Name: scalable generation of mature cerebellar organoids from human pluripotent stem cells and characterization by immunostaining
Uploaded: 2020-06-13T13:00:00
Description: Watch this Scientific Journal Video about Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining at JoVE.com

This work was supported by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project N. 007317, PD/BD/105773/2014 to T.P.S and PD/BD/128376/2017 to D.E.S.N.), projects co-funded by FEDER (POR Lisboa 2020&#8212;Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research grant LISBOA-01-0145-FEDER-029298. Funding was also received from the European Union&#39;s Horizon 2020 Research and Innovation Programme, under the Grant Agreement number 739572&#8212;The Discoveries Centre for Regenerative and Precision Medicine H2020-WIDESPREAD-01-2016-2017.

1. Passaging and maintenance of human iPSCs in monolayer culture
<ol>
	<li>Preparation of plates
	<ol>
		<li>Thaw the basement membrane matrix (see Table of Materials) stock at 4 &#176;C and prepare 60 &#956;L aliquots. Freeze the aliquots at -20 &#176;C.</li>
		<li>To coat the wells of a 6 well plate, thaw one aliquot of the basement membrane matrix on ice. Once thawed add 60 &#181;L to 6 mL of DMEM-F12. Gently resuspend by pipetting up and down.</li>
		<li>Add 1 mL of diluted basement membrane matrix...

