injected into the cerebrospinal fluid and are generallybeneficial167. When injected into the cerebrospinal fluid they can migrate intothe spinal cord grey matter where they give rise to cells that exhibit propertiesof astrocytes. They protect motor neurons, reduce inflammation and extendthe lifespan of ALS mice168. Similarly when human bone marrow derivedMSC are transplanted directly into the spinal cord, they decrease inflamma-tion, improve motor function and extend survival169.

One major limitation of all the pre-clinical studies presented here is the ex-clusive use of SOD1G93A mice and rats. Despite these shortcomings, NSC andMSC transplants are the most promising approaches for cell therapeutic strate-gies in ALS and this is reflected in the multitude of ongoing clinical studiescentred around their use141,167. Very little is known about the potentially ben-eficial effects of other neuroprotective stem cell types. bNCSC transplantedinto the spinal cord show cell protective and angiogenic properties and differ-entiate into both neurons and astrocytes170–174. These features render theminteresting candidates for the cell therapeutic treatment of ALS. Their effecton ALS motor neurons is presented in Paper V.

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1.3.3 Stem cell based in vitro disease modelling of ALSSince the advent of stem cell science and the development of efficient directeddifferentiation protocols, in vitro modelling of many neurological diseases hasbeen greatly improved. Not only is it now possible to gain defined neuronaland glial cell population from human stem cells and tissue allowing the fo-cused study of their contribution to complex disease phenotypes, they can alsobe genetically modified or directly collected from patients exhibiting geneticabnormalities. The development of highly efficient protocols to gain motorneurons and astrocytes from embryonic and induced pluripotent stem cells al-lowed 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 culturesshow 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 ofsmall molecules that revealed Kenpaullone, a GSK-3α/β and HGK inhibitorinvolved in neuronal apoptosis, that greatly improves motor neuron survival177.Most of the hallmarks identified in mESC derived SOD1G93A motor neuronsare also present in primary and iPS derived murine SOD1G93A motor neu-rons176,178 and have been corroborated in SOD1 mutated motor neurons de-rived from human embryonic and induced pluripotent stem cells177,179–181. Inaddition to the previously present cellular abnormalities, human iPS derivedSOD1G93A motor neurons revealed neurofilament dysregulation that could con-tribute to the degeneration of neurites and observed hyper-excitability of SOD1G93A motor neurons180,182.

Following the insights gained from the initial SOD1G93A mESC lines, theeffect of other common fALS mutations on motor neurons was elucidated.Motor neurons derived from iPS harbouring the TDP-43 mutation show re-duced neurite length, an abnormal protein level of TDP-43 and protein aggre-gation183,184. In addition, the cell line allowed the identification of anacardicacid in a high-throughput screen of small molecules. Anacardic acid is a his-tone acetyltransferase inhibitor, that ameliorates the reduced neurite length andabnormal levels of TDP-43184. Motor neurons derived from iPS harbouringthe C9orf72 mutation confirm glutamate excitotoxicity and hyper-excitabilityas a cause of neuronal death and revealed RNA toxicity and deficient nucle-oplasmatic transport to contribute to the C9orf72 pathology182,185–188. Lesswell understood are the cell pathological features of motor neurons derivedfrom iPS harbouring VAPB or FUS mutations, as so far only decreased levelsof VAPB and hyper-excitability of FUS motor neurons are reported in theselines182,189,190. Apart from the motor neuron pathology, also the neurotoxic ef-fect of astrocytes in ALS has been corroborated for SOD1 and C9orf72 usinga combination of stem cell derived motor neurons and fALS astrocytes from

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primary and stem cell sources191–196. In contrast, TDP-43 mutated astrocytesshow typical aggregation of TDP-43 but are not toxic to motor neurons197.

Taken together, the most common observation in in vitro stem cell derivedfALS afflicted motor neurons is protein aggregation, reduced survival of neu-rons, 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 featurethat is commonly linked to mitochondrial dysfunction in ALS. This is evenmore important as in a recent gene profiling study that analysed motor neu-rons derived from iPS cells from sALS patients, mitochondrial dysfunctionwas linked to the degeneration of motor neurons in most cases198.

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

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2. Aims of the study

The general aim of this thesis was to analyse the potential of neural progenitorsfor regenerative purposes in the spinal cord. Its major focus lies on neuralprogenitor transplantation for the recovery of sensory sensation and to a lesserextend on the potential benefits of neural progenitor transplantation for thetreatment 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 ofdorsal root avulsion in mice. (Paper I, reviewed in Paper II, Paper III)

• The second aim of this thesis was to analyse whether the observed hu-man neural progenitor induced sensory regeneration could be translatedto stem cells from alternative human sources and whether their benefi-cial effect could be improved by the co-administration of neurotrophicfactors. (Paper IV)

• The third aim of this thesis was to analyse the effect of murine neuralprogenitors on the survival of SOD1G93A motor neurons. (Paper V)

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3. Experimental procedures

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

3.2 Cell culture3.2.1 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 coupledwith the cytomegalovirus immediate-early enhancer) was introduced to hESCusing the transcription activator-like effector nuclease (TALEN) genome edit-ing technique180,200,201. The resulting CAG::hrGFP hESC line was patternedto 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, andthe defined small molecule mixture of GSK3 inhibitor CHIR99021, TGF/ Ac-tivin/ Nodal receptor inhibitor SB435142, bone morphogenic protein receptorALK2 inhibitor DMH1, and the caudalizing morphogen retinoic acid (RA) to-gether with the sonic hedgehog (SHH) agonist purmorphamine. Neuroepithe-lial cells were differentiated towards a spinal cord neural progenitor lineagein suspension culture. To induce directed differentiation, the defined smallmolecule mixture was replaced by higher concentrated RA and the SHH ag-onist Ag1.3. After seven days, spherical cell conglomerates had formed thatexpressed markers of ventral spinal cord neural progenitors. From here on,these conglomerates are referred to as human spinal cord neural progenitor(hNP) spheres.

