Name: differentiation of human pluripotent stem cells into insulin-producing islet clusters
Uploaded: 2023-06-23T14:00:00
Description: Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters at JoVE.com

We gratefully acknowledge the support from STEMCELL Technologies, Michael Smith Health Research BC, Stem Cell Network, JDRF, and the Canadian Institutes of Health Research. Jia Zhao and Shenghui Liang are recipients of the Michael Smith Health Research BC Trainee Award. Mitchell J.S. Braam is a recipient of the Mitacs Accelerate Fellowship. Diepiriye G. Iworima is a recipient of the Alexander Graham Bell Canada Graduate Scholarship and the CFUW 1989 Ecole Polytechnique Commemorative Award. We sincerely thank Dr. Edouard G. Stanley from MCRI and Monash University for sharing the Mel1 INS GFP/W line and the Alberta Diabetes Institute Islet Core for isolating and distributing human islets. We also acknowledge the support from the Life Sciences Institute Imaging and Flow Cytometry facilities at the University of British Columbia. Figure 1 was created with BioRender.com.

This protocol is based on work with hPSC lines, including H1, HUES4 PDXeG, and Mel1 INSGFP/W, in feeder-free conditions. A step-by-step procedure is detailed in this section, with supporting data from the differentiation of Mel1 INSGFP/W in the representative results section. We recommend that further optimization is needed when working with other hPSC lines that are not stated here. See the Table of Materials for details related to all reagents and solutions used in this protocol.<...

<ol>
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This paper describes a seven-stage hybrid protocol that allows for the generation of hPSC islets capable of secreting insulin upon glucose challenge within 40 days of culture in vitro. Among these multiple steps, efficient induction of definitive endoderm is believed to set an important starting point for the final differentiation outcomes18,27,28. In the manufacturer&#39;s protocol, a seeding density at 2.6 &#215; 10<s...

