Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

Rafael E. Flores-Obando; Mona M. Freidin; Charles K. Abrams

doi:10.3791/57543

JoVE Journal > Neuroscience

Neuroscience

Isolamento de Fima rápida e específica de oligodendrócitos primário de Mouse

Published: May 21, 2018

doi:

10.3791/57543

Rafael E. Flores-Obando¹, Mona M. Freidin², Charles K. Abrams²

¹Program in Molecular and Cellular Biology,State University of New York Downstate Medical Center, ²Department of Neurology and Rehabilitation,University of Illinois at Chicago

Summary

Descrevemos o isolamento de Fima de oligodendrócitos mouse principal, que permite o isolamento das células para cultura em vitro rápido e específico.

Abstract

O isolamento eficiente e robusto e a cultura de oligodendrócitos primários (OLs) é uma ferramenta valiosa para o estudo em vitro de desenvolvimento de oligodendróglias, bem como a biologia do desmielinizantes doenças como a esclerose múltipla e Doença de Pelizaeus-Merzbacher-como (PMLD). Aqui, apresentamos um simples e método de seleção eficiente para o isolamento de Fima da fase três O4⁺ preoligodendrocytes as células de filhotes de ratos neonatal. Desde que o imaturo OL constituem mais de 80% da matéria branca de cérebro de roedor no 7º dia pós-natal (P7) este método de isolamento não só garante alto rendimento celular, mas também o isolamento específico de OLs já se comprometeu a linhagem oligodendroglial, diminuindo o possibilidade de isolar células contaminantes como astrócitos e outras células do cérebro do rato. Este método é uma modificação das técnicas relatado anteriormente e fornece a pureza de preparação oligodendrocyte acima de 80% em cerca de 4 h.

Introduction

Oligodendrócitos (OLs) são células do sistema nervoso central (SNC)¹microambiental. O isolamento e a cultura de oligodendrócitos primários em um ambiente fortemente regulamentado é uma ferramenta valiosa para o estudo em vitro de desenvolvimento de oligodendróglias, bem como a biologia do desmielinizantes doenças como a esclerose múltipla². Isso requer um eficiente e robusto oligodendrocyte isolamento e cultura método³. Neste estudo, aproveitamos a expressão de um marcador de superfície de célula oligodendrocyte distintivo para implementar uma técnica de isolamento modificado que é rápida e específica.

Identificaram-se quatro fases distintas de maturação oligodendrocyte, cada um caracterizado pela expressão de marcadores de superfície celular distinto para cada estágio do desenvolvimento (Figura 1). Estes marcadores de superfície celular podem ser reconhecidos por anticorpos específicos⁴^,⁵e podem ser usados para isolar OLs em estágios específicos. Na primeira fase, as células precursoras de oligodendrocyte (OPCs) têm a capacidade de proliferar, migrar e especificamente expresso de fator de crescimento derivado de plaquetas do receptor (PDGF-Rα)⁶, gangliosídeos A2B5 proteoglycan NG2⁷^,⁸, polysialic ácido-neural celular adesão molécula⁹ e graxos-ácido-proteína obrigatória 7 (FABP7)¹⁰. OPCs têm morfologia bipolar com alguns processos curtos emanando os polos opostos do corpo celular, que é característica de células precursoras neurais¹¹.

Figura 1: expressão de marcadores de superfície celular durante o desenvolvimento do oligodendrocyte rato. OLs célula marcadores de superfície tais como A2B5 GalC (O1), NG2, O4 e PDGF-Rα podem ser usado para isolar especificamente oligodendrócitos no estágio de desenvolvimento específico usando anticorpos específicos. Por favor clique aqui para ver uma versão maior desta figura.

Na segunda etapa, OPCs dão origem a preoligodendrocytes em expresso na membrana da célula não apenas marcadores OPC, mas também o sulfatide (um galactolipid sulfatado) reconhecido pelo anticorpo O4¹²^,¹³e a proteína GPR17¹⁴, que persiste até o estágio imaturo oligodendrocyte (OL). Nesta fase, preoligodendrocytes estender processos curtos multipolares. Preoligodendrocytes são o grande palco OL em dia pós-Natal 2 (P2) na substância branca cerebral, do rato e do rato com uma pequena população de imaturos OLs¹⁵.

Durante a terceira fase, OLs imaturos continuam a expressar O4, perder a expressão de marcadores A2B5 e NG2 e expressar galactocerebroside C¹⁶. Nesta fase, OLs estão empenhados a linhagem oligodendroglial e tornar-se células pós mitóticas com ramos muito ramificado¹⁷^,¹⁸. OL imaturo constituem mais de 80% da matéria branca roedor no P7… e neste momento as primeiras células MBP⁺ são observadas¹⁵^,¹⁹^,²⁰^,²¹. Portanto, o isolamento de OLs no P7 poderia garantir celulares de alto rendimento.