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3.2.2 In vitro differentiation of human spinal cord neuralprogenitors [Paper I]

hNP spheres were dissociated and seeded onto poly-l-ornithin and lamininprecoated 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 neurotrophicfactor (GDNF) and ciliary neurotrophic factor (CNTF) were added to themedium. 10 and 21 days after initiation of terminal differentiation, cells werefixed using 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS).Cell populations present in hNP spheres and terminally differentiating humanspinal cord neural progenitors were analysed using immunocytochemistry.

3.2.3 Generation of transplantable murine boundary cap neuralcrest 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 flu-orescent protein variant DsRed.MST under the control of the CAG promoter(CAG::DsRed.MST, Jackson Laboratory,202) and from CAG::eGFP transgenicmice (Jackson Laboratory) were collected from the isolated cord and mechano-enzymatically dissociated. Single cells were cultivated in N2-medium contain-ing DMEM/F12 supplemented with N2, B27, epidermal growth factor (EGF)and basic fibroblast growth factor (FGF) until free floating neurospheres hadformed (for a detailed description of the establishment of these cell lines seeAldskogius et al. 203 and for their characterization Hjerling-Leffler et al. 170).bNCSC neurospheres were used for cell transplantation (Paper III), in vitrodifferentiation on poly-D-lysine and laminin precoated glass cover-slips inDMEM/F12 and Neurobasal supplemented with non-essential amino acids(NEAA), B27, N2 (Paper III) and co-culture with mESC derived motor neu-rons (Paper V).

3.2.4 Generation of transplantable human spinal cord neuralstem/progenitors derived from human foetal spinal cord[Paper VI]

Human foetal spinal cord cells were isolated from human first trimester spinalcord tissue (6-9 weeks of gestation) and maintained as free floating humanspinal cord neural stem/progenitor (hscNSPC) neurospheres. Cells were cul-tured in DMEM/F12 supplemented with glucose, Hepes, Heparin and N2 to-gether with neurotrophic factors EGF, fibroblast growth factor 2 (FGF2) andCNTF125.

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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 andlaminin precoated glass cover-slips in DMEM/F12 and Neurobasal supple-mented with NEAA, N2, B27 and neurotrophic factors GNDF and CNTF orneurotrophic factor peptide mimetics for GDNF (Gliafin204) and CNTF (Cin-trofin205) delivered by mesoporous silica particles206. 5 and 8 days after ini-tiation of terminal differentiation, hscNSPC were collected, ribonucleic acid(RNA) was isolated and RNA-to-cDNA synthesis was performed followingthe manufacturers protocol. Migration of differentiating hscNSPC was anal-ysed using wide-field microscopy, and an expression profile for multiple dif-ferentiation associated marker genes was established using qRT-PCR analysis.For used primers please see Table 3.1.

Table 3.1. Primers used for qRT-PCRTarget Forward Primer Reverse Primer

Genes of interestNestin 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 factormimetics [Paper IV]

Mesoporous silica with an average particle size of 12 µm and average poresize of 20 nm were characterized before206,207. Particles were loaded withGDNF and CNTF peptide mimetics via impregnation in water and succes-sive 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), thatinteracts with neural cell adhesion molecule (NCAM) and has neuritogenic

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

3.2.7 Generation of motor neuron cultures from murineembryonic stem cells [Paper V]

HB9::GFP mESC lines harbouring either the human SOD1G93A, human wildtype SOD1176 or expressing endogenous murine SOD1208 were establishedon a monolayer of mouse embryonic fibroblast cells in serum free propagationmedium containing KoDMEM, KoSerum-Replacement, GlutaMAX, NEAA,β -mercaptoethanol and penicillin streptomycin. Following a slightly adaptedversion of the original protocol for motor neuron differentiation208 (for detailsplease see Paper V), mESC lines were transferred to suspension culture disheswhere they formed embryoid bodies (EB) in neural differentiation mediumcontaining DMEM/F12, Neurobasal, GlutaMAX, neuronal cell culture sup-plements B27 and N2, β -mercaptoethanol and penicillin-streptomycin. Motorneuron differentiation of EBs was induced by the addition of RA and Ag1.3 toEB medium two days after the start of the suspension culture. EBs were dis-sociated after 7 days of culture and in some cases depleted of GLAST+ glialcells 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 extracellu-lar 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 insome experiments.

3.2.9 In vitro analysis of motor neuron and boundary cap neuralcrest 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-slipsin DMEM/F12 and Neurobasal supplemented with GlutaMAX, N2, B27 andneurotrophic factors GNDF and CNTF. Under these conditions, mESC-derivedmotor neurons mature over the course of one week indicated by the expressionof motor neuron specific markers208. Over the same time course, progressiveloss of motor neurons occurs208. The percentage of surviving motor neurons

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alone or in the presence of bNCSC was calculated for multiple time-pointsafter initiation of differentiation. In additional experiments, cultures weretreated with increasing levels of H2O2 and motor neuron and bNCSC survivalrates were calculated.

Figure 3.1. Overview of conducted experiments.

