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1. Seeding of astrocytes for reprogramming
NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).
<ol>
	<li>Prepare 24-well plates with poly-D-lysine coated glass coverslips in the same way culture flasks were prepared previously. To determine the number of plates needed, consider that astrocytes are plated at a density of 5-5.5 x 104 cells per well in a 24-well plate. Usually, isolating six spinal cords from postnatal day 2 (P2) mice yields around 1 x 106 cells.</li>
	<li>Aspirate media from the T25 culture flasks containing the cultured astrocytes and wash once with 1x Phosphate-buffered saline (PBS). Detach the astrocytes from the culture flask by adding 0.5 mL of 0.05% Trypsin/ Ethylenediaminetetraacetic acid (EDTA) and incubate at 37 &#176;C for 5 min. Gently tap the side of the flask to release cells from the culture flask surface and check detachment under a brightfield microscope using a 10X magnification.</li>
	<li>Stop trypsinization with 2.5 mL of astrocyte culture medium and collect cell suspension in 15 mL tubes. Centrifuge at 300 x g for 5 min, aspirate supernatant, and resuspend cells in 1 mL of astrocyte culture medium. Calculate cell concentration using a haemocytometer or an automated cell counting system.</li>
	<li>Based on the number of cells, dilute the cell suspension with fresh astrocyte medium to obtain a solution of 1-1.1 x 105 cells per mL. Supplement the medium with&#160; epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 10 ng/mL per factor. Add 500 &#181;L of cell suspension, equivalent to 5-5.5 x 104 cells, to each well of the previously prepared 24-well plates and culture cells at 37 &#176;C and 5% CO2.</li>
</ol>
2. Forced expression of transcription factors
NOTE: Before proceeding with the protocol, it is essential to properly design the experiment. In particular, it is important to always include a negative control for the reprogramming, namely a condition where no reprogramming factor is expressed. For instance, when using vectors carrying the cDNA for the reprogramming factor and a reporter (e.g., green fluorescent protein (GFP), DsRed), the negative control is represented by the same vector carrying only the reporter. When expressing multiple factors carrying different reporters, the negative control should be accordingly adjusted.
<ol>
	<li>The day after plating, inspect the 24-well plates to make sure cells have adhered to the coverslips.</li>
	<li>Based on the experimental aim and available resources, the forced expression of reprogramming factors can be achieved by viral transduction (see step 2.3) or DNA transfection (see step 2.4). 
	NOTE: The use of retrovirus or lentivirus requires the approval of government authorities and must be performed under a biological safety cabinet within a laboratory with safety level 2 (SL2).</li>
	<li>Reprogram the astrocytes by transducing the cells with the retrovirus or lentivirus carrying the genetic information to express the reprogramming factor(s) of interest as described below.
	<ol>
		<li>Transduce cells with a high virus titer of 1 x 1010-1 x 1012 particles/mL by adding 1 &#181;L of the cell suspension directly to the astrocyte medium. This ensures a high infection rate. Culture the cells with the astrocyte medium containing viral particles at 37 &#176;C for 24-36 h before proceeding with section 3, depending on the purpose of the experiment. 
		NOTE: Different promoters can be used to drive the expression of the transgenes (see also Representative Results). Constitutive promoters {e.g., cytomegalovirus (CMV), chicken &#946;&#8209;actin (CAG)} induce the expression of the transgene earlier than inducible promoters; however, both types of promoter types have been successfully used to reprogram cells into neurons.</li>
	</ol>
	</li>
	<li>DNA plasmids can also be introduced into astrocytes via DNA transfection as described below. 
	NOTE: This can be done under a biological safety cabinet approved for SL1.
	<ol>
		<li>Before transfection, obtain the transfection reagent, a plasmid DNA of the desired constructs, fresh astrocyte medium, and serum-reduced medium (see Table of Materials). Calculate the required amount of serum-reduced medium (see Table of Materials) by considering that each well of a 24-well plate requires 300 &#181;L of serum-reduced medium. Add the appropriate amount of serum-reduced medium to a 50 mL tube and warm it to 37 &#176;C.</li>
