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.

A patent including some of the findings in this report has been filed for by the University of Washington with inventors Nikolas Jorstad, Stefanie G. Wohl, and Thomas A. Reh. The patent is titled &#8216;&#8216;Methods and compositions to stimulate retinal regeneration.

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. The successful growth and reprogramming efficiency of the culture, however, depend on a variety of factors.
First, the age of the mouse plays an important role in successful cell growth. Therefore, if MG cultures are grown from wild-type mice or transgenic mice that resemble wild types such as reporter mice, the latest age to grow confluent MG monolayers is P12. At P12, all retinal neurons and the MG are differentiated, and no RPCs are present in the retina anymore. MG express mature MG markers such as glutamine synthetase or glutamate aspartate transporter (GLAST)20,23,41,42. Cultured P12 MG can still re-enter the cell cycle when exposed to EGF, but the number of cycles is limited albeit enough to form confluent cell layers in vitro. Nevertheless, it can occur that even P12 MG do not grow well because EGF or components in the medium may be degraded. Incomplete dissociation (chunks) can also result in less or delayed MG growth. A major cause for incomplete dissociation is inactivation of the DNase I used for retinal dissociation by harsh handling of the enzyme (fast pipetting, vortexing, etc.). Even if the cells grow well until passage, cells can be less confluent after passage. Reasons for this can be harsh handling during the passage process that leads to increased cell death or because the cells were seeded too sparsely. Since the glia do not grow much, cells should be plated in the same ratio as grown, not diluted (contrary to passaging procedures of immortalized cell lines). Of note, because MG cultures grow from the center to the periphery of a well, higher cell densities will always be found in the center. It is therefore imperative to check the margins of every well before passage to ensure that the whole well is covered by cells. Cell concentration measurements should be performed to ensure the processing of sufficient amounts of cells.
Additionally, primary cultures obtained from mice at P6 or younger will not contain any MG, since MG are just about to mature at this time point. This was shown via&#160;single-cell RNA-seq analysis43. Immunofluorescent labeling with mature MG markers at this age will also confirm this. The fraction of RPCs is, however, substantial at P6. Results should, therefore, be interpreted carefully. Moreover, markers such as the intermediate filament markers vimentin and nestin are not appropriate to identify primary MG since these markers are also expressed in astrocytes44,45,46, microglia47,48, endothelial cells, and pericytes49,50,51,52, and are not MG-specific. GFAP, expressed in retinal astrocytes but not in MG of undamaged retinas, is not a good marker for cultured MG. Although it is upregulated in dissociated MG due to the mechanical damage during dissociation, it is downregulated after 3 days in culture28.
Since MG share many RPC markers, the safest procedure to ensure a robust MG culture is to use mice at P12 or older mice. Although this protocol describes cultures obtained from P12 mouse MG, adult mouse MG can be cultured and reprogrammed27. However, more tissue per well needs to be plated (four to six retinas). This is because adult glia do not divide much, even if exposed to EGF and 10% serum. Reduced cell contacts will lead to cell death and cell degeneration (stretched-out cells, enlarged cells). Adult glia are a great tool for co-culture paradigms18 and cell density may not matter as much in these applications. For downstream applications such as transfection or transduction, however, confluent cell layers are a requirement.
Besides impaired growth, cell death can occur. If cell death occurs without any obvious signs, mycoplasma contamination should be considered (mycoplasma size 0.1-0.3 &#181;m) and a mycoplasma detection kit should be used. Keeping every sample separate (not pooling samples) can also reduce the risk of cross-contamination. However, larger bacteria can also be carried over from the animal and can cause contamination even though all work is done in a clean, sterile environment. Dipping the eyeball briefly in 70% ethanol and a thorough wash in an additional 10 cm Petri dish are recommended before dissecting the eye. To achieve a successful culture, it is imperative to work under sterile conditions since contamination&#160;can happen quickly. Once bacteria, yeast, or mold/fungus are found in a plate, even if not all wells are affected, the culture is lost. Contaminations will cause cell death (Supplementary Figure 1) and will affect the cells, resulting in non-representative data.
Cell death can also be caused by reagents required for downstream applications such as Poly-O (or Poly-D-Lysine) used to coat coverslips. These substances are toxic and thorough washes are required before Laminin is used. Coverslips should stick to the bottom of the well, not float inside a well. Air bubbles need to be avoided. Moreover, complete coating with Laminin is crucial to ensure cell adhesion to the coverslip. Lack of Laminin will also lead to less cell density and/or cell death.