3.3 Animal studies3.3.1 Dorsal root avulsion injury and cell transplantation [Paper

I,III,IV]Adult male nu/nu NMRI mice underwent dorsal root avulsion (DRA) andhuman spinal cord neural progenitor spheres or murine bNCSC neurospheretransplantation following previously established protocols57,172,173. Mice werekept under 2.5% isoflurane gas-anaesthesia while laminectomies were per-formed along the T13+L1 vertebrae of the left side. The dura was openedand the exposed L3-L5 dorsal roots were pulled one after another away fromthe spinal cord with steady force until they ruptured at the PNS-CNS inter-face. This led to a longitudinal dorsal spinal cord injury and destroyed thedorsal rootlets. Avulsed dorsal roots were repositioned to the site of avulsionand human and murine cell spheres were placed at the site of injury. Twogroups in Paper IV received 20 µg of mesoporous silica particles loaded withneurotrophic factor mimetics Gliafin and Cintrofin locally applied to the siteof 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 spinalcord. Mice were kept under 2.5% isoflurane gas-anaesthesia while partial

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Figure 3.2. Overview of dorsal root avulsion cell therapeutic approaches presented inthis thesis.

laminectomy along the C3-C5 vertebrae of the left side were performed. Thedura was opened and GLAST-enriched bNCSC and SOD1G93A MN cell sus-pensions 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 witheither cholera toxin subunit B (CTB) or lectin Griffonia Simplicifolia Agglu-tinin isolectin B4 (IB4)212,213. In naive and sham animals, CTB exclusivelylabels myelinated and IB4 exclusively labels unmyelinated sensory axons214.For tracer injections, animals were kept under 2.5% isoflurane gas-anaesthesiawhile the left sciatic nerve was exposed and 1% (wt/vol) tracer solution wasinjected through the epineurium of the nerve using a fine glass pipette, follow-ing the previously published procedure215. Spinal cord tissue was collectedthree days after injection of the tracer.

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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 [PaperII,III-V]

Mice received an overdose of anaesthetics and were then transcardially per-fused with saline followed by 4%PFA/14% picric acid solution. Cervicalor lumbar spinal cord tissue was collected, immersed in fixation solution forfour hours and transferred to cryo-protection in 15% sucrose in PBS at 4◦Covernight. In vitro grown cell spheres were collected from the culture dish andfixed 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 OCTcompound and flash frozen in a stream of CO2 gas or embedded in pulverizeddry ice and cryo-sections were prepared using a cryostat set to -22◦C. Spinalcord 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 block-ing solution containing bovine serum albumin, Triton X-100 and sodium azide(NaN3) in PBS. Mixed primary antibodies in blocking solution were applied inempirically determined concentrations and either incubated for four hours atroom temperature or at 4◦C overnight. Following PBS washing of tissue sec-tions, mixed secondary fluorophore-conjugated antibodies targeting the hostspecies of the primary antibodies were diluted to working concentration in

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

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

Gait analysisAnimals were tested with the CatWalk gait system that allows recording ofseveral aspects of gait by automated illumination, detection and recording ofthe 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 printarea, stride length, stance phase and swing phase) that are commonly affectedin models of neurotraumata217,218.

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

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

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slowly pulled back and the force at the time of release of the paw from the barwas recorded.

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Table 3.2. Antibodies used for immunohisto- and immunocytochemistryPrimary

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

Secondary

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

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3.4 Microscopy [Paper I,III-V]In vitro differentiated stem cells were imaged using either a Plan-Apochromat10x objective (numerical aperture (NA) 0.25) or a Plan-Apochromat 20x ob-jective (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 cellspheres were analysed using Zeiss LSM700, LSM710 and LSM780 confocallaser scanning microscopes. Images that contributed data to the same quanti-tative analysis were never collected with different microscopes. Single imageswere captured using a Plan-Apochromat 20x objective (NA 0.8), a LD LCIPlan-Apochromat 25x objective (NA 0.8) and a LD C-Apochromat 40x WCorr (NA 1.1) objective. For orthogonal projections, images were taken witha Plan-Apochromat 40x objective (NA 1.3) or a Plan-Apochromat 63x objec-tive (NA 1.4). Z-stacks were taken with an optical slice thickness of 1 µm atan 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 (NA0.8).

3.5 Image analysis [Paper I,III-V]For Paper I-IV, images of every 10th tissue section containing transplantedcells spanning along the L3-L5 segments of the spinal cord were processedusing Fiji ImageJ2220. For Paper V, every 5th tissue section along the C3-C5 segments was analysed. Double and triple labelled cells were countedin a semi-sterological manner in confocal z-stacks spanning the whole 14 µmtissue section (Paper I) or in single confocal images (Paper III and IV) usingthe Fiji ImageJ2 cell-counter plugin. High magnification z-stacks for doublelabelled 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. Ingeneral, images were background subtracted using the ImageJ implementedrolling ball algorithm and contrast enhanced to a saturation of 0.35, followedby automatic thresholding of the immuno-positive area using either Otsu’sor Tsai’s method221,222. In some analysis no suitable automatic thresholdingtechnique could be found and therefore a manual threshold had to be set. Inall cases, the site of dorsal root avulsion injury was set as the region of interest(ROI).

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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-Smirnovtest and for homoscedasticity using scatter plots of the raw data. When theseassumptions were violated, either the Mann-Whitney U test or for multiplecomparisons the Kruskal-Wallis analysis of variance followed by Dunn’s Mul-tiple Comparison test was used. When normality and homoscedasticity couldbe assumed and only two conditions were tested a two-sample Student’s t-testwas used, in cases where more then two conditions were analysed one-wayANOVA followed by Dunn’s Multiple Comparison test or Tukey’s test wasused. For data sets that consisted of two independent categorical variables,two-way ANOVA was used followed by Sidak’s multiple comparison. Theconfidence interval was stated at the 95% confidence level, placing statisticalsignificance at p < 0.05. Plotting of data and statistical analyses were per-formed in GraphPad Prism 6.

