Name: developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays
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Description: Watch this Scientific Journal Video about Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays at JoVE.com

We thank Elizabeth Benevides for proofreading the article. We thank Drs. John Hussman and Gene Blatt for their helpful discussions and comments.

1. Generation of Neural Progenitor Cells
<ol>
	<li>Maintain hiPSCs derived from fibroblasts and PBMCs in 6-well plates on a &#947;-irradiated mouse embryonic feeder (MEF) cell layer in human iPSC medium supplemented with small-molecules (see the Materials Table). 
	NOTE: Daily maintenance is a modified version of previously reported procedures1,16.
	<ol>
		<li>Plate 6 x 105 MEFs onto each well of a 6-well tissue culture grade pl...

<ol>
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P., Rubin, L., Eggan, K., Brock, M., Lipnick, S., Rao, M., Chang, S., Li, A., Noggle, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPSC-Derived+Dopamine+Neurons+Reveal+Differences+Between+Monozygotic+Twins+Discordant+for+Parkinson's+Disease.">iPSC-Derived Dopamine Neurons Reveal Differences Between Monozygotic Twins Discordant for Parkinson's Disease.</a> Cell Rep. 9 (4), 1173-1182 (2014).</li><li>Huang, S. 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=Tankyrase+inhibition+stabilizes+axin+and+antagonizes+Wnt+signalling.">Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling.</a> Nature. 461 (7264), 614-620 (2009).</li><li>Chambers, S. 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The protocol described here provides the conditions for differentiating a hiPSC source into a 3-D structure that recapitulates an early developmental stage of the frontal cortex. This procedure yields structures that can be interrogated for electrophysiology while also being amenable to microscopy. The final morphology of the SFEB resembles that of organotypic brain slice cultures and allows high quality detailed confocal imaging. This protocol can successfully generate SFEBs from both fibroblast and peripheral-blood mon...

We have previously reported a 3-D model system, generated from patient-derived human induced pluripotent stem cells (hiPSCs) that recapitulates some aspects of early cortical network development1. This 3-D model, a serum-free embryoid body (SFEB), improves on previous simple aggregation hiPSC models2,3. A growing body of work is revealing that 3-D structures like our SFEBs, approximate aspects of neural development commonly observed in vivo and at an earlier time point than observed in 2-dimensional (2-D)/monolayer hiPSC models4,5. Initial studies have been focused on the self-organizing complexity of 3-D bodies without demonstrating their physiological complexity2.
The protocol described here has been used on undifferentiated hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs). These cells are maintained on &#947;-irradiated mouse embryonic feeders (MEFs). These hiPSC colonies are manually cleaned of spontaneously differentiated cells, enzymatically harvested, and resuspended in medium containing Rho-Kinase inhibitor Y-27632 (ROCKi). Undifferentiated hiPSCs are subjected to dissociation and centrifugation before being transferred to 96-well low adhesion V-bottom plates. After plating, neural induction is initiated using dual SMAD inhibition (SB431542 and LDN193189 along with dickkopf 1 (DKK-1)) to drive an anterior-frontal forebrain neuronal-fate lineage6. After 14 days, the SFEBs are transferred to cell culture inserts in a 6-well plate. Once transferred, the round SFEBs begin to spread and thin, while maintaining local network connections as is often observed in hippocampal organotypic slice culture preparations using similar cell culture inserts1,7.
The use of an SFEB based 3-D platform in this format is amenable to the efficient production of cortical networks that may be interrogated using cell based physiological assays such as calcium imaging or electrophysiological assays such as single cell recordings or multi-electrode array (MEA)1. Although 3-D systems bear the markers of early cortical development, other studies have shown that these 3-D bodies may require longer incubation times to allow for the inherently slower pace of human tissue development8. This SFEB protocol successfully generates 3-D SFEBs from undifferentiated hiPSCs that captures aspects of the early development of the cortex.
The potential of SFEBs to model network aberrations in neurological disorders is a strength of this system. The hiPSCs derived from patient tissue can be grown into cells of the nervous system that are subject to assays relating to cell biology as well as concomitant gene expression. Human iPSCs are being used to ascertain the genetic profile of large groups of individuals with varying neurological disorders with complex etiologies such as autism spectrum disorder (ASD), schizophrenia9, Rett Syndrome10, and Alzheimer&#39;s disease11,12. Until recently, iPSC models were typically monolayer preparations that, while proficient in evaluating molecular interactions, were inadequate in deciphering the complex cellular interactions seen in vivo. Animal models have been the default substitute for recreating the whole-organ platform. These animal models are plagued by poor translation of findings and have limited ability to replicate human genetic profiles identified by large genetic screening studies. Thus, the development of 3-D systems from iPSCs adds a needed layer of complexity in human disease modeling13,14. The next step for 3D hiPSC platforms is to accommodate the large scale requirements of high throughput screening using cell based assays15.

