Name: primary cell cultures to study the regeneration potential of murine m&#252;ller glia after microrna treatment
Uploaded: 2022-03-28T13:00:00
Description: Watch this Scientific Journal Video about Primary Cell Cultures to Study the Regeneration Potential of Murine MÃ¼ller Glia after MicroRNA Treatment at JoVE.com

The authors thank Dr. Ann Beaton and all lab members for their input on the manuscript. Special thanks go to Drs. Tom Reh, Julia Pollak, and Russ Taylor for introducing MG primary cultures as a screening tool to S.G.W. during postdoctoral training at the University of Washington in Seattle. The study was funded by the Empire Innovation Program (EIP) Grant to S.G.W. and start-up funds from SUNY Optometry to S.G.W., as well as the R01EY032532 award from the National Eye Institute (NEI) to S.G.W.

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...

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 MG20,23,24. This method is a very robust and reliable technique and results can be easily reproduced if the protocol is followed correctly21,27</su...

The M&#252;ller 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 development1,2. This resemblance is assumed to be the reason for the naturally occurring MG-based neuronal regeneration in the fish retina after retinal damage3,4. During this process, MG re-enter the cell cycle and de-differentiate into RPCs that then differentiate into all six types of retinal neurons. Although this phenomenon occurs naturally in fish, mammalian MG do not convert into neurons5,6. They can, however, be reprogrammed. A variety of factors have been shown to reprogram MG into RPCs/neurons; among these factors is the basic helix-loop-helix (bHLH) transcription factor achaete-scute homolog 1 (Ascl1) that is involved in fish regeneration7,8. In mice, Ascl1 is only expressed in RPCs during retinogenesis but is absent in mature MG or retinal neurons9.
Reprogramming cells directly in vivo is not only methodologically challenging but also requires approval from an institutional animal care and use committee. To receive approval, preliminary data about the factor(s) used or altered, concentrations, off-target effects, underlying mechanisms, toxicity, and efficiency are required. Cell culture systems allow testing for these criteria before usage in in vivo models. Moreover, since MG only comprise about 5% of the entire retinal cell population10, MG cultures allow the study of their function11 as well their behavior, including migration12,13, proliferation14, stress reaction to injury/damage15,16, their interaction with other cell types such as microglia17 or retinal ganglion cells (RGCs)18, or their neurogenic potential19,20,21. Many researchers use immortalized cell lines for their studies since they have an unlimited proliferative potential and can be easily maintained and transfected. Primary cells, however, are preferable for biologically relevant assays than immortalized cells since they have true cell characteristics (gene and protein expression) and, more importantly, they represent a certain stage in development and therefore have an &#34;age&#34;. The age of an animal (and consequently of the cells obtained from an animal) is an especially crucial factor in cellular reprogramming since cell plasticity reduces with progressed stage of development22.
This protocol describes in detail how to reprogram primary MG with miRNAs as a current in vitro method for studying regeneration. This MG primary culture model was established in 2012 to evaluate cell proliferation characteristics of MG in P53 knock-out mice (trp53-/- mice)23. It was shown that cultured MG maintain their glial features (i.e., expression of S100&#946;, Pax6, and Sox2 proteins evaluated via immunofluorescent labeling), and that they resemble in vivo MG (microarray of FACS-purified MG)23. Shortly thereafter, glial mRNA and protein expression were validated and confirmed in a different approach using viral vectors20. A few years later, it was confirmed that the vast majority of cells found in these cultures are MG by using the MG-specific Rlbp1CreERT:tdTomatoSTOPfl/fl&#160;reporter mouse24. Moreover, quantification of the set of miRNAs in both FACS-purified MG and cultured primary MG showed that the levels of MG miRNAs (mGLiomiRs) do not vary much in cultured MG during the growth phase. Elongated culture periods, however, cause changes in miRNA levels and consequently in mRNA levels and protein expression since miRNAs are translational regulators25.