Pancreatic beta cells secrete insulin responding to rises in blood glucose levels. Patients lacking sufficient insulin production due to the autoimmune destruction of beta cells in type 1 diabetes (T1D)1, or due to beta cell dysfunction in type 2 diabetes (T2D)2, are typically treated with the administration of exogenous insulin. Despite this life-saving therapy, it cannot precisely match the exquisite control of blood glucose as achieved by dynamic insulin secretion from bona fide beta cells. As such, patients often suffer the consequences of life-threatening hypoglycemic episodes and other complications resulting from chronic hyperglycemic excursions. Transplantation of human cadaveric islets successfully restores tight glycemic control in T1D patients but is limited by the availability of islet donors and difficulties in purifying healthy islets for transplantation3,4. This challenge can, in principle, be solved by using hPSCs as an alternative starting material.
Current strategies for generating insulin-secreting islets from hPSCs in vitro often aim to mimic the process of embryonic pancreas development in vivo5,6. This requires knowledge of the responsible signaling pathways and timed addition of corresponding soluble factors to mimic critical stages of the developing embryonic pancreas. The pancreatic program initiates with the commitment into definitive endoderm, which is marked by transcription factors forkhead box A2 (FOXA2) and sex-determining region Y-box 17 (SOX17)7. Successive differentiation of definitive endoderm involves the formation of a primitive gut tube, patterning into a posterior foregut that expresses the pancreatic and duodenal homeobox 1 (PDX1)7,8,9, and epithelial expansion into pancreatic progenitors that co-express PDX1 and NK6 homeobox 1 (NKX6.1)10,11.
Further commitment to endocrine islet cells is accompanied by the transient expression of pro-endocrine master regulator neurogenin-3 (NGN3)12 and stable induction of key transcription factors neuronal differentiation 1 (NEUROD1) and NK2 homeobox 2 (NKX2.2)13. The major hormone-expressing cells, such as insulin-producing beta cells, glucagon-producing alpha cells, somatostatin-producing delta cells, and pancreatic polypeptide-producing PPY cells, are subsequently programmed. With this knowledge, as well as discoveries from extensive, high-throughput drug screening studies, recent advancements have enabled the generation of hPSC-islets with cells resembling beta cells capable of insulin secretion14,15,16,17,18,19.
Step-wise protocols have been reported for generating glucose-responsive beta cells6,14,18,19. Built upon these studies, the present protocol involves the use of a pancreatic progenitor kit for generating PDX1+/NKX6.1+ pancreatic progenitor cells in a planar culture, followed by microwell plate aggregation into uniform-sized clusters and further differentiation toward insulin-secreting hPSC-islets with the R-protocol in a static 3D suspension culture. Quality control analyses, including flow cytometry, immunostaining, and functional assessment, are performed for rigorous characterization of the differentiating cells. This paper provides a detailed description of each step of the directed differentiation and outlines the in vitro characterization approaches.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3,3&#8217;,5-Triiodo-L-thyronine (T3)</td>
			<td>Sigma</td>
			<td>T6397</td>
			<td>Thyroid hormone</td>
		</tr>
		<tr>
			<td>4% PFA solution</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td>Should be handled in fume hood</td>
		</tr>
		<tr>
			<td>96-Well, Ultralow Attachment, flat bottom</td>
			<td>Corning Costar (VWR)</td>
			<td>CLS3474</td>
			<td>Flat bottom; for static suspension culture in the last three stages</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>STEMCELL Technologies</td>
			<td>07920</td>
			<td>Dissociation reagent for Stage 4 cells</td>
		</tr>
		<tr>
			<td>Aggrewell400 plates</td>
			<td>STEMCELL Technologies</td>
			<td>34415</td>
			<td>400 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Aggrewell800 plates</td>
			<td>STEMCELL Technologies</td>
			<td>34815</td>