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4. Results and Discussion

4.1 Paper IHuman spinal cord neural progenitor (hNP) spheres expressed typical mark-ers of ventral spinal cord neural progenitors of the neural lineage. Long-termin vitro differentiation primarily gave rise to cholinergic and inhibitory neu-rons and astrocytes. When transplanted to the site of dorsal root avulsion(DRA), hNP cells nearly exclusively differentiated into inhibitory neurons andastrocytes. Up to five month after transplantation, engrafted hNP continued toshow expression of doublecortin, a marker typical for immature neurons. Thiscorroborates the previously observed long maturation time of human neuralprogenitors from human embryonic stem cells (hESC) and human inducedpluripotent stem cells (iPS) after transplantation to the rodent central nervoussystem (CNS)223,224.

It raises the question whether similarly long maturation times are expectedwhen human neural progenitors would be transplanted to the humans CNS.Doublecortin is a 40-kDa microtubule-associated protein expressed by neu-ral progenitors and post-mitotic neuronal precursors. It gets down-regulatedwith the appearance of markers of mature neurons and disappears shortly af-ter birth in mice and pigs225–227. In humans, doublecortin is prominent in thesub-ventricular zone up to 18 months after birth and from there on it is nearlyabsent from the brain228. In the adult murine CNS, doublecortin is associatedwith neurogenesis, while newly born neurons cease to express doublecortin atthe latest one month after their generation229. Taking the doublecortin expres-sion in the young human brain and adult murine CNS into consideration, thepresented maturation time here might lie within a reasonable time-frame. Themonth long maturation time of hNP might have implications on cell replace-ment strategies, as these aim to provide quick replacement of dying neuronsin neurodegenerative diseases and after injury to the CNS230. It suggests thattransplanting even further differentiated cells could increase the immediate ef-fect of neuronal cell therapy and in parallel would reduce the risk of teratomaformation by stem cell derived cell types231.

hNP intermingled with the dorsal root stump and interfered with the dorsalastrocytic scar and basal lamina. This resulted in the formation of openings inthe otherwise continuous spinal cord surface and were referred to as "gates" inPaper I. The formation of "gates" shares many properties with the previouslydescribed growth permissive "tissue bridges" formed by transplanted olfactoryensheathing cells (OEC) at the PNS-CNS interface112. Like hNP transplants,

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OEC directly contact endogenous astrocytes and show no migration into thespinal cord, but allow biotinylated dextran amine (BDA) labelled sensory ax-ons to transverse the OEC transplant and grow into the spinal cord112.

BDA is neuroanatomical tracer that is taken up by injured and uninjuredaxons and distributes by passive diffusion232,233. Thus, it does not allow todraw conclusions about the type of ingrowing sensory axons. Here, we per-formed transganglionic tracer injections of cholera toxin subunit B (CTB) orisolectin B4 (IB4) into the sciatic nerve resulting in the active transport oftracer to the neuronal soma in the dorsal root ganglion, and to myelinated andunmyelinated axonal projections of sensory neurons in the spinal cord, respec-tively212–214,234. Special care needs to be taken when CTB and IB4 are usedafter axotomy of the sciatic nerve, as CTB is transported by both myelinatedand unmyelinated fibres and IB4 might fail to be transported beyond the dor-sal root ganglion (DRG)235,236. This effect renders it difficult to analyse thetype of regenerating sensory fibres by transganglionic tracing alone. So far, noreport states whether sensory axons undergo similar changes in their ability totransport these tracers after dorsal root injury. Here, we could observe bothtracers inside the DRG and adjacent nerve fibres, whereas only CTB tracerwas found inside the ipsi-lateral spinal cord dorsal horn. Further, CTB positiveprofiles were found in both the superficial and deep dorsal horn, overlappingat least partially with dorsal horn areas that are innervated by proprioceptiveand mechanoreceptive input in uninjured animals10,237.

In addition to transganglionic tracing, both neurofilament 200kD (NF200)and calcitonin gene-related peptide (CGRP) expressing sensory fibres werefound in the transplant area. Of these, only NF200 expressing fibres were ableto cross the PNS-CNS interface through "gates" in the spinal cord surface.These were accompanied by fibres originating from the human transplant thatsuperficially grew into the dorsal spinal cord. The selective ingrowth of NF200positive fibres and the finding that only CTB traceable sensory axons werefound in the dorsal horn, leads to the conclusion that primarily myelinated,and not unmyelinated or peptidergic fibres regenerated across the PNS-CNSinterface.

The neuronal population of engrafted cells could function as synaptic relaysto restore the sensory circuitry after DRA. Foetal spinal cord tissue grafted intothe injured adult spinal cord is able to functionally reunite separated segmentsof the spinal cord238. This approach proves to be equally efficient for therestoration of sensory connectivity99,100. The observed functional reconnec-tion is attributed to host sensory axons passing through the foetal graft and theformation of a spinal cord-transplant relay99,100,239.

So far, a similar relay function of transplanted stem cell derived neurons torestore the sensory connectivity to the spinal cord has not been reported. Foetalneural stem cells transplanted to the murine spinal cord are able to establishsynapses and integrate structurally into the host’s motor circuitry147,148. Inaddition, murine embryonic stem cell derived motor neurons transplanted into

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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 thatstem cell derived neurons are able to integrate into the spinal cord circuitry.The complete absence of VGluT1 and VGluT2 positive terminals from areasof hNP engraftment suggests the absence of synaptic connectivity betweensensory fibres and the transplant, as primary sensory neurons are typicallyglutamatergic12. Together with the limited ingrowth of hNP fibres into thespinal cord, this renders it unlikely that human neurons functioned as relaysfor sensory axons and contributed to the observed sensorimotor improvement.

