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>

<ol>
	<li>Leenaars, C. H. C., 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=Animal+to+human+translation:+a+systematic+scoping+review+of+reported+concordance+rates.">Animal to human translation: a systematic scoping review of reported concordance rates.</a> Journal of Translational Medicine. 17 (1), 223 (2019).</li><li>Rhrissorrakrai, K., 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=Understanding+the+limits+of+animal+models+as+predictors+of+human+biology:+lessons+learned+from+the+sbv+IMPROVER+Species+Translation+Challenge.">Understanding the limits of animal models as predictors of human biology: lessons learned from the sbv IMPROVER Species Translation Challenge.</a> Bioinformatics. 31 (4), 471-483 (2015).</li><li>Hartung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thoughts+on+limitations+of+animal+models.">Thoughts on limitations of animal models.</a> Parkinsonism &amp; Related Disorders. 14, 81-83 (2008).</li><li>Ladewig, J., Koch, P., Brustle, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveling+Waddington:+the+emergence+of+direct+programming+and+the+loss+of+cell+fate+hierarchies.">Leveling Waddington: the emergence of direct programming and the loss of cell fate hierarchies.</a> Nature Reviews Molecular Cell Biology. 14 (4), 225-236 (2013).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Hamada, M., Malureanu, L. A., Wijshake, T., Zhou, W., van Deursen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+to+pluripotency+can+conceal+somatic+cell+chromosomal+instability.">Reprogramming to pluripotency can conceal somatic cell chromosomal instability.</a> PLoS Genetics. 8 (8), 1002913 (2012).</li><li>Stricker, S. H., Gotz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+neural+lineage+elaboration:+Implications+for+therapeutic+reprogramming.">Epigenetic regulation of neural lineage elaboration: Implications for therapeutic reprogramming.</a> Neurobiology of Disease. 148, 105174 (2020).</li><li>Tang, Y., Liu, M. L., Zang, T., Zhang, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Reprogramming+Rather+than+iPSC-Based+Reprogramming+Maintains+Aging+Hallmarks+in+Human+Motor+Neurons.">Direct Reprogramming Rather than iPSC-Based Reprogramming Maintains Aging Hallmarks in Human Motor Neurons.</a> Frontiers in Molecular Neuroscience. 10, 359 (2017).</li><li>Kelaini, S., Cochrane, A., Margariti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+adult+cells:+avoiding+the+pluripotent+state.">Direct reprogramming of adult cells: avoiding the pluripotent state.</a> Stem Cells Cloning. 7, 19-29 (2014).</li><li>Kim, E., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+and+biomaterials+for+controlling+cell+fate.">Direct reprogramming and biomaterials for controlling cell fate.</a> Biomaterials Research. 20, 39 (2016).</li><li>Kim, J., 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+reprogramming+of+mouse+fibroblasts+to+neural+progenitors.">Direct reprogramming of mouse fibroblasts to neural progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (19), 7838-7843 (2011).</li><li>Tian, C., 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+dermal+fibroblasts+into+neural+progenitor+cells+by+a+novel+cocktail+of+defined+factors.">Direct conversion of dermal fibroblasts into neural progenitor cells by a novel cocktail of defined factors.</a> Current Molecular Medicine. 12 (2), 126-137 (2012).</li><li>Mertens, J., Marchetto, M. C., Bardy, C., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+cell+reprogramming,+differentiation+and+conversion+technologies+in+neuroscience.">Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience.</a> Nature Reviews Neuroscience. 17 (7), 424-437 (2016).</li><li>Kim, J., Ambasudhan, R., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+lineage+reprogramming+to+neural+cells.">Direct lineage reprogramming to neural cells.</a> Current Opinion in Neurobiology. 22 (5), 778-784 (2012).</li><li>Ieda, 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+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li><li>Ambasudhan, R., 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+reprogramming+of+adult+human+fibroblasts+to+functional+neurons+under+defined+conditions.">Direct reprogramming of adult human fibroblasts to functional neurons under defined conditions.</a> Cell Stem Cell. 9 (2), 113-118 (2011).</li><li>Kogiso, T., Nagahara, H., Otsuka, M., Shiratori, K., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdifferentiation+of+human+fibroblasts+into+hepatocyte-like+cells+by+defined+transcriptional+factors.">Transdifferentiation of human fibroblasts into hepatocyte-like cells by defined transcriptional factors.</a> Hepatology International. 7 (3), 937-944 (2013).</li><li>Grath, A., Dai, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell+reprogramming+for+tissue+engineering+and+regenerative+medicine.">Direct cell reprogramming for tissue engineering and regenerative medicine.</a> Journal of Biological Engineering. 13, 14 (2019).</li><li>Meyer, K., 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+patient+fibroblasts+demonstrates+non-cell+autonomous+toxicity+of+astrocytes+to+motor+neurons+in+familial+and+sporadic+ALS.">Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (2), 829-832 (2014).</li><li>Lee, 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+Reprogramming+to+Human+Induced+Neuronal+Progenitors+from+Fibroblasts+of+Familial+and+Sporadic+Parkinson's+Disease+Patients.">Direct Reprogramming to Human Induced Neuronal Progenitors from Fibroblasts of Familial and Sporadic Parkinson's Disease Patients.</a> International Journal of Stem Cells. 12 (3), 474-483 (2019).</li><li>Meyer, K., 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=Glia-neuron+interactions+in+neurological+diseases:+Testing+non-cell+autonomy+in+a+dish.">Glia-neuron interactions in neurological diseases: Testing non-cell autonomy in a dish.</a> Brain Research. 1656, 27-39 (2017).</li><li>Verkhratsky, A., Parpura, V., Pekna, M., Pekny, M., Sofroniew, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+in+the+pathogenesis+of+neurodegenerative+diseases.">Glia in the pathogenesis of neurodegenerative diseases.</a> Biochemical Society Transactions. 42 (5), 1291-1301 (2014).</li><li>Ettle, B., Schlachetzki, J. C. M., Winkler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligodendroglia+and+Myelin+in+Neurodegenerative+Diseases:+More+Than+Just+Bystanders.">Oligodendroglia and Myelin in Neurodegenerative Diseases: More Than Just Bystanders.</a> Molecular Neurobiology. 53 (5), 3046-3062 (2016).</li><li>Ferraiuolo, L., 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=Oligodendrocytes+contribute+to+motor+neuron+death+in+ALS+via+SOD1-dependent+mechanism.">Oligodendrocytes contribute to motor neuron death in ALS via SOD1-dependent mechanism.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (42), 6496-6505 (2016).</li><li>Palpagama, T. H., Waldvogel, H. J., Faull, R. L. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; Supplement (50X), serum free</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td>For NPC and base media</td>
		</tr>
		<tr>
			<td>Cryogenic vials&#160; 1.2ML</td>
			<td>Corning</td>
			<td>CLS430658-500EA</td>
			<td>For freezing cell stocks</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX&#160; Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td>For fibroblast and Astrocyte media</td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX&#160; 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&#8217;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 &#956;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/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/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/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/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/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 ...

