L-myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation

Abstract

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.

Genetic Basis of Dilated Cardiomyopathy in Dogs and Its Potential as a Bidirectional Model

“…Retroviral transduction Viral, genomic integrating [80,81,[88][89][90][91][92] Lentiviral transduction Viral, genomic integrating [86,93,94] Sendai viral transduction Viral, non-integrating [84,85,95,96] Synthetic RNA transfection Nonviral, non-integrating [97] Episomal plasmids Nonviral, non-integrating [98] Initial protocols followed retroviral and lentiviral transgene integrating methods and the transgenes were eventually silenced after the iPSCs were fully reprogrammed. Due to concerns over the clinical applications with evidence from numerous studies showing that viral integrating methods can induce genomic integration and increase tumorigenic potential [99,100], other non-integrating methods have been extensively studied over the last few years.…”

“…be applied to both peripheral blood mononuclear cells (PBMCs) collected from whole blood samples as well as fibroblasts obtained from skin biopsies (Figure 3). Since the first successful attempt at reprogramming fibroblasts, several reports have described the generation of ciPSCs [78,[80][81][82][83][84][85][86][87], but the canine cell reprogramming protocols remain inconsistent and not well established [83]. Additionally, species-species differences involved in the reprogramming, development, and maturation of iPSCs need to be further elucidated.…”

“…Due to concerns over the clinical applications with evidence from numerous studies showing that viral integrating methods can induce genomic integration and increase tumorigenic potential [99,100], other non-integrating methods have been extensively studied over the last few years. Two studies recently successfully reprogrammed canine somatic cells into ciP-SCs [84,85] and one study reprogrammed canine PBMCs into ciPSCs using the Sendai viral transduction method [96]. This method is effective but much more expensive than other methods and the Sendai virus vectors can impair the regulation of reprogramming gene expression [97].…”

“…Lately, cells have been reprogrammed using an RNA transfection-based method, which is reportedly more reproducible and efficient for reprogramming [87,97]. In this strategy, canine fibroblasts were cotransfected with non-infectious, self-replicating, and integration-free Venezuelan equine encephalitis (VEE) RNA virus replicon that expresses four Since the first successful attempt at reprogramming fibroblasts, several reports have described the generation of ciPSCs [78,[80][81][82][83][84][85][86][87], but the canine cell reprogramming protocols remain inconsistent and not well established [83]. Additionally, species-species differences involved in the reprogramming, development, and maturation of iPSCs need to be further elucidated.…”

Opening the “Black Box” Underlying Barriers to the Use of Canine Induced Pluripotent Stem Cells: A Narrative Review

“…L-MYC has been used as a replacement reprogramming factor for c-MYC when generating ciPSCs [ 115 ] and has been shown to promote self-renewal but not tumorigenesis in canine fetal fibroblasts [ 115 ]. GLIS1 is another viable alternative to the oncogenic c-MYC and was used by Kim et al (2020) to generate ciPSCs [ 24 ].…”