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 ...

Research

JoVE Journal

Developmental Biology

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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 ...

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 ...

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

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 ...

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 ...

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 ...

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 ...

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 ...

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 ...