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Neuroscience

4.8K Views.  The Research Institute at Nationwide Children’s Hospital. 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. 

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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 ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into motor neuron is obtained by ectopic expression of alternative modules of transcription factors, namely Ngn2, Isl1 and Lhx3 (NIL) or Ngn2, Isl1 and Phox2a (NIP). NIL and NIP specify, respectively, spinal and cranial motor neuron identity. Our protocol starts with the generation of modified iPSC lines in which NIL or NIP are stably integrated in the genome via a piggyBac transposon vector. Expression of the transgenes is then induced by doxycycline and leads, in 5 days, to the conversion of iPSCs into MN progenitors. Subsequent maturation, for 7 days, leads to homogeneous populations of spinal or cranial MNs. Our method holds several advantages over previous protocols: it is extremely rapid and simplified; it does not require viral infection or further MN isolation; it allows generating different MN subpopulations (spinal and cranial) with a remarkable degree of maturation, as demonstrated by the ability to fire trains of action potentials. Moreover, a large number of motor neurons can be obtained without purification from mixed populations. iPSC-derived spinal and cranial motor neurons can be used for in vitro modeling of Amyotrophic Lateral Sclerosis and other neurodegenerative diseases of the motor neuron. Homogeneous motor neuron populations might represent an important resource for cell type specific drug screenings.

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

This protocol allows rapid and efficient conversion of induced pluripotent stem cells into motor neurons with a spinal or cranial identity, by ectopic expression of transcription factors from inducible piggyBac vectors.

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...