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«UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Sandra HOČEVAR ISOLATION AND CHARACTERIZATION OF PURE GLIAL CELL POPULATIONS FROM ADULT MOUSE CENTRAL ...»

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UNIVERSITY OF LJUBLJANA

BIOTECHNICAL FACULTY

Sandra HOČEVAR

ISOLATION AND CHARACTERIZATION OF PURE

GLIAL CELL POPULATIONS FROM ADULT

MOUSE CENTRAL NERVOUS SYSTEM WHITE

MATTER

M.Sc. Thesis

Ljubljana, 2014

UNIVERSITY OF LJUBLJANA

BIOTECHNICAL FACULTY

Sandra HOČEVAR

ISOLATION AND CHARACTERIZATION OF PURE GLIAL CELL

POPULATIONS FROM ADULT MOUSE CENTRAL NERVOUS

SYSTEM WHITE MATTER

M.Sc. Thesis

IZOLACIJA IN KARAKTERIZACIJA ČISTIH GLIJA CELIČNIH

POPULACIJ IZ BELINE CENTRALNEGA ŽIVČNEGA SISTEMA

ODRASLE MIŠI

Magistrsko delo Ljubljana, 2014 II Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014 The M. Sc. thesis is a completion of a Master Study Programme in Molecular Biology at Biotechnical Faculty, University of Ljubljana. The experimental part was carried out in the laboratory of Cellular Neurophysiology at the Faculty of Science in the School of Pharmacy and Biomedical Sciences, University of Portsmouth (United Kingdom).

The Council of the 1. and 2. study cycle appointed Professor Robert Zorec, PhD, as a supervisor, Professor Arthur Butt, PhD, as a co-supervisor and Professor Marko Kreft, PhD, as a reviewer.

Commission for assessment and defence:

President: prof. dr. Boris BULOG University of Ljubljana, Biotechnical Faculty, Department of Biology Member: prof. dr. Robert ZOREC University of Ljubljana, Faculty of Medicine Member: prof. dr. Arthur BUTT University of Portsmouth, Faculty of Science, School of Pharmacy and Biomedical Sciences Member: prof. dr. Marko KREFT University of Ljubljana, Biotechnical Faculty, Department of Biology

Date of defence:

The work is the result of my own research work. I agree with publishing of my work in full text on the internet page Digitalna knjižnica Biotehniške fakultete. I declare that the text in the electronic version is identical to the printed one.

Sandra HOČEVAR III Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

KEY WORDS DOCUMENTATION (KWD)

DN Du2 DC UDK 602.3:616.8(043.2)=111 CX glia/astrocytes/oligodendrocyte/adult mouse CNS/optic nerve/cell cultures/immunohistochemistry/MACS system/neurodegenerative diseases HOČEVAR, Sandra, B. Sc. (Biotech) AU AA ZOREC, Robert (supervisor)/BUTT, Arthur (co-supervisor) SI-1000 Ljubljana, Večna pot 111 PP PB University of Ljubljana, Biotechnical Faculty, Molecular Biology PY 2014

TI ISOLATION AND CHARACTERIZATION OF PURE GLIAL CELL

POPULATIONS FROM ADULT MOUSE CENTRAL NERVOUS SYSTEM

WHITE MATTER

DT M. Sc. Thesis (Master Study Programmes: Molecular Biology) NO X, 59 p., 10 tab., 14 fig., 109 ref.

LA en AL en/sl AB Glial cells are critical participants in the development, function and disease of the central nervous system (CNS). They play roles in brain homeostasis, synapse formation, myelin production, and provide the immune system for CNS. There are several in vitro glia studies which mostly include experiments on embryonic or young postnatal rodent brains. Therefore, it is important to find a way to isolate pure glia from adult mouse CNS, since all the major nerurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Multiple sclerosis (MS), occur in adulthood. With this aim, we tried to develop a reliable technique for isolating purified astrocytes and oligodendrocytes from adult mouse brain and optic nerve. Our experiments were based on two different methods of cell dissociation and isolation, using the Magnetic Activated Cell Sorter (MACS) system. We managed to dissociate, culture and characterise populations of oligodendrocytes, astrocytes and oligodendrocyte precursor cells (OPCs) (or oligodendrocyte-type 2 astrocyte progenitors-O-2A) from adult mouse CNS. Our optimised in-house dissociation method proved as useful and equally successful as the Neural Tissue Dissociation Kit (NTDK) method. However, cell isolation from adult tissue with MACS system turned out to be impracticable and remains difficult to perform.

