Name: organotypic cultures of adult human cortex as an ex vivo model for human stem cell transplantation and validation
Uploaded: 2022-12-09T13:20:00
Description: Watch this Scientific Journal Video about Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation at JoVE.com

This work is supported by grants from the Swedish Research Council, the Swedish Brain Foundation, the Swedish Stroke Foundation, Region Sk&#229;ne, The Thorsten and Elsa Segerfalk Foundation, and the Swedish Government Initiative for Strategic Research Areas (StemTherapy).

This protocol follows the guidelines approved by the Regional Ethical Committee, Lund, Sweden (ethical permit number 2021-07006-01). Healthy neocortical tissue was obtained from patients undergoing elective surgery for temporal lobe epilepsy. Informed consent was obtained from all patients.
NOTE: All the tissues obtained were processed regardless of their size. However, tissues smaller than 1-1.5 mm3 in size will be technically challenging to handle and section with a vibratome....

<ol>
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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=Human+brain+slice+culture:+A+useful+tool+to+study+brain+disorders+and+potential+therapeutic+compounds.">Human brain slice culture: A useful tool to study brain disorders and potential therapeutic compounds.</a> Neuroscience Bulletin. 35 (2), 244-252 (2019).</li><li>Verwer, R. W., 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=Injury+response+of+resected+human+brain+tissue+in+vitro.">Injury response of resected human brain tissue in vitro.</a> Brain Pathology. 25 (4), 454-468 (2015).</li><li>Verwer, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organogenesis+in+a+dish:+Modeling+development+and+disease+using+organoid+technologies.">Organogenesis in a dish: Modeling development and disease using organoid technologies.</a> Science. 345 (6194), 1247125 (2014).</li><li>Wang, Z., 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=Organoid+technology+for+brain+and+therapeutics+research.">Organoid technology for brain and therapeutics research.</a> CNS Neuroscience &amp; Therapeutics. 23 (10), 771-778 (2017).</li><li>Wang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neurological+diseases+with+human+brain+organoids.">Modeling neurological diseases with human brain organoids.</a> Frontiers in Synaptic Neuroscience. 10, 15 (2018).</li><li>Palma-Tortosa, S., Coll-San Martin, B., Kokaia, Z., Tornero, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+replacement+in+stem+cell+therapy+for+stroke:+Filling+the+gap.">Neuronal replacement in stem cell therapy for stroke: Filling the gap.</a> Frontiers in Cell and Developmental Biology. 9, 662636 (2021).</li></ol>

Obtaining hACtx slices of high enough quality is the most critical step in this protocol. Cortical tissue is obtained from epileptic patients undergoing resective surgery24. The quality of the resected tissue, as well as the exposure time of the tissue between resection and culture, is critical; the faster the tissue is transferred from the surgery room to the laboratory and cut, the more optimal the organotypic culture will be. Ideally, the tissue should be cut and transferred to...

