Name: developing hipsc derived serum free embryoid bodies for the interrogation of 3-d stem cell cultures using physiologically relevant assays
Uploaded: 2017-07-20T14:00:00.000+00:00
Duration: 10 min 43 s
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 plate in 300 &#956;L/well recommended medium (following manufacturer&#39;s protocol).</li>
		<li>After 48 h, replace MEFs medium with 300 &#956;L/well hiPSC medium for 1 h before adding hiPSCs.</li>
		<li>Quickly thaw hiPSCs in a 37 &#176;C water bath and slowly add 1 mL hiPSC medium dropwise. Transfer the hiPSCs and medium to a 15 mL conical tube. Add medium to bring the total volume to 5 mL. Centrifuge for 4 min at 129 x g, aspirate the supernatant, gently re-suspend the pellet in 1 mL hiPSC medium with ROCK inhibitor at 1:1,000 concentration.</li>
		<li>Plate the cell suspension (typically seeded at 300,000 cells/well) onto the MEF cell layer and incubate at 37 &#176;C, 5% CO2, 95% humidity.</li>
		<li>Monitor iPSC culture closely using a light microscope. After 48 h, remove cultures that have uneven edges, have grown to become cystic-like or appear yellowish-brown under the light microscope to minimize spontaneous differentiation. 
		NOTE: After 7 - 10 days, hiPSC colonies should form. Colonies should be round, with clearly defined borders and uniform cell densities, and devoid of loosely packed non-uniform cells.</li>
		<li>Manually select round hiPSC colonies to clear the culture of spontaneously differentiated cells. Expand the colonies by placing them on fresh MEF layers. Expand 1:3 every 7 days when cultures near confluency and freeze1,16.</li>
	</ol>
	</li>
	<li>After expansion and freezing cells (for backup), grow hiPSC colonies on plates until they reach 50 - 75% confluence.</li>
	<li>Seven-days post plating, use mild enzymatic treatment (5-10 min with 300 &#956;L of enzyme solution) and gentle trituration (2-4 times with a 1,000 &#181;L pipet) to harvest and prepare hiPSCs for neural precursor cell differentiation.</li>
	<li>Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and resuspend the pellet in 5 mL human iPSC medium. Transfer the cell suspension to a 0.1% gelatin coated 5 cm cell culture dish for 1 h at 37 &#176;C to eliminate MEFs and to maximize the hiPSC yield. Transfer the medium (with non-adherent cells) to another 5 cm gelatin coated dish.</li>
	<li>Transfer the non-adherent cells to a 15 mL centrifuge tube using a transfer pipet. Gently rinse the plates with 3 mL medium and add it to the 15 mL tube.</li>
	<li>Centrifuge the cell suspension at 129 x g for 4 min, aspirate the supernatant, and gently resuspend the pellet in medium. Determine the cell count using an automated cell counter. Plate 9,000 cells/well in a 96-well low-adhesion V-bottom plate.</li>
	<li>Centrifuge the plate at 163 x g for 3 min. Incubate the plate at 37 &#176;C, 95% humidity and 5% CO2. Using a pipette, change 50% medium every other day by removing half of the medium and replacing it with fresh chemically-defined differentiating medium 1 (DM1; Figure 1; Table of Materials) for 14 days.</li>
</ol>
2. Serum-Free Embryoid Body (SFEB) Induction (Figure 2)
<ol>
	<li>Place 40 &#181;m cell culture inserts into 6-well plates and add 1 mL DM1 medium at least 1 h prior to the addition of aggregates.</li>
	<li>On day 14, transfer the aggregates with 20 &#181;L medium using a 200 &#181;L wide mouth tip onto 40 &#181;m cell culture inserts and change the medium to DM2. 
	NOTE: Cell culture inserts are specialized inserts that sit slightly off the bottom of the well and consist of a polytetrafluoroethylene membrane suspended across a plastic frame. This membrane is biocompatible and can efficiently sustain nutrient and oxygen transport to the SFEBs, which are placed on top. They are commonly used with organotypic hippocampal slice cultures taken from mouse or rat7,17,18.
	<ol>
		<li>Transfer 4-6 aggregates to one cell culture insert and allow adequate space between the aggregates. Remove as much excess solution as possible using a fine pipette tip (e.g. 200 &#956;L tip). 
		NOTE: It is acceptable to briefly disturb the SFEB as they will move on the cell culture insert as the solution in removed from around the SFEBs.</li>
		<li>Maintain the cultures in 1 mL of DM2 at 37 &#176;C, 95% humidity and 5% CO2. Using a pipette, change 75% medium every other day by removing three quarters of the medium and replacing with three quarters fresh medium. For example, remove 750 &#956;L of medium and add 750 &#956;L of fresh medium.</li>
	</ol>
	</li>
	<li>After 14 days of culture, switch to DM3 medium with 75% medium change every other day and for another 16 days.</li>
	<li>To maintain the SFEBs on cell culture inserts beyond 30 days, change the medium every other day for 60, 90 and 120 days. 
	NOTE: The SFEBs will grow to about 1,000 &#956;m in diameter and are typically 100-150 &#956;m thick1.</li>
</ol>
3. Determining SFEB Composition
<ol>