In 2013, this MG culture model was used to test a variety of transcription factors with respect to their capability to reprogram MG into retinal neurons20. Ascl1 was found to be a very robust and reliable reprogramming factor. Overexpression of Ascl1 via viral vectors induced morphological changes, expression of neuronal markers, and the acquisition of neuronal electrophysiological properties. More importantly, the insights and results obtained from these first in vitro experiments were successfully transferred to in vivo applications22,26 demonstrating that primary MG cultures represent a solid and reliable tool for initial factor screenings and evaluation of glial features prior to in vivo implementation.
A few years ago, it was shown that the brain-enriched miRNA miR-124, which is also highly expressed in retinal neurons, can induce Ascl1 expression in cultured MG21. Ascl1 expression in living cells was visualized via an Ascl1 reporter mouse (Ascl1CreERT:tdTomatoSTOPfl/fl). A reporter mouse is a genetically engineered mouse that has a reporter gene inserted in its DNA. This reporter gene encodes for a reporter protein, which is in this study tdTomato, a red fluorescent protein. This reporter protein reports the expression of a gene of interest, in this case, Ascl1. In other words, cells that express Ascl1 will turn red. Since&#160;Ascl1&#160;is&#160;only&#160;expressed&#160;in&#160;RPCs9,&#160;this Ascl1CreERT:tdTomatoSTOPfl/fl mouse allows tracking of MG conversion into Ascl1 expressing RPCs, meaning converting cells will express red fluorescent tdTomato reporter protein. This is irreversible labeling since the DNA of these cells is altered. Consequently, any subsequent neuronal differentiation will be visualized because the tdTomato label remains in differentiating cells. If Ascl1 expressing MG-derived RPCs (with tdTomato label) differentiate into neurons, these neurons will still have their red label. This mouse, therefore, allows not only the labeling of MG-derived RPCs for live-cell imaging but also allows fate mapping and lineage tracing of these MG-derived (red) RPCs. More recently, the set of miRNAs in&#160;RPCs&#160;was&#160;identified&#160;and&#160;MG&#160;cultures&#160;of Ascl1CreERT:tdTomatoSTOPfl/fl RPC-reporter mice were used to screen and test the effect of these miRNAs on reprogramming capacity and efficiency27. One candidate, the RPC-miRNA miR-25, was found capable of reprogramming cultured MG into Ascl1 expressing (Ascl1-Tomato+) cells. These reprogrammed cells adopt neuronal features over time, including neuronal morphology (small somata and either short or long fine processes), expression of neuronal transcripts measured via scRNA-Seq, as well as expression of neuronal proteins validated via immunofluorescent labeling27.
Here, the protocol details how to grow and transfect MG from P12 mice adapted from the previous work21,24,27. Chosen for this protocol is the aforementioned miRNA miR-25, a miRNA highly expressed in RPCs, with low expression levels in MG or retinal neurons. In order to overexpress miR-25, murine miR-25 mimics, i.e., artificial miRNA molecules are used. As a control, mimics of a miRNA from Caenorhabditis elegans are chosen, that have no function in mammalian cells. Visualization of the conversion of MG into RPCs was accomplished via the RPC reporter mouse (Ascl1CreERT:tdTomatoSTOPfl/fl), a mouse with mixed background (C57BL/6, S129, and ICR strains). This culture can, however, be performed with all mouse strains, including wild-type strains. In the past few years, the original protocol has been modified to reduce growth phase duration and the overall culture period and ensure a more robust glia cell status and minimize the degree of cellular degeneration, which occurs in prolonged culture periods. The regular transfection time window was also extended from 3 h to 2 days. As mentioned before, although the current protocol describes MG cultures as a tool for regeneration studies, the method is not only useful for testing reprogramming factors, but can also be adapted for other applications, including studies about MG migratory or proliferative behavior, injury/cell damage related paradigms, and/or the identification of underlying mechanisms and pathways.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascl1-CreERT mouse Ascl1tm1.1(Cre/ERT2)Jejo/J</td>
			<td>Jax laboratories</td>
			<td>#012882</td>
			<td>Ascl1-CreERT mice were crossed with tdTomato mice</td>
		</tr>
		<tr>
			<td>tdTomato-STOPfl/fl mouse&#160; B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J</td>
			<td>Jax laboratories</td>
			<td>#007914</td>
			<td>Genotyping is requried to identify Ascl1CreER positive mice</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(Z)-4-Hydroxytamoxifen, &#8805;98% Z isomer</td>
			<td>Sigma-Aldrich</td>
			<td>H7904-5MG</td>
