Name: synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells
Uploaded: 2018-08-16T14:00:00
Description: Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

We would like to thank Dr. Erik Ullian (UCSF) for intellectual input on the design of these procedures, Dr. Michael Ward (NIH) for technical advice on iNeuron differentiation, and Saba Barlas for preliminary image analysis.

1. Cell Culture and Reagent Preparation
NOTE: The protocols in this section are written in the order in which they appear in the differentiation protocol (section 2). See the Table of Materials for materials and catalog numbers.
<ol>
	<li>Prepare coated plates for cell culture.
	<ol>
		<li>Dilute extracellular matrix (ECM) coating solution with DMEM/F12 media to prepare a 1 mg/mL stock solution. Aliquot the diluted ECM stock solution into 30 conical tubes of...

In this protocol, we describe a systematic method for the production of 3D spheres of neural cocultures. The spheres are composed of astrocytes and neurons, which are derived independently from hPSCs. Though not the focus of this protocol, the generation of pure populations of astrocytes from hPSCs28 is a critical step and can be technically challenging if performed without prior experience. This first step in the generation of these synaptic microcircuits should be performed with meticulous timin...

Astrocytes are a highly abundant glial cell type within the central nervous system (CNS) with a variety of functional responsibilities beyond structural support. Through secretion of soluble synaptogenic factors and extracellular matrix (ECM) components, astrocytes aid in the establishment and clustering of mature synapses during development1. They also play a critical role in maintaining the health and plasticity of synapses through extracellular signaling2,3,4,5, and contribute to long-term stability of homeostatic environments by regulating extracellular potassium and glutamate, as well as the secretion of energy substrates and ATP6,7,8. Finally, they can contribute to neurotransmission by influencing extrasynaptic currents9, and can indirectly influence activity through other cell types such as promoting myelination10. Importantly, because abnormality or dysfunction of astrocytes can lead to many neurodevelopmental syndromes and adult neuropathology, there is an obvious need to include astrocytes alongside neurons within engineered neural networks in order for an improved model of the endogenous brain environment. An integral characteristic of astrocytes is their ability to form dynamic interactions with neuronal synapses1,11,12. In the absence of glia, neurons form a limited number of synapses, which in general also lack functional maturity13.
Human astrocytes display morphological, transcriptional, and functional characteristics &#8212; such as increased size and complexity of branching, as well as species-specific genes &#8212; that are not recapitulated in rodents12,14,15. As a result, studies utilizing human pluripotent stem cell (hPSC)-derived neural cells have become widely accepted as a means of examining CNS-related diseases in vitro while developing novel therapies, injury models, and culture paradigms16,17. Furthermore, hPSCs permit the study of human synapse formation and function without the need for primary tissue18,19.
A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models of the human brain. There is a need for an appropriate platform to recapitulate its synaptic networks with high fidelity and reproducibility. Recently, interest has emerged in the production of 3D culture systems (broadly known as &#39;organoids,&#39; &#39;spheroids,&#39; or &#39;mini brains&#39;)20 to model complex three-dimensional (3D) structures at the cellular and macro levels. 3D culture systems retain ECM and cell-cell interactions that are normally absent or limited during typical 2D coculture paradigms21,22. An abundance of techniques exist for culturing 3D neural spheroids23,24,25; however, many require lengthy culture periods (months to years) for spontaneous development and layer preservation, with the user exhibiting very little control over the output.
Here, we illustrate a systematic method to rapidly and consistently bioengineer neural interactions among multiple cell types (pre-differentiated neurons and astrocytes) derived from hPSCs by assembling cells into sphere cocultures (&#39;asteroids&#39;)26 that recapitulate human-specific morphological complexities in 3D. This high-density neural system generates evenly-dispersed neural subtypes that take on mature properties over time and can be screened or assayed in a high-throughput manner. We demonstrate for the first time that human astrocytes induce synaptic network burst activity in these 3D cocultures. In addition, this protocol is easily adaptable to generate spheres of different sizes, to utilize cells specified to different regional identities of the CNS, and to study interactions of multiple other cell types as desired.

