Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment

Summary

Müller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

Abstract

Müller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

Introduction

The Müller glia (MG) are the predominant glia in the neural retina. They have similar functions compared to other glia in other parts of the central nervous system such as maintaining the water and ion homeostasis, nourishing neurons, and protecting the tissue. MG have another fascinating feature: although they are mature glia, they still express many genes expressed in retinal progenitor cells (RPCs) during late development^1,2. This resemblance is assumed to be the reason for the naturally occurring MG-based neuronal regeneration in the fish retina after retinal damage^3,

Protocol

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at SUNY College of Optometry.

NOTE: This culture protocol consists of three phases: growth, transfection, and conversion phase. A summary of the overall protocol with the timeline is given in Figure 1.

1. Preparation of media and all required reagents

NOTE: All steps need to be car.......

Discussion

This protocol describes how to grow MG from dissociated mouse retinas for reprogramming studies using miRNAs. As shown and confirmed in a variety of previous studies, the vast majority (80%-90%) of cells found in these cultures are MG^20,23,24. This method is a very robust and reliable technique and results can be easily reproduced if the protocol is followed correctly^21,27

Materials

Name	Company	Catalog Number	Comments
Animals
Ascl1-CreERT mouse Ascl1tm1.1(Cre/ERT2)Jejo/J	Jax laboratories	#012882	Ascl1-CreERT mice were crossed with tdTomato mice
tdTomato-STOPfl/fl mouse  B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J	Jax laboratories	#007914	Genotyping is requried to identify Ascl1CreER positive mice
Reagents
(Z)-4-Hydroxytamoxifen, ≥98% Z isomer	Sigma-Aldrich	H7904-5MG	reconstituted in ethanol, frozen aliquots
16 % Paraformaldehyde (PFA) aqueous solution	VWR	100504-782	2% PFA made with Phosphate-buffered saline (PBS), frozen aliquots
Alexa Fluor 488 - AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch Laboratories	711-546-152	dilution 1:500
Alexa Fluor 647 - AffiniPure F(ab')2 Fragment Donkey Anti-Goat IgG (H+L)	Jackson ImmunoResearch Laboratories	705-606-147	dilution 1:500
Anti-human Otx2 Antibody, R&D Systems	Fisher Scientific	AF1979	dilution 1:500
Anti-rabbit MAP2 antibody	Sigma-Aldrich	M9942-200UL	dilution 1:250
Anti-Red Fluorescent Protein (RFP) antibody	Antibodies-Online	ABIN334653	dilution 1:500
Ascorbic Acid	STEMCELL Technologies	72132	reconstituted in PBS, frozen aliquots
B-27 Supplement	Fisher Scientific	17-504-044	frozen aliquots
Brain Phys Neuronal Medium	STEMCELL Technologies	05790	used as neuronal medium in section 1.2, store at 4 °C (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831. 1643231638-1407032920.163831 5521&_gac=1.124727416.1643 231640.Cj0KCQiA_8OPBhDtAR IsAKQu0gbfxhGZMTOU9mHFY dHNsuLirnQzunvMEuS9wA08uY -26yfSbGvNhHEaArodEALw_wcB)
Click-iT EdU Alexa Fluor 647 Imaging Kit	Fisher Scientific	C10340	reconstitute following manual, 4°C
Dibutyryl-cAMP	STEMCELL Technologies	73886	reconstituted in Dimethyl sulfoxide (DMSO), frozen aliquots
Dimethyl Sulfoxide (DMSO)	Fisher Scientific	MT-25950CQC
Fetal Bovine Serum (FBS)	Fisher Scientific	MT35010CV	frozen aliquots
Gibco Opti-MEM Reduced Serum Medium, GlutaMAX Supplement	Fisher Scientific	51-985-034	store at 4 °C
Gibco TrypLE Express Enzyme (1X), phenol red	Fisher Scientific	12-605-028	used as solution containing trypsin, store at 4 °C
HBSS	Fisher Scientific	14-025-134	store at 4 °C