		<li>When the serum-reduced medium is warm, aspirate astrocyte medium from all wells and collect it in a 50 mL tube. Filter the collected astrocyte medium with a 0.45 &#181;M syringe filter to remove detached cells.</li>
		<li>Add an equal volume of fresh astrocyte medium to the filtered medium to obtain a solution sufficient to add 1 mL of astrocyte medium per well. Maintain the astrocyte conditioned medium in the incubator at 37 &#176;C until use (step 2.4.9). 
		NOTE: The culture medium is re-used as it contains several secreted factors that support the viability of the culture.</li>
		<li>Add 300 &#181;L of pre-warmed serum-reduced medium to each well and place the 24-well plate back to the incubator.</li>
		<li>Prepare solution A, consisting of DNA and serum-reduced medium. For each well, use a total of 0.6 &#181;g of DNA and add it to 50 &#181;L of serum-reduced medium. Store solution A at room temperature until use. 
		NOTE: Typically, a technical triplicate per condition is considered. Therefore, a mix sufficient for 3.5 reaction is prepared (e.g., 2.1 &#181;g of total DNA diluted in 175 &#181;L of serum-reduced medium) to ensure enough material to transfect three wells of a 24-well plate.</li>
		<li>Prepare solution B, composed of the transfection reagent and serum-reduced medium. For each well, add 0.75 &#181;L of transfection reagent to 50 &#181;L of serum-reduced medium. As this solution is common to all transfection conditions, prepare it in bulk to reduce the variability across transfections.</li>
		<li>Incubate solution B at room temperature for 5 min. Add solution B to solution A drop by drop at a 1:1 ratio and gently mix. Do not vortex. Incubate solution A+B for 20-30 min at room temperature under the hood.</li>
		<li>Repeat step 2.4.5-2.4.7 for all transfection conditions. After 20-30 min, add solution A+B to each well drop by drop for a final volume of 100 &#181;L. Gently shake the plate and place the cells back in the incubator at 37 &#176;C for 4 h.</li>
		<li>After 4 h, remove the transfection medium and add 1 mL of the pre-warmed astrocyte conditioned medium prepared in step 2.4.3. Maintain cells for 36-48 h before proceeding with section 3, depending on the purpose of the experiment.</li>
	</ol>
	</li>
</ol>
3. Reprogramming of astrocytes (7 days analysis)
<ol>
	<li>Prepare neuronal differentiation medium by adding 1 mL of B27-supplement to 49 mL of basic culture medium. After 24-48 h, depending on whether cells have been transduced or transfected (see steps 2.3 and 2.4, respectively), replace astrocyte medium with 1 mL of neuronal differentiation medium per well and culture cells at 37 &#176;C and 9% CO2. 
	NOTE: Cells can also be kept at 5% CO2 if no 9% CO2 incubator is available. However, neuronal reprogramming is more efficient under these conditions.</li>
	<li>Optional: To increase the reprogramming efficiency, supplement neuronal differentiation medium with Forskolin (final concentration of 30 &#181;M) and Dorsomorphin (final concentration of 1 &#181;M) when replacing the astrocyte medium to differentiation medium. When opting to treat cells with Forskolin and Dorsomorphin, provide a second dose of Dorsomorphin 2 days after the initial treatment. Add this second treatment directly to the culture medium.</li>
	<li>Direct reprogramming of murine astrocytes into neuronal cells normally occurs within 7 days after changing the medium (Figure 1). Hence, 7 days after the initiation of neuronal reprogramming, fix or collect cells for downstream analysis.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>13256029</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma Aldrich</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>PAN Biotech</td>
			<td>P30-3302</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (Transfection reagent)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM &#8211; GlutaMAX (serum-reduced medium)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P1149</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct neuronal reprogramming of mouse astrocytes into neurons