For identification of true reprogrammed glia, reporter mice and cell proliferation markers that allow cell tracking, such as EdU or BrdU, should be used. Since whole retinas are plated, neuronal survivors are present in all cultures. They are, however, almost completely removed after passage in most cases. Nevertheless, EdU (or BrdU) labeling should be performed to validate that neurons originated from proliferating MG/RPCs and are not neuronal survivors (post-mitotic). The small numbers of neurons found in the controls used here are neuronal survivors.
Interestingly, a few Ascl1-Tom+ cells are also present in the control treatment with slightly increasing numbers over time. These cells could be&#160;some rather immature MG express Ascl1 when isolated and cultured. The fraction of this Ascl1-Tom+ cell population is, however, small. Moreover, most of these Ascl1-Tom+ cells found in the control treatment do not express Otx2 and/or Map2 and keep a rather flat cell shape. No neuronal differentiation seems to occur under control conditions. Very delicate neuronal-like cells that appear to form networks are only found after miR-25 treatment.
The protocol described here was used in previous studies to test miRNAs for MG reprogramming21,27&#160;and&#160;has been modified slightly in the past years with respect to the well-plates to grow the glia. They are now grown in 12-well plates, instead of 6-well plates. Confluent monolayers can be obtained in 12-well plates after 4/5 days (versus 6/8 days with 6-well plates) and, therefore, the overall culture period that leads to cell alteration/degeneration is reduced. The transfection time window is also elongated. Regular transfection protocols have a 3 h transfection duration after which a complete medium change needs to be performed to ensure cell survival. All molecules are removed after this medium change. In the current protocol, the time window was extended and longer exposure to the miRNAs was allowed by adding 50% neuronal medium (supplemented with the factors required for Cre induction and cell proliferation tracking) to the transfection reagent medium. The cells were healthy and no problems were encountered with this modification. It appears that the transfection results are better with the elongated transfection time, but more data is required to confirm that observation.
Furthermore, all steps required for immunofluorescent labeling to perform confocal laser scanning microscopy are described here. Hence, the MG need to be passaged on glass coverslips. Since the coverslips are smaller than the area of a 24 well-plate, only about one-third of the cells that would cover a full well of a 24-well plate fits on the coated coverslip. For other applications such as qPCR, RNA-Seq, or western blots, cells are passaged in a 1:1 ratio, treated, and harvested at the desired time point.
As mentioned before, this culture system can also be used for other applications, for instance, to study glial behavior, including neuroprotective mechanisms, gliosis and/or to profile mRNA, miRNA, or proteins of cultured glia similar to the studies that used slightly different culture paradigms or species11,28,29,54. These applications would neither require neuronal medium to ensure neuronal survival after reprogramming, nor the addition of EdU to visualize cell proliferation. A low-serum medium without neuronal supplements should be used instead.
Although this method is robust and reliable, similar to all primary culture systems it has limitations; These are limited life span of the cells, progressive cell degeneration over time28, and the fact that it is a 2-dimensional, artificial culture system that cannot mimic the physiological context with regard to both, other retinal cell types and extracellular matrix. Therefore, after identifying top candidates and their most efficient concentrations in MG primary cultures, retinal explant cultures, a 3-dimensional culture system resembling the retinal tissue, should be performed. Explant cultures also have limited culture periods and cells in explants degenerate as well over time. However, they allow the study of MG in their natural 3-dimensional environment and can be performed at various ages reflecting the developmental stage of the animal23,32,55,56.
Taken together, this study reports about MG cultures used to monitor the potential of the RPC miRNA miR-25 to reprogram MG into neuronal-like cells, validated by immunofluorescent labeling. This protocol was used in the previous works21,24,27 and is a current in vitro method to study the regenerative potential of MG. Overall, MG primary cultures are a robust technique and allow reproducible results20,21. Although miRNA overexpression for MG reprogramming is described here exclusively, other factors such as small molecules, DNA (either plasmids or packed into viral vectors), antibodies for protein inhibition32, or specific compounds can be used/tested in MG primary cultures. There is also a broad spectrum of downstream applications, including miRNA profiling24, scRNA-seq27, RT-qPCR20,21, or western blots20. Moreover, MG primary cultures allow daily observation and surveillance of the cells. This can be a substantial advantage for determining time points, for instance, for the onset, duration, and/or end of a certain reaction or cell response. Migratory or proliferative behavior as well as injury/cell damage related paradigms (for example, global miRNA deletion in MG cultures)32 can be studied and analyzed in MG primary cultures to identify underlying mechanisms and pathways. Therefore, MG cultures are a very powerful tool for studying MG at molecular and cellular levels before implementation in in vivo systems.

