We thank all patients and healthy volunteers for donating critical samples to research studies. Moreover, we thank Rochelle Rodrigo for continuous expert technical assistance in tissue culture and Laura Ferraiuolo for independently confirming the technique in her lab as a collaborator. This work received funding from US National Institutes of Health Grants R01 NS644912-4 and RC2 NS69476-01 (to A.L.S.) and Packard Center for ALS Research Grant P2ALS and the Helping Link Foundation. K.M. also received funding from the Swiss National Science Foundation as well as the Young Investigator Development Award from the Muscular Dystrophy Association, The Rett Syndrome Research Trust and the Families of SCN2A foundations.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media</td>
			<td>Reagent</td>
			<td>Amount to mix</td>
			<td>Final concentration (%)</td>
		</tr>
		<tr>
			<td rowspan="3">Fibroblast media</td>
			<td>DMEM, high glucose, GlutaMAX</td>
			<td>500 mL</td>
			<td>89</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>50 mL</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="4">Base media</td>
			<td>DMEM/F12 + Glutamax</td>
			<td>500 mL</td>
			<td>97</td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td rowspan="4">Conversion media</td>
			<td>Base media</td>
			<td>50 mL</td>
			<td>99.9</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>1 &#181;L</td>
			<td>0.02 (20 ng/mL)</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>1 &#181;L</td>
			<td>0.02 (20 ng/mL)</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>50 &#181;L</td>
			<td>0.1 (5 &#181;g/mL)</td>
		</tr>
		<tr>
			<td rowspan="5">Neural Progenitor Cell (NPC) Media</td>
			<td>DMEM/F12 + Glutamax</td>
			<td>500 mL</td>
			<td>96.9</td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>10 uL</td>
			<td>0.002</td>
		</tr>
		<tr>
			<td rowspan="4">Astrocyte Media</td>
			<td>DMEM, high glucose, GlutaMAX</td>
			<td>500 mL</td>
			<td>89</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>50 mL</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>5 mL</td>
			<td>1</td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>1 mL</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td colspan="4">Media must be filtered after mixing all components. Conversion media (with factors) must be prepared fresh every week.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1. Media recipes for all cell types included in the protocol. Media should be filtered after mixing all components with either a sterile vacuum filtration system for big volumes or syringe and 0.2 um syringe filters. Conversion media (with factors) must be prepared fresh every week.
1. Direct conversion of adult human fibroblasts to neuronal progenitor cells
NOTE: A schematic timeline of this protocol can be reviewed in Meyer (2014)19.
<ol>
	<li>Use primary human skin fibroblasts that can be obtained from cell banks or skin biopsies31. Culture cells at 37 &#176;C in a 5% CO2 tissue culture incubator. Passage cells for at least 1-2 passages post thawing prior to use in the experiment. Once the cells are ready, coat a well of a 6-well plate with human fibronectin (1:200 diluted in PBS) at room temperature for 15-20 min.</li>
	<li>When the cells are ready to be plated, remove the fibronectin from the dish and immediately add cell solution or 2 mL of fibroblast media (Table 1) - do not let the dish dry out prior to adding media. Depending on how fast the cells grow, plate 150,000 (fast growing) - 200,000 (slow growing) fibroblasts in two wells of a human fibronectin coated 6-well plate each. 
	NOTE: The cells should be around 70% confluent for viral transduction to be able to keep them in the same plate for several more days after transduction. It can be beneficial to seed at two different densities in order to have a choice the next day for conversion.</li>
	<li>The day after seeding, check the fibroblasts under the microscope and verify cell density (between 70-85%) and even seeding throughout the well for optimal results.</li>
	<li>Transduce one well with all four retroviral vectors (Oct3/4, Klf4, Sox2 and c-Myc) - use a multiplicity of infection (MOI) of 10 for fast growing or 15 for slow growing fibroblasts (transduction efficiency should exceed 70% or more positive cells for each vector). Add regular fibroblast medium to viral mix to a total volume of 1 mL per well and incubate overnight at 37 &#176;C in a 5% CO2 tissue culture incubator. Leave the second well untreated and return cells to tissue culture incubator. 
	NOTE: Retroviral vectors can be purchased by a vendor or made in-house, protocols are available online32.</li>
	<li>The day after transduction, wash cells 1x with PBS and add 1 mL of fresh fibroblast medium.</li>
	<li>The next day, change medium to conversion medium. Wash cells 1x with PBS to get rid of the residual fibroblast medium, and then add 1 mL of conversion medium. Change the media of the untreated well to conversion media as well. 
	NOTE: Some morphological changes can be observed even by changing the media on the fibroblasts that did not receive retroviral vector treatment, thus having an untreated well (optional) will be helpful when determining effects of the viral vectors. Cells should start changing morphology 2-4 days after switching to conversion media.</li>
	<li>Observe cells under the microscope and watch for changes in morphology (Figure 1). Cell media should be replaced daily throughout the conversion process (1-2 mL of conversion media, Table 1) and for subsequent iNPC culture. Change medium by carefully removing 70% of medium from the well while making sure that the cells remain covered in the remaining 30% of media. 
	NOTE: Media with growth factors in it needs to be consumed within 1 week of preparation.
	<ol>
		<li>Continue to observe cells and change media daily (1-2 mL of conversion media).</li>
		<li>Some cell lines start forming loose elevated round formations growing in ball like structures or loose neuronal spheres (Supplemental Figure 1 and 19,33). Take care to not detach these cells. If the balls detach, they can be collected and re-plated in a new fibronectin coated well of a 6-well plate. 
		NOTE: If they are expanded on their own, the cells will have reduced diversity compared to the initial plate and may represent a distinctly different cell line. We recommend combining these cells with the rest of the converted cells at the next round of splitting.</li>
	</ol>
	</li>
	<li>After about 5-6 days in conversion media (up to 12 days for difficult cell lines) or when cells become very dense, passage them 1:2 or maximum 1:3. 