The 3rd, 4th and 5th lumbar DRG encompass nearly all the contribution ofsensory fibres from the sciatic nerve in mice and rats241,242. Following L3-L5DRA, animals showed a severe reduction of mechanoreceptive and sensorimo-tor 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 dermatomesin rodents243,244, animals showed only about 50% reduction in their abilityto react to a mechanical stimulus applied to this hind paw. The strength ofthe used van Frey filament (5.5g) could provide an explanation for the lackof complete desensitisation. Filaments of sufficient strength not only stimu-late the cutaneous hind paw but also minimally displace the leg, leading to asubstantial change in the nature of the stimulus219.

DRA of T13+L1 results in the development of bilateral allodynia of thehind paws61,62. In contrast, avulsion of dorsal and ventral roots (spinal rootavulsion) of L5 results in allodynia occurring only on the ipsi-lateral side66.Even transplantation of neural stem cells alone can lead to the developmentof allodynia122,245. These findings made it substantial to test for the develop-ment of allodynia in our DRA model, but we could not observe any develop-ment of hypersensitivity to noxious mechanical stimuli in either DRA aloneor animals receiving hNP transplants. The absence of allodynia in animalsundergoing L3-L5 DRA alone might be explained by the avulsion of multipleadjacent roots, as this results in a continuous longitudinal injury of the dorsalhorn along these segments. This outcome might resemble the more controlledmicro-surgical DREZotomy that relies on selective ablation of the dorsal roottransitional zone and reduces chronic pain after brachial plexus injury in pa-tients246,247.

Animals receiving hNP transplants showed improved mechanical nocicep-tive sensitivity from seven weeks after transplantation up to five months aftertransplantation. In addition, animals regained the majority of their strength togrip with the ipsi-lateral hind paw. Both tests record a motor response of thehind paw, this suggests that sensory fibres were conveying meaningful sensoryinformation into the spinal cord and were able to elicit an appropriate motorresponse. Five months after hNP transplantation, dorsal roots that were previ-ously avulsed were dissected close to the DRG during a second surgery. Thisresulted in the complete loss of observed sensorimotor improvements, indicat-ing that the behavioural improvements were caused by regenerating sensory

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fibres entering the spinal cord along previously avulsed dorsal roots and notby sprouting from neighbouring segments or trophic effects of the transplantedcells. Taken together, the improvement of mechanical nociceptive sensitivityand sensorimotor coordination, the expression of NF200 by ingrowing fibresand their uptake and transport of CTB as well as their projection pattern in thespinal cord suggests the regeneration of mechanoreceptive and/or propriocep-tive sensory axons innervating dorsal horn neurons.

After the findings presented in Paper I, a recent study showed that alsoOEC are able to form a tissue bridge after DRA avulsion248. In contrast tohNP transplants that primarily rescue NF200 positive axons and propriocep-tion, OEC transplants stimulate primarily peptidergic axons and ameliorate theDRA induced chronic pain. In theory, these two cell types would complementone another in their regenerative potential and it would be most interesting tosee the effect of their co-transplantation to a model of DRA.

4.2 Paper IIIDuring development, boundary cap neural crest stem cells (bNCSC) form theinterface between the spinal roots and cord22,23. This makes them particu-larly interesting for transplantation approaches to induce sensory ingrowth af-ter DRA. It allows a close match between the targeted tissue and the origin oftransplantation material, a clear advantage over the human spinal cord neuralprogenitors used in Paper I and IV that were either differentiated towards theventral spinal cord lineage in vitro (Paper I) or collected from primary tissueof the foetal spinal cord (Paper IV).

Another possible benefit of bNCSC transplantation is the use of allogenicinstead of xenogenic cells. Nevertheless, all transplantation approaches testedhere are expected to induce an immune response. hESC show reduced im-munogenicity by differential expression of major histology complexes and im-munomodulatory properties but it is unclear whether this transcends to hESCderived progenitors249. Therefore, both hNP spheres and hscNSPC neuro-spheres are expected to be rejected by the host. Allogenic transplantation ofbNCSC faces the same problem. For these reasons, immunodeficient micewere used in all studies and represent a major limitation of the studies pre-sented in this thesis. To overcome the problem of allogenic and xenogenictransplantation, future studies will have to focus on induced pluripotent cellsthat can be patterned to both bNCSC250–252 and spinal cord neural progen-itors253. The use of patient specific iPS and in vitro differentiation of thetherapeutic cell type of interest results in histocompatibility that is believed toovercome the need for immunosupression or -modulation254,255. More recentstudies challenge this view and so far it remains unclear whether iPS trans-plantation does not face the same challenges as transplanting hESC and theirprogeny256.

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Similar to hNP spheres, bNCSC transplanted to the site of DRA showedgood survival, integrated into the tissue and generated a gap in the glial scar ofthe injured spinal cord. Few transplanted cells even migrated into the spinalcord tissue. This suggested the formation of a tissue bridge similar to theone formed by hNP spheres. Tracing of injured sensory fibres revealed sen-sory axon outgrowth into the injured dorsal root and their termination at theinterface between dorsal root and bNCSC transplant, corroborating the pre-vious observation that bNCSC attract neurofilament 200kD positive sensoryfibres173. These fibres never crossed over into the spinal cord tissue.

Findings from Paper I and lessons from OEC induced sensory regenera-tion suggest glial progenitors to play a crucial role in the establishment of agrowth permissive tissue bridge. Here, we found one major difference be-tween hNP sphere formed tissue bridges that allowed ingrowth of sensoryfibres and those that were formed by bNCSC and did not allow ingrowth.bNCSC at the interface between injured dorsal root and bNCSC transplant ex-tensively co-expressed the glial marker GFAP and Mts1/S100A4. In contrast,hNP and hscNSPC transplants showed no detectable Mts1/S100A4 expres-sion (Paper III and Paper IV[data not shown]). In mice, Mts1/S100A4 wasexpressed by white matter astrocytes and became up-regulated in response toinjury. This finding is in line with previous studies in rats that describe in-tracellular Mts1/S100A4 as a marker of sensory outgrowth inhibiting reactivewhite matter astrocytes257. Intracellular Mts1/S100A4 reduces injury-inducedmigration and might contribute to the stabilization of the glial scar258. Surpris-ingly expression of Mts1/S100A4 could already be found in undifferentiatedbNCSC neurospheres, in contrast to hNP spheres that showed no detectableexpression. This suggests that for future transplantation approaches, specialemphasis should be placed on glial progenitors and their interaction with thelocal environment at the scarred spinal cord/spinal root interface.