<table border="0" cellpadding="0" cellspacing="0" width="1173">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:573px;">SFEB Neuronal Differentiation Cell culture Media. Reagents. Components&#160;</td>
			<td style="width:183px;"></td>
			<td style="width:164px;"></td>
			<td style="width:253px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">STEMdiff Neural Induction Medium (hiPSC Media)</td>
			<td>STEMCELL Technologies</td>
			<td>0-5835</td>
			<td>250ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">PluriQ ES-DMEM Medium (MEF Media)</td>
			<td>GlobalStem</td>
			<td>GSM-2001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM1 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-MEM/F-12 (1X), Glutamax liquid, 1:1</td>
			<td>Invitrogen</td>
			<td align="right">10565018</td>
			<td>385ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Knockout Serum Replacement</td>
			<td>Invitrogen</td>
			<td align="right">10828028</td>
			<td>20% 100ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MEM Non-Essential Amino Acids Solution 10 mM (100X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">11140050</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">2-Mercaptoethanol (1,000X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">21985023</td>
			<td>900ul</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM2 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">D-MEM/F-12 (1X), Glutamax liquid, 1:1</td>
			<td>Invitrogen</td>
			<td align="right">10565018</td>
			<td>500ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">N-2 Supplement (100X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">17502048</td>
			<td>10ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DM3 Media Components</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">NEUROBASAL Medium (1X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">21103049</td>
			<td>500ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">B-27 Supplement Minus Vitamin A (50X), liquid</td>
			<td>Invitrogen</td>
			<td align="right">12587010</td>
			<td>10ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Glutamax 200mM</td>
			<td>Invitrogen</td>
			<td align="right">35050061</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td align="right">15140122</td>
			<td>5ml</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Small Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thiazovivin</td>
			<td>Stemgent</td>
			<td>04-0017</td>
			<td>2uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">SB431542</td>
			<td>Stemgent</td>
			<td>04-0010-10</td>
			<td>1:1000 (10uM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dorsomorphin</td>
			<td>Stemgent</td>
			<td>04-0024</td>
			<td>1uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">LDN-193189</td>
			<td>Stemgent</td>
			<td>04-0074-10</td>
			<td>250nM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Y27632 (ROCKi)</td>
			<td>Stemgent</td>
			<td>04-0012-10</td>
			<td>10uM</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Recombinant Protiens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">DKK-1</td>
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			<td>120-30</td>
			<td>200ng/ml</td>
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			<td>Millicell</td>
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			<td>Evergreen</td>
			<td>222-8031-01V</td>
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			<td>A1110501</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher</td>
			<td align="right">10010023</td>
			<td>500 mL</td>
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		<tr height="22">
			<td height="22" style="height:22px;">Normal Donkey Serum</td>
			<td>Jackson Labs</td>
			<td>017-000-121</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Leibovitz&#39;s L-15 Medium</td>
			<td>ThermoFisher</td>
			<td align="right">11415114</td>
			<td>500 mL</td>
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		<tr height="22">
			<td height="22" style="height:22px;">DRAQ5 (Nuclear Marker)&#160;</td>
			<td>ThermoFisher</td>
			<td>65-0880-96</td>
			<td></td>
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			<td height="22" style="height:22px;">MEA Plates</td>
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			<td>M768-GL1-30Pt200</td>
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			<td align="right">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nestin</td>
			<td>Millipore</td>
			<td>MAB5326</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brn-2</td>
			<td>Protein tech</td>
			<td>14596-1-AP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VGLUT1&#160;</td>
			<td>Synaptic Systems&#160;</td>
			<td>135 303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pax6</td>
			<td>abcam</td>
			<td>ab5790</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calretinin&#160;</td>
			<td>abcam</td>
			<td>ab702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calbindin</td>
			<td>abcam</td>
			<td>ab11426</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoupTFII</td>
			<td>R&#38;D Systems</td>
			<td>PPH714700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nkx 2.1</td>
			<td>abcam</td>
			<td>ab12650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tuj1</td>
			<td>abcam</td>
			<td>ab41489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reelin</td>
			<td>Millipore</td>
			<td>MAB5364</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tbr1</td>
			<td>Millipore</td>
			<td>MAB2261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NFH</td>
			<td>Dako</td>
			<td>M0762</td>
			<td></td>
		</tr>
	</tbody>
</table>

developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

SFEBs grown using our technique yielded tissue with morphological characteristics that resemble an early developing cortical subventricular zone replete with extensive Tuj1-positive neurons, as well as neural progenitors (Figure 3A). Numerous developing cortical rosettes were observed in the outer layers and inner layers of the SFEB (Figure 3B). The outer edge of the SFEB resembles a developing cortical plate containing postmitot...

Watch this Scientific Journal Video about Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays at JoVE.com

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Research

JoVE Journal

Neuroscience

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