<table>
	<tbody>
		<tr>
			<td>6 well plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Olympus Plastics</td>
			<td>28-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma</td>
			<td>A6964-100ML</td>
			<td>Detachment solution</td>
		</tr>
		<tr>
			<td>AggreWell plate</td>
			<td>Stemcell Technologies</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>Stemcell Technologies</td>
			<td>7010</td>
			<td>Prevent cell adhesion to microwell plates</td>
		</tr>
		<tr>
			<td>Anti/anti</td>
			<td>Thermofisher</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermofisher</td>
			<td>17504044</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>BrainPhys neuronal medium</td>
			<td>Stemcell Technologies</td>
			<td>5790</td>
			<td>Neurophysiological basal medium alternative</td>
		</tr>
		<tr>
			<td>Circular glass coverslips</td>
			<td>Neuvitro</td>
			<td>GG-12-oz</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS10</td>
			<td>Stemcell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium with 10% DMSO</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermofisher</td>
			<td>10565-042</td>
			<td>With GlutaMAX supplement</td>
		</tr>
		<tr>
			<td>DMH-1</td>
			<td>Stemcell Technologies</td>
			<td>73634</td>
			<td>HAZARD: Toxic if swallowed. Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332100</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Doxycycline Hydrochloride (Dox)</td>
			<td>Sigma</td>
			<td>D3072-1ml</td>
			<td>HAZARD: Toxic for pregnant women. Working concentration: 2 ug/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-2 (FGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Working concentration: 10 ng/mL</td>
		</tr>
		<tr>
			<td>Fluoromount-G mounting solution</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Fisherbrand</td>
			<td>22-037-246</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Lampire Biological Laboratories</td>
			<td>7332500</td>
			<td>Working concentration: 5% in primary blocking buffer, 1% in secondary blocking buffer</td>
		</tr>
		<tr>
			<td>Hemacytometer or automatic cell counter</td>
			<td>Life Technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149-50KU</td>
			<td>Working concentration: 2 mg/mL</td>
		</tr>
		<tr>
			<td>Magnetic plate</td>
			<td>DLAB</td>
			<td>8030170200</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel membrane matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>ECM coating solution. Working concentration: 80 ug/ml. Prepare on ice and ensure that pipettes, tubes, and media are pre-chilled.</td>
		</tr>
		<tr>
			<td>MEA 2100 System</td>
			<td>Multichannel Systems</td>
			<td>MEA2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Thermofisher</td>
			<td>17502048</td>
			<td>Media Supplement</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Tissue embedding solution for cryosectioning</td>
		</tr>
		<tr>
			<td>Pap Pen (Aqua Hold)</td>
			<td>Scientific Device Laboratory</td>
			<td>9804-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Acros Organics</td>
			<td>169650025</td>
			<td>HAZARD: Toxic if inhaled. Working concentration: 4% in PBS</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Stemcell Technologies</td>
			<td>CA008-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-l-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Working concentration: 0.5 mg/mL</td>
		</tr>
		<tr>
			<td>Rectangular glass cover slips</td>
			<td>Fisherfinest Premium Superslip</td>
			<td>12-545-88</td>
			<td></td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stemcell Technologies</td>
			<td>5872</td>
			<td>Detachment and passaging reagent</td>
		</tr>
		<tr>
			<td>Rho-Kinase Inhibitor Y27632- (Y)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>Working concentration: 10 uM</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemcell Technologies</td>
			<td>72234</td>
			<td>Working concentration: 2 uM</td>
		</tr>
		<tr>
			<td>Spinner flasks</td>
			<td>Fisher Scientific</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Chemical</td>
			<td>S5-3</td>
			<td>Working concentration: 20% or 30% in PBS</td>
		</tr>
		<tr>
			<td>T25 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-207</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>T75 Culture Flask</td>
			<td>Olympus Plastics</td>
			<td>25-209</td>
			<td>Vented caps</td>
		</tr>
		<tr>
			<td>Terg-A-zyme</td>
			<td>Sigma</td>
			<td>Z273287-1EA</td>
			<td>Detergent. Working concentration: 1%</td>
		</tr>
		<tr>
			<td>TeSR-E8 basal medium</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Human pluripotent stem cell (hPSC) medium</td>
		</tr>
		<tr>
			<td>TeSR-E8 supplements</td>
			<td>Stemcell Technologies</td>
			<td>5940</td>
			<td>Supplements for human pluripotent stem cell medium</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td>Detergent for cell permeabilization. Working concentration: 0.25% in blocking buffer</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488</td>
			<td>Thermofisher</td>
			<td>A-11029</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>AlexaFluor 594</td>
			<td>Thermofisher</td>
			<td>A-11037</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ezrin</td>
			<td>Thermofisher</td>
			<td>MA5-13862</td>
			<td>Primary antibody; astrocytes perisynaptic</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Chemicon</td>
			<td>MAB360</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Aves</td>
			<td>GFP-1020</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Glt1</td>
			<td>Gift from Dr. Jeffrey Rothstein</td>
			<td>n/a</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Homer</td>
			<td>Synaptic Systems</td>
			<td>160 011</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>MAP2</td>
			<td>Synaptic Systems</td>
			<td>188 004</td>
			<td>Primary antibody; neurons</td>
		</tr>
		<tr>
			<td>PSD95</td>
			<td>Abcam</td>
			<td>ab2723</td>
			<td>Primary antibody; neurons, post-synaptic</td>
		</tr>
		<tr>
			<td>S100</td>
			<td>Abcam</td>
			<td>ab868</td>
			<td>Primary antibody; astrocytes</td>
		</tr>
		<tr>
			<td>Synapsin 1</td>
			<td>Synaptic Systems</td>
			<td>106 103</td>
			<td>Primary antibody; neurons, pre-synaptic</td>
		</tr>
		<tr>
			<td>TuJ1/&#946;3-tubulin (TUBB3)</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>Primary antibody; neurons</td>
		</tr>
	</tbody>
</table>