<ol>
	<li>Takahashi, 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=Induction+of+Pluripotent+Stem+Cells+from+Adult+Human+Fibroblasts+by+Defined+Factors.">Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Thomson, 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=Embryonic+Stem+Cell+Lines+Derived+from+Human+Blastocysts.">Embryonic Stem Cell Lines Derived from Human Blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Lancaster, M. 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=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Ishida, Y., 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=Vulnerability+of+Purkinje+Cells+Generated+from+Spinocerebellar+Ataxia+Type+6+Patient-Derived+iPSCs.">Vulnerability of Purkinje Cells Generated from Spinocerebellar Ataxia Type 6 Patient-Derived iPSCs.</a> Cell Reports. 17 (6), 1482-1490 (2016).</li><li>Liu, Y., Zhang, S. C. <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+cells+as+a+model+of+motoneuron+development+and+diseases.">Human stem cells as a model of motoneuron development and diseases.</a> Annals of the New York Academy of Sciences. 1198, 192-200 (2010).</li><li>Mariani, J., Coppola, G., Pelphrey, K. A., Howe, J. R., Vaccarino, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXG1-Dependent+Dysregulation+of+GABA/+Glutamate+Neuron+Differentiation+in+Autism+Spectrum+Disorders.">FOXG1-Dependent Dysregulation of GABA/ Glutamate Neuron Differentiation in Autism Spectrum Disorders.</a> Cell. 162 (2), 375-390 (2015).</li><li>Muguruma, K., Nishiyama, A., Kawakami, H., Hashimoto, K., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Organization+of+Polarized+Cerebellar+Tissue+in+3D+Culture+of+Human+Pluripotent+Stem+Cells.">Self-Organization of Polarized Cerebellar Tissue in 3D Culture of Human Pluripotent Stem Cells.</a> Cell Reports. 10 (4), 537-550 (2015).</li><li>Qian, X., 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=Generation+of+human+brain+region-specific+organoids+using+a+miniaturized+spinning+bioreactor.">Generation of human brain region-specific organoids using a miniaturized spinning bioreactor.</a> Nature Protocols. 13 (3), 565-580 (2018).</li><li>Aubry, 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=Striatal+progenitors+derived+from+human+ES+cells+mature+into+DARPP32+neurons+in+vitro+and+in+quinolinic+acid-lesioned+rats.">Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats.</a> Proceedings of the National Academy of Sciences. 105 (43), 16707-16712 (2008).</li><li>Gunhanlar, 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=A+simplified+protocol+for+differentiation+of+electrophysiologically+mature+neuronal+networks+from+human+induced+pluripotent+stem+cells.">A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.</a> Molecular Psychiatry. 23 (5), 1336-1344 (2018).</li><li>Hu, B. Y., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+spinal+motor+neurons+from+pluripotent+human+stem+cells.">Differentiation of spinal motor neurons from pluripotent human stem cells.</a> Nature Protocols. 4 (9), 1295-1304 (2009).</li><li>Jo, 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=Midbrain-like+Organoids+from+Human+Pluripotent+Stem+Cells+Contain+Functional+Dopaminergic+and+Neuromelanin-Producing+Neurons.">Midbrain-like Organoids from Human Pluripotent Stem Cells Contain Functional Dopaminergic and Neuromelanin-Producing Neurons.</a> Cell Stem Cell. 19 (2), 248-257 (2016).</li><li>Muguruma, 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=Ontogeny-recapitulating+generation+and+tissue+integration+of+ES+cell-derived+Purkinje+cells.">Ontogeny-recapitulating generation and tissue integration of ES cell-derived Purkinje cells.</a> Nature Neurosciences. 13 (10), 1171-1180 (2010).</li><li>Watson, L. M., Wong, M. M. K., Vowles, J., Cowley, S. A., Becker, E. B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simplified+Method+for+Generating+Purkinje+Cells+from+Human-Induced+Pluripotent+Stem+Cells.">A Simplified Method for Generating Purkinje Cells from Human-Induced Pluripotent Stem Cells.</a> The Cerebellum. 17 (4), 419-427 (2018).</li><li>Wang, 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=Differentiation+of+human+induced+pluripotent+stem+cells+to+mature+functional+Purkinje+neurons.">Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.</a> Science Reports. 5, 9232 (2015).</li><li>Tao, O., 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=Efficient+generation+of+mature+cerebellar+Purkinje+cells+from+mouse+embryonic+stem+cells.">Efficient generation of mature cerebellar Purkinje cells from mouse embryonic stem cells.</a> Journal of Neuroscience Research. 88 (2), 234-247 (2010).</li><li>Croughan, M. S., Giroux, D., Fang, D., Lee, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Single-Use+Bioreactors+for+Scale-Up+of+Anchorage-Dependent+Cell+Manufacturing+for+Cell+Therapies.">Novel Single-Use Bioreactors for Scale-Up of Anchorage-Dependent Cell Manufacturing for Cell Therapies.</a> Stem Cell Manufacturing. , 105-139 (2016).</li><li>Simunovic, M., Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryoids,+organoids+and+gastruloids:+new+approaches+to+understanding+embryogenesis.">Embryoids, organoids and gastruloids: new approaches to understanding embryogenesis.</a> Development. 144 (6), 976-985 (2017).</li><li>Lou, Y. R., Leung, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+organoids+for+biomedical+research+and+applications.">Next generation organoids for biomedical research and applications.