			<td>800 &#181;m diameter microwell plates</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat anti-Human FOXA2 (goat IgG)</td>
			<td>R&#38;D Systems</td>
			<td>IC2400G</td>
			<td>1:100 in flow cytometry; used for assaying Stage 1 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat IgG Isotype Control</td>
			<td>R&#38;D Systems</td>
			<td>IC108G</td>
			<td>1:100 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Mouse anti-Human SST (mouse IgG2B)</td>
			<td>BD Sciences</td>
			<td>566032</td>
			<td>1:250 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Mouse IgG2B Isotype Control</td>
			<td>R&#38;D Systems</td>
			<td>IC0041G</td>
			<td>1:500 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human C-peptide (mouse IgG1&#954;)</td>
			<td>BD Pharmingen</td>
			<td>565831</td>
			<td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human INS (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>565689</td>
			<td>1:2,000 in flow cytometry</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human NKX6.1 (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>563338</td>
			<td>1:33 in flow cytometry; used for assaying Stage 4 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse anti-Human SOX17 (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>562594</td>
			<td>1:50 in flow cytometry; used for assaying Stage 1 cells</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Mouse IgG1&#954; Isotype Control</td>
			<td>BD Sciences</td>
			<td>557714</td>
			<td>1:50 in flow cytometry</td>
		</tr>
		<tr>
			<td>ALK5i II</td>
			<td>Cayman Chemicals</td>
			<td>14794</td>
			<td>TGF-beta signaling inhibitor</td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>7010</td>
			<td>Microwell Rinsing Solution</td>
		</tr>
		<tr>
			<td>Assay chamber</td>
			<td>Cellvis</td>
			<td>D35-10-1-N</td>
			<td>For static GSIS and confocal imaging purposes</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP1600-100</td>
			<td>For immunostaining procedure</td>
		</tr>
		<tr>
			<td>CK19 antibody</td>
			<td>DAKO</td>
			<td>M0888</td>
			<td>1:50 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>For nuclear counterstaining</td>
		</tr>
		<tr>
			<td>DMEM/F12, HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td>Matrix diluting solution</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A21432</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A21447</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A31570</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A31571</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa Fluor 555</td>
			<td>Life technologies</td>
			<td>A31572</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A31573</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Donkey anti-sheep Alexa Fluor 647</td>
			<td>Life technologies</td>
			<td>A21448</td>
			<td>1:500 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td>Without Ca2+ and Mg2+</td>
		</tr>
		<tr>
			<td>ELISA, insulin, human</td>
			<td>Alpco</td>
			<td>80-INSHU-E01.1</td>
			<td>For human insulin measurement</td>
		</tr>
		<tr>
			<td>Fatty acid-free BSA</td>
			<td>Proliant</td>
			<td>68700</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>Fixation and Permeabilization Solution Kit</td>
			<td>BD Sciences</td>
			<td>554714</td>
			<td>Fix/Perm and 10x Perm/Wash solutions included</td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>7174</td>
			<td>For clump passaging hPSCs during maintenance culture</td>
		</tr>
		<tr>
			<td>Glucagon antibody</td>
			<td>Sigma</td>
			<td>G2654</td>
			<td>1:400 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>GLUT1 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA1-37782</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>GlutaMAX-I (100x)</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>L-glutamine supplement</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo Fisher Scientific</td>
			<td>G33-4</td>
			<td>For tissue clearing and mounting</td>
		</tr>
		<tr>
			<td>GSi XX</td>
			<td>Sigma Millipore</td>
			<td>565789</td>
