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Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo

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Abstract

Aims

New sources of insulin-secreting cells are strongly required for the cure of diabetes. Recent successes in differentiating embryonic stem cells, in combination with the discovery that it is possible to derive human induced pluripotent stem cells (iPSCs) from somatic cells, have raised the possibility that patient-specific beta cells might be derived from patients through cell reprogramming and differentiation. In this study, we aimed to obtain insulin-producing cells from human iPSCs and test their ability to secrete insulin in vivo.

Methods

Human iPSCs, derived from both fetal and adult fibroblasts, were differentiated in vitro into pancreas-committed cells and then transplanted into immunodeficient mice at two different stages of differentiation (posterior foregut and endocrine cells).

Results

IPSCs were shown to differentiate in insulin-producing cells in vitro, following the stages of pancreatic organogenesis. At the end of the differentiation, the production of INSULIN mRNA was highly increased and 5 ± 2.9 % of the cell population became insulin-positive. Terminally differentiated cells also produced C-peptide in vitro in both basal and stimulated conditions. In vivo, mice transplanted with pancreatic cells secreted human C-peptide in response to glucose stimulus, but transplanted cells were observed to lose insulin secretion capacity during the time. At histological evaluation, the grafts resulted to be composed of a mixed population of cells containing mature pancreatic cells, but also pluripotent and some neuronal cells.

Conclusion

These data overall suggest that human iPSCs have the potential to generate insulin-producing cells and that these differentiated cells can engraft and secrete insulin in vivo.

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Acknowledgments

This work was supported by the Italian Ministry of Health (Grant Rf-FSR-2008-1202373) and by EU (HEALTH-F5-2009-241883-BetaCellTherapy). SP is Ph.D. Student in Experimental and Translational Medicine, University of Insubria, Varese (Italy).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standard

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Human Rights disclosure

This article does not contain any studies with human subjects performed by any of the authors.

Author information

Authors and Affiliations

  1. Diabetes Research Institute, IRCCS San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milan, Italy

    Silvia Pellegrini, Alessia Mercalli, Raffaella Melzi, Lorenzo Piemonti & Valeria Sordi

  2. Stem Cells and Neurogenesis Unit, Division of Neuroscience, IRCCS San Raffaele Scientific Institute, 20132, Milan, Italy

    Federica Ungaro & Vania Broccoli

  3. Diabetes Unit, Department of Medicine, Surgery and Neuroscience, University of Siena, 53100, Siena, Italy

    Guido Sebastiani & Francesco Dotta

  4. Fondazione Umberto Di Mario ONLUS, c/o Toscana Life Sciences, 53100, Siena, Italy

    Guido Sebastiani & Francesco Dotta

Authors
  1. Silvia Pellegrini
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  2. Federica Ungaro
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  8. Lorenzo Piemonti
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Corresponding authors

Correspondence to Lorenzo Piemonti or Valeria Sordi.

Additional information

Managed by Massimo Porta.

Lorenzo Piemonti and Valeria Sordi have contributed equally to this work.

Electronic supplementary material

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592_2015_726_MOESM1_ESM.ppt

Figure 1S. Differentiation of iPSCs into insulin-producing cells. Quantification of NANOG, SOX17, HNF1b and NGN3 mRNAs during differentiation of iPSCs into insulin-producing cells by droplet digital PCR. Gene expression was analyzed at the steps of pluripotency (iPSC), definitive endoderm (DE), posterior foregut (PF), pancreatic endoderm (PE) and endocrine cells (EN). PCR amplification of iPSCs cDNAs was performed in an emulsion using gene specific primers and hydrolysis probes. The number of gene specific mRNA copies in each sample, corresponding to droplets fluorescing above background level (red line), was determined after droplets acquisition and counting on a QX100 instrument (Bio-Rad) and the QuantaSoft software v1.2.10 applying a correction algorithm based on the Poisson distribution. The number of gene specific mRNA copies per nanogram of total RNA in each sample of one representative experiment is shown. (PPT 228 kb)

592_2015_726_MOESM2_ESM.ppt

Figure 2S. Differentiation into insulin-producing cells of iPSCs reprogrammed from adult fibroblasts. Gene expression of adult fibroblast-derived iPSCs reprogrammed from adult fibroblasts was analyzed during differentiation at the steps of pluripotency, definitive endoderm, posterior foregut, pancreatic endoderm and endocrine cells. Gene expression analysis by Taqman of markers of pluripotency (OCT4 and NANOG), definitive endoderm (FOXA2 and SOX17), posterior foregut (HNF1b and PDX1), pancreatic endoderm (NGN3 and NKX2.2) and endocrine cells (INS) is shown. Normalized gene expression levels are reported with the highest expression set to 1 and all the others relative to this. Histograms represent mean of n=2 experiments ± SEM. (PPT 100 kb)

592_2015_726_MOESM3_ESM.ppt

Figure 3S: Functional characterization of iPSCs-derived cells after transplantation (tx) in NOD/SCID mice. Analysis of graft function by human C-peptide measurement after oral glucose test tolerance in sera of mice transplanted with iPSCs-derived-terminally differentiated (upper panel) or precursor cells (lower panel). (PPT 58 kb)

592_2015_726_MOESM4_ESM.ppt

Figure 4S: Characterization of iPSCs-derived beta cells. Protein expression analysis by flow cytometry of CD318 (CUB domain containing protein 1) and CD200 (OX-2 membrane glycoprotein). Dot plots of control unstained cells (left) and stained cells (right) are shown. Gate delimitates positive events. Percentage of positive cells of a representative experiment are reported. SSC: side scatter, PE: phycoerythrin, APC: allophycocyanin. (PPT 140 kb)

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Pellegrini, S., Ungaro, F., Mercalli, A. et al. Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo. Acta Diabetol 52, 1025–1035 (2015). https://doi.org/10.1007/s00592-015-0726-z

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