Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors

Abstract

Background: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions.

Methods: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion.

Results: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation.

Conclusions: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.

Keywords: Defined culture conditions; Diabetes; Differentiation; Induced pluripotent stem cells; Insulin producing cells; NKX6.1; PDX1; Reprogramming.

Fig. 1

The outline of the differentiation procedure of iPSC into insulin producing cells. Schematic representation of the procedure used for differentiation of iPS cells to insulin producing cells. Arrows indicate time points of induction of transcription factors expression with doxycycline (DOX). The experimental framework consisted of four stages: culture of iPS cells, generation of definitive endoderm cells, formation of pancreatic progenitor cells and development of mature insulin producing cells. The diagram presents composition of the media used at each step

Fig. 2

Optimisation of somatic cells culture conditions and transgenes delivery. a Cell number was monitored by xCELLigence system. 6000 of epithelial cells or fibroblasts were seeded on one well of the assay plate and maintained in corresponding culture media. Their impact on cell growth was monitored for 72 h and cell index (CI) values at the end of experiment are presented as a mean of three independent counts. b Comparison of transfection efficiencies obtained with use of FuGENE 6 reagent and synthesized L64-PEI copolymer. The expression plasmid pCXB-EmGFP was transiently introduced into HeLa cells. Transfection with both reagents was carried out in the presence of FBS and two days later, transfection efficiency was assessed by flow cytometry and fluorescence microscope. Scale bar 100 μm

Fig. 3

Reprogramming of human fibroblastic and epithelial cells into iPS cells with application of different protein components of the extracellular matrix. a Efficiencies of generation of iPS cells obtained from renal epithelial cells. b Colony forming efficiencies in the course of fibroblast reprogramming into iPS cells. c Exemplary results of alkaline phosphatase staining on iPS cells generated from renal epithelial cells grown on cell culture vessels coated with different proteins and extracellular matrix components

Fig. 4

Differentiation of pluripotent cells into definitive endoderm cells. a Expression of genes characteristic for definitive endoderm (CXCR4, FOXA2, SOX17) for examined differentiation protocols relative to undifferentiated iPSC samples. For the differentiation experiments cells were maintained in basal medium (RPMI-1640) with Activin A and CHIR99021 for 24 h, and in medium with Activin A alone for the next 48 h. In addition, the media contained: DE1—foetal bovine serum, DE2—human serum, DE3—insulin, transferrin and sodium selenite, DE4—albumin, insulin, transferrin, defined lipids concentrate and ascorbic acid. b Results of immunocytochemical analysis of the generated definitive endoderm cells showing cells positive for CXCR4 and SOX17 markers, and clusters of cells displaying E-cadherin expression. Cells were prepared as described in a. Scale bar 50 µm

Fig. 5

Insulin and C-peptide synthesis by generated insulin producing cells. a Doxycycline-regulated Pdx1 and Nkx6.1 expression. iPSC lines with stably integrated Pdx1 and Nkx6.1 transgenes under control of Tre3G promoter were induced by addition of doxycycline and stained with antibodies against Pdx1 and Nkx6.1. The negative control were iPS cells that were not treated with doxycycline. Scale bar 100 µm. b Insulin secretion in response to 2.5 mM glucose concentration after transgenes induction at selected steps of the differentiation procedure. Results of the ELISA were normalized to the total protein concentration and presented as picograms of produced insulin per milligram of the total protein content of insulin secreting cells. Control includes cells without introduced transgenes. Asterisk indicate statistically relevant difference between compared samples. c Immunocytochemical detection of insulin and C-peptide production in cells where transgene expression was switched on at indicated stages of differentiation. Scale bar 20 µm

Fig. 6

Structural and functional characterisation of established IPCs. Results of immunocytochemical and colorimetric characterisation of generated insulin producing cells. Pluripotent cell lines with stably introduced PDX1-VP16 and NKX6.1 transgenes were differentiated towards insulin producing cells. Transgenes expression was switched on by doxycycline at the stage of IPC progenitors or maturation of insulin producing cells. Differentiated cells were immunophenotyped with antibodies against MafA, Pax6, SLC30A8, somatostatin, tyrosine hydroxylase (all except TH counterstained with DAPI), and stained with dithizone. The images represent selected clusters of positive cells in the population of differentiated cells. Scale bar 100 µm