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 minimize the cellular alterations caused by the artificial environment over time24,28,29.
Retinal dissociation and growth phase of primary MG cultures 
The first MG can be spotted as early as day one in vitro using a light microscope. MG have a tubular, elongated shape and light gray cell bodies (Figure 3A-D, red arrowheads). Most of them are, however, covered by cellular debris at these early stages. MG from two retinas of one mouse, grown in one well of a 12-well plate, reach 90%-95% confluency within 4-5 days (Figure 3E,F). Over time, all neuronal debris will be cleared, and a dense cell layer will be present. Cells are not ready for passage if the bottom of the well is not completely confluent. However, passage should not be done later than 6 days in vitro since the glia lose their glial features in culture over time. Before transfection or any other treatment, cells need to be checked for density and vitality. Transfection should only be performed on 90%-95% confluent cells (Figure 3G). For immunofluorescent labeling and confocal microscopy analysis, cells need to be seeded on coated coverslips.
Early and late phase of the cell conversion period 
As early as 15 h post transfection and 4-Hydroxytamoxifen treatment, the first cells start to express faint red fluorescence, i.e., tdTomato red fluorescent reporter protein driven under the (activated) Ascl1 promotor. Ascl1 expressing cells are now further referred to as Ascl1-Tom+ cells. More robust Ascl1-Tom expression can be observed 2 days after transfection with increasing red fluorescence over time (Figure 4). The reprogramming effect becomes visible around 2 days in culture with many Ascl1-Tom+ cells per field found in the reprogramming miRNA treatment: shown here for control-miR and miR-25 (Figure 4B-B&#39;&#39;&#39; and Figure 4C-C&#39;&#39;&#39;, respectively). In both treatments, Ascl1-Tom+ cells are still rather round and relatively big, with a more glia/progenitor cell-like morphology. Moreover, the induction of the red fluorescent reporter does not influence the cell density of the culture (still 90%-95% confluent).
Three to five days after transfection (Figure 4A and Figure 5A), the increase in the number of Ascl1-Tom+ cells becomes more evident in the miR-25 treated wells (Figure 5B-D) showing a four-fold increase in number after miR-25 treatments compared to controls. Moreover, the first morphological changes become visible. These changes include reduction of cell soma size and the development of fine processes. While in control conditions the vast majority of cells are large and flat (glial/progenitor-like, Figure-&#160;5B-B&#39;&#39;), many cells with small nuclei and several small processes resembling retinal neurons appear in the miR-25 treatment (Figure- 5C-C&#39;&#39;). These neuronal-like cells represent approximately 70% of the total Ascl1-Tom population (15% in control treatment; Figure 5E). Cells are less dense due to cell conversion (smaller cells require less space). Interestingly, these neuronal-like cells even appear to form tiny networks (Figure&#160;5C&#39;&#39;). At this time, cells can be harvested for immunofluorescent labeling to confirm neuronal identity.
Confirmation of neuronal identity 
Cells are labeled with antibodies against microtubule-associated protein 2 (Map2), a marker for neuron-specific cytoskeletal proteins30,31, and against Orthodenticle homeobox 2 (Otx2) to validate neuronal identity32,33. Both markers were also used in previous reprogramming studies21,27. Otx2 is a transcription factor found in RPCs34,35,36, mature bipolar cells34,35,37,38,39, and photoreceptors35,36,37,38,40. DAPI nuclear labeling is used to counterstain the cultures. Immunofluorescent labeling shows little to absent Map2 or Otx2 expression in control conditions (Figure 6A-A&#39;&#39;&#39;,C). miR-25 treated samples, however, have many Map2+ and Otx2+ cells (Figure 6B-B&#39;&#39;&#39;). Quantification of confocal images shows that after miR-25 overexpression, about 40 neuronal cells per field are present as compared to five neuronal cells per field in controls (Figure 6C, field size: 630 &#181;m x 630 &#181;m). Images were taken with 20x objective. These neuronal cells constitute about 70% of the total Ascl1-Tom+ cell population of the miR-25 treated samples (control ~20%, Figure 6D). All neuronal-like Ascl1-Tom+ cells were Map2+ and Otx2+, confirming neuronal identity. Moreover, the absolute number of Otx2+ and Map2+ cells was higher after miR-25 treatments compared to control-miRNA treatment (miR-25: 60 cells per field; control-miR: 10 cells per field; Figure 6E).
