Efficient Modulation of TP53 Expression in Human Induced Pluripotent Stem Cells

Abstract

TP53 point mutations are found in 50% of all cancers and seem to play an important role in cancer pathogenesis. Thus, human induced pluripotent stem cells (hiPSCs) overexpressing mutant TP53 are a valuable tool for the generation of in vitro models of cancer stem cells or for in vivo xenograft models. Here, we describe a protocol for the alteration of gene expression in hiPSCs via overexpression of a mutant form of the TP53 (R249S) gene using lentiviral transduction. A high amount of TP53 protein is detected 1 week after transduction and antibiotic selection. Differentiation of transduced hiPSCs gives insight into better understanding cancer formation in different tissues and may be a useful tool for genetic or pharmacologic screening assays. © 2019 The Authors. Basic Protocol 1: Production and concentration of third‐generation lentivirus Support Protocol 1: Cloning of gene of interest into modulation vector Support Protocol 2: Preparation of DMEM GlutaMAX™ with 10% fetal bovine serum and 1% penicillin‐streptomycin Basic Protocol 2: Transduction of human induced pluripotent stem cells and selection of positively transfected cells Support Protocol 3: Preparation of Matrigel®‐coated plates Support Protocol 4: Preparation of mTeSR™1 medium

Different Calculation Strategies Are Congruent in Determining Chemotherapy Resistance of Brain Tumors In Vitro

“…The physical conducting of one-to-one comparative tests on healthy development in vitro models is favorable to predict toxicity, due to the power of real biologically acquired data. In this context, recently emerging synthetic cancer cell modeling technology, based on the introduction of cancer resembling genetic elements into healthy donor stem cells [32][33][34][35], offers a unique opportunity: given the step-wise introduction of multi-factorial alterations, one is able to model normal development, early stage malformation, and late stage disease progression in an isogenic, homogeneous single-cell of origin background. This setup may be more stable to score molecular associated, low adversity pharmacological perturbations as when relying on test matrices derived from different donors, as was the case in this study.…”

Progenitor cells derived from gene‐engineered human induced pluripotent stem cells as synthetic cancer cell alternatives for in vitro pharmacology

“…Each vector with our genes of interest (GOI; GLI1, c‐MYC, TP53R175H, and EGFRvIII) was cloned as previously described 18 . A detailed protocol can be found in the supplementary material.…”

“…Generation of lentiviral particles was done with a 3 rd generation lentiviral packaging system as described previously 18 and in the supplementary material.…”

“…12 As such, hiPSCs derived from healthy donors present an unlimited resource of cells to serve as receiver matrixes for genetic elements encoding for cancer-relevant transformations. Such healthy donorderived tumor models -herein referred to as hiPSC-oncogene models -manifest themselves as a sustainable alternative disease modeling strategy in the current cancer research community [13][14][15][16][17][18] complementing the portfolio of lab tools alongside the use of PD systems. It is speculated that iPSC models of cancer, derived through synthetic approaches or reprogramming of tumor cells, may be beneficial to homogeneously depict a cell of origin of cancers.…”

Testing the Stability of Drug Resistance on Cryopreserved, Gene-Engineered Human Induced Pluripotent Stem Cells

“…All our cells continually pass our quality control system [ 22 ] to exclude contamination, accumulation of karyotype abnormalities or loose of pluripotency. Overexpression of cMYC and TP53R175H in iPS11 was achieved according to the procedure described recently [ 23 ]. In our work, ethics follow general bioethics guidelines related to biomedical research, such as the Declaration of Helsinki.…”