	&#8203;NOTE: It is very important to keep the cells at a high density throughout the conversion process.</li>
</ol>
2. Conversion and iNPC splitting procedure
<ol>
	<li>Warm up cell detachment solution (e.g., Accutase) and coat an appropriate number of wells of a 6-well with fibronectin (1:200 in PBS, 1 mL of coating volume) for 15-20 min at room temperature. Fibronectin coating can be longer without negative impact, but care should be taken to not shorten the coating time.</li>
	<li>Wash cells carefully with PBS without detaching any cells (use the wall of the well to apply PBS gently to the cells). Carefully remove PBS with pipettes or by aspiration.</li>
	<li>Add 0.5 mL of cell detachment solution and incubate 2-3 min at 37 &#176;C in a tissue culture incubator. Check under the microscope to verify that most cells have detached. If all cells have come off, add 2 mL of fresh conversion media and gently pipette up and down 2-3 times to dissociate clumps (if required).</li>
	<li>Collect the cells in a 15 mL tube and add additional 3 mL of media to further dilute the cell detachment solution. Wash the well with the extra media first to ensure all cells have been collected.</li>
	<li>Centrifuge for 4 min at 200 x g at room temperature. 
	NOTE: Converting cells or iNPCs are extremely sensitive to the presence of cell detachment solution in the media. It is absolutely necessary to remove the residual enzyme by centrifugation and withdrawal of supernatant.</li>
	<li>Remove supernatant from cell pellet and resuspend the cells in 1 mL of fresh medium. Gently pipette up and down 2-3 times to resolve cell clumps. Remove fibronectin from coated 6-wells and add 1 mL of fresh media to each well immediately (do not let fibronectin dry). For a split ratio of 1:2, add 0.5 mL of the cell suspension in one well and the other 0.5 mL in a second well.
	<ol>
		<li>Culture cells at 37 &#176;C in a tissue culture incubator.</li>
	</ol>
	</li>
	<li>Distribute the cells by gently shaking the 6-well in north-south-east-west direction (not circular) and put in tissue culture incubator.</li>
	<li>Observe the cells under the microscope the next day and change media (1-2 mL). Change media every day until the cells are ready to be split again. 
	NOTE: At this point, the cell proliferation should start to accelerate, and cells should be ready for a second split within 2-3 days (4 days for very slow growing lines).
	<ol>
		<li>At this new split, seed one well of cells in conversion media and the other well in NPC media containing only FGF at increased concentration without EGF/heparin (Table 1). 
		NOTE: Some cell lines reach a stagnant state in conversion media after about 10 days and switching to NPC media might help with cell growth. In addition, NPCs have a more specific, smaller shape, when grown in NPC media19. At this point, the iNPCs are ready for differentiation and further use.</li>
	</ol>
	</li>
	<li>Keep changing media (1-2 mL) of the iNPCs daily.
	<ol>
		<li>Expand NPCs into 10 cm dishes by combining two or three confluent wells of a 6-well. The media volume for 10 cm dishes is typically 12-15 mL.</li>
		<li>At the next split, further expand these cells to three or four 10 cm dishes (12-15 mL of media) for generations of large numbers of cell stocks. 10 cm dishes are usually split at 1:3 or 1:4 every 3-4 days depending on proliferation rate.</li>
	</ol>
	</li>
</ol>
3. Generating induced astrocytes from NPCs
<ol>
	<li>To make astrocytes from fresh iNPCs, seed iNPCs (in 10 cm fibronectin coated plates) directly in 10 mL astrocyte medium (Table 1) so that they are around 10% confluency the following day. Culture cells at 37 &#176;C in a 5% CO2 tissue culture incubator. 
	NOTE: The recommended seeding density is usually 50 &#181;L of the cell resuspension of a 10 cm dish of NPCs, provided they are diluted to a final volume based on their split ratio (e.g., 3 mL for a 1:3 split, 4 mL for a 1:4 split, etc.).</li>
	<li>Change medium (10 mL astrocyte media) of the iAs three days after plating. Keep the cells differentiating for 5-7 days. 
	NOTE: iAs do not tolerate multiple passages well, therefore it is better to make fresh astrocytes every time you split the NPCs.</li>
	<li>To seed for experimentation, split the astrocytes using trypsin or cell detachment solution as described in steps 2.1-2.7. Recommended seeding densities are included in Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plate Type</td>
			<td>Astrocytes per Well</td>
		</tr>
		<tr>
			<td>384-well</td>
			<td>2500</td>
		</tr>
		<tr>
			<td>96-well</td>
			<td>10000</td>
		</tr>
		<tr>
			<td>24-well</td>
			<td>40000</td>
		</tr>
		<tr>
			<td>6-well</td>
			<td>150000</td>
		</tr>
	</tbody>
</table>
Table 2. Recommended seeding density of iAs per plate area. Typical number of iAs seeded to generate a monolayer on the most common tissue culture plates.
4. Preparing and defrosting portions for astrocyte differentiation from frozen NPC stocks (alternative to step 3)
NOTE: As an alternative to maintaining iNPCs in culture, astrocytes can also be produced directly from a frozen stock. To do so, the iNPCs are frozen in smaller portions. Table 3 shows suggested freezing and thawing volumes for portions to be defrosted into a 10 cm dish containing 10 mL of Astrocyte media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Portion Size</td>
			<td>Cell Suspension (&#181;L)</td>
			<td>Freezing media (&#181;L)</td>
			<td>Total volume (&#181;L)</td>
			<td>Defrosts Into</td>
		</tr>
		<tr>
			<td>4x</td>
			<td>400</td>
			<td>400</td>
			<td>800</td>
			<td>Two 10-cm dishes</td>
		</tr>
		<tr>
			<td>2x</td>
			<td>200</td>
			<td>200</td>
			<td>400</td>
			<td>One 10-cm dish</td>
		</tr>
	</tbody>
</table>
Table 3. Instructions on how to portion iNPC to generate iAs. Proportion of iNPC suspension and freezing media to generate portions. Note that the final DMSO concentration is 10% when cell suspension is mixed with freezing media.
<ol>
	<li>To make astrocytes from frozen iNPC stocks, remove portion stock vial from liquid nitrogen storage tank and quickly thaw at 37 &#176;C. As soon as cells are defrosted, pipette cell solution in a 15 mL tube containing 5 mL astrocyte media.</li>
	<li>Centrifuge for 4 min at 200 x g and room temperature. Remove the supernatant and resuspend in 1 mL of fresh astrocyte medium.</li>
	<li>Add 9 mL of fresh astrocyte medium to a fibronectin coated 10 cm dish and add 1 mL of cell solution. Gently distributing cells in north-south-east-west motion. Culture cells at 37 &#176;C in a 5% CO2 tissue culture incubator.</li>
</ol>