			<td>reconstituted in ethanol, frozen aliquots</td>
		</tr>
		<tr>
			<td>16 % Paraformaldehyde (PFA) aqueous solution</td>
			<td>VWR</td>
			<td>100504-782</td>
			<td>2% PFA made with Phosphate-buffered saline (PBS), frozen aliquots</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 - AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>711-546-152</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 - AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Goat IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>705-606-147</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-human Otx2 Antibody, R&#38;D Systems</td>
			<td>Fisher Scientific</td>
			<td>AF1979</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Anti-rabbit MAP2 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>M9942-200UL</td>
			<td>dilution 1:250</td>
		</tr>
		<tr>
			<td>Anti-Red Fluorescent Protein (RFP) antibody</td>
			<td>Antibodies-Online</td>
			<td>ABIN334653</td>
			<td>dilution 1:500</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>STEMCELL Technologies</td>
			<td>72132</td>
			<td>reconstituted in PBS, frozen aliquots</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17-504-044</td>
			<td>frozen aliquots</td>
		</tr>
		<tr>
			<td>Brain Phys Neuronal Medium</td>
			<td>STEMCELL Technologies</td>
			<td>05790</td>
			<td>used as neuronal medium in section 1.2, store at 4 &#176;C (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831. 
			1643231638-1407032920.163831 
			5521&#38;_gac=1.124727416.1643 
			231640.Cj0KCQiA_8OPBhDtAR 
			IsAKQu0gbfxhGZMTOU9mHFY 
			dHNsuLirnQzunvMEuS9wA08uY 
			-26yfSbGvNhHEaArodEALw_wcB)</td>
		</tr>
		<tr>
			<td>Click-iT EdU Alexa Fluor 647 Imaging Kit</td>
			<td>Fisher Scientific</td>
			<td>C10340</td>
			<td>reconstitute following manual, 4&#176;C</td>
		</tr>
		<tr>
			<td>Dibutyryl-cAMP</td>
			<td>STEMCELL Technologies</td>
			<td>73886</td>
			<td>reconstituted in Dimethyl sulfoxide (DMSO), frozen aliquots</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>MT-25950CQC</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>MT35010CV</td>
			<td>frozen aliquots</td>
		</tr>
		<tr>
			<td>Gibco&#160;Opti-MEM Reduced Serum Medium, GlutaMAX Supplement</td>
			<td>Fisher Scientific</td>
			<td>51-985-034</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Gibco&#160;TrypLE Express Enzyme (1X), phenol red</td>
			<td>Fisher Scientific</td>
			<td>12-605-028</td>
			<td>used as solution containing trypsin, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Fisher Scientific</td>
			<td>14-025-134</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Laminin mouse protein, natural</td>
			<td>Fisher Scientific</td>
			<td>23-017-015</td>
			<td>
			frozen aliquots, (https://cdn.stemcell.com/media/files/pis/10000000225-PIS_02.pdf?_ga=2.153046205.562651831. 
			1643231638-1407032920.163831 
			5521&#38;_gac=1.124727416.164323 
			1640.Cj0KCQiA_8OPBhDtARIsA 
			KQu0gbfxhGZMTOU9mHFYdHN 
			suLirnQzunvMEuS9wA08uY- 
			26yfSbGvNhHEaArodEALw_wcB)
			</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Fisher Scientific</td>
			<td>25-030-081</td>
			<td>frozen aliquots</td>
		</tr>
		<tr>
			<td>miRIDIAN microRNA Mimic Negative Control</td>
			<td>Horizon</td>
			<td>CN-001000-01-50</td>
			<td>reconstituted in RNase free water (200 &#181;M), frozen aliquots</td>
		</tr>
		<tr>
			<td>miRIDIAN microRNA Mouse mmu-miR-25-3p mimic</td>
			<td>Horizon</td>
			<td>C-310564-05-0050</td>
			<td>reconstituted in RNase free water (200 &#181;M), frozen aliquots</td>
		</tr>
		<tr>
			<td>N-2 Supplement</td>
			<td>Fisher Scientific</td>
			<td>17-502-048</td>
			<td>frozen aliquots</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Fisher Scientific</td>
			<td>21-103-049</td>
			<td>used for growth medium in section 1.1, store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Papain Dissociation System</td>
			<td>Worthington Biochemical</td>
			<td>LK003153</td>
			<td>reconstituted in Earle&#39;s Balanced Salt Solution, frozen aliquots</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td>frozen aliquots</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>20-012-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P4538-50MG</td>
			<td>reconstituted in steriled water, frozen aliquots</td>
		</tr>
		<tr>
			<td>Recombinant Human BDNF Protein</td>
			<td>R&#38;D Systems</td>
			<td>248-BDB-050/CF</td>
			<td>reconstituted in steriled PBS and FBS, frozen aliquots</td>
		</tr>
		<tr>
			<td>Recombinant Mouse EGF Protein</td>
			<td>Fisher Scientific</td>
			<td>2028EG200</td>
			<td>reconstituted in steriled PBS, frozen aliquots</td>
		</tr>
		<tr>
			<td>Recombinant Rat GDNF Protein</td>
