Name: differentiation of human pluripotent stem cells into pancreatic beta-cell precursors in a 2d culture system
Uploaded: 2021-12-16T14:45:00
Description: Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System at JoVE.com

This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and Qatar Biomedical Research Institute (QBRI) (no. 2016-003). This work is optimized for hESCs such as H1, H9, and HUES8. Blood samples were obtained from healthy individuals from Hamad Medical Cor...

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	<li>Rickels, M. R., Robertson, R. P. <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+in+humans:+Recent+progress+and+future+directions.">Pancreatic islet transplantation in humans: Recent progress and future directions.</a> Endocrine Reviews. 40 (2), 631-668 (2019).</li><li>Latres, E., Finan, D. A., Greenstein, J. L., Kowalski, A., Kieffer, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigating+two+roads+to+glucose+normalization+in+diabetes:+Automated+insulin+delivery+devices+and+cell+therapy.">Navigating two roads to glucose normalization in diabetes: Automated insulin delivery devices and cell therapy.</a> Cell Metabolism. 29 (3), 545-563 (2019).</li><li>Vaithilingam, V., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+and+encapsulation:+An+update+on+recent+developments.">Islet transplantation and encapsulation: An update on recent developments.</a> Review of Diabetic Studies. 8 (1), 51-67 (2011).</li><li>Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+different+types+of+diabetes+using+human+pluripotent+stem+cells.">Modeling different types of diabetes using human pluripotent stem cells.</a> Cellular and Molecular Life Sciences. 78 (6), 2459-2483 (2021).</li><li>Memon, B., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+therapy+for+diabetes:+Beta+cells+versus+pancreatic+progenitors.">Stem cell therapy for diabetes: Beta cells versus pancreatic progenitors.</a> Cells. 9 (2), 283 (2020).</li><li>Balboa, D., Iworima, D. G., Kieffer, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pluripotent+stem+cells+to+model+islet+defects+in+diabetes.">Human pluripotent stem cells to model islet defects in diabetes.</a> Frontiers in Endocrinology (Lausanne). 12, 642152 (2021).</li><li>Gaertner, B., Carrano, A. C., Sander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell+models:+Lessons+for+pancreatic+development+and+disease.">Human stem cell models: Lessons for pancreatic development and disease.</a> Genes &amp; Development. 33 (21-22), 1475-1490 (2019).</li><li>Abdelalim, E. M., Bonnefond, A., Bennaceur-Griscelli, A., Froguel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+as+a+potential+tool+for+disease+modelling+and+cell+therapy+in+diabetes.">Pluripotent stem cells as a potential tool for disease modelling and cell therapy in diabetes.</a> Stem Cell Reviews and Reports. 10 (3), 327-337 (2014).</li><li>Rezania, A., 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=Reversal+of+diabetes+with+insulin-producing+cells+derived+in+vitro+from+human+pluripotent+stem+cells.">Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.</a> Nature Biotechnology. 32 (11), 1121-1133 (2014).</li><li>Pagliuca, F. W., 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=Generation+of+functional+human+pancreatic+beta+cells+in+vitro.">Generation of functional human pancreatic beta cells in vitro.</a> Cell. 159 (2), 428-439 (2014).</li><li>Nair, G. G., 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=Recapitulating+endocrine+cell+clustering+in+culture+promotes+maturation+of+human+stem-cell-derived+&#946;+cells.">Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived &#946; cells.</a> Nature Cell Biology. 21 (2), 263-274 (2019).</li><li>Russ, H. A., 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=Controlled+induction+of+human+pancreatic+progenitors+produces+functional+beta-like+cells+in+vitro.">Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro.</a> EMBO Journal. 34 (13), 1759-1772 (2015).</li><li>Veres, A., 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=Charting+cellular+identity+during+human+in+vitro+&#946;-cell+differentiation.">Charting cellular identity during human in vitro &#946;-cell differentiation.</a> Nature. 569 (7756), 368-373 (2019).</li><li>Hrvatin, S., 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=Differentiated+human+stem+cells+resemble+fetal,+not+adult,+&#946;+cells.">Differentiated human stem cells resemble fetal, not adult, &#946; cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (8), 3038-3043 (2014).