Neurodegenerative disorders, such as Parkinson&#39;s disease, Alzheimer&#39;s disease, or ischemic stroke, are a group of diseases that share the common feature of neuronal malfunction or death. They are heterogeneous in terms of the brain area and neuronal population affected. Unfortunately, treatments for these diseases are scarce or of limited efficacy due to the lack of animal models that mimic what occurs in the human brain1,2. Stem cell therapy is one of the most promising strategies for brain regeneration3. The generation of neuronal progenitors from stem cells from different sources has been greatly developed in recent years4,5. Recent publications have shown that human induced pluripotent stem (iPS) cell-derived long-term self-renewing neuroepithelial-like stem (lt-NES) cells, following a cortical differentiation protocol and after intracortical transplantation in a rat model with ischemic stroke affecting the somatosensory cortex, generate mature cortical neurons. In addition, the graft-derived neurons received afferent and efferent synaptic connections from the host neurons, showing their integration into the rat neuronal network6,7. The graft-derived axons were myelinated and found in different areas of the rat brain, including the peri-infarct area, corpus callosum, and contralateral somatosensory cortex. Most importantly, iPS cell-derived transplantation reversed motor deficits in stroke animals7.
Even if animal models help to study transplant survival, neuronal integration, and the effect of the grafted cells on motor and cognitive functions, information about interaction between human cells (graft-host) is missing in this system8,9. For this reason, a combined method of long-term human brain organotypic culture with the ex vivo transplantation of human iPS cell-derived neuronal progenitors is described here. Human brain organotypic cultures obtained from neurosurgical resections are physiologically relevant 3D models of the brain that allow researchers to increase their understanding of the human central nervous system circuitry and the most accurate way of testing treatments for human brain disorders. However, not enough research has been done in this context, and in most cases, human hippocampal brain organotypic cultures have been used10,11. The cerebral cortex is affected by several neurodegenerative disorders, such as ischemic stroke12 or Alzheimer&#39;s disease13, so it is important to have a human cortical 3D system that allows us to expand our knowledge and to test and validate different therapeutic strategies. Several studies in the last few years have used cultures from adult human cortical (hACtx) tissue to model human brain diseases14,15,16,17,18,19; however, limited information is available in the context of stem cell therapy. Two studies have already demonstrated the feasibility of the system described here. In 2018, human embryonic stem cells programmed with different transcription factors and transplanted into hACtx tissue were shown to give rise to mature cortical neurons that could integrate into adult human cortical networks20. In 2020, the transplantation of lt-NES cells into the human organotypic system revealed their capacity to differentiate into mature, layer-specific cortical neurons with the electrophysiological properties of functional neurons. The grafted neurons established both afferent and efferent synaptic contacts with the human cortical neurons in the adult brain slices, as corroborated by rabies virus retrograde monosynaptic tracing, whole-cell patch-clamp recordings, and immuno-electron microscopy21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue Cutting and electrophysiology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath temperature controller&#160;</td>
			<td>Luigs &#38; Neumann</td>
			<td>TC0511354</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Merck</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen gas</td>
			<td>Air Liquide</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julaba FL 300</td>
			<td>9661012.03</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Patch-Clamp amplifier</td>
			<td>HEKA electronic</td>
			<td>EPC10</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-Triphosphate disodium salt</td>
			<td>Millipore</td>
			<td>371701</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>HEKA electronic</td>
			<td>Patchmaster 2x91</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>Sutter</td>
			<td>P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dish</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium D-gluconate</td>
			<td>ThermoFisher</td>
			<td>B25135</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber teat + glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Merck</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive: Acryl super glue</td>
			<td>Loctite</td>
			<td>2062278</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>RINSING SOLUTION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (without Ca, Mg, or PhenolRed)</td>
			<td>ThermoFisher Scientific</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Alvetex scaffold 6 well insert</td>
			<td>Reinnervate Ltd</td>
			<td>AVP004-96</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>ThermoFisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>BrainPhys without Phenol Red</td>
			<td>StemCell technologies</td>
			<td>#05791</td>
			<td>Referenced as neuronal medium in the text</td>
		</tr>
		<tr>
			<td>Filter units 250 mL or 500 mL</td>
			<td>Corning Sigma</td>
			<td>CLS431096/97</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15750037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax Supplement (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>Referenced as L-glutamine in the text</td>
		</tr>
		<tr>
			<td>Rubber teat + Glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GENERATION OF lt-NES cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol 50 mM</td>
			<td>ThermoFisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Free Recombinant EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Suplemment (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin Fraction V (7.5% solution)</td>
			<td>ThermoFisher Scientific</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopamine, V. calcifornicum</td>
			<td>Calbiochem</td>
			<td># 239803</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+) Glucose solution (45%)</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2438-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffer Saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-144</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential aminoacids solutions (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100 x)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Merk</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human BMP-4 Protein</td>
			<td>R&#38;D Systems</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Wnt-3a Protein</td>
			<td>R&#38;D Systems</td>
			<td>5036-WN</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean Trypsin Inhibitor, powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>17075029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile deionized water</td>
			<td>MilliQ</td>
			<td></td>
			<td>MilliQ filter system</td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.25%)</td>
			<td>Sigma</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT FOR CELL CULTURE&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable volume pipettes 10, 100, 200, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix ESC-qualified (Matrigel)</td>
			<td>Corning</td>
			<td>CLS354277-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich Centrifugen</td>
			<td>Rotina 420R</td>
			<td>5% CO2, 37 &#176;C</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoForma Steri-Cult CO2</td>
			<td>HEPA Class100</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell cutting tool 0.190-0.210 mm</td>
			<td>Vitrolife</td>
			<td>14601</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubes</td>
			<td>Sarstedt</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Glass Pasteur Pipettes 150 mm</td>
			<td>VWR</td>
			<td>612-1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipette tips 0.1-1000 &#160;&#181;L</td>
			<td>Biotix VWR</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Serological Pipettes 5, 10, 25, 50 mL</td>
			<td>Costar</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks Nunc</td>
			<td>ThermoFisher Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>IMMUNOHISTOCHEMISTRY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-mouse IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>715-545-151</td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-chicken IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>703-545-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 647-conjugated Streptavidin</td>
			<td>Jackson ImmunoReserach</td>
			<td>016-600-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Jackson ImmunoReserach</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>Merk Millipore</td>
			<td>AB16901</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-MAP2&#160;</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-chicken IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>703-165-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-goat IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>705-165-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-mouse IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>715-165-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Diazabicyclooctane (DABCO)</td>
			<td>Sigma Aldrich</td>
			<td>D27802</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>Goat anti-AIF1 (C-terminal)&#160;</td>
			<td>Biorad</td>
			<td>AHP2024</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Molecular Probes</td>
			<td></td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>Mouse anti-MBP&#160;</td>
			<td>BioLegend</td>
			<td>808402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SC123&#160;</td>
			<td>Stem Cells Inc</td>
			<td>AB-123-U-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Merk Millipore</td>
			<td>S30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>150127</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phospate Buffer Saline, KPBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Potassium dihydrogen Phospate (KH2PO4)</td>
			<td>Merk Millipore</td>
			<td>104873</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Potassium phospate dibasic (K2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-NeuN&#160;</td>
			<td>Abcam</td>
			<td>ab104225</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2&#160;</td>
			<td>Abcam</td>
			<td>ab109186</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-TMEM119&#160;</td>
			<td>Abcam</td>
			<td>ab185333</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>S2002-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Tri-Sodium Citrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher Scientific</td>
			<td>327371000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT FOR IMMUNOHISTOCHEMISTRY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 780</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides 76 mm x 26 mm</td>
			<td>VWR</td>
			<td>630-1985</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslips 24 mm x 60 mm</td>
			<td>Marienfeld</td>
			<td>107242</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software</td>
			<td>Zeiss</td>
			<td>ZEN Black edition</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber teat + Glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic cultures of adult human cortex as an ex vivo model for human stem cell transplantation and validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Following the described protocol, hACtx tissue from a patient with temporal lobe epilepsy was collected and processed, as explained above. A few slices were fixed after 24 h in culture to study the starting point of the host tissue. The analysis of different neural cell populations such as neurons (expressing NeuN and Map2, Figure 1A), oligodendrocytes (Olig2 and MBP, Figure 1B), and astrocytes (human-specific GFAP, also named STEM123, Figur...

Watch this Scientific Journal Video about Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation at JoVE.com

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

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Neuroscience

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