Na quarta e última fase de desenvolvimento de OL, maduros OLs expressam microambiental proteínas (proteína básica da mielina (MBP), proteína proteolipid (PLP), glicoproteína mielina associada (MAG) e mielina oligodendrocyte glicoproteína (MOG)²²^,²³ ^,²⁴^,²⁵^,²⁶. Nesta fase, maduros OLs estendem as membranas que compacta de forma enwrapping bainhas em torno dos axônios e são capazes de myelinate. Isso coincide com a observação de que, no cérebro do rato e do rato, MBP⁺ células tornam-se cada vez mais abundantes em P14¹⁹^,²⁰^,²¹.

Desde o primeiro isolamento do oligodendrocyte por Fewster e colegas em 1967²⁷, foram implementados diversos métodos para isolamento de OLs o roedor CNS incluindo immunopanning²⁸^,²⁹^,³⁰, fluorescência-ativado da pilha de classificação (FACS) explorando a célula antígenos de superfície específicos²⁸^,³¹, centrifugação gradiente diferencial³²^,³³^,³⁴^,³⁵e agitação método baseado na aderência diferencial de diferentes CNS glia³⁶^,³⁷. No entanto, os métodos de cultura existentes têm limitações, particularmente em termos de pureza, rendimento e tempo necessário para executar os procedimentos de³⁸. Portanto, mais eficientes métodos de isolamento de oligodendrócitos são necessários.

Neste trabalho, apresentamos um simples e método de seleção eficiente para o isolamento de Fima da fase três O4⁺ preoligodendrocytes as células de filhotes de ratos neonatal. Este método é uma modificação das técnicas relatado por Emery et al ³⁹ e Dincman et al . ⁴⁰ e fornece uma pureza de preparação do oligodendrocyte acima de 80% em cerca de 4 h.

Protocol

Os ratos utilizados neste estudo foram tratados de acordo com as diretrizes do número de protocolo de SUNY Downstate Medical Center divisão de laboratório Animal recursos (DLAR) 15-10492. Nota: Oligodendrócitos primários foram isolados de neonatal (P5-P7 selvagem-tipo C57Bl/6N) ratos. Nesta fase, o imaturos OLs constituem mais de 80% da matéria branca roedor, garantindo alto rendimento celular. Todo buffer e composições de reagente estão disponíveis no final da Tabela de mate…

Representative Results

O objetivo deste estudo foi estabelecer um método de isolamento melhorada para O4+ oligodendrócitos mouse principal, exigindo a mínimo possível manipulação das células alvo. Todo o procedimento de eutanásia dos filhotes para chapeamento das células em lamelas leva cerca de 4 h e dados mostrados aqui representam três experimentos independentes. Após a dissociação do tecido, uma média de 4,3 ± 0,46 x 107 células foram isolados para cada experimento inde…

Discussion

Nesta comunicação, apresentamos um método para a isolação eficiente das culturas do oligodendrocyte altamente purificada rato imaturo. Comparado com protocolos previamente publicados³⁹^,⁴⁰, esse método resultou em uma maior pureza com um nível muito inferior de astrócitos GFAP positivo e uma percentagem muito baixa de outras células não-caracterizada. É importante salientar que estas são imaturos OLs já se comprometeu a linhagem oligodendroglial. Assi…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Este estudo foi suportado por doações da sociedade nacional de esclerose múltipla (RG4591A1/2) e o National Institutes of Health (R03NS06740402). Os autores agradecer Dr. Ivan Hernandez e os seus membros de laboratório para fornecer conselhos, equipamentos e espaço de laboratório.

