Name: analysis of non-human primate pancreatic islet oxygen consumption
Uploaded: 2019-12-18T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

The authors would like to acknowledge the Vanderbilt High Throughput Screening Core for the use of their facilities, Agilent Biotechnologies, Dr. Paul Kievit (Oregon Health and Science University) for non-human primate islet isolations, and Eric Donahue (Vanderbilt University) for assistance with Figure 1. J.M.E. was supported by NIGMS of the National Institutes of Health under award number T32GM007347. M.G. was supported by the NIH/NIDDK (R24DK090964-06) and the Department of Veterans Affairs (BX003744).

1. Preparation of Microplate and Sensor Cartridge on the Day Prior to Running the Assay
Islets were isolated from three year old Japanese macaques as previously described13. This method is very similar to that used to isolate human islets from cadaver donors, but differs from mice, in which pancreata are often inflated with collagenase solution while the animal is under sedation and prior to organ removal. Islet retrieval was conducted in accordance with the guidelines of...

<ol>
	<li>Mulder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcribing+&#946;-cell+mitochondria+in+health+and+disease.">Transcribing &#946;-cell mitochondria in health and disease.</a> Molecular Metabolism. 6 (9), 1040-1051 (2017).</li><li>Maechler, P., Wollheim, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+signals+in+glucose-stimulated+insulin+secretion+in+the+beta+cell.">Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell.</a> The Journal of Physiology. 529 (Pt 1), 49-56 (2000).</li><li>Curry, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon+Potentiation+of+Insulin+Secretion+by+the+Perfused+Rat+Pancreas.">Glucagon Potentiation of Insulin Secretion by the Perfused Rat Pancreas.</a> Diabetes. 19 (6), 420 (1970).</li><li>Song, G., Pacini, G., Ahr&#233;n, B., D'Argenio, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon+Increases+Insulin+Levels+by+Stimulating+Insulin+Secretion+Without+Effect+on+Insulin+Clearance+in+Mice.">Glucagon Increases Insulin Levels by Stimulating Insulin Secretion Without Effect on Insulin Clearance in Mice.</a> Peptides. 88, 74-79 (2017).</li><li>Vergari, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+inhibits+glucagon+release+by+SGLT2-induced+stimulation+of+somatostatin+secretion.">Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.</a> Nature Communications. 10 (1), 139 (2019).</li><li>Watts, M., Ha, J., Kimchi, O., Sherman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+glucagon+secretion:+the+&#946;/&#945;/&#948;+model.">Paracrine regulation of glucagon secretion: the &#946;/&#945;/&#948; model.</a> American Journal of Physiology--Endocrinology and Metabolism. 310 (8), E597-E611 (2016).</li><li>Sweet, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+measurement+of+oxygen+consumption+by+pancreatic+islets.">Continuous measurement of oxygen consumption by pancreatic islets.</a> Diabetes Technology &amp; Therapeutics. 4 (5), 661-672 (2002).</li><li>. Agilent Seahorse XF Instruments Overview and Selection Guide Available from: <a target="_blank" href="https://www.agilent.com/en/products/cell-analysis/seahorse-xf-instruments-selection-guide">https://www.agilent.com/en/products/cell-analysis/seahorse-xf-instruments-selection-guide</a> (2019)</li><li>Wikstrom, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+assay+for+islet+respiration+reveals+uncoupling+of+rodent+and+human+islets.">A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.</a> PLoS One. 7 (5), e33023 (2012).</li><li>Taddeo, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+islet+respirometry+reveals+functional+diversity+within+the+islet+population+of+mice+and+human+donors.">Individual islet respirometry reveals functional diversity within the islet population of mice and human donors.</a> Molecular Metabolism. 16, 150-159 (2018).</li><li>Conrad, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAFB+transcription+factor+impacts+islet+alpha-cell+function+in+rodents+and+represents+a+unique+signature+of+primate+islet+beta-cells.">The MAFB transcription factor impacts islet alpha-cell function in rodents and represents a unique signature of primate islet beta-cells.</a> American Journal of Physiology--Endocrinology and Metabolism. 310 (1), E91-E102 (2016).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity:+interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Elsakr, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Western-style+diet+affects+offspring+islet+composition+and+function+in+a+non-human+primate+model+of+maternal+over-nutrition.">Maternal Western-style diet affects offspring islet composition and function in a non-human primate model of maternal over-nutrition.</a> Molecular Metabolism. , (2019).</li><li>Soutar, M. P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FBS/BSA+media+concentration+determines+CCCP's+ability+to+depolarize+mitochondria+and+activate+PINK1-PRKN+mitophagy.">FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.</a> Autophagy. , 1-10 (2019).</li><li>Hirshberg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+Islet+Transplantation+Using+the+Nonhuman+Primate+(Rhesus)+Model+Predicts+That+the+Portal+Vein+Is+Superior+to+the+Celiac+Artery+as+the+Islet+Infusion+Site.">Pancreatic Islet Transplantation Using the Nonhuman Primate (Rhesus) Model Predicts That the Portal Vein Is Superior to the Celiac Artery as the Islet Infusion Site.</a> Diabetes. 51 (7), 2135-2140 (2002).</li><li>Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data.">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Methods in Enzymology. 547, 309-354 (2014).</li><li>Papas, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+Oxygen+Consumption+Rate+(OCR)+Dose+Predicts+Insulin+Independence+in+Clinical+Islet+Autotransplantation.">Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.</a> PLoS One. 10 (8), e0134428 (2015).</li></ol>