			<td>Notch inhibitor</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>ITS-X (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>51500056</td>
			<td>Insulin-Transferrin-Selenium-Ethanolamine; medium supplement</td>
		</tr>
		<tr>
			<td>LDN193189&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>72147</td>
			<td>BMP antagonist</td>
		</tr>
		<tr>
			<td>MAFA antibody</td>
			<td>Abcam</td>
			<td>ab26405</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Matrigel, hESC-qualified</td>
			<td>Thermo Fisher Scientific</td>
			<td>08-774-552</td>
			<td>Extracellular matrix for vessel surface coating</td>
		</tr>
		<tr>
			<td>MCDB131 medium</td>
			<td>Life technologies</td>
			<td>10372019</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>mTeSR1 Complete Kit</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>stem cell medium and 5x supplement included</td>
		</tr>
		<tr>
			<td>N-Cys (N-acetyl cysteine)</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Antioxidant</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6297</td>
			<td>Medium supplement</td>
		</tr>
		<tr>
			<td>NEUROD1 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF2746</td>
			<td>1:20 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>NKX6.1 antibody</td>
			<td>DSHB</td>
			<td>F55A12-c</td>
			<td>1:50 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Pancreatic polypeptide antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF6297</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>D8662</td>
			<td>With Ca2+ and Mg2+</td>
		</tr>
		<tr>
			<td>PDX1 antibody</td>
			<td>Abcam</td>
			<td>ab47267</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human GCG (mouse IgG1&#954;)</td>
			<td>BD Sciences</td>
			<td>565860</td>
			<td>1:2,000 in flow cytometry; used for assaying Stage 7 cells</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human NKX6.1 (mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>563023</td>
			<td>1:250 in flow cytometry</td>
		</tr>
		<tr>
			<td>PE Mouse anti-Human PDX1 (mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>562161</td>
			<td>1:200 in flow cytometry; used for assaying Stage 4 cells</td>
		</tr>
		<tr>
			<td>PE Mouse IgG1&#954; Isotype Control</td>
			<td>BD Sciences</td>
			<td>554680</td>
			<td>1:2,000 in flow cytometry</td>
		</tr>
		<tr>
			<td>PE Mouse-Human Chromogranin A (CHGA, mouse IgG1k)</td>
			<td>BD Sciences</td>
			<td>564563</td>
			<td>1:200 in flow cytometry</td>
		</tr>
		<tr>
			<td>R428&#160;</td>
			<td>Cayman Chemicals</td>
			<td>21523</td>
			<td>AXL tyrosine kinase inhibitor</td>
		</tr>
		<tr>
			<td>Retinoid acid, all-trans</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Light-sensitive</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer, 10x</td>
			<td>Sigma</td>
			<td>20-188</td>
			<td>For hormone extraction</td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Sigma</td>
			<td>S4572</td>
			<td>SHH inhibitor</td>
		</tr>
		<tr>
			<td>SLC18A1 antibody</td>
			<td>Sigma</td>
			<td>HPA063797</td>
			<td>1:200 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Somatostatin antibody</td>
			<td>Sigma</td>
			<td>HPA019472</td>
			<td>1:100 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>STEMdiff Pancreatic Progenitor Kit</td>
			<td>STEMCELL Technologies</td>
			<td>05120</td>
			<td>Basal media and supplements included</td>
		</tr>
		<tr>
			<td>Synaptophysin antibody</td>
			<td>Novus</td>
			<td>NB120-16659</td>
			<td>1:25 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>For permeabilization</td>
		</tr>
		<tr>
			<td>Trolox&#160;</td>
			<td>Sigma Millipore</td>
			<td>648471</td>
			<td>Vitamin E analog</td>
		</tr>
		<tr>
			<td>TrypLE Enzyme Express</td>
			<td>Life technologies</td>
			<td>12604-021</td>
			<td>cell dissociation enzyme reagent for single cell passaging hPSCs</td>
		</tr>
		<tr>
			<td>Trypsin1/2/3 antibody</td>
			<td>R&#38;D Systems</td>
			<td>AF3586</td>
			<td>1:25 in whole mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72304</td>
			<td>ROCK inhibitor</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>Sigma</td>
			<td>Z0251</td>
			<td>Medium supplement</td>
		</tr>
	</tbody>
</table>