Laminin mouse protein, natural	Fisher Scientific	23-017-015	frozen aliquots, (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831. 1643231638-1407032920.163831 5521&_gac=1.124727416.164323 1640.Cj0KCQiA_8OPBhDtARIsA KQu0gbfxhGZMTOU9mHFYdHN suLirnQzunvMEuS9wA08uY- 26yfSbGvNhHEaArodEALw_wcB)
L-Glutamine	Fisher Scientific	25-030-081	frozen aliquots
miRIDIAN microRNA Mimic Negative Control	Horizon	CN-001000-01-50	reconstituted in RNase free water (200 µM), frozen aliquots
miRIDIAN microRNA Mouse mmu-miR-25-3p mimic	Horizon	C-310564-05-0050	reconstituted in RNase free water (200 µM), frozen aliquots
N-2 Supplement	Fisher Scientific	17-502-048	frozen aliquots
Neurobasal Medium	Fisher Scientific	21-103-049	used for growth medium in section 1.1, store at 4 °C
Papain Dissociation System	Worthington Biochemical	LK003153	reconstituted in Earle's Balanced Salt Solution, frozen aliquots
Penicillin Streptomycin	Fisher Scientific	15-140-122	frozen aliquots
Phosphate-buffered saline (PBS)	Fisher Scientific	20-012-043
Poly-L-ornithine hydrobromide	Sigma-Aldrich	P4538-50MG	reconstituted in steriled water, frozen aliquots
Recombinant Human BDNF Protein	R&D Systems	248-BDB-050/CF	reconstituted in steriled PBS and FBS, frozen aliquots
Recombinant Mouse EGF Protein	Fisher Scientific	2028EG200	reconstituted in steriled PBS, frozen aliquots
Recombinant Rat GDNF Protein	Fisher Scientific	512GF010	reconstituted in steriled PBS, frozen aliquots
Rhodamine Red 570 - AffiniPure F(ab')2 Fragment Donkey Anti-Rat IgG (H+L)	Jackson ImmunoResearch Laboratories	712-296-150	dilution 1:1,000
Slide Mounting Medium	Fisher Scientific	OB100-01
Transfection Reagent (Lipofectamine 3000)	Fisher Scientific	L3000015	store at 4 °C
plasticware/supplies
0.6 mL microcentrifuge tube	Fisher Scientific	50-408-120
1.5 mL microcentrifuge tube	Fisher Scientific	50-408-129
10 µL TIP  sterile filter  Pipette Tips	Fisher Scientific	02-707-439
100 µL TIP  sterile filter Pipette Tips	Fisher Scientific	02-707-431
1000 µL TIP sterile filter Pipette Tips	Fisher Scientific	02-707-404
2.0 mL microcentrifuge tube	Fisher Scientific	50-408-138
20 µL TIP  sterile filter Pipette Tips	Fisher Scientific	02-707-432
Adjustable-Volume Pipettes (2.5, 10, 20, 100, 200, & 1000 µL)	Eppendorf	2231300008
Disposable Transfer Pipets	Fisher Scientific	13-669-12
Multiwell Flat-Bottom Plates with Lids, No. of Wells=12	Fisher Scientific	08-772-29
Multiwell Flat-Bottom Plates with Lids, No. of Wells=24	Fisher Scientific	08-772-1
PIPET  sterile filter 10ML Disposable Serological Pipets	Fisher Scientific	13-676-10J
PIPET  sterile filter 50ML Disposable Serological Pipets	Fisher Scientific	13-676-10Q
PIPET  sterile filter 5ML Disposable Serological Pipets	Fisher Scientific	13-676-10H
Powder-Free Nitrile Exam Gloves	Fisher Scientific	19-130-1597B
Round coverslips (12 mm diameter, 0.17 - 0.25 mm thickness)	Fisher Scientific	22293232
Vacuum Filter, Pore Size=0.22 µm	Fisher Scientific	09-761-106
equipment
1300 B2 Biosafety cabinet	Thermo Scientific	1310
All-in-one Fluorescence Microscope Keyence BZ-X 810	Keyence	9011800000
Binocular Zoom Stereo Microscope	Vision Scientific	VS-1EZ-IFR07
Disposable Petri Dishes (100 mm diameter)	VWR	25384-088
Dumont #5 Forceps - Biologie/Titanium	Fine Science Tools	11252-40
Dumont #55 Forceps - Biologie/Inox	Fine Science Tools	11255-20
Dumont #7 curved Forceps - Biologie/Titanium	Fine Science Tools	11272-40
Eppendorf Centrifuge 5430 R	Eppendorf	2231000508
Fine Scissors-sharp	Fine Science Tools	14058-11
McPherson-Vannas Scissors, 8 cm	World Precision Instruments	14124
Metal bead bath	Lab Armor	74309-714
Nutating Mixer, Electrical=115V, 60Hz, Speed=24 rpm	VWR	82007-202
Silicone coated dissection Petri Dish (90 mm diameter)	Living Systems Instrumentation	DD-ECON-90-BLK-5PK
Tweezers, economy #5	World Precision Instruments	501979
Water Jacketed CO2 Incubator	VWR	10810-744