Name: a neurite outgrowth assay and neurotoxicity assessment with human neural progenitor cell-derived neurons
Uploaded: 2020-08-06T15:40:00
Description: Watch this Scientific Journal Video about A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons at JoVE.com

This research was funded by NIMAD research grant (940714) awarded to MAF.

Ethics Statement: Fetal specimens were received from the Birth Defects Research Laboratory at the University of Washington in Seattle through a tissue distribution program supported by the National Institute of Health (NIH). The Birth Defects Research Laboratory obtained appropriate written informed consent from the parents and the procurement of tissues was monitored by the Institutional Review Board of the University of Washington. All the work was performed with approval by the Human Subject Research Office at the Uni...

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This protocol is one of the few published papers describing the test for neurite length upon treatment with test compounds. Furthermore, we describe how to use hNPCs for a neurite outgrowth assay and neurotoxicity assessment. By utilizing this neurite outgrowth assay and neurotoxicity assessment on hNPCs-derived neurons, the neurogenic potential of a category of epigenetic small-molecule compounds, HDAC inhibitors, in inducing neurite outgrowth is demonstrated22. Furthermore, in another paper pres...

Neurite growth is a process fundamental to the formation of the neuronal network and nerve regeneration1,2. Following an injury, neurite outgrowth plays a key role in regeneration of the nervous system. Neurite outgrowth is also an important element of the extracellular signaling in inducing neuronal regenerative activities to enhance the outcomes for neurodegenerative disorders and neuronal injury3,4,5,6.
By maintaining their differentiation potential in producing various neural lineages, human neural progenitor cells (hNPCs) could provide a model system for studies of central nervous system (CNS) function and development7,8,9. High translational potential and physiological relevance of hNPCs as a primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screenings. However, the maintenance and scaling of the primary cell models for high-throughput assays could be time-consuming and labor-intensive10,11,12,13.
In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment is another application using the hNPC-derived neurons. There are thousands of commercial chemical compounds that are either not examined or with poorly understood neurotoxicity potential. Therefore, more reliable and effective screening experiments to assess, distinguish, and rank compounds based on their potential to elicit developmental neurotoxicity is in high demand14. The increase in prevalence and incidence of neurological disorders along with the abundance of untested compounds in the environment necessitates the development of more trustworthy and efficient experiments to identify hazardous environmental compounds that may pose neurotoxicity15.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.

<table>
	<tbody>
		<tr>
			<td>4-well Glass Chamber Slides</td>
			<td>Sigma</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>R37117</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;-Tubulin III</td>
			<td>Thermo</td>
			<td>MA1-118X</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 - minus vitamin A</td>
			<td>Thermo</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>PeproTech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A8531</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo</td>
			<td>Promega</td>
			<td>G7572</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell</td>
			<td>Corning</td>
			<td>432000</td>
			<td>Cell freezing containers ensuring standardized controlled-rate -1&#8451;/minute cell freezing in a -80&#8451; freezer</td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>StemCell Technologies</td>
			<td>7930</td>
			<td>Cryopreservation medium containing 10% DMSO</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>34869-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF</td>
			<td>Gibco</td>
			<td>PHG6015</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Thermo</td>
			<td>FB002</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>PeproTech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-alanyl-L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Thermo</td>
			<td>50062Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Calbiochem</td>
			<td>375095</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid</td>
			<td>Sigma</td>
			<td>A92902-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-lysine</td>
			<td>Sigma</td>
			<td>L5501</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>StemCell Technologies</td>
			<td>5854</td>
			<td>Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>71376</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc 384-Well Polystyrene White Microplates</td>
			<td>Thermo</td>
			<td>164610</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermo</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly&#8208;L&#8208;lysine</td>
			<td>Sigma</td>
			<td>P5899-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>Cell dissociation reagent containing proteolytic and collagenolytic enzymes</td>
		</tr>
		<tr>
			<td>Synaptophysin</td>
			<td>Thermo</td>
			<td>MA5-14532</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Sigma</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

a neurite outgrowth assay and neurotoxicity assessment with human neural progenitor cell-derived neurons

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect.
Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model.
The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology.&#34;

The protocol presented in the manuscript has successfully been used in two recently published papers22,23. Figure 3 demonstrates the use of hNPCs-derived neurons in examining the effect of HDAC inhibitors as epigenetic compounds on the extension of neurites as a marker for neurite outgrowth and subsequent neurogenic ability of small molecule compounds.
Furthermore, in Figure 4...

Watch this Scientific Journal Video about A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons at JoVE.com

A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons

The presented protocol describes a method for a neurite outgrowth assay and neurotoxicity assessment of small molecule compounds.