Taken together, results demonstrate that MG cultures can be grown and reprogrammed with miRNAs. miR-25 supplementation induces Ascl1-Tom expression in primary MG. Many MG convert into RPCs that adopt a neuronal morphology and express the neuronal markers Map2 and Otx2 after a few days.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63651/63651fig01.jpg" /> 
Figure 1: Experimental design and time course of a primary M&#252;ller glia culture. (A) Schematic of the Ascl1CreERT:tdTomatoSTOPfl/fl&#160;mouse, a retinal progenitor reporter mouse used to track the conversion of M&#252;ller glia (MG) into retinal progenitor cells (RPCs). (B) Time course of the culture periods consisting of growth phase (blue, 0-4/5 days in vitro, div), transfection phase (purple, 5/6-7/8 div), and MG conversion phase (yellow, starts 7/8 div). Growth phase: dissociated retinas (two retinas of one mouse) are grown in one well of a 12-well plate in a growth medium. Around day 4/6, cells are passaged into a 24-well plate that contains coated coverslips. Transfection phase: 5-7 div (1 day after passage) cells are transfected with miRNAs for 2 days in transfection medium. Cre recombinase is activated with 4-Hydroxytamoxifen (4-OHT). Cell proliferation is tracked with EdU. MG conversion phase starts 1 day after transfection. Cells are now grown in the neuronal medium until harvest (6-7 days post transfections, dpTF). <a href="https://www.jove.com/files/ftp_upload/63651/63651fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63651/63651fig02.jpg" /> 
Figure 2: Retinal dissociation and genotyping. (A) Eye cup with removed cornea, lens, iris, and vitreous. (B) Isolated retinas; retinal pigment epithelial (RPE) cells are removed after a thorough wash. (C) 12-well plate with dissociated retinas (two retinas per well). (D)&#160;Cutout of a genotyping gel image example. Genotyping is required to identify the mice that have Ascl1 driven Cre recombinase expression and will be used for tracking cell conversion. Scale bars: 1 mm. <a href="https://www.jove.com/files/ftp_upload/63651/63651fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63651/63651fig03.jpg" /> 
Figure 3: Primary M&#252;ller glia during the growth phase. (A) Time course of culture periods during the growth phase (0-4/5 days in vitro (div)) highlighted in blue.&#160;(B-G) Live images (phase) of M&#252;ller glia (MG) during growth phase after 1 div (B), 2 div after the medium change (C), 3 div (D), 4 div before passage (E,F) and 5 div, 1 day after passage and before transfection (G). MG cell bodies are indicated by red arrowheads. After 3 div, cultures are 60%-80% confluent (D), after 4-5 div, cultures are 90%-100% confluent and ready to be passaged (E-F). After passage on coverslips, cell cultures need to be 80%-90% confluent for subsequent transfection (G). Scale bars: 50 &#181;m (A-D), 100 &#181;m (E), 200 &#181;m (F). <a href="https://www.jove.com/files/ftp_upload/63651/63651fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63651/63651fig04.jpg" /> 
Figure 4: Early stages in the M&#252;ller glia conversion phase. (A) Time course of culture periods during conversion phase highlighted in yellow (starts 7/8 days in vitro (div)). The time point of analysis shown in this figure is indicated by the red star: 7 div, 2 days post-transfection (dpTF). (B-C&#39;&#39;&#39;) Live images of M&#252;ller glia (MG) cultures in phase and red fluorescence, combined or single red fluorescence. Red fluorescence visualizes Ascl1 expressing MG (tdTomato+, abbreviated Ascl1-Tom), 2 days after transfection with either control miRNA mimics (control-miR, B-B&#39;&#39;&#39;) or miR-25 mimics (C-C&#39;&#39;&#39;). Areas in B/B&#39; and C/C&#39; are shown in higher magnification in B&#39;&#39;/B&#39;&#39;&#39;, C&#39;&#39;/C&#39;&#39;&#39;. Scale bars 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63651/63651fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63651/63651fig05.jpg" /> 
