A Highly Optimized Protocol for Reprogramming Cancer Cells to Pluripotency Using Nonviral Plasmid Vectors

Abstract

In spite of considerable interest in the field, reprogramming induced pluripotent stem cells (iPSCs) directly from cancer cells has encountered considerable challenges, including the extremely low reprogramming efficiency and instability of cancer-derived iPSCs (C-iPSCs). In this study, we aimed to identify the main obstacles that limit cancer cell reprogramming. Through a detailed multidimensional kinetic optimization, a highly optimized protocol is established for reprogramming C-iPSCs using nonviral plasmid vectors. We demonstrated how the initial cancer cell density seeded could be the most critical factor ultimately affecting C-iPSCs reprogramming. We have consistently achieved an unprecedented high C-iPSC reprogramming efficiency, establishing stable colonies with typical iPSC morphology, up to 50% of which express the iPSC phenotypic (Oct3/4, Sox2, Nanog) and enzymatic (alkaline phosphatase) markers. Furthermore, established C-iPSC lines were shown to be capable of forming teratomas in vivo, containing cell types and tissues from each of the embryonic germ layers, fully consistent with their acquisition of pluripotency. This protocol was tested and confirmed in two completely unrelated human lung adenocarcinoma (A549) and mouse melanoma (B16f10) cancer cell lines and thus offers a potentially valuable method for generating effectively virus-free C-iPSCs for future applications.

Optimization of episomal reprogramming for generation of human induced pluripotent stem cells from fibroblasts

“…However, we found that reprogramming efficiency decreased when the total vector concentration was too high. Previous studies have shown that cell density is critical for reprogramming (Zhao et al 2015 ; Sia et al 2016 ). Thus, to optimize cell density, we plated BJ cells transfected with 9 µg episomal vectors onto Matrigel coated 6-well dishes at a density ranging from 1.0 × 10 4 to 3.0 × 10 5 cells per well.…”

Immortalized murine fibroblast cell lines are refractory to reprogramming to pluripotent state

“…Several other reports also showed refractoriness of mouse cancerous cell lines to the reprogramming [ 3 , 43 ]. At the same time, it was shown that primary mouse carcinoma cells and malignant melanoma cell line remain amenable to reprogramming to iPSCs [ 10 , 29 , 30 ]. Also it was shown that immortalized mouse fibroblast cells m5S can be efficiently reprogrammed via cell fusion with ESCs [ 44 ].…”

“…Important to note that human primary cancer cell lines could be reprogrammed to iPSCs more efficiently than mouse ones [ 22 , 26 , 29 , 56 – 60 ]. Several studies have reported generation of iPSCs from primary chronic myeloid leukemias, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors, indicating that B-cell origin and leukemic fusion gene were not reprogramming barriers [ 28 , 58 , 61 ].…”

“…The reprogramming inefficiency and relative instability of the cancer-derived iPSCs became the main issues [ 28 , 29 ]. On the other hand, referring to the published data, iPSCs have been generated only from few mouse primary cancer cell lines, suggesting that mouse cancer cells are mainly refractory to reprogramming in standard condition [ 29 , 30 ]. It is known that multiple passages and culture conditions lead to significant abnormalities in chromosome numbers and stability.…”

Murine melanoma cells incomplete reprogramming using non‐viral vector

“…Therefore, we supposed that the use of less invasive non‐viral vector would help to produce more viable cells, thus increasing reprogramming efficiency. This method was successfully used previously for human adipose tissue stem cells reprogramming and for cancer cells . Using non‐viral vector, we reprogrammed highly heterogeneous population of melanoma cells into less aggressive Murine Melanoma Reprogrammed Cancer Cells (MMRCC).…”