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

<ol>
	<li>Kuriakose, D., Xiao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+treatment+of+stroke:+Present+status+and+future+perspectives.">Pathophysiology and treatment of stroke: Present status and future perspectives.</a> International Journal of Molecular Sciences. 21 (20), 7609 (2020).</li><li>Armstrong, M. J., Okun, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+Parkinson+disease:+A+review.">Diagnosis and treatment of Parkinson disease: A review.</a> The Journal of the American Medical Association. 323 (6), 548-560 (2020).</li><li>Lindvall, O., Kokaia, Z., Martinez-Derrano, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+for+human+neurodegenerative+disorders-How+to+make+it+work.">Stem cell therapy for human neurodegenerative disorders-How to make it work.</a> Nature Medicine. 10, 42-50 (2004).</li><li>Reubinoff, B. E., 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=Neural+progenitors+from+human+embryonic+stem+cells.">Neural progenitors from human embryonic stem cells.</a> Nature Biotechnology. 19 (12), 1134-1140 (2001).</li><li>Chandrasekaran, A., 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=Comparison+of+2D+and+3D+neural+induction+methods+for+the+generation+of+neural+progenitor+cells+from+human+induced+pluripotent+stem+cells.">Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.</a> Stem Cell Research. 25, 139-151 (2017).</li><li>Tornero, D., 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=Synaptic+inputs+from+stroke-injured+brain+to+grafted+human+stem+cell-derived+neurons+activated+by+sensory+stimuli.">Synaptic inputs from stroke-injured brain to grafted human stem cell-derived neurons activated by sensory stimuli.</a> Brain. 140 (3), 692-706 (2017).</li><li>Palma-Tortosa, S., 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=Activity+in+grafted+human+iPS+cell-derived+cortical+neurons+integrated+in+stroke-injured+rat+brain+regulates+motor+behavior.">Activity in grafted human iPS cell-derived cortical neurons integrated in stroke-injured rat brain regulates motor behavior.</a> Proceedings of the National Academy of Sciencesof the United States of America. 117 (16), 9094-9100 (2020).</li><li>Robinson, N. B., 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=The+current+state+of+animal+models+in+research:+A+review.">The current state of animal models in research: A review.</a> International Journal of Surgery. 72, 9-13 (2019).</li><li>Akhtar, 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+flaws+and+human+harms+of+animal+experimentation.">The flaws and human harms of animal experimentation.</a> Cambridge Quarterly Healthcare Ethics. 24 (4), 407-419 (2015).</li><li>Gonzalez-Ramos, A., 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+stem+cell-derived+GABAergic+neurons+functionally+integrate+into+human+neuronal+networks.">Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks.</a> Scientific Reports. 11, 22050 (2021).</li><li>Noraberg, 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=Organotypic+hippocampal+slice+cultures+for+studies+of+brain+damage,+neuroprotection+and+neurorepair.">Organotypic hippocampal slice cultures for studies of brain damage, neuroprotection and neurorepair.</a> Current Drug Targets. CNS &amp; Neurological Disorders. 4 (4), 435-452 (2005).</li><li>Delavaran, H., 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=Proximity+of+brain+infarcts+to+regions+of+endogenous+neurogenesis+and+involvement+of+striatum+in+ischaemic+stroke.">Proximity of brain infarcts to regions of endogenous neurogenesis and involvement of striatum in ischaemic stroke.</a> European Journal of Neurology. 20 (3), 473-479 (2013).</li><li>Sabuncu, M. 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=The+dynamics+of+cortical+and+hippocampal+atrophy+in+Alzheimer+disease.">The dynamics of cortical and hippocampal atrophy in Alzheimer disease.</a> Archives of Neurology. 68 (8), 1040-1048 (2011).</li><li>Eugene, E., 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=An+organotypic+brain+slice+preparation+from+adult+patients+with+temporal+lobe+epilepsy.">An organotypic brain slice preparation from adult patients with temporal lobe epilepsy.</a> The Journal of Neuroscience Methods. 235, 234-244 (2014).</li><li>Mendes, N. D., 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=Free-floating+adult+human+brain-derived+slice+cultures+as+a+model+to+study+the+neuronal+impact+of+Alzheimer's+disease-associated+A&#946;+oligomers.">Free-floating adult human brain-derived slice cultures as a model to study the neuronal impact of Alzheimer's disease-associated A&#946; oligomers.