Figure 5: End stages in the M&#252;ller glia conversion phase. (A) Time course of culture periods during the conversion phase highlighted in yellow (starts 7/8 days in vitro (div)).&#160;The time point of analysis shown in this figure is indicated by the red star: 10 div, 5 days post-transfection (dpTF). (B-C&#39;&#39;) Live images of M&#252;ller glia (MG) cultures in phase and red fluorescence, combined or single red fluorescence. Red fluorescence visualizes Ascl1 expressing MG (tdTomato+, abbreviated Ascl1-Tom), 5 days after transfection (pTF) with either control miRNA mimics (control-miR, B-B&#39;&#39;) or miR-25 mimics (C-C&#39;&#39;). Insets in B&#39;/C&#39; are shown in higher magnification in B&#39;&#39;/C&#39;&#39;, respectively. (D) The absolute number of Ascl1-Tomato+ cells per field (10x; 1440 &#181;m x 1080 &#181;m) after control-miRNA or miR-25 treatment, 2- and 5-days post transfection (dpTF; n =1, five images per well are counted; mean &#177; S.D.). (E) Percentage of neuronal-like Ascl1-Tomato+ cells of total Ascl1-Tomato+ cells per field (10x; 1440 &#181;m x 1080 &#181;m) after control-miRNA or miR-25 treatment, 5 days post transfection (5dpTF, n = 1, mean &#177; S.D.). Scale bars: 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63651/63651fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63651/63651fig06.jpg" /> 
Figure 6: Confirmation of the neuronal identity of reprogrammed cells. (A-A&#39;&#39;&#39;;B-B&#39;&#39;&#39;)&#160;Immunofluorescent labeling of primary mouse M&#252;ller glia (MG) cultures 5 days post transfection treated with control miRNA mimics (control-miR, A-A&#39;&#39;&#39;) or miR-25 mimics (B-B&#39;&#39;&#39;). Cells were fixed with 2% paraformaldehyde (PFA) and incubated with antibodies against RFP to label tdTomato, Map2, and Otx2 to label neurons. DAPI nuclear labeling (blue) was used to stain all cell nuclei. (C-E)&#160;Quantification (n = 1; five images per coverslip are counted; mean &#177; S.D.) of the absolute number of Ascl1-Tom+Otx2+ Map2+ cells per field (C), percentage of Ascl1-Tom+Otx2+Map2+ cells of total Ascl1-Tom+ cells (D), and the absolute number of Otx2+Map2+ cells per field (E). Results show a higher number of neurons in the miR-25 treatment compared to controls. Field size: 630 &#181;m x 630 &#181;m. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63651/63651fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Cell death and bacterial contamination. (A-A&#39;&#39;) Live images of M&#252;ller glia (MG) cultures 3 div contaminated with bacteria. Inset 1 in A is shown in higher magnification in A&#39; and displays atrophic, dead glia. Inset 2 in A is shown in A&#39;&#39; with alive glia. Scale bars:100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63651/Supplement Figure 1_ Kang-Wohl Jove 2021-12-6.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Primary Cell Cultures to Study the Regeneration Potential of Murine MÃƒÂ¼ller Glia after MicroRNA Treatment at JoVE.com

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 ...

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Developmental Biology

3.0K Views.  The State University of New York. 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.

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

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used therapeutically, in the near future, to restore function to damaged organs. Nevertheless, several technical issues, including the highly invasive procedure of isolating MSCs and the inefficiency surrounding their amplification, currently hamper the potential clinical use of these therapeutic modalities. Herein, we introduce a highly efficient method for the generation of dedifferentiated fat cells (DFAT), MSC-like cells. Interestingly, DFAT cells can be differentiated into several cell types including adipogenic, osteogenic, and chondrogenic cells. Although other groups have previously presented various methods for generating DFAT cells from mature adipose tissue, our method allows us to produce DFAT cells more efficiently. In this regard, we demonstrate that DFAT culture medium (DCM), supplemented with 20% FBS, is more effective in generating DFAT cells than DMEM, supplemented with 20% FBS. Additionally, the DFAT cells produced by our cell culture method can be redifferentiated into several tissue types. As such, a very interesting and useful model for the study of tissue dedifferentiation is presented.

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...