Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications

Abstract

Background: Differentiation of patient-specific induced pluripotent stem cells (iPS) helps researchers to study the individual sensibility to drugs. However, differentiation protocols are time-consuming, and not all tissues have been studied. Few works are available regarding pancreatic exocrine differentiation of iPS cells, and little is known on culturing and cryopreserving these cells.

Methods: We differentiated the iPS cells of two pediatric Crohn's disease patients into pancreatic progenitors and exocrine cells, adapting and shortening a protocol for differentiating embryonic stem cells. We analyzed the expression of key genes and proteins of the differentiation process by qPCR and immunofluorescence, respectively. We explored the possibility of keeping differentiated cells in culture and freezing and thawing them to shorten the time needed for the differentiation. We analyzed the cell cycle of undifferentiated and differentiated cells by flow cytometry.

Results: The analysis of mRNA levels of key pancreatic differentiation genes PDX1 and pancreatic amylase indicate that iPS cells were successfully differentiated into pancreatic exocrine cells with expression of PDX1 (one way ANOVA p < 0.0001), and the two isoforms of amylase (one way ANOVA p < 0.05) significantly higher in exocrine cells in comparison to iPS cells. Differentiation efficiency was also confirmed by immunofluorescence analysis of PDX1 and amylase. We confirmed the possibility of shortening the time necessary for obtaining pancreatic cells without losing differentiation efficiency. Pancreatic progenitors and exocrine cells were maintained in culture and cryopreserved. Interestingly, the stemness marker OCT4 resulted significantly lower after subculturing (OCT4 p < 0.001; one-way ANOVA) and after freezing and thawing procedures (p < 0.05, one-way ANOVA) suggesting a reduction of undifferentiated stem cells leading to a purer population of pancreatic progenitor cells. Also, the stemness marker NANOG resulted lower after passaging, corroborating this result.

Conclusions: In this work, we optimized the generation of patient-specific pancreatic differentiated cells and laid the foundation for creating a bank of patient-specific pancreatic lines exploitable for tailored pharmacological assays.

Trial registration: The study was approved by the Ethical Committee of the Institute of Maternal and Child Health IRCCS Burlo Garofolo, with approval number 1556 (internal ID RC 44/22).

Keywords: Crohn’s disease.; Human induced pluripotent stem cells; Pancreatic exocrine cells; Pancreatic progenitors; Patient-specific model.

Fig. 1

Protocol for the differentiation of human iPS cells into pancreatic exocrine cells

Fig. 2

Real-time PCR analysis showing the dynamics of mRNA expression for several key markers of human iPS cells differentiated to pancreatic exocrine cells applying the protocol reported in Fig. 1. Comparison between human iPS cells and differentiated counterpart (one-way ANOVA OCT4 p < 0.0001; one-way ANOVA PDX1 p < 0.0001; one-way ANOVA AMY2A p < 0.05; one-way ANOVA AMY2B p < 0.05; Dunnett’s multiple comparisons test, *: p < 0.05; ***: p < 0.001; ****: p < 0.0001). DE, definitive endoderm; PP pancreatic progenitors, EX exocrine cells

Fig. 3

Double immunostaining shows that positive cells for PDX1 (red) and amylase (green) are present in pancreatic exocrine cells obtained after human iPS cell differentiation (A, B). Negative control, represented by undifferentiated human iPS cells (C). In blue the nuclear staining (DAPI) is represented

Fig. 4

Real-time PCR analysis showing the dynamics of mRNA expression for several key markers of pancreatic differentiation. Comparison between pancreatic exocrine cells differentiated for 4 and 20 days and the healthy pancreatic ductal cell line H6C7 (one-way ANOVA PDX1 p < 0.05; Tukey’s multiple comparisons test *: p < 0.05)

Fig. 5

Double immunostaining showing positive cells for PDX1 (red) and amylase (green) pancreatic exocrine cells obtained after human iPS cell differentiation for 4 (A) or 20 days (B). In blue the nuclear staining (DAPI) is represented. Scale bar: 20 μm (A), 50 μm (B)

Fig. 6

Real-time PCR analysis of mRNA expression for key markers of differentiation. Analysis of mRNA level of pancreatic progenitors maintained in culture (pancreatic progenitors) and re-plated at half density two times (passed x1, passed x2). One-way ANOVA OCT4 p < 0.001; Dunnett’s multiple comparison test ***: p < 0.001

Fig. 7

Real-time PCR analysis shows mRNA expression dynamics for several key markers of pancreatic differentiation. Comparison of mRNA level of pancreatic progenitors after thawing and passaging. One-way ANOVA OCT4 p < 0.05; Dunnett’s multiple comparison test *: p < 0.05

Fig. 8

Real-time PCR analysis shows mRNA expression dynamics for several key markers of pancreatic differentiation. Comparison of mRNA levels of pancreatic exocrine cells in culture after thawing

Fig. 9

Analysis of cell cycle measured by propidium iodide uptake of human iPS cells, definitive endoderm, and pancreatic progenitor cells (G0 one-way ANOVA p < 0.05, G2/M one-way ANOVA p < 0.01, Dunnett’s multiple comparison test *: p < 0.05; **: p < 0.01)