			<td>Fisher Scientific</td>
			<td>512GF010</td>
			<td>reconstituted in steriled PBS, frozen aliquots</td>
		</tr>
		<tr>
			<td>Rhodamine Red 570 - AffiniPure F(ab&#39;)2 Fragment Donkey Anti-Rat IgG (H+L)</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>712-296-150</td>
			<td>dilution 1:1,000</td>
		</tr>
		<tr>
			<td>Slide Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>OB100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfection Reagent (Lipofectamine 3000)</td>
			<td>Fisher Scientific</td>
			<td>L3000015</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>plasticware/supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>50-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>50-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L TIP&#160; sterile filter&#160; Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-439</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;L TIP&#160; sterile filter Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-431</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;L TIP sterile filter Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-404</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>50-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L TIP&#160; sterile filter Pipette Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable-Volume Pipettes (2.5, 10, 20, 100, 200, &#38; 1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>2231300008</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Transfer Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-669-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Flat-Bottom Plates with Lids, No. of Wells=12</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Flat-Bottom Plates with Lids, No. of Wells=24</td>
			<td>Fisher Scientific</td>
			<td>08-772-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPET&#160; sterile filter 10ML Disposable Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10J</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPET&#160; sterile filter 50ML Disposable Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10Q</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPET&#160; sterile filter 5ML Disposable Serological Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-676-10H</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder-Free Nitrile Exam Gloves</td>
			<td>Fisher Scientific</td>
			<td>19-130-1597B</td>
			<td></td>
		</tr>
		<tr>
			<td>Round coverslips (12 mm diameter, 0.17 - 0.25 mm thickness)</td>
			<td>Fisher Scientific</td>
			<td>22293232</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filter, Pore Size=0.22 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>09-761-106</td>
			<td></td>
		</tr>
		<tr>
			<td>equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1300 B2 Biosafety cabinet</td>
			<td>Thermo Scientific</td>
			<td>1310</td>
			<td></td>
		</tr>
		<tr>
			<td>All-in-one Fluorescence Microscope Keyence BZ-X 810</td>
			<td>Keyence</td>
			<td>9011800000</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular Zoom Stereo Microscope</td>
			<td>Vision Scientific</td>
			<td>VS-1EZ-IFR07</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes (100 mm diameter)</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps - Biologie/Titanium</td>
			<td>Fine Science Tools</td>
			<td>11252-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps - Biologie/Inox</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 curved Forceps - Biologie/Titanium</td>
			<td>Fine Science Tools</td>
			<td>11272-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>2231000508</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors-sharp</td>
			<td>Fine Science Tools</td>
			<td>14058-11</td>
			<td></td>
		</tr>
		<tr>
			<td>McPherson-Vannas Scissors, 8 cm</td>
			<td>World Precision Instruments</td>
			<td>14124</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal bead bath</td>
			<td>Lab Armor</td>
			<td>74309-714</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutating Mixer, Electrical=115V, 60Hz, Speed=24 rpm</td>
			<td>VWR</td>
			<td>82007-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone coated dissection Petri Dish (90 mm diameter)</td>
			<td>Living Systems Instrumentation</td>
			<td>DD-ECON-90-BLK-5PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers, economy #5</td>
			<td>World Precision Instruments</td>
			<td>501979</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Jacketed CO2 Incubator</td>
			<td>VWR</td>
			<td>10810-744</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary cell cultures to study the regeneration potential of murine m&#252;ller glia after microrna treatment