</a> Biotechnology Advances. 36 (1), 132-149 (2018).</li><li>Silva, T. P., 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=Design+Principles+for+Pluripotent+Stem+Cell-Derived+Organoid+Engineering.">Design Principles for Pluripotent Stem Cell-Derived Organoid Engineering.</a> Stem Cells International. 2019, 1-17 (2019).</li><li>Silva, T. P., 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=Maturation+of+human+pluripotent+stem+cell-derived+cerebellar+neurons+in+the+absence+of+co-culture.">Maturation of human pluripotent stem cell-derived cerebellar neurons in the absence of co-culture.</a> Frontiers of Bioengineering and Biotechnology. , (2020).</li><li>Burridge, P. 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=A+universal+system+for+highly+efficient+cardiac+differentiation+of+human+induced+pluripotent+stem+cells+that+eliminates+interline+variability.">A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability.</a> PLoS One. 6 (4), 18293 (2011).</li><li>Watanabe, 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=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Smith, J. 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=Inhibition+of+Activin/Nodal+signaling+promotes+specification+of+human+embryonic+stem+cells+into+neuroectoderm.">Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm.</a> Developmental Biology. 313 (1), 107-117 (2008).</li><li>Yaguchi, Y., 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=Fibroblast+growth+factor+(FGF)+gene+expression+in+the+developing+cerebellum+suggests+multiple+roles+for+FGF+signaling+during+cerebellar+morphogenesis+and+development.">Fibroblast growth factor (FGF) gene expression in the developing cerebellum suggests multiple roles for FGF signaling during cerebellar morphogenesis and development.</a> Developmental Dynamics. 238 (8), 2058-2072 (2008).</li><li>Fischer, T., 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=Fgf15-mediated+control+of+neurogenic+and+proneural+gene+expression+regulates+dorsal+midbrain+neurogenesis.">Fgf15-mediated control of neurogenic and proneural gene expression regulates dorsal midbrain neurogenesis.</a> Developmental Biology. 350 (2), 496-510 (2011).</li><li>Bagri, 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=The+chemokine+SDF1+regulates+migration+of+dentate+granule+cells.">The chemokine SDF1 regulates migration of dentate granule cells.</a> Development. 129 (18), 4249-4260 (2002).</li><li>Bardy, 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=Neuronal+medium+that+supports+basic+synaptic+functions+and+activity+of+human+neurons+in+vitro.">Neuronal medium that supports basic synaptic functions and activity of human neurons in vitro.</a> Proceedings of the National Academy of Sciences. 112 (20), 2725-2734 (2015).</li><li>Bauwens, C. 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=Control+of+Human+Embryonic+Stem+Cell+Colony+and+Aggregate+Size+Heterogeneity+Influences+Differentiation+Trajectories.">Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories.</a> Stem Cells. 26 (9), 2300-2310 (2008).</li><li>Bratt-Leal, A. M., Carpenedo, R. L., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+embryoid+body+microenvironment+to+direct+embryonic+stem+cell+differentiation.">Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation.</a> Biotechnology Progress. 25 (1), 43-51 (2009).</li><li>Miranda, C. 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=Spatial+and+temporal+control+of+cell+aggregation+efficiently+directs+human+pluripotent+stem+cells+towards+neural+commitment.">Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment.</a> Biotechnology Journal. 10 (10), 1612-1624 (2015).</li><li>Miranda, C. C., Fernandes, T. G., Diogo, M. M., Cabral, J. 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=Scaling+up+a+chemically-defined+aggregate-based+suspension+culture+system+for+neural+commitment+of+human+pluripotent+stem+cells.">Scaling up a chemically-defined aggregate-based suspension culture system for neural commitment of human pluripotent stem cells.</a> Biotechnology Journal. 11 (12), 1628-1638 (2016).</li><li>Bardy, 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=Microcarrier+Suspension+Cultures+for+High-Density+Expansion+and+Differentiation+of+Human+Pluripotent+Stem+Cells+to+Neural+Progenitor+Cells.">Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells.</a> Tissue Engineering Part C Methods. 19 (2), 166-180 (2013).</li><li>Rigamonti, 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=Large-Scale+Production+of+Mature+Neurons+from+Human+Pluripotent+Stem+Cells+in+a+Three-Dimensional+Suspension+Culture+System.">Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.</a> Stem Cell Reports. 6 (6), 993-1008 (2016).</li><li>Arora, 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=A+process+engineering+approach+to+increase+organoid+yield.">A process engineering approach to increase organoid yield.</a> Development. 144 (6), 1128-1136 (2017).</li><li>Gimeno, L., Martinez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+chick+Fgf19+and+mouse+Fgf15+orthologs+is+regulated+in+the+developing+brain+by+Fgf8+and+Shh.">Expression of chick Fgf19 and mouse Fgf15 orthologs is regulated in the developing brain by Fgf8 and Shh.</a> Developmental Dynamics. 236 (8), 2285-2297 (2007).</li></ol>