IV Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

KLJUČNA DOKUMENTACIJSKA INFORMACIJA (KDI)

ŠD Du2 DK UDK 602.3:616.8(043.2)=111 glija/astrociti/oligodendrociti/CŽS odrasle miši/optični živec/celične KG kulture/imunohistokemija/MACS sistem/nevrodegenerativne bolezni HOČEVAR, Sandra, dipl. bioteh. (UN) AV SA ZOREC, Robert (mentor)/BUTT Arthur (somentor) SI-1000 Ljubljana, Večna pot 111 KZ Univerza v Ljubljani, Biotehniška fakulteta, Molekulska biologija ZA LI 2014

IZOLACIJA IN KARAKTERIZACIJA ČISTIH GLIJA CELIČNIH POPULACIJ





IN

IZ BELINE CENTRALNEGA ŽIVČNEGA SISTEMA ODRASLE MIŠI

Magistrsko delo (Magistrski študij- 2. stopnja Molekulska biologija) TD OP X, 59 str., 10 pregl., 14 sl., 109 vir.

IJ en JI en/sl Celice glija so pomembne udeleženke v razvoju, funkciji in boleznih centralnega AI živčnega sistema (CŽS). Igrajo vloge pri homeostazi v možganih, produkciji mielina in zagotavljajo imunski sistem CŽS. Večina študij glije in vitro vključuje poskuse na embrionalnih ali mladih postnatalnih možganih glodalcev. Ker pa se večina poglavitnih nevrodegenerativnih bolezni, kot so Alzheimerjeva bolezen, Parkinsonova bolezen in multipla skleroza, pojavljajo pri odraslem človeku, je pomembno, da najdemo način za izolacijo čiste glije iz CŽS odrasle miši. S tem namenom smo poskušali razviti zanesljivo tehniko izolacije čistih astrocitov in oligodendrocitov iz možganov in optičnega živca odrasle miši. Naši poskusi so temeljili na dveh različnih disociacijskih metodah in izolaciji z magnetno pogojenim ločevanjem celic (ang. MACS). Iz CŽS odrasle miši nam je uspelo disociirati, kultivirati in karakterizirati populacije oligodendrocitov, astrocitov in prekurzorskih celic oligodendrocitov (ang. OPCs) (ali oligodendrocit-astrocit tipa 2- O-2A). Dokazali smo, da je naša optimizirana disociacijska metoda enako uspešna kot disociacijska metoda z uporabo kompleta za disociacijo živčnega tkiva (ang. NTDK). Vendar pa se je izolacija celic s sistemom MACS iz odraslega tkiva izkazala za težavno in se je ne uporablja.

V Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

TABLE OF CONTENTS

KEY WORDS DOCUMENTATION (KWD)

KLJUČNA DOKUMENTACIJSKA INFORMACIJA (KDI)

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABBRIVATIONS AND SYMBOLS

1 INTRUDUCTION

1.1 AIM OF STUDY

1.2 HYPOTHESIS

2 LITERATURE REVIEW

2.1 BACKGROUND OF GLIA

2.2 ASTROCYTES

2.2.1 Astrocyte morphology and functions

2.2.2 Astrocyte markers

2.2.3 Astrocyte pathology

2.2.3.1 Reactive astrogliosis

2.2.3.2 Alzheimer’s disease

2.2.3.3 Parkinson’s disease

2.3 OLIGODENDROCYTES

2.3.1 Oligodendrocyte morphology and myelin functions

2.3.2 Oligodendrocyte progenitor cells (OPCs)

2.3.3 NG2-glia

2.3.4 Oligodendrocyte markers

2.3.5 Oligodendrocyte pathology

2.3.5.1 Multiple sclerosis

2.4 MICROGLIA

2.5 ADULT NEURAL STEM CELLS

2.6 Wnt/GSK-3β PATHWAY

2.7 OPTIC NERVE

2.8 In vitro CULTURING OF GLIAL CELLS FROM ADULT MOUSE CNS................ 18 VI Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014 3 MATERIALS AND METHODS