The study of islet oxygen consumption has previously been hampered by the spherical shape of islets, their lack of adherence to culture surfaces, and the number of islets required per well. In this protocol, we highlight the efficacy of the 96-well spheroid microplate for measuring islet oxygen consumption on a small number of islets and demonstrate a technique for handling and loading islets which is technically feasible and produces consistent results.
In order for islets to adhere to the bo...

In order to maintain normal blood glucose levels, the pancreatic &#946; cell must sense elevations in glucose and secrete insulin accordingly. The coupling of insulin secretion with glucose levels is directly linked to glucose metabolism and the production of ATP through mitochondrial oxidative phosphorylation. Thus, mitochondria play a critical role in stimulus-secretion coupling1. Assessing &#946;-cell mitochondrial function can reveal defects that lead to impaired insulin secretion. The secretion of glucagon by pancreatic &#945; cells is also closely tied to mitochondrial function2. Although immortalized islet cell lines have proven useful for some types of assays, the physiology of these cells does not accurately recapitulate whole islet function, as illustrated by the potentiation of insulin secretion by glucagon3,4 and the inhibition of glucagon secretion by insulin/somatostatin5,6 in intact islets. This demonstrates the need for measuring oxygen consumption using whole, intact islets.
Techniques for the measurement of islet cell respirometry have evolved over time, from the use of oxygen-sensitive fluorescent dyes7 to solid-state sensors that directly measure oxygen consumption8. Initially designed for monolayer, adherent cells, commonly used cell culture plate systems have proven to be ineffective for pancreatic islets. As islets do not naturally adhere to the wells, they are prone to being pushed to the periphery of the culture well resulting in inaccurate measurement of oxygen consumption9. To combat this problem, specialized 24-well plates with a central depression that could contain islets were developed9. However, the 24-well plate system was limited by the large number of islets required (50-80 per well) and the number of conditions that could be tested simultaneously10. The recent development of 96-well microplates designed specifically for extracellular flux analysis in spheroids has overcome these barriers, enabling the measurement of islet respirometry with 20 or fewer islets per well10.
Here, we demonstrate the use of this system to measure oxygen consumption in islets from the Japanese macaque (Macaca fuscata), an animal model with similar islet biology to humans11,12. In this protocol, 15 macaque islets are analyzed per well. In our hands, 15 islets per well produced higher baseline oxygen consumption than fewer islets, with robust activation and repression of respiration in response to pharmacologic manipulation. We highlight the steps to prepare for the assay, an effective method for consistent loading of islets at the center of each well, and common challenges when performing this assay.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture dish, 60 mm X 15 mm style</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak Cell and Tissue Adhesive</td>
			<td>Corning</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td>For glucose solution, 200 mg/ml, sterile filetered</td>
		</tr>
		<tr>
			<td>Disposable reservoirs (sterile), 25 ML</td>
			<td>Vistalab</td>
			<td>3054-1033</td>
			<td>for loading multichannel pipet</td>
		</tr>
		<tr>
			<td>EZFlow Sterile 0.45 &#956;m PES Syringe Filter, 13 mm</td>
			<td>Foxx Life Sciences</td>
			<td>371-3115-OEM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200 mM (100x)</td>
		</tr>
		<tr>
			<td>Multichannel pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>94410810</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette, 15-1250 &#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>4672100BT</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>P20, P200, and P1000 pipettes</td>
			<td>Eppendorf</td>
			<td>2231000602</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Probe</td>
			<td>Hanna Instruments</td>
			<td>HI2210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 20 &#956;L, 200 &#956;L, 1000 &#956;L</td>
			<td>Olympus</td>
			<td>24-404, 24-412, 24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF Base Media</td>
			<td>Agilent</td>
			<td>103334-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF Cell Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103015-100</td>
			<td>Includes Oligomycin, FCCP, and Rotenone/Antimycin A</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td>S7800B</td>
			<td>Including prep station with 37 &#176;C non-CO2 incubator</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Spheroid Fluxpak Mini</td>
			<td>Agilent</td>
			<td>102905-100</td>
			<td>Includes sensor cartridge, spheroid microplate, and calibrant</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>100 mM (100x)</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZX9</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (sterile), 5 mL</td>
			<td>BD</td>
			<td>309603</td>
			<td>For sterile filtration</td>
		</tr>
		<tr>
			<td>Water (sterile)</td>
			<td>Sigma</td>
			<td>W3500-500mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of non-human primate pancreatic islet oxygen consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

To load islets into microplate, 15 islets should be aspirated in 15 &#181;L of media, as shown in Figure 1A. Islets will naturally settle toward the bottom of the pipet tip within a few seconds. Then, the pipet tip is lowered to the bottom of the well. The tip is very slightly lifted, and a small volume (about 5 &#181;L) is pipetted out along with the islets. This technique results in consistent placement of islet at the bottom of the micropl...

Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

This method allows for the quantification of oxygen consumption from small numbers of islets from relevant species, such as non-human primates and humans, allowing for assessment of mitochondrial function. This technique can be performed with small numbers of primary islets, increasing the technical replicates, allowing for the testing of multiple conditions, and improving the rigor and reproducibility of the assay. This technique assesses mitochondrial health, a major determinate of islet function and insulin secretion that is often impaired in diabetes and is a significant predictor of successful islet transplantation.It is essential to make sure that the islets are localized to the center of the wells following transfer. Failure to do so results in inconsistent oxygen consumption measurements. Demonstrating this procedure will be, Valerie Ricciardi, an undergraduate student working in my laboratory.The day before the assay, add 200 microliters of cell and tissue adhesive to 2.8 milliliters of 0.1 molar sodium bicarbonate solution and add 20 microliters of the resulting solution to the bottom of each well of a 96 well spheroid microplate. Ensure that any air bubbles are removed from the pipette tip. When all of the wells have been loaded, place the plate in a 37 degree Celsius non-carbon dioxide incubator.After one hour, aspirate the cell adhesive solution from the plate within a sterile environment and wash each well two times with 400 microliters of 37 degree Celsius sterile water. Then allow the wells to air dry for 30 to 40 minutes before covering the plate for overnight storage at four degrees Celsius. To hydrate the sensor cartridge, in a sterile environment open the sensor cartridge package and remove the contents.Place the sensor cartridge upside down next to the utility plate and fill each well of the utility plate with 200 microliters of sterile water. When all of the wells have been loaded, lower the sensor cartridge into the utility plate and place the assembled sensor cartridge and utility plate into the 37 degree Celsius non-carbon dioxide incubator overnight. On the day of the assay, remove the sensor cartridge lid and decant the water from the wells of the utility plate.Add 200 milliliters of prewarmed calibrant to each well and replace the sensor cartridge lid. Then place the sensor cartridge back into the incubator for about one hour. To prepare the islets for transfer, hand pick the islets into a 60 by 15 millimeter cell culture dish containing assembled medium to a near 100%purity.When all of the islets have been collected, load each well of the prepared spheroid microplate with 175 microliters of assembled medium and use a P20 pipette set to 15 microliters to aspirate 15 islets in 15 microliters of medium from the cell culture dish. To seed the islets onto the spheroid microplate, lower the pipette tip to the bottom of the well, barely lift the tip, and slowly dispense about five microliters of medium. To load the islets into the wells, allow the islets to settle in the pipette tip before gently releasing them directly above the bottom of the well center in a small volume of medium.Confirm that all of the islets have left the pipette tip, occasionally checking the spheroid microplate under the microscope to verify that the islets are seeded at the bottom of the plate and not stuck to the sides of the wells. When all of the islets have been transferred, place the microplate in the 37 degree Celsius non-carbon dioxide incubator. Before initiating the assay procedure, load Port A of the sensor cartridge with 20 microliters of freshly prepared 45 micromolar oligomycin, Port B with 22 microliters of freshly prepared 10 micromolar FCCP, and Port C with 25 microliters of freshly prepared 25 micromolar rotenone AA.Next, program an extracellular flux analyzer assay for a 30 minute baseline respiration period, 42 minute oligomycin measurement period, 30 minute FCCP period, and 30 minute rotenone AA period.Then run the assay following the instructions on the screen for calibrating the sensor and inserting the microplate. For islet delivery to the assay plate, 15 islets should be aspirated in 15 microliters of medium as illustrated. This technique will result in the consistent placement of islets at the bottom center of each microplate well allowing for accurate oxygen consumption measurements.In this representative experiment showing the oxygen consumption of individual wells, the wells were poorly loaded resulting in a very low baseline level of oxygen consumption and little to no response to FCCP. In this experiment, most of the wells demonstrated a significant baseline respiration and response to drugs. However, two wells exhibited no response to rotenone, suggesting that the drug was not properly released into the well.These wells were excluded from further analyses. Here, the results from a successful assay in which the wells were properly loaded with islets and correctly injected with drugs can be observed. It is beneficial to prepare the reagents and plates the day prior to performing the assay to allow the islets to be used shortly after they are isolated or received.Following the assay, the islets can be recovered for protein or DNA extraction to normalize the oxygen consumption data or to quantify the mitochondrial DNA content. The ability to measure mitochondrial function in small numbers of primary primate islets allows the testing of multiple drugs and other manipulations in a single biological replicate. Individuals working with primary primate islets should be sure to be trained in the proper and safe handling with these tissues.In particular, the use of appropriate personal protective equipment and the proper disposal of these samples.

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Developmental Biology

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

This protocol describes a detailed methodology for detecting H2O2 localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Analysis of Epididymal Protein Synthesis and Secretion

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.