	<li>Detach the SFEBs from the insert by gently pipetting medium using a 200 &#181;L wide mouth pipette tip. To transfer the SFEBs, use a wide mouth pipette tip to gently suction the bodies into the pipet tip. After loading SFEB into the pipet tip, gently transfer to 12-well plates and wash with 300 &#956;L phosphate buffered saline (PBS) per well. 
	NOTE: SFEBs will be suspended in solution in the 12-well plates. This is important to allow full penetration of the fixative, the blocking solution, and antibodies. If using multiple SFEBs for staining, up to 10 SFEBs can be added per well of a 12 well plate. This will conserve solutions and antibodies.</li>
	<li>Fix SFEBs with 300 &#956;L per well 4% paraformaldehyde at room temperature for 30-45 min. Wash fixed SFEBs twice with 300 &#956;L PBS, 3-5 min each wash. 
	Caution: Wear appropriate personal protective equipment when handling paraformaldehyde. 
	NOTE: Probe for immunoreactivity to markers to determine maturity, cell type etc. For marking neurons in this study chicken-Tuj1 was used at 1:500, for additional markers please see Table 2 and references1,16.</li>
	<li>Permeabilize and block with 0.1% Triton-X100 in PBS and 10% normal donkey serum (blocking solution; 300 &#956;L per well) for 1 h at room temperature.</li>
	<li>Remove the blocking solution and treat SFEBs with 300 &#956;L primary antibodies in blocking solution. Incubate at 4 &#176;C overnight. Wash SFEBs thrice (3-5 min per wash) in 300 &#956;L PBS containing 0.1% Triton-X.</li>
	<li>Prepare secondary antibodies 1:1000 in blocking solution. Incubate SFEBs for 1 h at room temperature with secondary antibodies. Wash SFEBs with 300 &#956;L PBS, 3 times for 3-5 min each. 
	NOTE: At this stage SFEBs can be prepared for imaging.</li>
	<li>To image, pipette SFEBs onto a glass slide using a wide-bore pipette. Remove excess solution around the SFEB using gentle suction. 
	NOTE: For similar staining conditions, multiple SFEBs can be placed on the same glass slide.</li>
	<li>Add a drop of mounting medium on the SFEBs, place a glass coverslip, and gently press to ensure that there are no air bubbles. Allow the mounting medium to harden and proceed to imaging and quantification (step 3.8). 
	NOTE: After the mounting medium hardens, the SFEBs are ready for imaging.</li>
	<li>Perform cell quantification by taking multiple regions of interest (ROI) across the SFEB and using a z-stack combined with a maximal projection image through each ROI on a standard confocal microscope as described in references1,16. 
	NOTE: Whole SFEBs can be quantified using image-based tiling (see references1,16 for details). Since the SFEBs thin to approximately 100 &#956;m, there is minimal scattering of light in the tissue and thus there is no need for 2-photon microscopy. Earlier uses of this protocol with fibroblast-derived iPSCs produced a SFEB fate map that revealed complex and diverse structure with subpopulations of interneurons with transcriptional identities that resembled CGE and anterior forebrain fates1,16.</li>
</ol>
4. Recording Neuronal activity in SFEBs Using MEAs
<ol>
	<li>Prepare 12-well MEA plates as per manufacturer&#39;s instructions.</li>
	<li>Wash the MEA plates 3 times for 5 min with sterile H2O under aseptic conditions to clean. Wash for 5 min with 75% ethanol and then with 100% ethanol to sterilize the plate.</li>
	<li>Bake the plate inverted in an oven for 4-5 h at 50 &#176;C to complete the sterilization process.</li>
	<li>Add 500 &#181;L 0.2% polyethyleneimine solution (PEI) to each well and incubate 1 h at room temperature. Aspirate PEI and wash the wells 4x with sterile distilled H2O. Air dry in the hood overnight.</li>
	<li>Prepare a 20 &#181;L/mL solution of laminin in L15 medium and add 10 &#181;L laminin to the center of each well. 
	NOTE: Do not coat the surrounding reference and grounding electrodes.</li>
	<li>Add sterile dH2O to the surrounding reservoirs to prevent medium or laminin evaporation. Incubate the plate for 1 h at 37 &#176;C.</li>
	<li>Add SFEBs (see sections 1 and 2) to the laminin coated MEA plates by gently suctioning them into a wide mouth pipette tip and transfer them. Add 200 &#181;L DM2 medium to each well and incubate overnight at 37 &#176;C, 95% humidity and 5% CO2.</li>
	<li>Add an additional 200 &#181;L medium after 24 h and allow it to recover for 30 min at 37 &#176;C, 95% humidity, 5% CO2 before reading the plate.</li>
	<li>Place the MEA plate in the plate reader (preheated to 37 &#176;C) to record neuronal activity. Record activity for 10 min with the associated MEA recording software. See reference19 for details.</li>
	<li>Generate raw data-continuous streams and raster plots of neuronal activity using statistical analysis software19. 
	NOTE: MEA recordings are taken 7 days post SFEB plating. For this study, recordings were taken for 10 min to detect network burst activity, continuous trace, and raster plots. SFEBs can be maintained long-term on MEA plates and can be recorded over longer periods of time using environmentally controlled conditions (Figure 6).</li>
</ol>