Source: Hersbach, B. A. et al., <a href="https://app.jove.com/v/64175">Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes.</a> J. Vis. Exp. (2022) 
This video showcases the direct reprogramming of astrocytes expressing transforming factors into neurons. Transformation factors trigger a cascade of molecular events facilitating astrocyte-to-neuron conversion. The process is further supported by supplying specific agents and supplements through a neuronal differentiation medium.

<img alt="Figure 1" src="/files/ftp_upload/22368/22368_Figure1.jpg" /> 
Figure 1: Overview of astrocyte culture and reprogramming. (A)Timeline of astrocyte-to-neuron direct conversion. Each black line represents an important step in the protocol. (B) Representative brightfield images of cultured spinal cord-derived astrocytes after 7 days in culture. Pictures were taken using a brightfield microscope and 10x objective. Scale bar represents 100 &#181;m. (C) Representative brightfield images of spinal cord astrocytes 1 day after re-plating at a density of 5.5 x 104 cells per well in a 24-well plate. Images were taken using a brightfield microscope and a 10x objective. Scale bar represents 100 &#181;m. (D) Immunofluorescence image of a &#946;III-tub, Sox9, GFAP triple staining on astrocytes fixed 1 day after plating to demonstrate culture purity. Cells were fixed in 4% paraformaldehyde for 10 min and washed twice with 1x PBS. Cells were blocked using a 3% BSA, 0.5% Triton-X 100 in 1x PBS solution. Primary antibodies were diluted at the proper concentration (e.g., anti-GFAP 1:250; anti-&#946;III-tub 1:250; anti-Syp1 1:500) in blocking solution and incubated for 2 h at room temperature. Cells were washed three times with 1x PBS and incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature. Coverslips were washed three times with 1x PBS before mounting with Aqua Poly/Mount. Images were acquired using an epifluorescence microscope and a 40x objective. Scale bar represents 20 &#181;m. (E) Immunofluorescence image of a &#946;III-tub, DsRed double staining to demonstrate astrocyte to neuron conversion with Ascl1 after 7 DPT. Protocol of immunofluorescence and image acquisition was as described above. Scale bar represents 20 &#181;m. (F) Immunofluorescence images of a &#946;III-tub, DsRed, Synaptophysin 1 (Syp1) triple staining to demonstrate neuronal maturity after 21 DPT of reprogramming with Ascl1. Protocol of immunofluorescence and image acquisition was as described above. Scale bar represents 20 &#181;m.

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

1. Seeding of astrocytes for reprogramming
NOTE: The following steps have to be performed under a biological safety cabinet with a safety level 1 (SL1).

	...

Begin with a multi-well plate containing genetically altered astrocytes. These cells are adhered to a coverslip placed in an astrocyte medium.
These genetically modified cells express transformation factors that force their transition into neurons. This process is called neuronal reprogramming.
Take a neuronal differentiation medium containing specific molecules and the B27 supplement. Together, these components provide essential nutrients and factors for differentiation.
Replace the astrocyte medium with the neuronal differentiation medium.
Incubate the astrocytes for a required duration.
During incubation, the transformation factors expressed by the astrocytes trigger a sequence of molecular events that enable the conversion of an astrocyte into a neuron.
Converted neurons have smaller cell bodies and elongated processes, similar to typical neurons.

direct-neuronal-reprogramming-of-mouse-astrocytes-into-neurons

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Advanced Neuronal Models

55 Views. Source: Hersbach, B. A. et al., Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes. J. Vis. Exp. (2022) This video showcases the direct reprogramming of astrocytes expressing transforming factors into neurons. Transformation factors trigger a cascade of molecular events facilitating astrocyte-to-neuron conversion. The process is further supported by supplying specific agents and supplements through a neuronal differentiation medium. 

Video: Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons - Concept

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

JoVE Journal

Developmental Biology

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

Transcript

Direct Neuronal Reprogramming of Mouse Astrocytes into Neurons

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

Direct Reprogramming of Mouse Fibroblasts into Melanocytes