</li><li>Rezania, A., 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=Maturation+of+human+embryonic+stem+cell-derived+pancreatic+progenitors+into+functional+islets+capable+of+treating+pre-existing+diabetes+in+mice.">Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.</a> Diabetes. 61 (8), 2016-2029 (2012).</li><li>Rezania, A., 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=Enrichment+of+human+embryonic+stem+cell-derived+NKX6.1-expressing+pancreatic+progenitor+cells+accelerates+the+maturation+of+insulin-secreting+cells+in+vivo.">Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.</a> Stem Cells Journal. 31 (11), 2432-2442 (2013).</li><li>Rezania, A., 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=Enrichment+of+human+embryonic+stem+cell-derived+NKX6.1-expressing+pancreatic+progenitor+cells+accelerates+the+maturation+of+insulin-secreting+cells+in+vivo.">Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo.</a> Stem Cells Journal. 31 (11), 2432-2442 (2013).</li><li>Bruin, J. 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=Characterization+of+polyhormonal+insulin-producing+cells+derived+in+vitro+from+human+embryonic+stem+cells.">Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.</a> Stem Cell Research. 12 (1), 194-208 (2014).</li><li>Toyoda, T., 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=Cell+aggregation+optimizes+the+differentiation+of+human+ESCs+and+iPSCs+into+pancreatic+bud-like+progenitor+cells.">Cell aggregation optimizes the differentiation of human ESCs and iPSCs into pancreatic bud-like progenitor cells.</a> Stem Cell Research. 14 (2), 185-197 (2015).</li><li>Memon, B., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+differentiation+of+human+pluripotent+stem+cells+into+pancreatic+progenitors+co-expressing+PDX1+and+NKX6.1.">Highly efficient differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.</a> Methods in Molecular Biology. , (2020).</li><li>Memon, B., Karam, M., Al-Khawaga, S., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+differentiation+of+human+pluripotent+stem+cells+into+pancreatic+progenitors+co-expressing+PDX1+and+NKX6.1.">Enhanced differentiation of human pluripotent stem cells into pancreatic progenitors co-expressing PDX1 and NKX6.1.</a> Stem Cell Research &amp; Therapy. 9 (1), 15 (2018).</li><li>Aigha, I. I., Memon, B., Elsayed, A. K., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+pluripotent+stem+cells+into+two+distinct+NKX6.1+populations+of+pancreatic+progenitors.">Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors.</a> Stem Cell Research &amp; Therapy. 9 (1), 83 (2018).</li><li>Ali, G., 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=Keratinocytes+derived+from+patient-specific+induced+pluripotent+stem+cells+recapitulate+the+genetic+signature+of+psoriasis+disease.">Keratinocytes derived from patient-specific induced pluripotent stem cells recapitulate the genetic signature of psoriasis disease.</a> Stem Cells and Development. 29 (7), 383-400 (2020).</li><li>Elsayed, A. 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=Generation+of+a+human+induced+pluripotent+stem+cell+line+(QBRIi009-A)+from+a+patient+with+a+heterozygous+deletion+of+FOXA2.">Generation of a human induced pluripotent stem cell line (QBRIi009-A) from a patient with a heterozygous deletion of FOXA2.</a> Stem Cell Research. 42, 101705 (2020).</li><li>Davenport, C., Diekmann, U., Budde, I., Detering, N., Naujok, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior-posterior+patterning+of+definitive+endoderm+generated+from+human+embryonic+stem+cells+depends+on+the+differential+signaling+of+retinoic+acid,+Wnt-,+and+BMP-signaling.">Anterior-posterior patterning of definitive endoderm generated from human embryonic stem cells depends on the differential signaling of retinoic acid, Wnt-, and BMP-signaling.</a> Stem Cells. 34 (11), 2635-2647 (2016).</li><li>Schroeder, I. S., 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=Induction+and+selection+of+Sox17-expressing+endoderm+cells+generated+from+murine+embryonic+stem+cells.">Induction and selection of Sox17-expressing endoderm cells generated from murine embryonic stem cells.</a> Cells Tissues Organs. 195 (6), 507-523 (2012).