The need for large cell numbers as well as defined culture conditions to generate specific cell types for drug screening and regenerative medicine applications has been driving the development of scalable culture systems. In recent years, several groups have reported the scalable generation of neural progenitors and functional neurons32,33,34, providing significant advances in the development of new models for neurodegenerative ...

The emergence of human pluripotent stem cells (PSCs) represents a excellent tool for regenerative medicine and disease modeling, because these cells can be differentiated into most cell lineages of the human body1,2. Since their discovery, PSC differentiation using diverse approaches has been reported to model different diseases, including neurodegenerative disorders3,4,5,6.
Recently, there have been reports of 3D cultures derived from PSCs resembling human cerebral structures; these are called brain organoids3,7,8. The generation of these structures from both healthy and patient-specific PSCs provides a valuable opportunity to model human development and neurodevelopmental disorders. However, the methods used to generate these well-organized cerebral structures are difficult to apply for their large-scale production. To produce structures that are large enough to recapitulate tissue morphogenesis without necrosis inside the organoids, protocols rely on the initial neural commitment in static conditions, followed by encapsulation in hydrogels and subsequent culture in dynamic systems3. However, such approaches may limit the potential scale-up of organoid production. Even though efforts have been made to direct PSC differentiation to specific regions of the central nervous system, including cortical, striatal, midbrain, and spinal cord neurons9,10,11,12, the generation of specific brain regions in dynamic conditions is still a challenge. In particular, the generation of mature cerebellar neurons in 3D structures has yet to be described. Muguruma et al. pioneered the generation of culture conditions that recapitulate early cerebellar development13 and recently reported a protocol that allows for human embryonic stem cells to generate a polarized structure reminiscent of the first trimester cerebellum7. However, the maturation of cerebellar neurons in the reported studies requires the dissociation of the organoids, sorting of cerebellar progenitors, and coculture with feeder cells in a monolayer culture system7,14,15,16. Therefore, the reproducible generation of the desired cerebellar organoids for disease modeling under defined conditions is still a challenge associated with culture and feeder source variability.
This protocol presents optimal culture conditions for 3D expansion and efficient differentiation of human PSCs into cerebellar neurons using single-use vertical wheel bioreactors (see Table of Materials for specifications), hereafter called bioreactors. Bioreactors are equipped with a large vertical impeller, which in combination with a U-shaped bottom, provide a more homogeneous shear distribution inside the vessel, allowing gentle, uniform mixing and particle suspension with reduced agitation speeds17. With this system, shape and size-controlled cell aggregates can be obtained, which is important for a more homogeneous and efficient differentiation. Moreover, a larger number of iPSC-derived organoids can be generated in a less laborious manner.
The main feature of the organoids, which are 3D multicellular structures usually formed from stem cells, is the self-organization of different cell types that forms specific shapes like those seen in human morphogenesis18,19,20. Therefore, organoid morphology is an important criterion to be evaluated during the differentiation process. Cryosectioning of organoids and further immunostaining of organoid slices with a specific set of antibodies allow for the spatial visualization of molecular markers to analyze cell proliferation, differentiation, cell population identity, and apoptosis. With this protocol, by immunostaining organoid cryosections, an initial efficient neural commitment is observed by the 7th day of differentiation. During differentiation, several cell populations with cerebellar identity are observed. After 35 days in this dynamic system, the cerebellar neuroepithelium organizes along an apicobasal axis, with an apical layer of proliferating progenitors and basally located postmitotic neurons. During the maturation process, from days 35&#8211;90 of differentiation, distinct types of cerebellar neurons can be seen, including Purkinje cells (Calbindin+), granule cells (PAX6+/MAP2+), Golgi cells (Neurogranin+), unipolar brush cells (TBR2+), and deep cerebellar nuclei projection neurons (TBR1+). Also, a nonsignificant amount of cell death is observed in the generated cerebellar organoids after 90 days in culture.
In this system, human iPSC-derived organoids mature into different cerebellar neurons and survive for up to 3 months without the need for dissociation and feeder coculture, providing a source of human cerebellar neurons for disease modeling.