M&#252;ller 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.

This protocol describes how to grow MG from P12 mouse retinas and how to reprogram these cells with miR-25 into retinal neurons using the Ascl1CreERT:tdTomatoSTOPfl/fl RPC reporter mouse. This method was used in previous work reporting in detail other suitable miRNAs (mimics or inhibitors, as single molecules or in combination) to reprogram MG into RPC that then adopt neuronal cell characteristics27. This method has been modified to grow cultures faster and thus mini...

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Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller 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.

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

This protocol allows to study the potential and the capability of Muller glia to convert into retinal progenitor cells after treatment with specific factors such as microRNAs. The advantage of this technique is that microRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications. Helping to demonstrate the procedure will be Seoyoung Kang, a PhD student from the laboratory of Stefanie Wohl.First, dip the removed eyeball briefly into a tube with ethanol to avoid carryover of bacteria from the animal. Then, wash the eyeball briefly in the 10 centimeter Petri dish before placing it into the 24 well plate on ice. Once the eyeballs are removed and cleaned, place one eye in the dissection dish placed under a dissecting microscope with a light source.Now fix one eyeball by grabbing the optic nerve and the surrounding connective tissue around the sclera with Dumont 5 fine forceps and press it carefully against the dissection dish. Next, make a hole in the cornea center using a 30 gauge needle to allow easier access for the venous scissors. Then, dissect the cornea around the ciliary body using venous scissors and carefully remove the cornea, lens, iris and vitreous body with Dumont 5 fine forceps.Later dissect the sclera with venous scissors until the optic nerve is reached and carefully extract the retina using Dumont 5 fine forceps. Now use a second Dumont 5 fine forceps to push against the retina and completely remove the vitreous body. Transfer the retinas cut about 2.5 centimeters of the tip of a sterile transfer pipette to enlarge the diameter.Now using this tip, pick up whole retinas without damaging the tissue and transfer the retinas into a new sterile Petri dish with cold HBSS and rock the dish. Next, using a new sterile transfer pipette, carefully push the retinas around to wash off retinal pigment epithelial cells. Immediately place the isolated retina in a new clean well of the 24 well plate filled with one milliliter of HBSS while keeping the 24 well plate on the ice during the dissection.Prepare Papain DNase I dissociation mixture as described in the manuscript. Now with an enlarged tip transfer pipette, pick up the retinas. Wait until the retinas settle at the bottom of the tip and release the retinas without excessive HBSS into the tube containing Papain DNase I mixture.Next, place the tube on a nutator in the incubator and incubate for 10 minutes at 37 degrees Celsius and with 5%carbon dioxide. Next, dissociate the cells by carefully pipetting up and down with a one milliliter pipette. After cells are dissociated, add 275 microliters of ovomucoid protease inhibitor from the Papain dissociation kit to neutralize the Papain and mix gently by pipetting up and down.Place tubes into a centrifuge and spin at four degrees Celsius for eight minutes at a relative centrifugal force of 300. Add epidermal growth factor to the calculated volume of growth medium pre warmed at 37 degrees Celsius. Remove the tubes carefully from the centrifuge.Without touching the pellet at the bottom of the tube, remove the