</li><li>Nostro, M. C., 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=Efficient+generation+of+NKX6-1++pancreatic+progenitors+from+multiple+human+pluripotent+stem+cell+lines.">Efficient generation of NKX6-1+ pancreatic progenitors from multiple human pluripotent stem cell lines.</a> Stem Cell Reports. 4 (4), 591-604 (2015).</li><li>Elsayed, A. K., Younis, I., Ali, G., Hussain, K., Abdelalim, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+development+of+pancreatic+beta+cells+derived+from+human+iPSCs+with+FOXA2+deficiency.">Aberrant development of pancreatic beta cells derived from human iPSCs with FOXA2 deficiency.</a> Cell Death &amp; Disease. 12 (1), 103 (2021).</li><li>Mfopou, J. K., Chen, B., Mateizel, I., Sermon, K., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noggin,+retinoids,+and+fibroblast+growth+factor+regulate+hepatic+or+pancreatic+fate+of+human+embryonic+stem+cells.">Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells.</a> Gastroenterology. 138 (7), 1-14 (2010).</li><li>Mfopou, J. K., De Groote, V., Xu, X., Heimberg, H., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+and+other+soluble+factors+from+differentiating+embryoid+bodies+inhibit+pancreas+development.">Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development.</a> Stem Cells. 25 (5), 1156-1165 (2007).</li><li>Gattazzo, F., Urciuolo, A., Bonaldo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix:+A+dynamic+microenvironment+for+stem+cell+niche.">Extracellular matrix: A dynamic microenvironment for stem cell niche.</a> Biochimica et Biophysica Acta. 1840 (8), 2506-2519 (2014).</li><li>Watt, F. M., Huck, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+extracellular+matrix+in+regulating+stem+cell+fate.">Role of the extracellular matrix in regulating stem cell fate.</a> Nature Reviews Molecular Cell Biology. 14 (8), 467-473 (2013).</li><li>Shih, H. P., Panlasigui, D., Cirulli, V., Sander, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+signaling+regulates+collective+cellular+dynamics+to+control+pancreas+branching+morphogenesis.">ECM signaling regulates collective cellular dynamics to control pancreas branching morphogenesis.</a> Cell Reports. 14 (2), 169-179 (2016).</li><li>Raza, A., Ki, C. S., Lin, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+matrix+properties+on+growth+and+morphogenesis+of+human+pancreatic+ductal+epithelial+cells+in+3D.">The influence of matrix properties on growth and morphogenesis of human pancreatic ductal epithelial cells in 3D.</a> Biomaterials. 34 (21), 5117-5127 (2013).</li><li>Lin, H. Y., 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=Fibronectin+and+laminin+promote+differentiation+of+human+mesenchymal+stem+cells+into+insulin+producing+cells+through+activating+Akt+and+ERK.">Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK.</a> Journal of Biomedical Science. 17, 56 (2010).</li><li>Boretti, M. I., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+extracellular+matrix+and+3D+morphogenesis+on+islet+hormone+gene+expression+by+Ngn3-infected+mouse+pancreatic+ductal+epithelial+cells.">Effect of extracellular matrix and 3D morphogenesis on islet hormone gene expression by Ngn3-infected mouse pancreatic ductal epithelial cells.</a> Tissue Engineering Part A. 14 (12), 1927-1937 (2008).</li><li>Boretti, M. I., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+cell+clustering+enhances+islet+beta+cell+formation+from+human+cultures+enriched+for+pancreatic+ductal+epithelial+cells.">Induced cell clustering enhances islet beta cell formation from human cultures enriched for pancreatic ductal epithelial cells.</a> Tissue Engineering. 12 (4), 939-948 (2006).</li><li>Beattie, G. M., Rubin, J. S., Mally, M. I., Otonkoski, T., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+proliferation+and+differentiation+of+human+fetal+pancreatic+islet+cells+by+extracellular+matrix,+hepatocyte+growth+factor,+and+cell-cell+contact.">Regulation of proliferation and differentiation of human fetal pancreatic islet cells by extracellular matrix, hepatocyte growth factor, and cell-cell contact.</a> Diabetes. 45 (9), 1223-1228 (1996).</li><li>Gage, B. K., Webber, T. D., Kieffer, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+cell+seeding+density+influences+pancreatic+endocrine+development+during+in+vitro+differentiation+of+human+embryonic+stem+cells.">Initial cell seeding density influences pancreatic endocrine development during in vitro differentiation of human embryonic stem cells.</a> PLoS One. 8 (12), 82076 (2013).</li></ol>