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides ...

The presented protocol describing neurite outgrowth assay that is highly relevant to human biology and neurotoxicity assessment of a small molecule compounds. High translational potential and physiological relevance of human neural progenitor cells as the primary human cell model offer a considerable advantage in neurite outgrowth-related drug discovery screening and neurotoxicity assessment. Utilizing the presented protocol, human induced pluripotent stem cell and human embryonic stem cell-derived neurons can also be used as to alternative cell sources for the neurite outgrowth assay and neurotoxicity assessment.Begin by collecting the media with floating neurospheres and transferring it to a 50 milliliter conical tube. Centrifuge the tube at 300 to 400 times g for three minutes. Then carefully aspirate the supernatant.and resuspend the spheres in 500 microliters of defrosted cell dissociation reagent. Incubate the spheres at 37 degrees Celsius for five to 15 minutes. Then add five to 10 milliliters of pre-warmed culture media and centrifuge the tube at 300 to 400 times g for five minutes to sediment the neurospheres.Aspirate the supernatant and add two milliliters of cultural media pipetting up and down with a 1000 microliter pipette until the neurospheres form a single cell suspension. After counting the cells, add two to 3 million cells to a T25 flask in 10 milliliters of culture media. Replace half the culture media every three days.Use commercially available cryopreservation medium to freeze the cells. Transfer the neurospheres to a 50 milliliter tube and allow the spheres to settle at the bottom. Use a 200 or 1000 microliter pipette tip to transfer the big neurospheres to a new 50 milliliter tube before passaging.Centrifuge the remaining neurospheres at 300 to 400 times g for three minutes and carefully remove the supernatant. Resuspend up to a thousand spheres in one milliliter of cryopreservation reagent and transfer it to a cryo tube. Store the tube overnight at minus 80 degrees Celsius, then move it to liquid nitrogen for long-term storage.To measure neuronal outgrowth, open the image in Fiji and select Analyze, Tools and ROI Manager. Right-click on the fifth icon in the tool bar Straight and switch to Freehand Line. Optionally, double-click on the same icon to change the line width to 10.Then trace the longest neurite beginning near the cell body and extending to the tip. Press Control and T then F to add the measurement to the ROI Manager and highlight the measured neuron. Select all numbers in the ROI.Click on Measure, then copy and paste the measurements into a spreadsheet. To measure fluorescence intensity of labeled cells, click on the fourth icon in the toolbar Freehand Selections and draw the shape of the cell. Click Analyze and Set Measurements.Then make sure that Area, Mean gray value, Min max gray value and Integrated density are selected. Click Analyze and measure. Select the region next to the cell as background.And then click analyze and measure. Select all measure data and copy, paste it into a spreadsheet. To coat the plate add 30 microliters of poly-L-lysine into each well of a 384-well plate and incubate the plate at room temperature for one hour.Wash it twice with PBS and let it dry for approximately 30 minutes. Add 30 microliters of laminine to each well, then incubate the plate at 37 degrees Celsius for two hours. Repeat the washes with PBS and proceed with plating the cells.Add 20, 000 single cell neurospheres in 25 microliters of differentiation media to each well of the 384-well plate and incubate the plate at 37 degrees Celsius for five days. Hight precision pipetting skills are required for plating the exact number of neurospheres that's all segregated into single cells. Therefore, differentiating the neurospheres into neurons before plating is recommended to achieve more reproducible results.After the incubation, add five microliters of test compound to each well at six times the desired concentration. And incubate the cells for another 24 hours. To perform the cell viability essay, add 30 microliters of luminescent reagent to each well.And leave the plate on a shaker for two minutes. Centrifuge the plate at 300 to 400 times g for 30 seconds, then incubate the plate at room temperature for 10 minutes, protecting it from light. After the incubation, measure the luminescence on a microplate reader.The human neural progenitor cell-derived neurons were used to examine the effect of HDAC inhibitors as small molecule epigenic compounds for the extension of neurites and subsequent neurogenic ability of small molecule compounds. The neurotoxicity of the HDAC inhibitors was also assessed after differentiating the neural progenitor cells in 384-well plates, showing the potential to scale the protocol for a higher number of compounds. In a different study, measurement of the neuronal cell fluorescence intensity was used to quantify the abundance of histone H4 lysine five acetylation as a histone marker after treating the neurons with epigenetic modifier compounds.Additionally, this protocol has been used to visualize a pre-synaptic protein, synaptophysin, to check for possible synaptogenic effects of small molecule compounds. Using the presented method, neurites number, intensity, length, and width, together with synaptic characteristics, including pre and post-synaptic protein modifications could be reliably measured.

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Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

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

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

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

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