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M., Kwakowsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Microglia+and+Astrocytes+in+Huntington's+Disease.">The Role of Microglia and Astrocytes in Huntington's Disease.</a> Frontiers in Molecular Neuroscience. 12, 258 (2019).</li><li>Maezawa, I., Swanberg, S., Harvey, D., LaSalle, J. M., Jin, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome+astrocytes+are+abnormal+and+spread+MeCP2+deficiency+through+gap+junctions.">Rett syndrome astrocytes are abnormal and spread MeCP2 deficiency through gap junctions.</a> Journal of Neuroscience. 29 (16), 5051-5061 (2009).</li><li>Hautbergue, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SRSF1-dependent+nuclear+export+inhibition+of+C9ORF72+repeat+transcripts+prevents+neurodegeneration+and+associated+motor+deficits.">SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits.</a> Nature Communications. 8, 16063 (2017).</li><li>Almad, A., Maragakis, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stocked+toolbox+for+understanding+the+role+of+astrocytes+in+disease.">A stocked toolbox for understanding the role of astrocytes in disease.</a> Nature Reviews Neurology. 14 (6), 351-362 (2018).</li><li>Gatto, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+converted+astrocytes+retain+the+ageing+features+of+the+donor+fibroblasts+and+elucidate+the+astrocytic+contribution+to+human+CNS+health+and+disease.">Directly converted astrocytes retain the ageing features of the donor fibroblasts and elucidate the astrocytic contribution to human CNS health and disease.</a> Aging Cell. , (2020).</li><li>Caiazzo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+conversion+of+fibroblasts+into+functional+astrocytes+by+defined+transcription+factors.">Direct conversion of fibroblasts into functional astrocytes by defined transcription factors.</a> Stem Cell Reports. 4 (1), 25-36 (2015).</li><li>Vangipuram, M., Ting, D., Kim, S., Diaz, R., Schule, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+punch+biopsy+explant+culture+for+derivation+of+primary+human+fibroblasts.">Skin punch biopsy explant culture for derivation of primary human fibroblasts.</a> Journal of Visualized Experiments. (77), e3779 (2013).</li><li>Pear, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transfection+methods+for+preparation+of+high-titer+retroviral+supernatants.">Transient transfection methods for preparation of high-titer retroviral supernatants.</a> Current Protocols in Molecular Biology. , 11 (2001).</li><li>Boczonadi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+glycyl-tRNA+synthetase+impair+mitochondrial+metabolism+in+neurons.">Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.</a> Human Molecular Genetics. 27 (12), 2187-2204 (2018).</li><li>Rinaldi, F., Motti, D., Ferraiuolo, L., Kaspar, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+content+analysis+in+amyotrophic+lateral+sclerosis.">High content analysis in amyotrophic lateral sclerosis.</a> Molecular and Cellular Neuroscience. 80, 180-191 (2017).</li></ol>

