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Stem cells are undifferentiated cells that divide and produce different cell types. Ordinarily, cells that have differentiated into a specific cell type are terminally differentiated; however, scientists have found a way to reprogram these mature cells so that they dedifferentiate and return to an unspecialized, proliferative state. These cells are pluripotent like embryonic stem cells—able to produce all cell types—and are called induced pluripotent stem cells (iPSCs).
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Under normal conditions, most adult cells remain in a non-proliferative state unless stimulated by internal or external factors to replace lost cells. Abnormal cell proliferation is a condition in which the cell's growth exceeds and is uncoordinated with normal cells. In such situations, cell division persists in the same excessive manner even after cessation of the stimuli, leading to persistent tumors. The tumor arises from the damaged cells that replicate to pass the damage to the...
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RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells
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Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells.

Constanze Uhlmann1, Lisa-Maria Kuhn2, Julia Tigges3

  • 1Department of Neurosurgery, University Hospital Düsseldorf, Düsseldorf, Germany.

Current Protocols in Stem Cell Biology
|December 29, 2019
PubMed
Summary

This study details a protocol for genetically modifying human induced pluripotent stem cells (hiPSCs) to overexpress mutant TP53. This advancement aids in creating better cancer models for research and drug screening.

Keywords:
TP53hiPSClentiviral transductionmolecular alteration

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cancer Research

Background:

  • TP53 gene mutations are prevalent in approximately 50% of human cancers, significantly contributing to cancer development.
  • Mutant TP53 is crucial for understanding cancer pathogenesis and developing therapeutic strategies.
  • Human induced pluripotent stem cells (hiPSCs) offer a promising platform for modeling diseases, including cancer.

Purpose of the Study:

  • To establish a reliable protocol for lentiviral transduction of hiPSCs to overexpress a specific mutant TP53 (R249S).
  • To enable the generation of advanced in vitro and in vivo models for cancer stem cells and xenografts.
  • To facilitate genetic and pharmacologic screening assays for cancer research.

Main Methods:

  • Lentiviral vector construction for TP53 (R249S) gene delivery.
  • Transduction of hiPSCs with the lentiviral vector.
  • Antibiotic selection to isolate successfully modified hiPSCs.
  • Verification of mutant TP53 protein expression via Western blot or similar techniques.

Main Results:

  • Successful overexpression of mutant TP53 protein in hiPSCs was confirmed one week post-transduction and selection.
  • The protocol provides a high yield of genetically modified hiPSCs expressing the target mutant protein.
  • The methodology is robust and reproducible for generating TP53-mutated hiPSC lines.

Conclusions:

  • The described protocol effectively generates hiPSCs overexpressing mutant TP53 (R249S) using lentiviral transduction.
  • These modified hiPSCs serve as a valuable tool for creating in vitro cancer stem cell models and in vivo xenograft models.
  • This approach supports further research into cancer formation and aids in the development of novel screening assays for therapeutic interventions.