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The authors have no competing financial interests.

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><tbody><tr><td>&lt;strong&gt;SFEB Neuronal Differentiation Cell culture Media. Reagents. Components&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>STEMdiff Neural Induction Medium (hiPSC Media)</td><td>STEMCELL Technologies</td><td>0-5835</td><td>250 mL</td></tr><tr><td>PluriQ ES-DMEM Medium (MEF Media)</td><td>GlobalStem</td><td>GSM-2001</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM1 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-MEM/F-12 (1x), Glutamax liquid, 1:1</td><td>Invitrogen</td><td>10565018</td><td>385 mL</td></tr><tr><td>Knockout Serum Replacement</td><td>Invitrogen</td><td>10828028</td><td>20% 100 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>MEM Non-Essential Amino Acids Solution 10 mM (100x), liquid</td><td>Invitrogen</td><td>11140050</td><td>5 mL</td></tr><tr><td>2-Mercaptoethanol (1,000x), liquid</td><td>Invitrogen</td><td>21985023</td><td>900 &amp;mu;L</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM2 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-MEM/F-12 (1x), Glutamax liquid, 1:1</td><td>Invitrogen</td><td>10565018</td><td>500 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>N-2 Supplement (100x), liquid</td><td>Invitrogen</td><td>17502048</td><td>10 mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;DM3 Media Components&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NEUROBASAL Medium (1x), liquid</td><td>Invitrogen</td><td>21103049</td><td>500 mL</td></tr><tr><td>B-27 Supplement Minus Vitamin A (50x), liquid</td><td>Invitrogen</td><td>12587010</td><td>10 mL</td></tr><tr><td>Glutamax 200 mM</td><td>Invitrogen</td><td>35050061</td><td>5 mL</td></tr><tr><td>Pen/Strep</td><td>Invitrogen</td><td>15140122</td><td>5 mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Small Molecules&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thiazovivin</td><td>Stemgent</td><td>04-0017</td><td>2 &amp;mu;M</td></tr><tr><td>SB431542</td><td>Stemgent</td><td>04-0010-10</td><td>1:1,000 (10 &amp;mu;M)</td></tr><tr><td>Dorsomorphin</td><td>Stemgent</td><td>04-0024</td><td>1 &amp;mu;M</td></tr><tr><td>LDN-193189</td><td>Stemgent</td><td>04-0074-10</td><td>250 nM</td></tr><tr><td>Y27632 (ROCKi)</td><td>Stemgent</td><td>04-0012-10</td><td>10 &amp;mu;M</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Recombinant Protiens&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DKK-1</td><td>Peprotech</td><td>120-30</td><td>200 ng/mL</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Components/Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture inserts 0.4 &amp;mu;M, 30mm Diameter</td><td>Millicell</td><td>PICM0RG50</td><td></td></tr><tr><td>Mouse Embryonic Fibroblasts</td><td>GlobalStem</td><td>GSC-6301G</td><td></td></tr><tr><td>96 well V bottom w/Lids</td><td>Evergreen</td><td>222-8031-01V</td><td></td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>ThermoFisher</td><td>A1110501</td><td></td></tr><tr><td>TritonX-100</td><td>ThermoFisher</td><td>85111</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>ThermoFisher</td><td>10010023</td><td>500 mL</td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Labs</td><td>017-000-121</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>ThermoFisher</td><td>11415114</td><td>500 mL</td></tr><tr><td>DRAQ5 (Nuclear Marker)</td><td>ThermoFisher</td><td>65-0880-96</td><td></td></tr><tr><td>MEA Plates</td><td>Axion Biosystems</td><td>M768-GL1-30Pt200</td><td></td></tr><tr><td>6 well flat bottom</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Nestin</td><td>Millipore</td><td>MAB5326</td><td></td></tr><tr><td>Brn-2</td><td>Protein tech</td><td>14596-1-AP</td><td></td></tr><tr><td>VGLUT1</td><td>Synaptic Systems</td><td>135 303</td><td></td></tr><tr><td>Pax6</td><td>abcam</td><td>ab5790</td><td></td></tr><tr><td>Calretinin</td><td>abcam</td><td>ab702</td><td></td></tr><tr><td>Calbindin</td><td>abcam</td><td>ab11426</td><td></td></tr><tr><td>CoupTFII</td><td>R&amp;amp;D Systems</td><td>PPH714700</td><td></td></tr><tr><td>Nkx 2.1</td><td>abcam</td><td>ab12650</td><td></td></tr><tr><td>Tuj1</td><td>abcam</td><td>ab41489</td><td></td></tr><tr><td>Reelin</td><td>Millipore</td><td>MAB5364</td><td></td></tr><tr><td>Tbr1</td><td>Millipore</td><td>MAB2261</td><td></td></tr><tr><td>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 ...