The authors have nothing to disclose.

In summary, the direct conversion is a fast and easy method to generate induced neuronal progenitor cells from patient skin fibroblasts. This method is advantageous because of its speed as well as the large number of cells generated that are easy to maintain. Moreover, increasing evidence suggests that direct reprogramming methods remove less epigenetic marks compared to classic iPSC reprogramming technology, therefore leaving the disease environment more intact4,7,8. The differentiation of iNPCs to astrocytes is very straight forward and highly reproducible even between multiple independent laboratories19,29,33. iNPCs can be frozen in small portions and thawed directly for differentiation purposes, which helps reduce variation between experiments since passage numbers can be kept similar. Astrocytes play a crucial role in disease progression in many neurological diseases and are therefore an interesting cell type to work with. The astrocytes can be used for mechanistic studies, metabolic studies or drug testing assays and are usually grown as monolayers. Moreover, astrocytes can be combined in co-culture systems with mouse or human neurons to assess the impact of astrocytes on neuronal survival and morphology, both of which are good readouts for drug testing34.
In order to successfully use this protocol, a few points and potential limitations need to be kept in mind. The human skin fibroblasts for example vary greatly in their cell division rate as well as response to viral vectors. Since these are not standardized cell lines, they are as variable as mankind itself, each line is different. As for many reprogramming methods, it is easier to reprogram fibroblasts that have a moderate to fast cell division rate versus cells that are barely replicating. The replication rate can be impacted by the skin biopsy quality and processing itself, by the passage number of the fibroblasts as well as handling. When working with primary skin fibroblasts, it is important to not let the cells get too dense to avoid induction of senescence. If the fibroblasts do not grow well, it is best to try a new stock or a lower passage, although in some cases, the disease itself could also play a role. Cell division can sometimes be improved by using 15% or 20% FBS in the fibroblast media compared to the standard 10%.
The quality of the reprogramming viral vectors is of high importance for success. In our hands, retroviral vectors were more efficient compared to lentiviral vectors or other non-integrating viruses; however, this could be dependent on the viral vector quality and purity. Commercial retroviral vector kits usually specify the viral vector concentration and often also a multiplicity of infection for a certain cell line. It is important to keep in mind that primary fibroblasts differ from the cell line used by the companies to determine viral vector uptake. Furthermore, even when ordering the same commercial kit, variation between batches is common. Moreover, the uptake of viral vectors also varies between fibroblast cell lines. Some lines could be more sensitive and therefore transduced cells could die, while other cell lines might be more resistant to viral uptake. Thus, determining transduction efficiency for a given cell line can be important especially if the cell line is growing slowly or previous conversion attempts failed. In our hands, the best way to evaluate how much of a specific retroviral vector is needed for conversion is to perform an immunofluorescent staining with several dilutions of the viral vector. The number of cells expressing the transgene for each vector can then be counted, with the aim of a transduction efficiency of 70% or higher. In our experience, similar MOIs can be used for most cell lines however the MOI can be adjusted if necessary.
During the conversion process, it is critical to not split the cells too early. The time until the cells are ready to be split depends on multiple factors including the primary cell line and its characteristics, the quality of the viral vector used and the media quality. Importantly, the success rate of the conversion is much higher when the cells are kept at high density. Even if the cells start to change morphology, it is better to leave the cells in the first well longer than splitting them prematurely. If the split occurs too early the conversion can halt in its progress and lead the cells to differentiate back to fibroblast-like shape and behavior. Furthermore, diluting them too early can result in more elongated cell bodies compared to the small and compact cell bodies of the NPCs observed previously. Thus, the first splits should always be conservative; it is better to keep the cells too dense with a 1:1 or 1:2 split compared to diluting them too much. Some cell death is expected following the initial split. Of note, the cells always look worse (flatter) the first day after splitting, which is normal. Best judgments on cell shape should be made 2-3 days later. Importantly, the quality of the growth factors and media components is crucial as well. It is important to prepare small amounts of media containing growth factors so that the fully prepared media will only be used for 5-7 days maximum. Do not keep the media at room temperature or 37&#176;C for extended periods of time. Use quickly and refrigerate as soon as the media change is performed. In addition, keeping the volume of media low at 1 mL instead of 2 mL can help especially for cell lines that are more resistant to converting. If the cells consume the media rapidly, which can be observed by a change in media color form red to yellow (acidification rate), adjust the volume of the media to up to 2 mL. Rapid media consumption is a positive sign and often observed at later stages of conversion alongside increased proliferation.
NPCs are also very sensitive to the presence of accutase in the media and it is very important to keep the accutase incubation time short. We recommend an incubation period of 2-4 minutes, check cells frequently under the microscope to see when they have detached. It is essential to centrifuge the cells after splitting to remove the enzyme mix from the media. It is also important to keep the passage number in mind as cell lines can change upon extended culturing leading to alteration of expression profiles. Thus, it is important to freeze many cell stocks at early passages. Cells should be frozen in 10% DMSO and 90% NPC media and stored long term in liquid nitrogen. Due to the lack of serum in this media it is critical to ensure a slow freeze using freezing chambers and once frozen overnight, immediately transfer to liquid nitrogen. While no clonal selection is performed in this protocol, it is possible that extended culturing and passaging leads to natural selection of an initial cell population with favorable growth and survival rate. Therefore, it is important to keep passage number and handling in mind when performing experiments. We prefer to use lower passages for experiments, if possible, we do not exceed passaging the cell lines for more than 20 times. Since NPCs grow rapidly, large numbers of stocks can be frozen at lower passages for experiments. Cell lines with particularly high growth rates are at higher risk of media acidification. Thus, as cell lines grow with different speed, the amount of media might need to be adjusted based on proliferation. If the cells are continuously left in highly acidified media, it can influence the health of both control and disease cell lines. This causes even healthy astrocytes to become reactive, affecting their use in further differentiation and experiments.
For astrocyte differentiation, it is important to keep the cells at low density as high density will prevent the cells from differentiating and they will keep proliferating at higher rates. Thus, seeding density needs to be adjusted for each cell line dependent on their proliferation rate. We recommend trying different seeding densities and perform stainings/RT-PCR with astrocyte markers to ensure proper differentiation. IAs quality can be assessed alongside healthy controls using co-culture assays with neurons. Provided disease pathology does not lead to a reactive phenotype, neurons should remain viable in contact with iAs for several days. Astrocyte morphology can vary greatly between individual cell lines and can be impacted by the disease phenotype as well. Moreover, in diseases where astrocytes are heavily impacted, they might show a reactive phenotype with large, elongated extensions.