4.3 Paper IVhscNSPC grafted to the site of DRA remained immature up to two monthsafter transplantation, indicated by expression of Sox2 and Olig2 and showedcontinuous proliferation. In contrast to previous findings that neurotrophicfactor mimetics for glial derived neurotrophic factor (GDNF) and ciliary neu-rotrophic factor (CNTF) lead to increased differentiation of transplanted stemcells206, co-transplantation of hscNSPC and neurotrophic factor mimetics hadno effect on the observed differentiation pattern or proliferation of hscNSPC.The overall immaturity of hscNSPC and their unaltered differentiation profilesafter co-transplantation with neurotrophic factor mimetics renders it unlikelythat immaturity is required for hscNSPC to promote sensory axon regenera-tion.

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hscNSPC transplants of both conditions differed only in one aspect, theirdegree of integration into the injured spinal cord tissue. hscNSPC are trans-planted to the surface of the injured spinal cord, thus human cells found insidethe spinal cord have to originate from migratory hscNSPC. Extensive migra-tion was observed when hscNSPC were transplanted alone. Here, Olig2 pos-itive human cells were found in the ipsi-lateral and contra-lateral gray matterand migrated deep into the ventral horn. In contrast, Olig2 positive humancells 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 giveprimarily rise to motor neurons and oligodendrocytes259. hscNSPC trans-planted to the site of DRA may have the potential to serve as a replacementfor lost oligodendrocytes and spinal motor neurons in the ipsi-lateral ventralhorn 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 riseto oligodendrocytes125. This suggests that either the way of delivery or morelikely differences in the initial local environment have an effect on the survivalof oligodendrocyte progenitors or the differentiation of hscNSPC. Due to thelack of studies that systematically compare ways of human neural progenitordelivery to the spinal cord, this question needs to remain unanswered.

Following injection into healthy spinal cord grey matter, human spinal cordneural progenitors are able to migrate far away from their initial site andspread both into gray and white matter areas260. When degenerative CNStissue is present, neural stem cells show a remarkable ability to migrate to-wards the lesion site261–265, an effect that is attributed to cues provided bythe lesion environment261,262,266,267. The local administration of neurotrophicfactor mimetics might disturb this environment enough to no longer stimulatedirected migration of transplanted cells and could explain the reduced integra-tion of hscNSPC transplants into the spinal cord tissue.

hscNSPC transplantation allowed CTB traceable sensory fibres to grow intothe injured dorsal horn. Interestingly, administration of neurotrophic factormimetics alone led to limited ingrowth of CTB traceable sensory fibres. Localapplication of the neurotrophic factor GDNF enhances the regenerative drivein damaged sensory neurons and ameliorates the inhibitory effect of reactiveastrocytes87,268. Thus, the local application of the GDNF peptide mimetic tothe site of DRA could explain this finding.

The combination of neurotrophic factor mimetics and hscNSPC grafts didnot promote regeneration. Analysis of the spinal cord revealed strong GFAPexpression at the site of DRA. When hscNSPC were transplanted alone, thesite of injury showed reduced expression of murine GFAP but increased pres-ence of human GFAP. In contrast, when cells were transplanted together withneurotrophic factor mimetics this effect was diminished and the majority of theinjured dorsal horn showed strong murine GFAP expression. Further, DRA ledto a 2.5 fold increase in neural/glial antigen 2 (NG2) immunoreactivity com-

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pared to naive animals that was not present in animals receiving hscNSPCalone. Co-transplantation with neurotrophic factor mimetics negated this ef-fect 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 growthinhibiting chondroitin sulfate proteoglycans (CSPG)37, but its role as an in-hibitory proteoglycan is disputable. Knock-out of NG2 fails to amelioratesensory axonal ingrowth and NG2 positive cells are permissive to neurite out-growth of DRG neurons in vitro32,269. More recent evidence suggests a rolefor NG2 positive cells in the immobilization of regenerating sensory axonsafter dorsal root injury44.

Taken together, the negative synergistic effect on sensory regeneration ob-served when hscNSPC were combined with neurotrophic factor mimetics sug-gests that successful regeneration of CTB traceable sensory fibres by humanspinal cord neural progenitors is dependent on transplant integration into thespinal cord tissue. Further, hscNSPC provide a growth promoting environmentfor sensory fibres by reducing the presence of growth inhibitory componentstypical for the spinal cord glial scar.

4.4 Paper VThe primary goal of this study was to test the effect of bNCSC on the survivalof SOD1G93A motor neurons with the future goal to use bNCSC in cell ther-apeutic transplantation experiments. SOD1G93A motor neurons show reducedsurvival in vitro176. This finding could be corroborated here. In line withthe well established neurotoxic role of SOD1G93A astrocytes176,191,193,270, re-moval of SOD1G93A astrocytes increased SOD1G93A motor neuron survival byapproximately 75%. SOD1G93A motor neuron survival can be improved by theaddition of healthy astrocytes alone176. Depending on the used culture con-ditions, bNCSC can differentiate into astrocytes and neurons (171 and PaperVI) or peripheral neurons and Schwann cells170,271. This made it necessaryto 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 stemcell derived astrocytes, purified in the same way as bNCSC astrocytes, did notincrease the survival of SOD1G93A motor neurons.

