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Generation of pancreatic β cells from CD177+ anterior definitive endoderm

Nature Biotechnology volume 38, pages 1061–1072 (2020)Cite this article

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Abstract

Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive β-like cells in vitro compared with cells from unsorted differentiation cultures.

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Fig. 1: Identification of CD177+ and CD275+ ADE subpopulations.
Fig. 2: Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures.
Fig. 3: CD177+ ADE efficiently differentiates into PDX1+/NKX6.1+ pancreatic progenitors.
Fig. 4: Inhibition of canonical WNT secretion promotes pancreatic differentiation.
Fig. 5: CD177+ ADE efficiently differentiates into NKX6.1+/INS+ β-like cells.
Fig. 6: CD177+-enriched ADE generates more mature β-like cells in vitro.
Fig. 7: Functional characterization of the US-β-cells and CD177-β-cells.

Data availability

Additional data supporting the findings of this study are available from the corresponding author upon request.

Code availability

All microarray data are available at GEO under the accession number GSE113791.

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Acknowledgements

We thank A. Rezania for providing hMAFA and hGLUT1 antibodies. We thank the Alberta Diabetes Institute IsletCore and Olle Korsgren from Uppsala University for human islets. We are grateful to I. Burtscher, A. Bastidas-Ponce, C. Salinno and M. Tarquis-Medina for their invaluable inputs and technical assistance. We thank D. M. Thomson and M. Catani for reading and correction of the manuscript. This work was supported by the HumEN consortium funded by European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement no. 602889 (http://www.hum-en.eu/). This project has received funding from the European Union’s Horizon 2020 researchand innovation programme under grant agreement no. 874839. This work was also funded in part by the German Center for Diabetes Research (DZD e.V.) and the Helmholtz Alliance ‘Aging and Metabolic Programming, AMPro’. P.U.M. was funded by a postdoctoral grant from the HumEN consortium, EU. M.S., S.P. and K.S. are supported by the Helena Graduate School and Technische Universität München. S.K. is an employee of Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. The project was funded by the German Federal Ministry of Education and Research (project number 01EK1607A).

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Author notes

  1. These authors contributed equally: Pallavi U. Mahaddalkar, Katharina Scheibner.

Authors and Affiliations

  1. Institute of Diabetes and Regeneration Research, Helmholtz Diabetes Center, Helmholtz Zentrum München, Neuherberg, Germany

    Pallavi U. Mahaddalkar, Katharina Scheibner, Sandra Pfluger,  Ansarullah, Michael Sterr, Julia Beckenbauer & Heiko Lickert

  2. Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany

    Pallavi U. Mahaddalkar, Michael Sterr & Heiko Lickert

  3. Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany

    Martin Irmler & Johannes Beckers

  4. Chair of Experimental Genetics, School of Life Sciences Weihenstephan, Technische Universität München, Freising, Germany

    Johannes Beckers

  5. German Center for Diabetes Research (DZD), Neuherberg, Germany

    Johannes Beckers & Heiko Lickert

  6. Miltenyi Biotec, Bergisch Gladbach, Germany

    Sebastian Knöbel

  7. β-Cell Biology, Technische Universität München, School of Medicine, Klinikum Rechts der Isar, Munich, Germany

    Heiko Lickert

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Contributions

Sandra Pfluger and Ansarullah contributed equally to this work. P.U.M. and H.L. wrote the manuscript. P.U.M. and H.L. designed experiments, and analyzed and interpreted the results. K.S., S.P. and A. performed experiments and analyzed data. M.S. provided bioinformatics assistance and analyzed the results. J. Beckenbauer provided technical assistance. S.K. designed, performed and analyzed the antibody screen. J. Beckers and M.I. performed and analyzed the microarray chips. P.U.M. and H.L. conceived the project.

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Correspondence to Heiko Lickert.

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Competing interests

Helmholtz Zentrum München and Miltenyi Biotec own a patent (International Application no. PCT/EP2018/065779), ‘Methods for purifying endoderm and pancreatic endoderm cells derived from human embryonic stem cells’, using CD177 and CD275 antibodies for enrichment or depletion of pancreatic and liver progenitors).

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Integrated supplementary information

Supplementary Figure 1 Screening strategy for the identification of endoderm subpopulations.

(a) Screening work flow for the initial screen. (b-d) Representative FACS plots for CD177 and CD275 (b) labelling of differentiated day 4 DE cells with known endoderm markers (FOXA2 and CXCR4) revealed definitive endoderm (FOXA2+/CXCR4+) and mes-endoderm (FOXA2low/CXCR4-) subpopulations. (c-d) CD177 and CD275 expression profiles reveal different endoderm subpopulations (n = 1). (e) Immunofluorescent staining for CER1 with FOXA2 in DE cultures (n = 3, biologically independent experiments). Scale bar, 25 µm. (f) FACS analysis for CD275+/CER1+ and CD177+/CER1+ ADE cell populations at day 4 DE. (g-h) qPCR quantification for the mRNA expression of FOXA2 and SOX17 (g), CER1 and HHEX (h) in enriched CD177+ and CD275+ ADE subpopulations (ANOVA, (e-h) n = 3, biologically independent experiments). Data is represented as mean ±s.e.m; P<0.05 and P<0.01. Statistically non-significant results are not indicated in the figure.

Supplementary Figure 2 Percentage of CD177+ and CD275+ ADE subpopulations induced in different hESC and hiPSC lines.