Materials

10ml serological pipets	Fisher Scientific	13-676-10J
10ml syringe Luer-Loc tip	BD, Becton Dickinson	309604
15ml conical tubes	Falcon	352097
24-well tissue culture plates	Falcon	353935
40µm cell strainer	Fisher Scientific	22368547
50ml conical tubes	Falcon	352098
5ml serological pipets	Fisher Scientific	13-676-10H
60mm tissue culture plates	Falcon	353002
70µm cell strainer	Fisher Scientific	22363548
Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody	Invitrogen	A11001
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody	Invitrogen	A21042
Alexa Fluor 488 goat anti-rabbit IgM (H+L) secondary antibody	Invitrogen	A11008
Alexa Fluor 594 goat anti-chicken IgG (H+L) secondary antibody	Invitrogen	A11042
Anti-O4 beads- Anti-O4MicroBeads	Miltenyi Biotec	130-094-543
Apo-Transferrin human	Sigma	T1147
Autofil complete bottle top filter assembly, 0.22um filter, 250ml	USA Scientific	6032-1101
Autofil complete bottle top filter assembly, 0.22um filter, 250ml	USA Scientific	6032-1102
B27 Supplement	Invitrogen	17504-044
Boric acid	Sigma	B7660
Bovine Growth Serum (BGS)	GE Healthcare Life Sciences	SH30541.03
BSA	Fisher Scientific	BP-1600-100
CNTF	Peprotech	450-50
d-Biotin	Sigma	B4639
Desoxyribonuclease I (DNAse I)	Worthington	LS002007
EDTA	Fisher Scientific	S311
Epifluorescence microscope with an Olympus DP70 camera	Olympus	Bx51
Feather disposable scalpels	Andwin Scientific	EF7281C
Forskolin	Sigma	F6886
German glass coverslips, #1 thickness, 12mm diameter round	NeuVitro	GG-12-oz
GFAP antibody	Aves	GFAP
Glucose	Fisher Scientific	D16-1
GlutaMAX	Invitrogen	35050-61
Insulin	Invitrogen	12585-014
Magnetic separator stand – MACS multistand	Miltenyi Biotec	130-042-303
Magnetic separator-MiniMACS separator	Miltenyi Biotec	130-042-302
Millex PES 0.22µm filter unit	Millipore	SLG033RS
Mounting media- Prolong Gold with DAPI	Thermo Fisher	P36930
N-acetyl-cysteine (NAC)	Sigma	A8199
Natural mouse laminin	Invitrogen	23017-015
Neurobasal Medium A	Invitrogen	10888-022
Neurotrophin-3 (NT-3)	Peprotech	450-03
NG2 antibody	Millipore	AB5320
Papain	Worthington	LS003126
PBS without Ca2⁺ and Mg2⁺	Sigma	D5652
PDGF	Peprotech	100-13A
Petri dishes	Falcon	351029
Poly-D-Lysine	Sigma	P6407
Primocin	Invivogen	ant-pm-2
Progesterone	Sigma	P8783
Putrescine	Sigma	P5780
Selection column-LS columns	Miltenyi Biotec	130-042-401
Sodium Selenite	Sigma	S5261
Trace elements B	Corning	25-000-CI
Triiodothyronine (T3)	Sigma	T6397
Triton-X	Sigma	T8787
Trypan Blue Solution	Corning	25-900-CI
Tween 20	Sigma	P1379
B27NBMA			487.75 mL Neurobasal Medium A; 10 mL B27 Supplement; 1 mL Primocin; 1.25 mL Glutamax; Filter sterilize and store at 4 °C until use.
B27NBMA + 10% BGS			27 mL B27NBMA; 3 mL Bovine growth serum
CNTF solution stock (10 µg/ml; 1000X)			Order from Peprotech (450-50). Make up at 0.1 to 1 mg/ml according to Manufacturer’s instruction (may vary from lot to lot) in buffer (e.g. DPBS + 0.2% BSA). Store at -80 °C. Working solution (10 µg/ml, 1000X) 1. Make on 0.2% BSA (Fisher scientific BP-1600-100) in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 10µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80 °C.
d-Biotin stock solution (50 µg/ml; 5000X)			Resuspend d-Biotin (Sigma-B4639) in double-distilled H2O at 50 µg/ml (e.g. 2.5 mg in 50 ml of ddH2O). Resuspension might take fair amount of agitation/vortexing, or mild warming briefly at 37°C. If the d-Biotin still will not solubilize, it is fine to make up a less concentrated (e.g. 10µg/ml), and to add a higher volume to the B27NBMA (1/1000), instead of 1/5000). Store at 4°C.
DNase I stock solution			1. Dissolve at 12,500 U Deoxyribonuclease I / ml in HBSS chilled on ice. 2. Filter sterilize on ice 3. Aliquot at 200 µl and freeze overnight at -20°C. 4. Store aliquots at -20 to -30°C.
Dulbecco’s Phosphate Buffered Saline (w/o Ca2+ and Mg2+)			Dissolve pouch in 1 Liter of water to yield 1 liter of medium at 9.6 grams of powder per liter of medium. Store at 2-8 °C.