Oxidative stress is a common pathological component of both sALS andfALS and appears at least in spinal cord and brain stem areas272,273. It re-mains unclear whether oxidative stress is a major mechanism behind ALS orsymptomatic for ALS, but it is tightly linked to excitotoxicity, mitochondrialand cytoskeletal dysfunction and protein aggregation, all of which are neu-rodegenerative processes that are involved in ALS273,274. Oxidative stress isinduced by reactive oxygen species (ROS) that are a constant side product

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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 as-trocyte deprived wild type motor neuron cultures and SOD1G93A motor neu-ron cultures. Interestingly, SOD1G93A motor neurons were more vulnerableto oxidative stress than wild type motor neurons. So far, no clear explana-tion exists why motor neurons are more burdened by oxidative stress thanother cell types277. The addition of bNCSC astrocytes to SOD1G93A motorneurons resulted in an increase of motor neuron survival. It remains unclearwhether bNCSC astrocytes exert this function by paracrine effects, as bNCSCare 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 oxidativestress had no effect on bNCSC survival.

Co-transplantation of bNCSC astrocytes and SOD1G93A motor neurons tothe 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 neu-rons from injection induced stress and/or from the neuroinflammatory envi-ronment caused by the transplantation induced insult of the spinal cord. Apartfrom the aforementioned properties of bNCSC, unpublished results from Pa-per VII suggest that bNCSC are able to protect spinal neurons from excito-toxic damage, whereas the underlying mechanism remains unknown. This isparticularly interesting as ALS is characterised by hyper-excitability of spinalneurons and glutamate excitotoxicity both of which contribute to the increasedvulnerability 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 over90%279,280. Reduced expression of GLT-1 might have a negative impact onthe spinal neurons ability to cope with increased extracellular levels of gluta-mate281. Transplanted embryonic glial-restricted progenitors are able to ame-liorate glutamate excitotoxicity in ALS mice by increasing the expression ofGLT-1157. In addition to their proposed cell-protective properties, transplantedbNCSC astrocytes might be able to act in a similar way. Transplantation ex-periments of purified bNCSC astrocytes into the spinal cord of ALS animalswill hopefully shed light onto these questions.

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5. Conclusions

• Human spinal cord neural progenitor transplantation partially restoressensorimotor functions of dorsal root avulsed mice (Paper I).

• Transplantation of human spinal cord neural progenitors from either hu-man embryonic stem cells or foetal abortion tissue promotes regenera-tion of sensory axons by providing a growth permissive environment atthe site of dorsal root injury (Paper I+IV).

• Engrafted human spinal cord neural progenitors from both human em-bryonic stem cell and foetal sources show robust survival and differenti-ation towards glial cells and neurons but distinctively different migrationpatterns and levels of maturity (Paper I+IV).

• Murine boundary cap neural crest stem cells are not able to promotesensory recovery, possibly due to their contribution to the axonal growthinhibitory glial scar (Paper III).

• Peptide mimetics of the neurotrophic factors GDNF and CNTF appliedto the site of dorsal root avulsion lead to ingrowth of sensory axons. Incontrast, combinatorial therapy of human spinal cord neural progenitorsand peptide mimetics results in synergistic effects that prevents ingrowth(Paper IV).

• Murine boundary cap neural crest stem cells promote survival of amyo-trophic lateral sclerosis effected motor neurons and protect them fromH2O2 induced oxidative stress (Paper V).

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6. Future aims

6.1 Stimulation of sensory axon ingrowth by celltherapeutic means

Before the treatment of brachial plexus injuries using human spinal cord neuralprogenitor transplantation can be envisioned, the cell type(s) responsible forthe observed ingrowth of CTB positive fibres needs to be determined. BothhNP and hscNSPC transplants give rise to a multitude of different neural celltypes of varying stages of maturity and it remains unclear what combinationof factors is crucial for the observed improvements. The results from papersI, III and IV suggest that modification of the glial scar and the direct contactbetween transplanted cells and the scarred spinal cord is required for the for-mation of a growth permissive tissue bridge. The tissue bridge has to reachthrough the scar to allow sensory axons to grow into the spinal cord gray mat-ter. A sub-population of transplanted cells that later gives rise to astrocytesplays a major role in the formation of the tissue bridge. It remains unclearwhether the co-transplanted neuronal cell populations are functional or evennecessary for the observed effect.

For these reasons, future studies should focus on the isolation of astro-cytic progenitor populations. As presented in Paper III, the presence of un-favourable astrocytic progenitor types already at the stage of neurosphere cul-ture could be most crucial for the pre-evaluation of ideal cell therapeutic can-didates. To achieve this goal, currently available cell separation techniqueslike immunopanning or magnetic activated cell sorting (MACS) to purify as-trocytic progenitor populations followed by single cell transcriptomal profilingcould help to distinguish between cell populations282. In addition, this wouldsupport the identification of specific markers that allow the formulation of bet-ter differentiation protocols or purification attempts before transplantation .