This work describes an enhanced protocol for generating pancreatic progenitors from hPSCs with a high co-expression of PDX1 and NKX6.1. Dissociation and replating of the hPSC-derived endoderm at half density on fresh matrix resulted in higher PDX1 and NKX6.1 in hPSC-derived pancreatic progenitors.
Although the growth factor cocktail for each stage is highly similar to P1-ND27, it has been shown that a more extended Stage 3 treatment including FGF and retinoid signaling ...

Diabetes is a complex metabolic disorder affecting millions of people globally. Supplementation of insulin is considered the only treatment option for diabetes. More advanced cases are treated with beta cell replacement therapy, achieved through transplantation of either whole cadaveric pancreas or islets1,2. Several issues surround transplantation therapy, such as limitation with the availability and quality of the tissue, invasiveness of transplantation procedures in addition to the continuous need for immunosuppressants. This necessitates the need for discovering novel and alternative options for beta cell replacement therapy2,3. Human pluripotent stem cells (hPSCs) have recently emerged as a promising tool for understanding human pancreas biology and as a non-exhaustive and potentially a more personalized source for transplantation therapy4,5,6,7. hPSCs, including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), have a high self-renewal capacity and give rise to any tissue type of the human body. hESCs are derived from the embryo&#39;s inner cell mass, and hiPSCs are reprogrammed from any somatic cell4,8.
Directed differentiation protocols are optimized to generate pancreatic beta cells from hPSCs that sequentially direct hPSCs through pancreatic developmental stages invitro. These protocols generate hPSC-derived islet organoids. While they have greatly improved at increasing the proportion of pancreatic beta cells therein, the efficiency of protocols is highly variable. It does not increase to more than ~40% of NKX6.1+/INSULIN+ or C-PEPTIDE + cells5,9,10,11,12,13. However, the generated beta cells are not entirely identical to the adult human beta cells in terms of their transcriptional and metabolic profiles and their response to glucose4,5,14. The hPSC-derived beta cells lack gene expression of key beta cell markers such as PCSK2, PAX6, UCN3, MAFA, G6PC2, and KCNK3 compared to adult humans islets5. Additionally, the hPSC-derived beta cells have diminished calcium signaling in response to glucose. They are contaminated with the co-generated polyhormonal cells that do not secrete appropriate amounts of insulin in response to increasing glucose levels5. On the other hand, hPSC-derived pancreatic progenitors, which are islet precursors, could be generated more efficiently in vitro compared to beta cells and, when transplanted in vivo, could mature into functional, insulin-secreting beta cells15,16. Clinical trials are currently focused on demonstrating their safety and efficacy upon transplantation in T1D subjects.
Notably, expression of the transcription factors PDX1 (Pancreatic and Duodenal Homeobox 1) and NKX6.1 (NKX6 Homeobox 1) within the same pancreatic progenitor cell is crucial for commitment towards a beta cell lineage5. Pancreatic progenitors that fail to express NKX6.1 give rise to polyhormonal endocrine cells or non-functional beta cells17,18. Therefore, a high co-expression of PDX1 and NKX6.1 in the pancreatic progenitor stage is essential for ultimately generating a large number of functional beta cells. Studies have demonstrated that an embryoid body or 3D culture enhances PDX1 and NKX6.1 in pancreatic progenitors where the differentiating cells are aggregated, varying between 40%-80% of the PDX1+/NKX6.1+ population12,19. However, compared to suspension cultures, 2D differentiation cultures are more cost-effective, feasible, and convenient for application on multiple cell lines5. We recently showed that monolayer differentiation cultures yield more than up to 90% of PDX1+/NKX6.1+ co-expressing hPSC-derived pancreatic progenitors20,21,22. The reported method conferred a high replicating capacity to the generated pancreatic progenitors and prevented alternate fate specifications such as hepatic lineage21. Therefore, herein, this protocol demonstrates a highly efficient method for the differentiation of hPSCs to pancreatic beta-cell precursors co-expressing PDX1 and NKX6.1. This method utilizes the technique of dissociating hPSC-derived endoderm and manipulating the cell density, followed by an extended FGF and Retinoid signaling as well as Hedgehog inhibition to promote PDX1 and NKX6.1 co-expression (Figure 1). This method can facilitate a scalable generation of hPSC-derived pancreatic beta-cell precursors for transplantation therapy and disease modeling.