differentiation of human pluripotent stem cells into insulin-producing islet clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

We developed a hybrid strategy to differentiate stem cells into insulin-secreting hPSC-islets in seven steps, which utilizes a pancreatic progenitor kit for the first four stages in planar culture, followed by a modified protocol built upon a previously reported method6 in a static suspension culture for the last three stages (Figure 1). With this protocol, ensuring a near confluency (90%-100%) culture at 24 h after cell seeding (Stage 0) is critical for initiating an...

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters at JoVE.com

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

This protocol uses a static suspension culture strategy for differentiating pancreatic progenitors into islet clusters, reduces the stresses that cells experience during shaking culture, and greatly improves differentiation success. The hardware protocol is beginner-friendly, and save time for optimization of the first four stages. Personnel with basic stem cell culture techniques can reproduce this protocol with a good chance of generating functional islet-like clusters.To begin, precoat culture wells with hESC-qualified matrigel diluted in ice-cold DMEM/F12 and place the plate in a humidified 37-degrees-Celsius 5%carbon dioxide incubator for 30 minutes. Aspirate the spent medium from the hPSC culture and rinse once with DPBS. Dissociate hPSC culture into single cells with cell dissociation enzyme at 37 degrees Celsius for three to five minutes.After dissociation, rinse the cells by adding warm DMEM/F12 medium and transfer them into a 15-or 50-milliliter conical tube. Centrifuge the cell suspension at 300 G for five minutes and resuspend the pellet in a stem cell complete medium containing 10-micromolar Y-27632. Then, perform live cell counting using a hemocytometer.After counting aspirate diluted matrigel and immediately seed live cells at 1.6 to 1.8 times 10 to the five per square centimeter density on matrigel-coated wells. Incubate the cells in a humidified incubator for 24 hours. Next, on stage one, day one, prepare stage 1A medium.Replace the spent medium with stage 1A medium and incubate for 24 hours. The next day, replace the spent medium from the wells with freshly prepared stage 1B medium. On stage two, day one, prepare stage 2A medium and replace the medium from wells with stage 2A medium.Incubate for 24 hours before adding stage 2B medium into the wells;An stage three, day one, aspirate the spent medium from wells, then add freshly prepared stage 3 medium and incubate in a humidified incubator. On stage four, day one, replace the spent medium with freshly prepared stage 4 medium and incubate in a humidified incubator. On stage four, day five, pretreat the microwells with 500 microliters of anti-adherence rinsing solution per well.Centrifuge the microwell plate at 1, 300 G for five minutes and observe under a microscope to ensure no air bubbles are trapped in microwells. Next, aspirate the rinsing solution from the wells and rinse once with one milliliter of DPBS. Then, add one milliliter of aggregation medium to each well.After removing the spent medium from the pancreatic progenitor cultures, wash the culture once with DPBS before dissociating into single cells with dissociation reagent at 37 degrees Celsius for 10 to 12 minutes. Rinse the cells with warm DMEM/F12 and transfer them into a tube for centrifugation at 300 G for five minutes. Resuspend the pellet in the aggregation medium and perform live cell counting.Seed 2.4 to 3.6 million cells per well and add an aggregation medium to each well to achieve a final volume of two milliliters per well. Using a P-1000 pipette tip, gently pipette the cells up and down for even distribution. Centrifuge the cell suspension at 300 G for five minutes to capture the cells in the microwells and incubate in a humidified incubator for 24 hours to initiate aggregation.After aggregation, gently pipette the aggregates up and down several times to float any non-aggregated cells. Once the aggregates settle, carefully remove the spent medium containing floating non-aggregated cells without aspirating aggregates. Dispense fresh stage 5 medium onto the surface of the microwell plate to dislodge aggregates from the microwells, and transfer them into the ultra low-attachment six-well plate.After collecting all aggregates in the ultra low-attachment six wells, resuspend them in stage 5 medium to adjust the density to 20 clusters per 100 microliters. Then, using a multichannel pipette, dispense 50 microliters of stage 5 medium into each well of an ultra-low attachment ULA flat-bottom 96-well plate. Fill the corner and edge wells with 200 microliters of DPBS.Gently mix cluster resuspension every time before dispensing. Then, add 100 microliters of the cluster resuspension into each well of the ULA flat-bottom 96-well plate. Place the culture plates on a level surface in a humidified incubator for 24 hours.The next day or on stage five, day two, using a multi-channel pipette, gently remove 90 microliters of the spent medium from each well and refresh with 100 microliters of stage 5 medium. On stage six, day one, replace the 90 microliters of spent medium with 100 microliters of stage 6 medium per well. On stage seven, day one, remove the spent medium and add 100 microliters of stage 7 medium per well.In this study, using microwell plates, thousands of pancreatic progenitor clusters with uniform size and morphology were generated at a time, and the average aggregation efficiency was around 74%Importantly, expression of PDX1 and NKX6.1 was maintained at high levels in these clusters post-aggregation. Insulin expressing cells were gradually induced within endocrine clusters, as indicated by increased insulin GFP signals in living cultures over time. Cluster size increases from an average diameter of 150 microns to 220 microns between stages five and seven.Characterization of cell composition shows that stage seven hPSC islets generated by the hybrid protocol were primarily endocrine, and comprised of four major islet cell types. This hybrid protocol generated largely monohormonal islet cells and a minority of bihormonal cells. Differentiating beta cells express NKX6.1, MAFA, NEUROD1, PDX1, and GLUT1.Furthermore, 60%insulin and PDX1 double-positive cells and 50%C-peptide and NKX6.1 double-positive cells were induced in stage seven cultures. In vitro, static glucose-stimulated insulin secretion assays demonstrated that stage seven hPSC islets secrete high insulin levels in response to high glucose and direct depolarization. The total insulin content of hPSC islets is approximately half the amount of cadaveric islets.The multi-well-based static culture system for generating stem cell-derived islets facilitates various in situ assays, and can serve as a suitable platform for protocol development and small-scale screening studies. To minimize cluster fusion during a static culture in 96-well plate, do not tilt the plate when performing the medium change. Always keep the plate horizontal on a level surface.

differentiation-human-pluripotent-stem-cells-into-insulin-producing

Research

JoVE Journal

Developmental Biology

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

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 ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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 ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

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 ...

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 ...