(a) Endoderm differentiation scheme from hESCs toward DE/ADE. (b) FACS plots represent the percentage of CXCR4+/CD177+ and CXCR4+/CD275+ subpopulations in hH1, hMEL1-NKX6.1, HMGUi001-A hiPSC, HUES8 and H9 at DE/ADE stage. (c) Quantification of flow cytometry data from ((b,c) n = 3, biologically independent experiments).

Supplementary Figure 3 Induction efficiency of CD177+ and CD275+ ADE shows variation using different protocols.

(a-c) Adaptation of previously published endoderm differentiation protocols from hESCs. (d) FACS quantification for the percentage of total population expressing CXCR4 in DE cells derived from HMGUi001-A hiPSC using 3 different endoderm induction protocols (n = 2 biologically independent experiments). (e) FACS quantification for the percentage of cells expressing CXCR4+/CD177+ and CXCR4+/CD275+ in DE generated using previously published protocols (n = 3 (HMGUi001-A hiPSC), n = 2 (H9) biologically independent experiments). Data is represented as mean ±s.e.m.

Supplementary Figure 4 Differentiation of enriched CD177+, CD275+ and CXCR4+ ADE subpopulations toward liver and pancreas fate.

(a) Expression of CD177-, CD275-, and CXCR4 during differentiation of hESCs toward pancreatic β-like cells. (b) Liver differentiation protocol. (c) qPCR quantification of the expression of early liver progenitor markers HHEX, TTR and AFP in enriched ADE subpopulations (ANOVA, n = 3, biologically independent experiments). (d) Immunofluorescent staining of pancreatic progenitor cells derived from enriched ADE subpopulations for the co-expression of posterior foregut marker GATA6 and PDX1. (e) Immunofluorescent staining of pancreatic progenitor cells derived from enriched ADE subpopulations for the co-expression of lung marker SOX2, intestinal marker CDX2 and PDX1 ((d, e) n = 3, biologically independent experiments). Data is represented as mean ±s.e.m; P<0.05 and P<0.01. Statistically non-significant results are not indicated in the figure. Scale bars, 25µm.

Supplementary Figure 5 CD177+ ADE positively correlates with PP1 induction.

(a) Pancreatic induction protocol. (b) FACS analysis of H1, HMGUi001-A hiPSC, HUES8 and MEL1-NKX6.1 for PDX1 at S3 stage. (c) Quantification of CD177+ cells generated at S1 and PDX1+ cells generated at S3 stage showing correlation between CD177 and PDX1 induction (n = 10 (H1), n = 7 (MEL1-NKX6.1), n = 9 (HMGUi001-A), n = 9 (HuES8), n = 8 (H9)). Data is represented as mean ±s.e.m.

Supplementary Figure 6 H1 hESC pancreatic and endocrine differentiations of CD177+- and US-DE.

(a) Overview of differentiation protocol used to generate CD177-/US-β-cells. (b-e) Immunostainings for INS and NKX6.1 (b), GCG, INS and SST (c) and C-peptide and GLUT1 (d), INS, MAFA and NKX6.1 (e) in CD177- and US- β-cells ((b-e) n = 3, biologically independent experiments). Scale bars, 50 µm. (f) Representative flow cytometry contour plots of S4 and S7 cells generated from CD177+- and US-ADE/DE cells on H1 line and stained for indicated markers. (g,h) Percentage of cells expressing indicated markers (two-sided unpaired Student’s t-test, (g) n = 3, (h) n=13, n = 17 for INS+, biologically independent experiments). Data is represented as mean ±s.e.m; P<0.05 and P<0.01. Statistically non-significant results are not indicated in the figure.

Supplementary Figure 7 Comparison of 2D and 3D culture system on pancreatic differentiation.

(a) Overview of differentiation protocol used. (b) Comparison of PDX1+/NKX6.1+ generated from CD177+- and CXCR4+-ADE in 2D and 3D settings (two-sided unpaired Student’s t-test, n = 3 biologically independent experiments). (c) Morphology of CD177- and CXCR4-β-cells; DAPI (blue) and E-CAD (green). Scale bars, 20 µm. Graph represents the size of the aggregates in µm (two-sided unpaired Student’s t-test, n = 15 aggregates from 3 biologically independent experiments). Data is represented as mean ±s.e.m, P<0.05. Statistically non-significant results are not indicated in the figure.

Supplementary Figure 8 H1 hESC-derived CD177+-ADE generates more functional β-like cells in vitro.

(a) Insulin content of US-β-cells and CD177-β-cells (two-sided unpaired Student’s t-test, n = 10, biologically independent experiments). (b) Comparison of insulin secretion of US-β-cells and CD177-β-cells in sequential static GSIS (two-sided unpaired Student’s t-test, n = 5, biologically independent experiments). (c,d) Insulin secretion in response to dynamic glucose, Ex-4 and KCl challenges in a perifusion system on US-β-cells (c) and CD177-β-cells (d) ((c,d) n = 5, biologically independent experiments). Data is represented as mean ±s.e.m; P<0.05 and P<0.01. Statistically non-significant results are not indicated in the.

Supplementary Figure 9 CD177+ and CD275+ ADE receive differential Wnt signaling.

(a,b) Original Western blot of WNT/PCP components such as DVL2 (a) and p-JNK (b) in ADE subpopulations as shown in Fig. 2h.

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Mahaddalkar, P.U., Scheibner, K., Pfluger, S. et al. Generation of pancreatic β cells from CD177+ anterior definitive endoderm. Nat Biotechnol 38, 1061–1072 (2020). https://doi.org/10.1038/s41587-020-0492-5

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