Understanding underlying disease mechanisms is critical for neurological diseases as it aids in the development of potential therapeutic strategies. While animal models have historically been the gold standard for researching diseases of the nervous system, the direct translation of potential therapies into a clinical setting often shows limited success1,2,3. Reasons for the lack of translation are lack of variability of genetic background and mutations in mice, incomplete display of disease phenotypes and variation in drug sensitivity or dosing in human patients that are not pictured well in inbred mouse strains. Furthermore, for many rare neurological disorders, no or few animal models are available. Studying disease mechanisms directly in relevant human cell types could facilitate research and improve translation to the clinic. For CNS disorders, primary human cells are difficult to retrieve and represent a very limited source as biopsies are invasive or can only be retrieved post-mortem at the end-stage of the disease. In the past 15 years, the development of cellular reprogramming technology has rapidly expanded the ability to model human neurological and neurodegenerative diseases in vitro.
Traditional methods of cell reprogramming involve reprogramming fibroblasts or other cell types into induced pluripotent stem cells (iPSCs) capable of differentiating into cell types from all three germ layers4. Takahashi and Yamanaka were the first to show that re-expression of four stem cell transcription factors is sufficient to redirect somatic cells to become iPSCs5. iPSCs can then be further differentiated into specific cell types. However, the drawback of this process is that it is labor intensive and time consuming to generate and isolate stable stem cell clones. Somatic mutations or specific aberrancies of the mother cell are also maintained in the stem cell clone and can impact results of the study6. In addition, increasing evidence suggests that the reprogramming process to stem cells erases valuable epigenetic changes that could influence cellular disease mechanisms4,7,8. In recent years, researchers focused on modified reprogramming methods allowing a more direct generation of different cell types while bypassing the pluripotent stem cell state9,10,11,12 (reviewed in 13,14). Initial breakthroughs revealed in vitro combinatorial reprogramming of fibroblasts to cardiomyocytes15, neurons16 and hepatocytes17 by ectopic expression of multiple lineage-specific transcription factors or microRNAs18. This was followed by studies directly reprograming cells to model neurological disorders19,20. As mentioned above, such direct reprogramming or conversion protocols have potential advantages over classical iPSC protocols including speed and the ablation of the clonal isolation step. Moreover, evidence suggests that these protocols allow to maintain a larger amount of epigenetic information related to the age of the patient and likely the same holds true for disease relevant markers7,8.
To date, most in vitro research on neurological disorders has been focused primarily on neurons. However, it is well known that other cell types such as astrocytes, microglia and oligodendrocytes play a critical role in disease pathology and progression of neurodegenerative disorders such as Alzheimer&#39;s disease (AD), Amyotrophic Lateral Sclerosis (ALS), Parkinson&#39;s disease (PD), Huntington&#39;s Disease (HD), Multiple Sclerosis (MS), and other neurological pathologies such as Rett syndrome (RTT), sleep disorders, addiction, epilepsy, lysosomal storage disorders, depression, migraine and pathological pain21,22,23,24,25,26. Astrocytes are the most abundant cell type in the central nervous system (CNS) and until recently, they have been broadly neglected from a pathological point of view7. They are now known to have a critical role in supporting almost all homeostatic functions of the healthy CNS. Additionally, it is apparent that they have an important pathological impact and are very valuable in understanding disease mechanism and evaluating therapeutic strategies19.
In the current description, we detail a protocol for direct conversion of human patient fibroblasts into induced Neuronal Progenitor Cells (iNPCs) using retroviral vectors expressing the Yamanaka reprogramming factors (Klf4, Oct3/4, Sox2 and c-Myc) and subsequent exposure to neuralizing media. Previous studies have used different combinations of transcriptional factors for direct reprogramming to neural cells (reviewed in 14), but the factors used in the described protocol are broadly available either as plasmids for in house production of viral vectors, or as commercially available ready to go viral reprogramming kits. The resulting iNPCs can be further differentiated into induced neurons (iNs)27, oligodendrocytes (iOs)24&#160;and astrocytes (iAS)19 to study their role in neurological diseases. Our protocol does not involve time-consuming generation of iPSCs which most available protocols utilize for derivation of human astrocytes28. Approximately 98% to 100% of directly reprogrammed iNPCs can differentiate into GFAP-positive astrocytes29 as compared to only 2% using direct conversion method from fibroblasts to astrocytes30. Recently, a comparative transcriptomic study using our described reprogramming method has shown that iNPCs reprogrammed from donor fibroblasts can retain ageing features at transcriptional and functional level similar to age matched postmortem astrocytes and primary astrocytes29. Thus, this conversion protocol is a powerful tool to investigate disease mechanisms and evaluate novel therapeutic approaches for age-related neurodegenerative and neurological disorders.
Here we describe the protocol utilized to generate iNPCs and further differentiation into iAs, which are the most suited for larger scale small molecular screening assays. Disease mechanisms and drug testing with iAs can be done using different methodologies such as co-cultures with neurons or metabolic and biochemical analysis. The advantage of this system is the speed and ease of maintenance of these cell lines compared to iPSCs. Moreover, iNPCs can be frozen in small portions for iAs differentiation which facilitates drug testing under constant conditions over extended periods of time. Overall, comparison of larger sample numbers becomes possible this way which opens the door to evaluate therapeutic potential of compounds in a larger and more representative patient population.