3.1 MATERIALS

3.1.1 Animals

3.1.2 Agents

3.1.3 Equipment

3.2 METHODS

3.2.1 Dissection of mouse brain and optic nerve

3.2.2 Tissue dissociation of mouse brain and optic nerve

3.2.2.1 In-house dissociation method

3.2.2.2 Neural Tissue Dissociation Kit (P) and (T)

3.2.3 Magnetic Activated Cell Sorting (MACS) of O4+ cells

3.2.3.1 Magnetic labelling

3.2.3.2 Magnetic separation

3.2.4 Cell culturing

3.2.5 Immunohistochemistry

4 RESULTS

4.1 DISSOCIATED GLIAL CELS OF ADULT MOUSE CNS

4.1.1 Dissociated cells of optic nerve

4.1.2 Dissociated cells of forebrain

4.1.3 Dissociated cells of cerebellum

4.2 ISOLATED GLIAL CELLS

4.2.1 Isolated cells of young brain

4.2.2 Isolated cells of adult forebrain

5 DISCUSSION

6 CONCLUSION

7 SUMMARY

7.1 POVZETEK

8 REFERENCES

AKNOWLEDGEMENTS

VII Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

LIST OF TABLES

Tab. 1: Astrocyte markers

Tab. 2: Oligodendrocyte markers

Tab. 3: List of chemicals

Tab. 4: Primary antibodies

Tab. 5: Secondary antibodies

Tab. 6: Culture media recipes

Tab. 7: List of equipment

Tab. 8: Dissociation solution for brain and optic nerve

Tab. 9: Enzyme Mix I for NTDK (P) and (T) tissue dissociation

Tab. 10: Enzyme Mix II for NTDK (P) and (T) tissue dissociation

VIII Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

LIST OF FIGURES

Fig. 1: Neural cell types

Fig. 2: Glia-neuron interactions

Fig. 3: Astrocyte subtypes

Fig. 4: The oligodendrocyte and the mature myelin sheath

Fig. 5: Schematic of progenitor types and lineages in the adult brain SVZ

Fig. 6: Wnt/β-catenin signalling

Fig. 7: Principle of the MACS Separation

Fig. 8: Dissociated glial cells from the optic nerve of adult mouse, using in-house dissociation method

Fig. 9: Dissociated glial cells from the optic nerve of adult mouse, using NTDK dissociation method

Fig. 10: Comparison of in-house and NTDK dissociation method

Fig. 11: Dissociated astrocytes from adult mouse forebrain

Fig. 12: Dissociated glial cells from adult mouse forebrain

Fig. 13: Dissociated glial cells from cerebellum of transgenic GFAP-eGFP adult mouse. 37 Fig. 14: Isolated cells from young mouse brain

IX Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014

–  –  –

1 INTRUDUCTION Our central nervous system (CNS) consists of neurons and glial cells. The defining characteristic of a neuron is its ability to transmit rapid electrical signals in the form of action potentials, whereas glial cells are unable to generate action potentials but instead surround and ensheath neuronal cell bodies, axons and synapses throughout the nervous system (Allen and Barres, 2009). Neurons and glia have a common origin in neuronal precursor cells derived from the embryonic germ layer known as the neuroectoderm.

Astrocytes, oligodendrocytes and ependymal cells are cells of neural origin and often referred to as macroglial or neuroglial cells. The remaining 5-10 % of glia consist of microglia, which are a part of immune system and have non-neuronal (mesodermal) origin.

In the peripheral nervous system (PNS), the main class of glia is represented by Schwann cells which enwrap and myelinate peripheral axons (Verkhratsky and Butt, 2007).

Figure 1: Neural cell types. CNS: Central nervous system, PNS: peripheral nervous system (Verkhratsky and Butt, 2007: 4) Glial cells play multiple roles in adult neurogenesis, ranging from functioning as neural precursor cells to the key determinants of neurogenic permissiveness (Morrens et al., 2012). Astrocytes are fundamental for brain homeostasis, are critical at the brain-blood Hočevar S. Isolation and characterization of pure glial cell populations from adult mouse CNS white matter.

M.Sc. Thesis. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2014 barrier (Heneka et al., 2009) and provide structural and metabolic support to the nervous system (Haydon and Carmignoto, 2006).



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