</a> The Journal of Neuroscience Methods. 307, 203-209 (2018).</li><li>Kalmbach, B. E., 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=Signature+morpho-electric,+transcriptomic,+and+dendritic+properties+of+human+layer+5+neocortical+pyramidal+neurons.">Signature morpho-electric, transcriptomic, and dendritic properties of human layer 5 neocortical pyramidal neurons.</a> Neuron. 109 (18), 2914-2927 (2021).</li><li>Barth, 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=Microglial+inclusions+and+neurofilament+light+chain+release+follow+neuronal+alpha-synuclein+lesions+in+long-term+brain+slice+cultures.">Microglial inclusions and neurofilament light chain release follow neuronal alpha-synuclein lesions in long-term brain slice cultures.</a> Molecular Neurodegeneration. 16 (1), 54 (2021).</li><li>Almeida, 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=Neural+infection+by+oropouche+virus+in+adult+human+brain+slices+induces+an+inflammatory+and+toxic+response.">Neural infection by oropouche virus in adult human brain slices induces an inflammatory and toxic response.</a> Frontiers in Neuroscience. 15, 674576 (2021).</li><li>Schwarz, 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=Human+cerebrospinal+fluid+promotes+long-term+neuronal+viability+and+network+function+in+human+neocortical+organotypic+brain+slice+cultures.">Human cerebrospinal fluid promotes long-term neuronal viability and network function in human neocortical organotypic brain slice cultures.</a> Scientific Reports. 7, 12249 (2017).</li><li>Miskinyte, G., 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+human+fibroblasts+to+functional+excitatory+cortical+neurons+integrating+into+human+neural+networks.">Direct conversion of human fibroblasts to functional excitatory cortical neurons integrating into human neural networks.</a> Stem Cell Research &amp; Therapy. 8 (1), 207 (2017).</li><li>Gronning Hansen, 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=Grafted+human+pluripotent+stem+cell-derived+cortical+neurons+integrate+into+adult+human+cortical+neural+circuitry.">Grafted human pluripotent stem cell-derived cortical neurons integrate into adult human cortical neural circuitry.</a> Stem Cells Translational Medicine. 9 (11), 1365-1377 (2020).</li><li>Falk, A., 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=Capture+of+neuroepithelial-like+stem+cells+from+pluripotent+stem+cells+provides+a+versatile+system+for+in+vitro+production+of+human+neurons.">Capture of neuroepithelial-like stem cells from pluripotent stem cells provides a versatile system for in vitro production of human neurons.</a> PLoS One. 7 (1), 29597 (2012).</li><li>Avaliani, 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=Optogenetics+reveal+delayed+afferent+synaptogenesis+on+grafted+human-induced+pluripotent+stem+cell-derived+neural+progenitors.">Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.</a> Stem Cells. 32 (12), 3088-3098 (2014).</li><li>Engel, 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=Practice+parameter:+temporal+lobe+and+localized+neocortical+resections+for+epilepsy.">Practice parameter: temporal lobe and localized neocortical resections for epilepsy.</a> Epilepsia. 44 (6), 741-751 (2003).</li><li>Qi, X. 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. 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=Altered+loyalties+of+neuronal+markers+in+cultured+slices+of+resected+human+brain+tissue.">Altered loyalties of neuronal markers in cultured slices of resected human brain tissue.</a> Brain Pathology. 26 (4), 523-532 (2016).</li><li>Xu, L., Wang, J., Ding, Y., Wang, L., Zhu, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+knowledge+of+microglia+in+traumatic+spinal+cord+injury.">Current knowledge of microglia in traumatic spinal cord injury.</a> Frontiers in Neurology. 12, 796704 (2021).</li><li>Jones, R. S., da Silva, A. B., Whittaker, R. G., Woodhall, G. L., Cunningham, M. O. <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+slices+for+epilepsy+research:+Pitfalls,+solutions+and+future+challenges.">Human brain slices for epilepsy research: Pitfalls, solutions and future challenges.</a> Journal of Neuroscience Methods. 260, 221-232 (2016).</li><li>Schwarz, 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=Long-term+adult+human+brain+slice+cultures+as+a+model+system+to+study+human+CNS+circuitry+and+disease.">Long-term adult human brain slice cultures as a model system to study human CNS circuitry and disease.</a> Elife. 8, 48417 (2019).</li><li>Lancaster, M. A., Knoblich, J. 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>

The authors declare no conflicts of interest.