synaptic microcircuit modeling with 3d cocultures of astrocytes and neurons from human pluripotent stem cells

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

When performed properly, this protocol will produce defined populations of functional cocultures of astrocytes28,33,34 and neurons35 generated from hPSCs (Figure 1A-1C), as detailed previously26 and described here in steps 2.1&#8211;2.2. This stepwise procedure, with the use of microwell plates, is expe...

Watch this Scientific Journal Video about Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells at JoVE.com

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

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

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

This method can help answer key questions in neuroscience regarding how intercellular communication between multiple human neural cell types contributes to synaptic circuit formation and function. This technique rapidly and systematically generates three-dimensional coculture spheres of functionally-mature astrocytes and neurons from human pluripotent stem cells. These can be utilized for rigorous experimental investigation.The reproducibility and scalability of this protocol is a defined alternative to the emerging organoid technologies. These show promise for novel drug development and cellular-engraftment therapy to promote neuroregeneration after injury or disease. Begin by seating clusters of HPSCs in two milliliters of HPSC medium containing the rho-kinase inhibitor Y-27632 into each well of an ECM-coated six-well plate.Maintain the stem cells until they are about 50%confluent, then change the medium to neural medium containing SB431542 and DMH1 to promote neural induction. When cells are about 95%confluent, split each well one-to-six into new ECM-coated wells. On day 14 dissociate the cells with detachment solution, and transfer the contents of each well to a non-coated T25 flask with medium containing Y-27632 to promote the formation of aggregates.To generate astrocyte progenitors and astrocytes in spontaneously-formed 3D aggregates, switch to neural medium containing EGF and FGF2, and feed every three to four days until astrocyte identity is confirmed at four to six months. By default, this protocol produces dorsal cortical astrocytes. However, astrocyte subtypes can be regionally specified by the addition of patterning morphogens, if desired.Once a week, when dark centers appear, collect the spheres by centrifugation, then gently dissociate the H-astro aggregates with detachment solution. Only replate those spheres that do not spontaneously attach to avoid passaging non-CNS and non-neural cells. After four to six months of expansion, confirm cell identity in either freeze and cryo-preservation medium as per manufacturer's instructions, or use immediately for experimentation.For the generation of nontransgenic neuronal progenitors in neurons, or H-neurons, seed day-14 HPSC-derived neural progenitors into ECM-coated six-well plates with two milliliters of neural medium and Y-27632 per well. If working with doxycycline-inducible iNeurons, add neural medium containing doxycycline when the cells are about 35%confluent to induce differentiation and activate gene expression. After two days, feed transgenic and nontransgenic lines with two milliliters of neural media per well per day.Continue until the cells are ready to lift at 70%confluency. For cocultures, first gently dissociate H-astros from 3D aggregates with detachment solution. Then proceed to dissociate H-neurons, or iNeurons, from a 2D monolayer.First, remove the medium from the six-well plate, and add 500 microliters of detachment solution to each well containing monolayer cultures. Incubate at 37 degrees Celsius for five minutes. Following the incubation, gently add two to three milliliters of DMEM-F12 medium to remove the