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 exoc...
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BMC
2024-12-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-024-04068-6 |
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| author | Elena Genova Paola Rispoli Yue Fengming Johkura Kohei Matteo Bramuzzo Roberta Bulla Marianna Lucafò Rosalba Monica Ferraro Giuliana Decorti Gabriele Stocco |
| author_facet | Elena Genova Paola Rispoli Yue Fengming Johkura Kohei Matteo Bramuzzo Roberta Bulla Marianna Lucafò Rosalba Monica Ferraro Giuliana Decorti Gabriele Stocco |
| author_sort | Elena Genova |
| collection | DOAJ |
| description | 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). |
| format | Article |
| id | doaj-art-012f9754029e4e2ebca9dbe5c9d6bf07 |
| institution | DOAJ |
| issn | 1757-6512 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | BMC |
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| series | Stem Cell Research & Therapy |
| spelling | doaj-art-012f9754029e4e2ebca9dbe5c9d6bf072025-08-20T02:39:51ZengBMCStem Cell Research & Therapy1757-65122024-12-0115111410.1186/s13287-024-04068-6Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applicationsElena Genova0Paola Rispoli1Yue Fengming2Johkura Kohei3Matteo Bramuzzo4Roberta Bulla5Marianna Lucafò6Rosalba Monica Ferraro7Giuliana Decorti8Gabriele Stocco9Institute for Maternal and Child Health - IRCCS Burlo GarofoloDepartment of Medicine, Surgery and Health Sciences, University of TriesteDepartment of Histology and Embryology, Shinshu University School of MedicineDepartment of Histology and Embryology, Shinshu University School of MedicineInstitute for Maternal and Child Health - IRCCS Burlo GarofoloDepartment of Life Sciences, University of TriesteDepartment of Life Sciences, University of TriesteAngelo Nocivelli Institute for Molecular Medicine, Department of Molecular and Translational Medicine, University of BresciaInstitute for Maternal and Child Health - IRCCS Burlo GarofoloInstitute for Maternal and Child Health - IRCCS Burlo GarofoloAbstract 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).https://doi.org/10.1186/s13287-024-04068-6Human induced pluripotent stem cellsPancreatic progenitorsPancreatic exocrine cellsPatient-specific modelCrohn’s disease. |
| spellingShingle | Elena Genova Paola Rispoli Yue Fengming Johkura Kohei Matteo Bramuzzo Roberta Bulla Marianna Lucafò Rosalba Monica Ferraro Giuliana Decorti Gabriele Stocco Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications Stem Cell Research & Therapy Human induced pluripotent stem cells Pancreatic progenitors Pancreatic exocrine cells Patient-specific model Crohn’s disease. |
| title | Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications |
| title_full | Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications |
| title_fullStr | Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications |
| title_full_unstemmed | Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications |
| title_short | Time-efficient strategies in human iPS cell-derived pancreatic progenitor differentiation and cryopreservation: advancing towards practical applications |
| title_sort | time efficient strategies in human ips cell derived pancreatic progenitor differentiation and cryopreservation advancing towards practical applications |
| topic | Human induced pluripotent stem cells Pancreatic progenitors Pancreatic exocrine cells Patient-specific model Crohn’s disease. |
| url | https://doi.org/10.1186/s13287-024-04068-6 |
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