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

Content Provider	Springer Nature : BioMed Central
Author	Genova, Elena Rispoli, Paola Fengming, Yue Kohei, Johkura Bramuzzo, Matteo Bulla, Roberta Lucafò, Marianna Ferraro, Rosalba Monica Decorti, Giuliana Stocco, Gabriele
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).
Related Links	https://stemcellres.biomedcentral.com/counter/pdf/10.1186/s13287-024-04068-6.pdf
Ending Page	14
Page Count	14
Starting Page	1
File Format	HTM / HTML
ISSN	17576512
DOI	10.1186/s13287-024-04068-6
Journal	Stem Cell Research & Therapy
Issue Number	1
Volume Number	15
Language	English
Publisher	BioMed Central
Publisher Date	2024-12-18
Access Restriction	Open
Subject Keyword	Stem Cells Cell Biology Regenerative Medicine Tissue Engineering Biomedical Engineering and Bioengineering Human induced pluripotent stem cells Pancreatic progenitors Pancreatic exocrine cells Patient-specific model Crohn’s disease Regenerative Medicine/Tissue Engineering
Content Type	Text
Resource Type	Article
Subject	Cell Biology Medicine Biochemistry, Genetics and Molecular Biology Molecular Medicine
Journal Impact Factor	7.1/2023
5-Year Journal Impact Factor	7.9/2023

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