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	<tbody>
		<tr>
			<td>15 mL, conical, centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>20X TBS Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28360</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plates, flat bottom with lid</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL, conical, centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>6- well culture plates, multidish</td>
			<td>Thermo Scientific</td>
			<td>140685</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>0-7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC</td>
			<td>Reconstituted in 4 mM HCl</td>
		</tr>
		<tr>
			<td>Anti NKX6.1 antibody, mouse monoclonal</td>
			<td>DSHB</td>
			<td>F55A12-C</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-PDX1 antibody, guinea pig polyclonal</td>
			<td>Abcam</td>
			<td>ab47308</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining</td>
		</tr>
		<tr>
			<td>B27 minus Vit A</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, heat shock fraction, fatty acid free</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>ThermoFisher</td>
			<td>41965047</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td></td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>ThermoFisher</td>
			<td>PHG0313</td>
			<td>Reconstituted in 0.1% BSA in PBS</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG</td>
			<td>Reconstituted in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Sigma</td>
			<td>23491-45-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12 (1X)</td>
			<td>Gibco</td>
			<td>12660-012</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut SR serum replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid (vitamin C)</td>
			<td>Sigma</td>
			<td>A92902</td>
			<td>Reconstituted in distilled water</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Aliquot the thawed stock and freeze at -20C.</td>
		</tr>
		<tr>
			<td>MCDB131</td>
			<td>ThermoFisher</td>
			<td>10372019</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SOX17</td>
			<td>ORIGENE</td>
			<td>CF500096</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>85850</td>
			<td>Mix the basal media with supplement. Aliquot and store at -20 &#176;C for longer time or at 4 &#176;C for instant use</td>
		</tr>
		<tr>
			<td>Nalgene filter units, 0.2 &#181;m PES</td>
			<td>ThermoFisher</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>72340</td>
			<td>Reconstituted in distilled water</td>
		</tr>
		<tr>
			<td>NOGGIN</td>
			<td>R&#38;D</td>
			<td>6057-NG</td>
			<td>Reconstituted in 0.1% BSA in PBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>ChemCruz</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable vacuum aspirator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-FOXA2</td>
			<td>Cell signaling technology</td>
			<td>3143</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining</td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Rock inhibitor (Y-27632)</td>
			<td>ReproCell</td>
			<td>04-0012-02</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Round Bottom Polystyrene FACS Tubes with Caps, STERILE</td>
			<td>Stellar Scientific</td>
			<td>FSC-9010</td>
			<td></td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Sigma Aldrich</td>
			<td>S4572</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex</td>
			<td>ThermoFisher</td>
			<td>A3349401</td>
			<td>Mix the basal media with supplement. Aliquot and store at -20 &#176;C for longer time or at 4 &#176;C for instant use</td>
		</tr>
		<tr>
			<td>TALI Cellular Analysis Slide</td>
			<td>Invitrogen</td>
			<td>T10794</td>
			<td></td>
		</tr>
		<tr>
			<td>Tali image-based cytometer automated cell counter</td>
			<td>Invitrogen</td>
			<td>T10796</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE 100 mL</td>
			<td>ThermoFisher</td>
			<td>12563011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 results show that optimized protocol P2-D (Figures 1A) enhanced pancreatic progenitor differentiation efficiency by upregulating PDX1 and NKX6.1 co-expression (Figure 2A,B, and Figure 3A). In particular, the results showed that dissociation of endodermal cells and their replating on fresh membrane matrix along with a longer duration of Stage 3 enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors (op...

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System at JoVE.com

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

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.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