Forskolin stock solution (4.2 mg/ml; 1000X)			Add 1 ml of sterile DMSO to 50 mg Forskolin in bottle (Sigma-F6886) and pipette until resuspended. Transfer to a 15 ml centrifuge tube and add 11 ml of sterile DMSO to bring to 4.2 mg/ml. Aliquot (e.g. 20 µl) and store at -20°C.
Hank’s balanced salts (HBSS) (Sigma			1. Measure 900 ml of water (temperature 15-20 °C) in a cylinder and stir gently. 2. Add the power and stir until dissolved. 3. Rinse original package with a small amount of water to remove all traces of the powder. 4. Add to the solution in step 2. 5. Add 0.35 gr of sodium bicarbonate (7.5% w/v) for each liter of final volume. 6. Keep stirring until dissolved. 7. Adjust the pH of the buffer while stirring to 0.1-0.3 units below pH= 7.4 since it may rise during filtration. The use of 1N HCl or 1N NaOH is recommended to adjust the pH. 8. Add additional water to bring the final volume to 1L. 9. Sterilize by filtration using a membrane with a porosity of 0.22 microns. 10. Store at 2-8 °C.
Insulin stock solution (4000 µg/ml)			Thaw the bottle and aliquot 25 µl per microcentrifuge tube and store at -20°C.
Laminin solution			Slowly thaw laminin in the cold (2°C to 8°C) to avoid gel formation. Then, aliquot into polypropylene tubes. Store at 5° C to -20° C in aliquots (e.g. 20 µl) and do not freeze/thaw repeatedly. Laminin may be stored at these temperatures for up to six months.
Magnetic Cell Sorting (MCS) Buffer			Prepare the solution containing phosphate-buffered saline (PBS), pH 7.2, and 0.5% bovine serum albumin (BSA), 0.5 mM EDTA, 5µg/ml Insulin, 1 g/L Glucose. Sterilize and degas by filtration the buffer by passing it through a 0.22 µm Millex filter. Store the buffer at 4°C until use
N-Acetyl-L-cysteine (NAC) stock solution (5mg/ml; 1000X)			Dissolve N-Acetyl-L-cysteine (Sigma-A8199) at 5 mg/ml in DMEM (e.g. 50 mg NAC in 10 ml B27NBMA). Filter sterilize and aliquot (e.g. 20 µl). Store at -20°C.
NT3 stock solution (1 µg/ml; 1000X)			Master stock: Order from Peprotech (450-03). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot), in buffer (e.g. DPBS + 0.2% BSA). Store at -80°C. Working stock (1µg/ml; 1000X): 1. Make on 0.2% BSA in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 1 µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80°C.
PDGF stock solution (10 µg/ml; 1000X)			Master stock: Order from Peprotech (100-13A). Make up at 0.1 to 1 mg/ml according to manufacturer’s instructions (may vary from lot to lot) in buffer (e.g. DPBS) + 0.2% BSA). Store at -80°C. Working stock (1µg/ml; 1000X): 1. Make on 0.2% BSA in DPBS solution and filter sterilize. 2. Dilute master stock aliquot to 1µg/ml in sterile, chilled 0.2% BSA/DPBS. 3. Aliquot (e.g. 20µl/tube) and snap freeze in liquid nitrogen. 4. Store aliquots at -80°C.
Poly-D-lysine (1mg/ml; 100X)			Resuspend poly-D-lysine, molecular weight 70-150 kD (Sigma P6407) at 0.5mg/ml in 0.15M boric acid pH 8.4 (e.g. 50mg in 50ml borate buffer). Filter sterilize and aliquot (e.g. 100µl/tube). Store at -20°C. Prior to use, dilute the 100X stock (1mg/ml) to 50 µg/ml in sterile water.
Oligodendrocyte proliferation media			see Supplementary Table 1
Oligodendrocyte differentiation media			see Supplementary Table 1
Sato supplement (100X)			see Supplementary Table 1
References: the list of reagents and recipes were adopted from the protocols previously described by Emery et. al. 2013 (Emery, B. & Dugas, J. C. Purification of oligodendrocyte lineage cells from mouse cortices by immunopanning. Cold Spring Harb Protoc. 2013 (9), 854-868, doi:10.1101/pdb.prot073973, (2013)) and Dincman et. al. (Dincman, T. A., Beare, J. E., Ohri, S. S. & Whittemore, S. R. Isolation of cortical mouse oligodendrocyte precursor cells. J Neurosci Methods. 209 (1), 219-226, doi:10.1016/j.jneumeth.2012.06.017, (2012))

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Flores-Obando, R. E., Freidin, M. M., Abrams, C. K. Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes. J. Vis. Exp. (135), e57543, doi:10.3791/57543 (2018).

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