Name: Video: Preparation of iMEF Feeder Cells and Reprogramming Transduction for Induced Pluripotent Stem Cells
Uploaded: 2024-02-02T13:30:00
Description: 61 Views. Preparation of iMEF Feeder Cells and Reprogramming Transduction for Induced Pluripotent Stem Cells

preparation of imef feeder cells and reprogramming transduction for induced pluripotent stem cells

Reprogramming PDAC and Pancreatic Cells into Induced Pluripotent Stem Cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal malignancies, and early diagnosis remains challenging due to the asymptomatic nature of the disease. The majority of PDAC patients are diagnosed at the advanced metastatic stage when very limited treatment options are available1,2. This is mainly due to the lack of reliable biomarkers for the earlier stages, such as those that could be conveniently detected as proteins released into the bloodstream.
PDAC can disseminate very early during its progression, and a better prognosis has been linked to early cancer detection when PDAC is localized in the pancreas3. However, less than a tenth of PDAC patients are diagnosed with a favorable prognosis, allowing for surgical resection. Nonetheless, those few with resectable tumors are also prone to tumor recurrence within 12 months4.
In the past five decades, remarkable improvements have been made in surgical techniques, patient care, and treatment modalities5,6. However, the 5-year survival rate in surgically resected PDAC patients has barely risen to 17%. Nonetheless, this is still better than that in non-resected patients, which has remained almost unchanged (0.9%)4,7. Chemotherapy is the only other alternative PDAC treatment. Yet, this option is very limited as the great majority of PDAC patients exhibit strong resistance to chemotherapy medications such as Gemcitabine7,8. Other drugs, such as Erlotinib, are only available to a small group of PDAC patients with specific mutations, most of whom show Erlotinib resistance9. The adverse side effects associated with chemotherapy in most PDAC patients are yet another disadvantage of this treatment10. Recently, promising strategies have shown that immune checkpoint inhibitors (ICIs) and small molecule kinase inhibitors (SMKIs) can be effective in treating PDAC, but durable responses to these targeted therapies remain limited to a minority of patients11,12. Overall, the discovery of PDAC-specific early biomarkers can pave new avenues for early diagnosis and treatment.
PDAC develops from pancreatic intraepithelial neoplasms (PanIN) precursor lesions that result from non-invasive pancreatic duct epithelial proliferations13,14. While the formation of PanIN is initiated by oncogene mutations such as KRAS, additional genetic and epigenetic alterations are required for the progression to PDAC. It has been projected that the progression of PanIN through the different stages into invasive PDAC takes about 10 years13,15,16,17. This timeframe provides a great opportunity to benefit from early PDAC diagnosis. Therefore, extensive research has been carried out to establish tumor xenograft animal models and organoid cultures to study PDAC progression18,19,20,21. These models have been very useful for studying the invasive stages of PDAC, although not the transition from the early PanIN phases. It is, therefore, important to develop experimental models that can recapitulate the early progression of PanIN stages to enable the discovery of early detection biomarkers.
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) using the four transcription factors OCT4, SOX2, KLF4, and c-MYC (OSKM) has illustrated the extent of cellular plasticity22. Cancer cell plasticity has been well-documented, and reprogramming human cancer cells into iPSCs has been successfully used to reset cells to their original cellular state, removing many of the epigenetic insults that have accumulated during cancer progression23,24,25,26,27,28,29. The possibility of using this reprogramming strategy to manipulate cancer cell identity has, therefore, presented great promise in treating cancer30,31. Indeed, we have previously shown that the differentiation of iPSCs derived from PDACs can recapitulate PDAC progression through the early PanIN stages32. By identifying genes and pathways specific to the early-to-intermediate stages of PDAC, candidate biomarkers were identified that can be clinically used for early PDAC diagnosis32,33. However, the biomarkers discovered using a single iPSC line showed limited coverage in the majority of PDAC patients32. The challenges of generating iPSC lines from other PDAC patients have halted the ability to discover more reliable biomarkers. This is due to many technical factors, including the heterogeneity of OSKM delivery, as only a small portion of human primary PDAC cells contained all four factors and responded successfully to reprogramming. Here, a detailed protocol is presented for reprogramming primary PDAC cells using a more efficient and consistent dual lentiviral delivery of OSKM.

Author Spotlight: Reprogramming Cancer Cells to iPSCs to Study Disease Progression and Treatment Targets

Reprogramming Pancreatic Ductal Adenocarcinoma to Pluripotency

Preparation of iMEF Feeder Cells and Reprogramming Transduction for Induced Pluripotent Stem Cells

preparation-imef-feeder-cells-reprogramming-transduction-for-induced

Research

JoVE Journal

Cancer Research

61 Views. Preparation of iMEF Feeder Cells and Reprogramming Transduction for Induced Pluripotent Stem Cells

Video: Preparation of iMEF Feeder Cells and Reprogramming Transduction for Induced Pluripotent Stem Cells

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has proved highly valuable for research and clinical applications. Interestingly, iPSC reprogramming of cancer cells, such as pancreatic ductal adenocarcinoma (PDAC), has been shown to revert the invasive PDAC phenotype and override the cancer epigenome. The differentiation of PDAC-derived iPSCs can recapitulate PDAC progression from its early pancreatic intraepithelial neoplasia (PanIN) precursor, revealing the molecular and cellular changes that occur early during PDAC progression. Therefore, PDAC-derived iPSCs can be used to model the earliest stages of PDAC for the discovery of early-detection diagnostic markers. This is particularly important for PDAC patients, who are typically diagnosed at the late metastatic stages due to a lack of reliable biomarkers for the earlier PanIN stages. However, reprogramming cancer cell lines, including PDAC, into pluripotency remains challenging, labor-intensive, and highly variable between different lines. Here, we describe a more consistent protocol for generating iPSCs from various human PDAC cell lines using bicistronic lentiviral vectors. The resulting iPSC lines are stable, showing no dependence on the exogenous expression of reprogramming factors or inducible drugs. Overall, this protocol facilitates the generation of a wide range of PDAC-derived iPSCs, which is essential for discovering early biomarkers that are more specific and representative of PDAC cases.

The present protocol describes the reprogramming of Pancreatic Ductal Adenocarcinoma (PDAC) and normal pancreatic ductal epithelial cells into induced pluripotent stem cells (iPSCs). We provide an optimized and detailed, step-by-step procedure, from preparing lentivirus to establishing stable iPSC lines.

The generation of induced pluripotent stem cells (iPSCs) using transcription factors has been achieved from almost any differentiated cell type and has ...