This optimized protocol enhanced the generation of pancreatic beta cell precursors from human pluripotent stem cells, which can be used for cell therapy for diabetes and investigating molecular mechanisms underlining diabetes biogenesis. This is a feasible technique as it is used for 2D monolayer cultures. It is easy to apply, and is consistent across multiple control and patient-derived pluripotent stem cell lines.These beta cell precursors when transplanted in a diabetic patient can mature into insulin secreting beta cells, and can potentially reverse hyperglycemia. Therefore, the increased percentages of pancreatic progenitors generated using our protocol can be used for cell therapy for diabetes. This enhanced protocol can be used for studying early human pancreatic development in healthy individuals as well as in diabetic patients, which is challenging due to a shortage of tissue samples.When the human pluripotent stem cell or hPSC colonies have reached 70 to 80%confluence, wash them twice with warm PBS inside the tissue culture hood. Then aspirate the buffer using a portable vacuum aspirator. Next, add stage one differentiation medium containing CHIR-99021 to the colonies, and incubate the plate at 37 degrees Celsius for 24 hours.The next day, replace the spent media with stage one differentiation medium without CHIR-99021. After aspirating the spent medium and washing the definitive endoderm cells twice with warm PBS, swirl the plate to remove any cell debris. Then fix the cells by adding 4%paraformaldehyde, and place the plate on a two-dimensional shaker.After fixation, wash the cells with TRIS-buffered saline containing 0.5%Tween, and place the plate on the shaker. Then permeabilize the fixed cells by adding a generous amount of PBS containing 0.5%Triton X-100 and place the plate back on the shaker. Next, add freshly prepared blocking buffer to the permeabilized cells, and incubate the plate for one hour on the shaker.Then add diluted primary antibodies to the blocked cells, and place the plate on the shaker at four degrees Celsius with gentle shaking. The following day after aspirating the primary antibody solution, wash the wells three times with TBST for 10 minutes each on the shaker. Next, add the secondary antibodies.Cover the plate with aluminum foil to protect from light, and place the plate on the shaker at room temperature for one hour. Then aspirate the secondary antibody solution, and wash the stained wells with TBST on the shaker for 10 minutes, keeping the plate covered with foil. Next, to stain the nuclei, add Hoechst solution to the wells and place the plate on the shaker.After two to three minutes, aspirate the Hoechst solution and rinse the wells twice with PBS. Finally, add PBS to the stained cells, and image them using an inverted fluorescence microscope in the dark. On day one of stage two, dissociate the hPSC derived endodermal cells by first washing them with warm PBS, and then adding one milliliter of warm enzyme solution per well of a six well plate.Place the plate at 37 degrees Celsius and 5%carbon dioxide for three to five minutes or until the cells begin to detach from one another. Dissociate the detached sheets or monolayer of cells in the wells, and collect the detached cells together in a 15 milliliter polypropylene tube using basal stage half media. Then spin the cells down, discard the supernatant, and resuspend the pellet using one milliliter of sterile PBS.After counting the cells using an automated counter, spin the cells again, and discard the supernatant. Then resuspend the pellet in the appropriate volume of stage two differentiation medium containing a Rock inhibitor. Plate the resuspended cells on one to 50 membrane matrix coated plates and incubate them at 37 degrees Celsius and 5%carbon dioxide.After 24 hours, replace the media with stage two differentiation medium without the Rock inhibitor. At the end of stage four, detach cells as demonstrated earlier, and collect them in a 15 milliliter tube. Then spin the cells, discard the supernatant, wash the cells with PBS, and dissociate them into single cells.After counting the cells using an automated cell counter, spin the cells again and resuspend the pellet in 200 microliters of chilled PBS. Next, add two milliliters of chilled 80%ethanol drop wise with the tube on a vortex set low to medium speed. Close the cap tightly, and place the tube slightly tilted on the shaker at four degrees Celsius overnight.The next day, spin the cells and wash the pellet with PBS to dissociate any clumps of fixed cells. Then block the fixed cells for at least one hour at room temperature or four degrees Celsius overnight on the shaker. Next, to stain for the pancreatic progenitor markers, distribute 200, 000 cells per condition including appropriate isotype controls, unstained, and secondary antibody controls in a 96 well V bottom plate.Then spin the plate briefly, and flip the plate with a swift motion to discard the supernatant without losing the pellets. Next, incubate the cells in primary antibodies for at least two hours at room temperature on a shaker with gentle shaking. Then wash the stained cells three times with TBST by pipetting up and down in the wells.Centrifuge the cells again, discard the supernatant, and add secondary antibodies to the cells. After a 30 minute incubation at room temperature, wash the stained cells twice with TBST. Then spin the plate, collect the stained cells in 100 microliters of PBS in light protected fax tubes, and run the samples on a flow cytometry machine.Compared to the non-optimized method, the optimized protocol enhanced pancreatic progenitor differentiation efficiency by upregulating PDX 1 and NKX 6.1 co-expression. In particular, the disassociation of endodermal cells and their replating on fresh membrane matrix, along with a longer duration of stage three, enhanced NKX 6.1 expression in hPSC derived pancreatic progenitors compared to the non-dissociated protocol. Pancreatic progenitors generated using the optimized protocol also increased numbers of SOX9 positive cells compared to the non-dissociated method.Further, the optimized method also generated a higher proportion of proliferative NKX 6.1 positive cells that co-expressed the proliferation marker Ki67. In order to enhance the efficiency, it is crucial to dissociate the HbA1 derived endoderm and then extend the duration of stage three treatment with FGF amyloid signaling and Hedgehog inhibition. Scalable generation of pancreatic progenitors using this enhanced protocol has facilitated studies on improving efficiency and maturation of human pluripotent stem cell derived pancreatic beta cells and allowed modeling of different type of diabetes.

Research

JoVE Journal

Developmental Biology

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

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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 ...

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

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 ...

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 ...

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 ...

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 ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...