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 the cell culture lab within the first few hours after collection. The oxygenation of the tissue during this process also improves the quality of the slices. In this regard, the bigger the tissue sample, the lower the concentration of oxygen reaching the core and, thus, the lower the viability if the tissue is not cut in time. If the quality of the host tissue is not optimal, the validation of stem cell therapies will not be possible. 
When the cortical tissue is transferred from the human brain to a plate, some limitations must be taken into account. During the cutting process, a large number of axons are dissected, inducing neuronal damage that will lead to inflammatory processes such as microglia activation25. For this reason, even if the tissue is considered healthy when located in the human brain, the cell behavior in culture could be different due to the partial damage suffered during the resection and preparation of the organotypic sections26,27. Changes in the microglia population could be monitored using different techniques, including the measurement of the released cytokine levels or the assessment of morphological changes28. Importantly, although microglia activation was observed at 2 weeks in culture as a result of the change in environment from a whole organ to ex vivo culture conditions, the neurons were still viable at this timepoint, as shown by the neuronal staining and the analysis of their electrophysiological properties. Thicker tissue slices are better preserved; however, the penetration of nutrients to the inner part of the slice is affected, resulting in partial tissue death. For this reason, 300 &#181;m is the optimal thickness for organotypic culture. 
Organotypic cultures of hACtx tissue have clear advantages compared to other 3D culture methods such as organoids or spheroids. The source is a fully developed human brain, meaning that the cellular and matrix environment, as well as the maturation status of the different cell populations, are the same as those generally found in the adult human brain25,29,30. Organoids are more similar to fetal tissues, which is optimal for some research fields such as the modeling of developmental disorders31 but not, for example, for the study of neurodegenerative diseases, which affect mainly the adult population and have a late onset32,33. Most importantly, the organotypic culture of human tissue is, to date, the only human system that allows the validation of cell therapies.
Most of the knowledge about stem cell transplantation for neuronal replacement in neurodegenerative disorders is derived from in vivo animal modeling. Even though these systems are extremely valuable for assessing the modulatory effect of transplanted cells in the damaged host circuitry, unfortunately, therapies tested in this setup normally fail when translated to the clinic due to the clear differences between rodents and humans34. For this reason, the organotypic culture of hACtx tissue is an excellent strategy for modeling human neurodegenerative diseases that affect the cortex due to the possibility to study the interactions between human neural populations and the preservation of the human brain structure25.
In summary, this combined methodology of long-term organotypic cultures of the adult human cortex and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors is a promising strategy for the validation of stem cell-based therapies, which can facilitate the clinical translation of neuronal replacement strategies for stimulating functional recovery in the damaged brain.