<table>
	<tbody>
		<tr>
			<td>3MM paper</td>
			<td>WHA3030861</td>
			<td>Merck</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A6964 - 500mL</td>
			<td>Sigma</td>
			<td>cell detachment medium</td>
		</tr>
		<tr>
			<td>Anti-BARHL1 Antibody</td>
			<td>HPA004809</td>
			<td>Atlas Antibodies</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Calbindin D-28k Antibody</td>
			<td>CB28</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 Antibody</td>
			<td>M4403</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-N-Cadherin Antibody</td>
			<td>610921</td>
			<td>BD Transduction</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-NESTIN Antibody</td>
			<td>MAB1259-SP</td>
			<td>R&#38;D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-OLIG2 Antibody</td>
			<td>MABN50</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PAX6 Antibody</td>
			<td>PRB-278P</td>
			<td>Covance</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-SOX2 Antibody</td>
			<td>MAB2018</td>
			<td>R&#38;D</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TBR1 Antibody</td>
			<td>AB2261</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TBR2 Antibody</td>
			<td>ab183991</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TUJ1 Antibody</td>
			<td>801213</td>
			<td>Biolegend</td>
			<td></td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>T1147</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>BrainPhys Neuronal Medium N2-A &#38; SM1 Kit</td>
			<td>5793 - 500mL</td>
			<td>Stem cell tecnhnologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemically defined lipid concentrate</td>
			<td>11905031</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips 24x60mm</td>
			<td>631-1575</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallization-purified BSA</td>
			<td>5470</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>10236276001</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibutyryl cAMP</td>
			<td>SC- 201567B -500mg</td>
			<td>Frilabo</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>32500-035</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>A3840001</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>G9391</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Copling Jar</td>
			<td>E94</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax I</td>
			<td>10566-016</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>MB014001</td>
			<td>NZYtech</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F12</td>
			<td>21765029</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Episomal iPSC Line</td>
			<td>A18945</td>
			<td>ThermoFisher</td>
			<td>iPSC6.2</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>12440046</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>91077C</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>iPS DF6-9-9T.B</td>
			<td></td>
			<td>WiCell</td>
			<td></td>
		</tr>
		<tr>
			<td>Iso-pentane</td>
			<td>PHR1661-2ML</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>A-92902</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>354230</td>
			<td>Corning</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>Monothioglycerol</td>
			<td>M6154</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>475904</td>
			<td>Millipore</td>
			<td>mounting medium</td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>85850 -500ml</td>
			<td>Stem cell technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>17502048</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>12348017</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>158127</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-0.1 Single-Use Vessel</td>
			<td>SKU: IA-0.1-D-001</td>
			<td>PBS Biotech</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-MINI MagDrive Base Unit</td>
			<td>SKU: IA-UNI-B-501</td>
			<td>PBS Biotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human BDNF</td>
			<td>450-02</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human bFGF/FGF2</td>
			<td>100-18B</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human FGF19</td>
			<td>100-32</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human GDNF</td>
			<td>450-10</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human SDF1</td>
			<td>300-28A</td>
			<td>Peprotech</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK inhibitor Y-27632</td>
			<td>72302</td>
			<td>Stem cell technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>S4317</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>S7903</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Microscope slides</td>
			<td>12372098</td>
			<td>ThermoFisher</td>
			<td>adhesion microscope slides</td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>25608-930</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL 1M</td>
			<td>T3038-1L</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>9002-93-1</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>P1379</td>
			<td>Sigma</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>15575020</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
	</tbody>
</table>

scalable generation of mature cerebellar organoids from human pluripotent stem cells and characterization by immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

The protocol was initiated by promoting cell aggregation using the 0.1 L bioreactors (Figure 1A). Single cell inoculation of the iPSCs was performed, with 250,000 cells/mL seeded in 60 mL of medium with an agitation speed of 27 rpm. This was defined as day 0. After 24 h, the cells efficiently formed spheroid-shaped aggregates (day 1, Figure 1B), and the morphology was well-maintained until day 5, with a gradual increase in size, demonstrating a high degree of ho...

Watch this Scientific Journal Video about Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining at JoVE.com

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Research

JoVE Journal

Bioengineering

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...