For the presented cell therapeutic approach to be considered in a clinicalsetting, the culture and pre-differentiation of stem cells needs to comply withgood manufacturing practice (GMP) standards. Cell therapeutic candidatesshould be gathered from patient derived iPS cells to minimise the requiredimmunosupression after transplantation. First steps into this direction havealready been taken283,284, for example the stem cell differentiation protocolused in Paper I can be directly used for the differentiation of iPS cells180, anda modified xenogenic-free culture system of human iPS derived spinal cordneural progenitors has been established285. If the collection of an astrocyticprogenitor population of the spinal lineage is the main goal, differentiation

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

6.2 Boundary cap neural crest stem cells for celltherapeutic transplantation in amyotrophic lateralsclerosis

The experiments in Paper V have shown that the beneficial effects of bound-ary cap neural crest stem cells (bNCSC) are not limited to pancreatic isletcells174,289, and while they do not promote sensory regeneration as presentedin Paper III, they are safe to be used for spinal cord transplantation172,173. Inline with their better studied effect on β -cells278, the initial results suggest thattransplanted bNCSC have to be in proximity to their target, similar to the ben-eficial effect of neuronal stem cells transplanted to ALS animals149,150. Thisincreases the challenge of cell delivery as treatment will most likely requiremultiple intra-spinal injections. It remains unclear whether in a transplanta-tion setting bNCSC would act in a similar way as reported in Paper V. Tocorroborate their beneficial effect, bNCSC need to be injected into the spinalcord of ALS mice. A pilot study is currently under way and initial results arepromising but not yet conclusive.

So far, it remains unknown how bNCSC improve ALS motor neuron sur-vival in vitro. Future studies, should focus on elucidating the mechanism be-hind the improvement of SOD1G93A motor neuron survival in vitro and onadditional aspects of the cytopathology of ALS. A multitude of different stemcell 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 paracrineeffects of bNCSC in co-culture and after transplantation. Taken the extensiveamount of cell therapeutic ALS studies into consideration141,167, the secre-tory profile of bNCSC could be of major interest. First attempts to elucidatewhether direct contact between bNCSC and SOD1G93A motor neurons is nec-essary, suggest that direct contact is required. So far, these assays lack con-clusive proof, but if the initial observation holds true, special emphasis shouldbe placed on cell-cell adhesion molecules and their effect on SOD1G93A motorneuron survival. Interestingly, multiple members of the cadherin and collagegene families are dysregulated in mice carrying a fALS-associated FUS muta-tion290. These could be interesting candidates for a directed analysis as bothgene families are involved in synapse formation, axonal guidance and motorneuron differentiation291.

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7. Sammanfattning på svenska

Skador och neurodegenerativa sjukdomar i ryggmärgen kan leda till förlamn-ing och förlust av känsel. Stamcellstransplantation kan återställa förloradeförbindelser som förmedlar sensorisk information och skydda ryggmärgscellerfrån degeneration. I allmänhet kan nerver återväxa efter skada men de kaninte växa in i den ärrade ryggmärgen. Tre olika typer av stamceller, som gerupphov till ryggmärgsceller, undersöktes med avseende på deras förmåga attövervinna denna växthämmande ärrvävnad. Stamcellerna transplanterades tilldet skadade området och växt av skadade sensoriska nerver analyserades. Avde tre typer av spinala stamceller som undersöktet, möjliggjorde de celler medursprung 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 frånembryonala stamceller förbättrade känseln markant hos ryggmärgsskadadedjur. I syfte att ytterligare förbättra den observerade nervväxten kombineradesbehandling med humana ryggmärgsceller från fetal vävnad med tillförsel avfaktorer vilka stimulerar återväxt av sensoriska fibrer. I kontrast mot förvänt-ningarna förlorade de transplanterade humana ryggmärgscellerna sin förmågaatt understödja sensoriska fibrer och att tillåta inväxt genom ryggmärgsärret.Den sista typen av ryggmärgsstamcell som prövades erhölls från platsen förde sensoriska fibrernas inväxt hos friska embryonala möss. Dessa celler, vilkakallas "boundary cap neural crest stem cells" (bNCSC), kunde inte stimulerainväxt 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 oxidativstress. Oxidativ stress är en degenerativ process, vilken är typisk för motorneu-ronsjukdomen amyotrofisk lateral skleros.

Denna avhandling erbjuder första beviset för att transplantation av humanaryggmärgsceller kan stödja sensoriska nervers återväxt in i den skadade ryg-gmärgen. Avhandlingen lyfter också fram bNCSC som intressanta kandidaterför stamcellsbaserad behandling av amyotrofisk lateral skleros.

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8. Acknowledgment

I am grateful to my supervisor Elena Kozlova for inspiring discussions on- andoff-topic and to allow me to pursue this work in her laboratory. I thank HåkanAldskogious for providing the small nudges that are sometimes necessary togo "beyond" and his profound knowledge. Thanks to my co-supervisor ÅsaMackenzie to always have an ear for a PhD-student’s troubles. Special thanksto Robert Fredriksson, you might not have been an official co-supervisor butI surely see you as one. I am indebted to Niclas König and Carl Trolle, notonly for their surgical expertise that made Paper I, III and IV possible but alsofor the great scientific and non-scientific discourses we had over the years andthe fantastic teamwork. I thank Svitlana Vasylovska for the great companyand all the shared experience. Ninnie Abrahamsson, I thank you for yourinvaluable technical assistance, resourceful introduction to stem cell cultureand to always keep me updated on the intricate social network of our work-ing environment. Thank you Tanya Aggarwal for our constructive discussionsand your caring that goes beyond the time spent in the lab. Special thanks goto Emilia Lekholm for a great collaboration and for enduring my mundanecomplains about PhD life. Thank you Patrik Ivert for lending us your surgicalskills and to Ronald Deumens for the great collaboration, your good spirit andshared 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 Iam 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 ongoingeducation; Ali Inan El-Naggar, it was a pleasure teaching you stem cell cul-ture and discussing scientific theory. Last but not least, I thank my parentsElke and Heiner for giving me all the tools to get this far and my brotherCedric for keeping me on my "mental" toes. Finally, I thank the two mainpillars of my life, my wife Steffi and my son Joshua. To you: "As I leave thesecond most important job I could ever hold, I cherish even more the first - asa husband and father." (Gordon Brown, 1951)

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1365

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A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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