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><tbody><tr><td>&lt;strong&gt;Tissue Cutting and electrophysiology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate magnesium salt</td><td>Sigma</td><td>A9187</td><td></td></tr><tr><td>Bath temperature controller&amp;nbsp;</td><td>Luigs &amp;amp; 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&#039;-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&amp;nbsp;</td><td></td></tr><tr><td>Vibratome&amp;nbsp;</td><td>Leica</td><td>VT1200 S</td><td></td></tr><tr><td>&lt;strong&gt;RINSING SOLUTION&lt;/strong&gt;</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>&lt;strong&gt;MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE&lt;/strong&gt;</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>&lt;strong&gt;GENERATION OF lt-NES cells&lt;/strong&gt;</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&#039;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&amp;amp;D Systems</td><td>314-BP-010</td><td></td></tr><tr><td>Recombinant Human Wnt-3a Protein</td><td>R&amp;amp;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>&lt;strong&gt;EQUIPMENT FOR CELL CULTURE&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adjustable volume pipettes 10, 100, 200, 1000 &amp;micro;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 &amp;deg;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 &amp;nbsp;&amp;micro;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>&lt;strong&gt;IMMUNOHISTOCHEMISTRY&lt;/strong&gt;</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&amp;nbsp;</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)&amp;nbsp;</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&amp;nbsp;</td><td>BioLegend</td><td>808402</td><td></td></tr><tr><td>Mouse anti-SC123&amp;nbsp;</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>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Distilled water</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Potassium dihydrogen Phospate (KH2PO4)</td><td>Merk Millipore</td><td>104873</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Potassium phospate dibasic (K2HPO4)</td><td>Sigma Aldrich</td><td>P3786</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Sodium chloride (NaCl)</td><td>Sigma Aldrich</td><td>S3014</td><td></td></tr><tr><td>Rabbit anti-NeuN&amp;nbsp;</td><td>Abcam</td><td>ab104225</td><td></td></tr><tr><td>Rabbit anti-Olig2&amp;nbsp;</td><td>Abcam</td><td>ab109186</td><td></td></tr><tr><td>Rabbit anti-TMEM119&amp;nbsp;</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>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Distilled water</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Tri-Sodium Citrate</td><td>Sigma Aldrich</td><td>S1804-500G</td><td></td></tr><tr><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 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&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT FOR IMMUNOHISTOCHEMISTRY&lt;/strong&gt;</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, Figure 1C) showed optimal preservation of the tissue.
The next step was to study how the culture conditions affect the neuronal viability in human tissue. For this purpose, the staining of NeuN and Map2 was performed after 2 weeks of culture. At the studied timepoint, the expression of both these neuronal markers was still present in the tissue (Figure 2A). Additionally, electrophysiological recordings were performed to assess the functionality. The recordings using whole-cell patch clamp showed that the neurons had sustained RMP (&#8722;70 mV on average) and membrane input resistance (Ri) (300 M&#8486; on average), comparable to neurons from acute preparations21,23. Overall, the cells were slightly less active than in fresh tissue, although the majority of the cells were still able to fire at least one (Figure 2B-E), if not multiple, action potentials (APs, Figure 2F-I), and fast inward sodium and slow outward potassium currents were present upon step current injections in voltage-clamp mode (Figure 2C-E,G-I). Taken together, these recordings indicated that the neurons in organotypic cultures were relatively healthy and exhibited typical neurophysiological intrinsic properties.
Furthermore, the effect of culture on microglia activation was assessed by Tmem119 and Iba1 staining in 24 h (Figure 3A) and 2 week cultured (Figure 3B) tissue. As expected, some changes in microglial appearance were observed. After 2 weeks in culture, they became less ramified and acquired a more activated morphology compared to acute tissue.
After characterizing the host tissue, the transplantation of the lt-NES cell-derived progenitors was performed as follows. The GFP-lt-NES cells were differentiated for 7 days and grafted into 1 week cultured hACtx tissue (Figure 4A). An overview of the transplantation was observed using immunohistochemistry with GFP (Figure 4B,C). The results were compared with those of previous transplantations in tissue that was poorly preserved due to there being a longer time window between resection and plating. The images show that, in the optimal system, 4 weeks after ex vivo transplantation, the grafted GFP-lt-NES cells exhibited extended neurites and extensive and complex arborizations throughout the whole organotypic culture (Figure 4B). The poorly preserved tissue did not allow for successful transplantation due to the poor host connectivity. Barely any grafted cell survived; moreover, debris and unspecific labeling of antibodies on dead cells were broadly observed throughout the human slice (Figure 4C).
Regarding the electrophysiological properties of the grafted cells, it was found that, in the case of successful transplantation, the cells became not only morphologically but also functionally active mature neurons with repetitive and often spontaneous APs, fast inward sodium and slow outward potassium currents, and a certain level of synaptic activity, indicating functional integration of the graft with the host tissue 4 weeks post grafting (Figure 4D-H).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64234/64234fig01.jpg" /> 
Figure 1: Characterization of the different cell populations in the hACtx tissue after 24 h in culture. Representative confocal images of hACtx tissue showing the presence of (A) neurons (expressing NeuN and Map2), (B) oligodendrocytes (Olig2 and MBP), and (C) astrocytes (human-specific GFAP [STEM123]). Nuclear staining (Ho: Hoechst, blue) is included in the individual and merged panels. Scale bar = 20 &#181;m. The white arrows indicate colocalization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64234/64234fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64234/64234fig02.jpg" /> 
Figure 2: Characterization and electrophysiological properties of hACtx neurons after 2 weeks in organotypic culture. (A) Representative confocal images of hACtx tissue showing NeuN and Map2 expression. (B-I) Examples of the cortical neurons recorded in 2-week-old hACtx tissue. (B,F) Biocytin labeling of the recorded neuron (red), together with nuclear staining (Ho: Hoechst, blue), included in a merged panel. Scale bars = 20 &#181;m. Whole-cell patch-clamp recording traces showing examples of a cell with (C-E) single, or (G-I) multiple APs. APs were induced either by (C,G) a 250 pA step, or (D,H) a 0 to 300 pA ramped current injection at RMP. The insets indicate a magnified view of one of the APs in each case. (E,I) Inward sodium and outward potassium currents were observed in both cell examples at the voltage depolarization steps applied from &#8722;70 mV in 10 mV increments in voltage-clamp mode. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64234/64234fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64234/64234fig03.jpg" /> 
Figure 3: Characterization of the microglia population in the hACtx tissue. Confocal images of the hACtx tissue showing the expression of Iba1 and Tmem119 after (A) 24 h and (B) 2 weeks in culture. Nuclear staining (Ho: Hoechst, blue) is included in the individual and merged panels. Scale bar = 20 &#181;m. The white arrows indicate colocalization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64234/64234fig03large.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/64234/64234fig04.jpg" /> 
Figure 4: Overview and electrophysiological properties of lt-NES cell-derived neurons 4 weeks after the ex vivo transplantation of GFP-lt-NES cells. (A) Experimental design. Diff = differentiation. (B,C) Representative confocal pictures of grafted GFP-lt-NES cells in (B) well-preserved and (C) poorly preserved hACtx tissue. Scale bars = 50 &#181;m. (D) Example trace of a graft-derived neuron spontaneously firing APs. The inset indicates a magnified view of one of the APs. Repetitive APs could be induced by a (E) step (50 pA) or (F) ramped (0 to 300 pA) injection of depolarizing current from the potential of &#8722;70 mV. (G) Inward sodium and outward potassium currents were induced by 10 mV depolarizing steps in voltage-clamp mode from a holding potential of -70 mV. (H) In voltage-clamp mode, spontaneous postsynaptic currents (sPSCs) could be observed in the graft-derived neurons at a holding potential of &#8722;70 mV. The insets indicate a magnified view of some of the sPSCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64234/64234fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