<table><tbody><tr><td>100mm x 2mm Style dish, Cell culture treated, Nonpyrogenic</td><td>Corning</td><td>430167</td><td>Tissue culture</td></tr><tr><td>15 ml conical screw cap centrifuge tubes, copolymer polypropylene</td><td>USA Scientific</td><td>1475-1611</td><td>Used for lifting and centrifuge cells</td></tr><tr><td>50mL Conical Centrifuge Tubes</td><td>USA Scientific</td><td>1500-1811</td><td>Media preparation</td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco</td><td>15240062</td><td>Antibiotic with antifungal activity for media preparation</td></tr><tr><td>B-27&amp;nbsp; Supplement (50X), serum free</td><td>Invitrogen</td><td>17504044</td><td>For NPC and base media</td></tr><tr><td>Cryogenic vials&amp;nbsp; 1.2ML</td><td>Corning</td><td>CLS430658-500EA</td><td>For freezing cell stocks</td></tr><tr><td>DMEM, high glucose, GlutaMAX&amp;nbsp; Supplement, pyruvate</td><td>Gibco</td><td>10569010</td><td>For fibroblast and Astrocyte media</td></tr><tr><td>DMEM/F-12, GlutaMAX&amp;nbsp; supplement</td><td>Gibco</td><td>10565042</td><td>For NPC and base media</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2438-50ML</td><td>For freezing cell stocks</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>14190136</td><td>Referred in protocol as PBS. For fibronectin dilution and cell wash</td></tr><tr><td>EZ Retrovirus iPSC Reprogramming Kit</td><td>ALSTEM</td><td>RF101</td><td>Retrovirus containing the Yamanaka factors. Virus can also be made in-house.</td></tr><tr><td>Fetal Bovine Serum, certified</td><td>Gibco</td><td>16000-036</td><td>Referred in protocol as FBS . For Fibroblast and Astrocyte media</td></tr><tr><td>Fisherbrand Sterile Syringes for Single Use</td><td>Fisher</td><td>14-955-461</td><td>Filter media (50mL)</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3149-10KU</td><td>Referred in protocol as Heparin. Used in conversion media. Powder diluted in ultrapure water. Final stock concentration of 5000X</td></tr><tr><td>Human Plasma Fibronectin Purified Protein</td><td>Millipore Sigma</td><td>FC010-10MG</td><td>Referred in protocol as Fibronectin, used in 1:200 dilution for coating.</td></tr><tr><td>Isopropanol (technical grade)</td><td>Fisher Scientific</td><td>S25372A</td><td>For freezing cell stocks</td></tr><tr><td>Mr. Frosty</td><td>Thermo Fisher</td><td>5100-0001</td><td>For freezing cell stocks</td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502048</td><td>For Astrocyte, NPC and base media</td></tr><tr><td>Recombinant Human EGF</td><td>Preprotech</td><td>AF-100-15</td><td>Referred in protocol as EGF, used in conversion media. Powder diluted in PBS, final concentration of 1mg/mL, stored in small frozen aliquots</td></tr><tr><td>Recombinant Human FGF-basic</td><td>Preprotech</td><td>100-18B</td><td>Referred in protocol as FGF, used in NPC and conversion media. Powder diluted in PBS, final concentration of 1mg/mL, stored in small frozen aliquots</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Gibco</td><td>A1110501</td><td>Referred in protocol as Accutase. Used for lifting during the conversion process and NPCs.</td></tr><tr><td>Stericup Quick Release-GP Sterile Vacuum Filtration System</td><td>Millipore Sigma</td><td>S2GPU05RE</td><td>Media filtration</td></tr><tr><td>Tissue culture plate, 6 well, Flat bottom with Low evaporation lid</td><td>Fisher</td><td>08-772-1B</td><td>Tissue culture</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Invitrogen</td><td>25300062</td><td>Lifting of Fibroblasts and Astrocytes</td></tr><tr><td>Whatman Puradisc 25 syringe filters, 0.2 &amp;mu;m, PES sterile</td><td>Millipore Sigma</td><td>80-2502</td><td>Filter media (50mL)</td></tr></tbody></table>

in vitro modeling for neurological diseases using direct conversion from fibroblasts to neuronal progenitor cells and differentiation into astrocytes

