Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic “Emergency Exit” Switch

Abstract

Abstract:Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Use of 3D Organoids as a Model to Study Idiopathic Form of Parkinson’s Disease

“…After establishing iPSCs lines, they were characterized according to current standards [1,19]. The generated cell lines expressed alkaline phosphatase ( Figure 2a) and endogenous pluripotency markers, such as NANOG, OCT3, and telomerase (TERT) at the mRNA level ( Figure 2b).…”

Carbon Fibers as a New Type of Scaffold for Midbrain Organoid Development

“…The expression of neuron-specific class III β-tubulin ( TUJ1 ) was relatively high in all types of organoids ( Figure 4 D) on day 17, however, it significantly increased on day 49 in organoids growing on PLGA scaffold (2.27 fold) and even more on CF scaffold (4.12 fold) compared to the control ( Figure 4 D). The level of gene expression for tyrosine hydroxylase ( TH ), the characteristic marker of mDA neurons [ 2 ], was the highest in piPSC CF organoids on both day 17 and day 49, and as much as 8.75 times higher than in piPSC FL organoids on day 17 and 6.85 higher than on day 49 in piPSC FL organoids ( Figure 4 E). piPSC PLGA organoids also showed an increase in TH gene expression (2.84 fold) relative to control on day 49.…”

“…The media were changed after 2 days. Organoids were grown as described previously in [ 2 ]. Briefly, after 4 days, the detached colonies formed EBs, which were then transferred to a V-shaped plate and suspended in a medium for the induction of neurogenesis for 4 days (DMEM/F12:Neurobasal 1:1 (ThermoFisher), 1:100 N2 supplement (ThermoFisher), 1:50 B27 without vitamin A (ThermoFisher), 1% glutamine (ThermoFisher), 1% minimum essential media-nonessential amino acid (ThermoFisher), and 0.1% β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 1 μg/mL heparin (Sigma-Aldrich), 10 μM SB431542 (Sigma-Aldrich), 200 ng/mL Noggin (PeproTech, Inc., London, UK), 0.8 μM CHIR99021 (Sigma-Aldrich), penicillin/streptomycin 100 U/mL (ThermoFisher), and 10 μM ROCK inhibitor Y27632 (ThermoFisher)).…”

“…Induced pluripotent stem cells (iPSCs) have made it possible to generate human neurons, including dopaminergic neurons. This has created favorable conditions for research on the human brain, its development, and related diseases [ 1 , 2 ]. In combination with 3D organoid cell culture technology, it is possible to obtain structures resembling individual brain regions [ 3 ].…”

The Challenge of Bringing iPSCs to the Patient

“…There are several novel strategies that seek to overcome some of the pitfalls previously mentioned, shedding light on the possibility of widespread utilization of iPSCs in the clinics. For instance, the introduction of a suicide gene based on the herpes simplex virus thymidine kinase could incorporate some sort of "emergency switch" which could be activated by a non-toxic drug when the iPSCs were not needed anymore or in case of a problem, such a tumorigenic risk [103]. Alternatively, Deuse and colleagues have tried to overcome the problem of immunogenicity by inactivating some key components of the major histocompatibility complex (MHC) class I and II with CRISPR/Cas9 technology and overexpressing the transmembrane protein CD47 [104].…”