attached cells.Then, collect the cells in medium in a 15-milliliter conical tube, and centrifuge at 300 times G for one minute. Aspirate the supernatant, add one milliliter of fresh medium, and pipette up and down gently with a 1, 000-microliter micro pipette to achieve a single-cell suspension. Count each cell type using a hemocytometer or automated cell-counter.Add the desired ratio of dissociated H-astros and H-neurons, or iNeurons, in a total volume of two milliliters to each well of a microplate. Centrifuge the plate at 100 times G for three minutes, and return to the incubator for two days. After one day, densely-packed microspheres should have formed in the plate.Use a 1, 000-microliter micro pipette to gently remove spheres from the micro wells. Lightly apply force to the bottom of micro wells with medium to remove any additional adhered spheres. After collecting all spheres in a 15-milliliter conical tube, allow to settle.Then wash the spheres by first aspirating the old medium, and then adding at least two milliliters of fresh medium. Add the spheres to a spinner flask containing 50 to 60 milliliters of medium. Place the flask in the incubator on a magnetic stir plate set at 60 RPM.A minimum of three weeks in culture is needed for synapse formation. Begin by coating the surface of each multielectrode array, or MEA, with one milliliter of polyornithine to render the surface hydrophilic, and incubate at 37 degrees Celsius for a minimum of four hours. Following the incubation, remove the polyornithine, and wash the surface of each MEA with deionized water.Then add one milliliter of extracellular matrix, and return to the incubator for a minimum of four hours. When ready, remove the extracellular matrix, then apply the spheres in 1.5 milliliters of medium onto the MEA surface, ensuring that the spheres are positioned on top of the electrode array. Allow to adhere for two days.To measure the electrical activity of neural spheres, place the MEA on a temperature-controlled head stage. Use a five-Hertz highpass filter, and a 200-Hertz lowpass filter, and a spike threshold of five times standard deviation, to record spontaneous electrical activity. Save raw data, including spike frequency and amplitude for statistical analysis.Neural-cell-type restricted protein markers, such as MAP2 for neurons and GFAP for astrocytes, visually demonstrate the evenly-dispersed arrangement and maturity of astrocytes in neurons within 3D spheres. A maximum projection of astrocyte morphology and branching is shown in a representative 3D sphere with H-astros sparsely labeled with membrane-bound GFP. Mature H-astros express markers including GFAP.Even though iNeurons express pre and postsynaptic proteins, as shown in the image on the left, synaptic density is significantly enhanced by the presence of H-astros and coculture. During recordings, healthy spheres of iNeurons placed on MEAs will spontaneously elicit voltage spikes greater than 40 microvolts with consistent firing frequencies. Coculture spheres display greater network connectivity with the presence of H-astros, resulting in an increase of synchronous network bursts of spikes.Following this procedure, other methods such as calcium imaging can be performed in order to measure the physiological response of astrocytes to synaptic activity. In addition, transplantation into animal models can be conducted to investigate connectivity with preexisting synaptic circuits. By following this bioengineering approach, one can incorporate extracellular biomaterials and additional cell types such as oligodendrocytes, microglian, and ethelial cells in specific ratios in order to model the complex signaling that occurs in the brain.

synaptic-microcircuit-modeling-with-3d-cocultures-astrocytes-neurons

Research

JoVE Journal

Developmental Biology

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...