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely impacts the study of cell type specific contributions to disease. Moreover, animal models usually do not reflect variability in mutations and disease courses seen in human patients. Reprogramming methods for generation of induced pluripotent stem cells (iPSCs) have revolutionized patient specific research and created valuable tools for studying disease mechanisms. However, iPSC technology has disadvantages such as time, labor commitment, clonal selectivity and loss of epigenetic markers. Recent modifications of these methods allow more direct generation of cell lineages or specific cell types, bypassing clonal isolation or a pluripotent stem cell state. We have developed a rapid direct conversion method to generate induced Neuronal Progenitor Cells (iNPCs) from skin fibroblasts utilizing retroviral vectors in combination with neuralizing media. The iNPCs can be differentiated into neurons (iNs) oligodendrocytes (iOs) and astrocytes (iAs). iAs production facilitates rapid drug and disease mechanism testing as differentiation from iNPCs only takes 5 days. Moreover, iAs are easy to work with and are generated in pure populations at large numbers. We developed a highly reproducible co-culture assay using mouse GFP+ neurons and patient derived iAs to evaluate potential therapeutic strategies for numerous neurological and neurodegenerative disorders. Importantly, the iA assays are scalable to 384-well format facilitating the evaluation of multiple small molecules in one plate. This approach allows simultaneous therapeutic evaluation of multiple patient cell lines with diverse genetic background. Easy production and storage of iAs and capacity to screen multiple compounds in one assay renders this methodology adaptable for personalized medicine.

This protocol allows the rapid and easy generation of iNPCs directly from human skin fibroblasts using retroviruses containing the Yamanaka factors. This method allows bypassing the stem cell state and the need for clonal selection, thereby avoiding clonal variation. Important steps to keep in mind while cells are undergoing the conversion process are the fibroblast seeding density, media pH and keeping the cells at an optimal confluency. Examples of optimal splitting confluency and morphology changes during the conversion process can be found in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62016/62016fig01.jpg" /> 
Figure 1. Representative images of the conversion process after adding the retroviral mix of Yamanaka factors. Fibroblasts were seeded and transduced twenty-four hours later with Yamanaka factors. Two days post virus (DPV), media was changed to conversion media. Cells were cultured in conversion media until they were ready to passage (13 DPV). Cells were seeded post passage to reach 80% confluence the following day (14 DPV). Subsequent passages maintained this density until neuronal progenitor cells begin rapidly dividing. Scale bar is 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62016/62016fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After the conversion process is complete, the NPCs will show strongly reduced expression or no expression of fibroblast markers and morphology, and express NPC specific cell markers (Figure 2). Moreover, they can also be used for generating different cell lines, like iNs, iOs, and iAs.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62016/62016fig02.jpg" /> 
Figure 2. Cells that undergo the conversion process express cell-specific markers. Patient fibroblasts (A), induced Neuronal Progenitor Cells (iNPCs (B) and induced Astrocytes (iAs (C) cells were seeded on glass coverslips in a 24 well tissue culture treated plate. Cells were immunostained for: fibroblast specific cell markers (A), Vimentin (green) and TE7 (red), iNPC specific cell marker (B), Nestin (red), and iAs cell marker (C) GFAP (purple) and iNPC marker Nestin (green). Scale bar is 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62016/62016fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In our experience, iAs in particular are very valuable for drug and disease mechanism testing as they can be generated in pure populations (98% or more GFAP positive cells29) at reproducible large numbers. iAs can be differentiated from iNPCs by taking a small aliquot of cells during splitting or a previously frozen iNPC portion and directly plating in astrocyte media. Important considerations during this process are maintaining the iNPCs initial seeding density low (Figure 3 and Figure 4), as a high density has been shown to hinder the differentiation process (Figure 4) and paying additional attention to the media pH, as acidification can activate even healthy astrocytes.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62016/62016fig03.jpg" /> 
Figure 3. Representative images of the iAs generation process from iNPCs. iNPCs are seeded in Astrocyte media (Table 1) at a low seeding density. A good seeding density is about 10% on the first day post seeding (DPS) (left); however, this density can be adjusted according to the growth rate of cells. Typical iAs morphology can be observed after 5 DPS (middle). In some cases, aberrant astrocyte morphology can be observed, with long, spiky extensions. This change is indicative of astrocyte activation and can be secondary to disease state or incorrect culturing techniques (right). Scale bar is 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62016/62016fig03large.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/62016/62016fig04.jpg" /> 
Figure 4. iNPC seeding density affects the iAs differentiation efficiency. Control iNPC lines were seeded at low (A) and high (B) density in Astrocyte media to demonstrate effects of seeding density on differentiation. 5 DPS, the low-density line expressed astrocyte-specific markers, while the high-density line shows a mixture of iNPC and iAs markers with a marked iNPC-like morphology. Scale bar is 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62016/62016fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1. Additional figures of the conversion process after adding the retroviral mix of Yamanaka factors. Images of the conversion process at 12 DPV (1 day before passage) and 19 DPV (6 days after passage). Note the difference on morphology between cells before and after passage, at 12 DPV cells have a ball-like structure morphology. After a passage and several days on conversion media (table 1) cells start displaying a NPC-like morphology. Scale bar is 200 &#181;m.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62016/Supplemental 1. Additional Images Conversion.tif" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes at JoVE.

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities.