supernatant carefully and entirely. Now resuspend the cell pellet with 500 microliters of epidermal growth factor supplemented growth medium. Then, transfer the cell suspension into one well of the labeled 12 well plate.Rinse the tube with another 500 microliters of the epidermal growth factor supplemented growth medium and add it to the well. Rock the well plate carefully then place the plate into the incubator at 37 degrees Celsius with carbon dioxide. Begin by checking for 90%to 100%cell confluency.Next, remove the medium and add one milliliter of cold HBSS to wash the well. Gently rock the plate and remove HBSS without any left traces. Later, add 500 microliters of a pre warmed trypsin containing solution to detach the cells from the well.Rock gently and incubate for two minutes in 37 degrees Celsius incubator. After moving the plate from the incubator to the bio safety cabinet, aspirate the trypsin containing solution while tilting. Disperse it carefully and slowly over the well several times until the cells detach completely.Next, transfer this cell suspension to a sterile 1.5 milliliter tube and put tubes into the centrifuge. Spin at 300 times g for eight minutes at four degrees Celsius and bring tubes back into the bio safety cabinet. Remove the supernatant without touching the pellet.Now carefully resuspend the cell pellet by adding 600 microliters of pre warmed growth medium and pipetting up and down approximately 30 to 40 times. Finally, seed 100 microliters of the obtained cell suspension in the center of six coated cover slips of the 24 well plate. Place the plate on the incubator and let the cells settle for subsequent transfection using micro RNA mimics.The figure shows control conditions five days after transfection with a few Ascl1-Tomato positive cells present. After a miR-25 treatment however, many more Ascl1-Tomato positive cells are found. The quantification revealed a fourfold increase in the number of Ascl1-Tomato positive cells in the miR-25 treated wells compared to controls.Most importantly, the vast majority of the Ascl1-Tomato positive cells found in the riR-25 treated wells adopted a neuronal morphology. Features of this neuronal morphology included reduced cell soma size, the development of fine processes and the formation of tiny networks. The quantification revealed that about 70%of all Ascl1-Tomato positive cells had neuronal features.Immunofluorescent labeling shows that Ascl1-Tomato positive cells with neuronal morphology express the neural markers OTX2 and MAP2, confirming neuronal identity. The quantification of these cells revealed that after miR-25 overexpression, about 40 Ascl1-Tomato positive neurons per field are present as compared to five neuronal cells per field in controls. The identified neuronal cells constitute about 70%of the total Ascl1-Tomato positive cell population of the miR-25 treated samples.Furthermore, quantification of the absolute number of OTX2 and MAP2 coexpressing neurons showed that after miR-25 treatment, about 60 neurons per field were found as compared to 10 neurons per field in controls. It is imperative to work in a clean and sanitized environment with clean and sterile tools and to work carefully by avoiding any harsh treatment. Downstream applications can be for instance, protein quantification, DNA or RNA analysis, or electrophysiological studies.This technique helped us to explore initial questions about nuclear reprogramming which belongs to the field of regenerative medicine. And there is a long range of factors and conditions that can be tested in order to explore new questions.

Research

JoVE Journal

Developmental Biology

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Methods for the Study of Regeneration in Stentor

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