organotypic-cultures-adult-human-cortex-as-an-ex-vivo-model-for-human

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.3K Views.  Lund University. Organotypische culturen van de menselijke volwassen cortex zijn tot nu toe het enige systeem voor het testen van stamceltherapieën voor beschadigde cortex dat het mogelijk maakt om de interactie tussen menselijke geënte en menselijke gastheercellen te bestuderen. Een therapie getest in diermodellen faalt meestal wanneer vertaald naar de klinische instellingen vanwege de verschillen tussen knaagdier- en menselijke cellen. Hier kunnen we getransplanteerde cellen, overleving, differentiatie en...

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

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Developmental Biology

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Medicine

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

2D and 3D Human Induced Pluripotent Stem Cell-Based Models to Dissect Primary Cilium Involvement during Neocortical Development

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older centriole during the G1/G0 phase of the cell cycle, while they disassemble as the cells re-enter the cell cycle at the G2/M phase boundary. They function as signal hubs, by detecting and transducing extracellular signals crucial for many cell processes. Similar to most cell types, all neocortical neural stem and progenitor cells (NSPCs) have been shown harboring a PC allowing them to sense and transduce specific signals required for the normal cerebral cortical development. Here, we provide detailed protocols to generate and characterize two-dimensional (2D) and three-dimensional (3D) cell-based models from human induced pluripotent stem cells (hIPSCs) to further dissect the involvement of PC during neocortical development. In particular, we present protocols to study the PC biogenesis and function in 2D neural rosette-derived NSPCs including the transduction of the Sonic Hedgehog (SHH) pathway. To take advantage of the three-dimensional (3D) organization of cerebral organoids, we describe a simple method for 3D imaging of in toto immunostained cerebral organoids. After optical clearing, rapid acquisition of entire organoids allows detection of both centrosomes and PC on neocortical progenitors and neurons of the whole organoid. Finally, we detail the procedure for immunostaining and clearing of thick free-floating organoid sections preserving a significant degree of 3D spatial information and allowing for the high-resolution acquisition required for the detailed qualitative and quantitative analysis of PC biogenesis and function.

We present detailed protocols for the generation and characterization of 2D and 3D human induced pluripotent stem cell (hIPSC)-based models of neocortical development as well as complementary methodologies enabling qualitative and quantitative analysis of primary cilium (PC) biogenesis and function.

Primary cilia (PC) are non-motile dynamic microtubule-based organelles that protrude from the surface of most mammalian cells. They emerge from the older ...

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

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

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