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...

in-vitro-modeling-for-neurological-diseases-using-direct-conversion

Nanyang Technological University

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JoVE Journal

Neuroscience

4.9K Views.  The Research Institute at Nationwide Children’s Hospital. We gebruiken dit protocol om neurale voorlopercellen te maken van huidfibroblasten van patiënten en neurologische en neurodegeneratieve ziekten te bestuderen. We hebben het gevoel dat dit protocol veel voordelen heeft ten opzichte van klassieke IPS-technologie, waaronder de snelheid, maar in bepaalde gevallen ook een ernstiger ziektefenotype krijgt. We bestuderen neurologische en neurodegeneratieve ziekten en met behulp van huidmonsters kunnen we ook de diversiteit van de patiënten echt eva...

Video: In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Developmental Biology

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

Bioengineering

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

Efficient and Scalable Generation of Human Ventral Midbrain Astrocytes from Human-Induced Pluripotent Stem Cells

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal degeneration has diverse causes in Parkinson's, including non-cell autonomous mechanisms mediated by astrocytes. Throughout the CNS, astrocytes are essential for neuronal survival and function, as they maintain metabolic homeostasis in the neural environment. Astrocytes interact with the immune cells of the CNS, microglia, to modulate neuroinflammation, which is observed from the earliest stages of Parkinson's, and has a direct impact on the progression of its pathology. In diseases with a chronic neuroinflammatory element, including Parkinson's, astrocytes acquire a neurotoxic phenotype, and thus enhance neurodegeneration. Consequently, astrocytes are a potential therapeutic target to slow or halt disease, but this will require a deeper understanding of their properties and roles in Parkinson's. Accurate models of human ventral midbrain astrocytes for in vitro study are therefore urgently required.
We have developed a protocol to generate high purity cultures of ventral midbrain-specific astrocytes (vmAstros) from hiPSCs that can be used for Parkinson's research. vmAstros can be routinely produced from multiple hiPSC lines, and express specific astrocytic and ventral midbrain markers. This protocol is scalable, and thus suitable for high-throughput applications, including for drug screening. Crucially, the hiPSC derived-vmAstros demonstrate immunomodulatory characteristics typical of their in vivo counterparts, enabling mechanistic studies of neuroinflammatory signaling in Parkinson's.

Here, we present a method for reproducible generation of ventral midbrain patterned astrocytes from hiPSCs and protocols for their characterization to assess phenotype and function.

In Parkinson's disease, progressive dysfunction and degeneration of dopamine neurons in the ventral midbrain cause life-changing symptoms. Neuronal ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) pathophysiology in vitro. Multi-electrode array (MEA) recordings are a means to record electrical field potentials from large populations of neurons and analyze network activity over time. It was previously demonstrated that the presence of hiPSC-A that are differentiated using techniques to promote a spinal cord astrocyte phenotype improved maturation and electrophysiological activity of regionally specific spinal cord hiPSC-motor neurons (MN) when compared to those cultured without hiPSC-A or in the presence of rodent astrocytes. Described here is a method to co-culture spinal cord hiPSC-A with hiPSC-MN and record electrophysiological activity using MEA recordings. While the differentiation protocols described here are particular to astrocytes and neurons that are regionally specific to the spinal cord, the co-culturing platform can be applied to astrocytes and neurons differentiated with techniques specific to other fates, including cortical hiPSC-A and hiPSC-N. These protocols aim to provide an electrophysiological assay to inform about glia-neuron interactions and provide a platform for testing drugs with therapeutic potential in ALS.

We describe a method for differentiating spinal cord human induced pluripotent-derived astrocytes and neurons and their co-culture for electrophysiological recording.

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through multipotent intermediates. This technique not only holds great promises in the field of disease modeling, as it allows to convert, for example, fibroblasts for patients suffering neurodegenerative diseases into neurons, but also represents a promising alternative for cell-based replacement therapies. In this context, a major scientific breakthrough was the demonstration that differentiated non-neural cells within the central nervous system, such as astrocytes, could be converted into functional neurons in vitro. Since then, in vitro direct reprogramming of astrocytes into neurons has provided substantial insights into the molecular mechanisms underlying forced identity conversion and the hurdles that prevent efficient reprogramming. However, results from in vitro&#160;experiments performed in different labs are difficult to compare due to differences in the methods used to isolate, culture, and reprogram astrocytes. Here, we describe a detailed protocol to reliably isolate and culture astrocytes with high purity from different regions of the central nervous system of mice at postnatal ages via magnetic cell sorting. Furthermore, we provide protocols to reprogram cultured astrocytes into neurons via viral transduction or DNA transfection. This streamlined and standardized protocol can be used to investigate the molecular mechanisms underlying cell identity maintenance, the establishment of a new neuronal identity, as well as the generation of specific neuronal subtypes and their functional properties.

Here we describe a detailed protocol to generate highly enriched cultures of astrocytes derived from different regions of the central nervous system of postnatal mice and their direct conversion into functional neurons by the forced expression of transcription factors.

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

3D Bioprinted Cocultures of iPSC-Derived Neurons and Astrocytes

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D models&#160;do not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Author Spotlight: Advancing 3D Cell Modeling &#8211; A High-Throughput Approach for Neural Cocultures 

Here, we present a protocol to produce 3D-bioprinted cocultures of iPSC-derived neurons and astrocytes. This coculture model, generated within a hydrogel scaffold in 96- or 384-well formats, demonstrates high post-print viability and neurite outgrowth within 7 days and shows the expression of maturity markers for both cell types.

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

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