developing-hipsc-derived-serum-free-embryoid-bodies-for-interrogation

Research

JoVE Journal

Neuroscience

8.8K Views.  The Hussman Institute for Autism. 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.

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Bioengineering

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Developmental Biology

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 ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

Biochemistry

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Biology

Matrix Comparison for Efficient Human Intestinal Organoid Generation from iPSCs

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

Author Spotlight: Advancing Organoid Generation for Drug Development Using iPSCs

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

Differentiation of Induced Pluripotent Stem Cells into Neural Progenitor Cells

Source: Phillips, A. W., et al. <a href="https://app.jove.com/v/55799">Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays.</a> J. Vis. Exp. (2017).
The video demonstrates a technique for differentiating human induced pluripotent stem cells (hiPSCs) into neural progenitor cells (NPCs). Initially, the hiPSCs are cultured on a feeder layer of mouse embryonic fibroblasts (MEFs). Subsequently, the cells are detached and transferred to a biopolymer-coated plate to allow the MEFs to adhere to and separate from the hiPSCs. The suspended hiPSCs are then transferred to a V-bottom plate and centrifuged to form aggregates. Finally, the addition of a differentiation medium induces the differentiation of hiPSCs within the aggregate into NPCs.

Różnicowanie indukowanych pluripotencjalnych komórek macierzystych w neuronalne komórki progenitorowe

1. Generation of Neural Progenitor Cells 

	Maintain hiPSCs derived from fibroblasts and peripheral blood mononuclear cells (PBMCs) in 6-well plates on a ...