The impact of transposable element activity on therapeutically relevant human stem cells

Abstract

Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

Keywords: Adult stem cells; Genomic destabilization; LINE-1; Methylation; Pluripotent stem cells; Regenerative medicine; Restriction; Transposable elements.

Fig. 1

Human stem cell-based clinical studies conducted worldwide (Status: October 2018; www.clinicaltrials.gov). a Interventional clinical trials (Phase I or phase I/II) applying adult stem cell types (mesenchymal stem cells, MSCs; hematopoietic stem cells, HSCs; neuronal stem cells, NSCs) or their differentiated derivatives, or embryonic stem cell (ESC)-or induced pluripotent stem cell (iPSC)-derived differentiated cells. Numbers in brackets indicate the number of individual trials based on the respective stem cell type. b Interventional clinical trials and observational studies that are currently ongoing or in preparation use therapeutic derivatives of ESCs (blue lettering) or iPSCs (green lettering) to treat ophthalmic, urological, blood, cardiac and genital diseases, neurological disorders and cancers/neoplasms. Numbers in brackets represent the number of clinical trials and/or observational studies initiated to treat the respective disease or disorder

Fig. 2

Retrotransposons in the human genome. Currently, only LINE-1 (L1), Alu and SVA elements are verifiably still mobilized in humans. A full length HERV-K provirus is ~ 9.5 kb long, codes for group-specific antigen (Gag), protease (Pro), polymerase (Pol) and envelope (Env) proteins and is flanked by ~ 1-kb long terminal repeats (LTRs) with the 5’LTR including the HERV-K promoter. A functional full length L1 element is ~ 6 kb in length, harbours three open reading frames (ORF0, ORF1 and ORF2) at which ORF1 and ORF2 are separated by a 63-bp noncoding spacer region. The 5′ untranslated region (5’UTR) harbours both sense and antisense promoter. Alu elements comprise ~ 280-300 bp, are composed of two 7SL-RNA derived monomers separated by an A-rich connector (A₅TACA₆), contain an internal A and B box promoter, and end in a poly A tail (A_n). SVA elements are ~ 0.7–4 kb long, consist of a 5′ hexamer repeat that can be variable in length ((CCCTCT)n), two Alu fragments in antisense orientation, a GC-rich variable number of tandem repeats (VNTR) region, a SINE-R sequence derived from an HERV-K10 element and a poly A tail following a polyadenylation signal. The length of an intact SVA can vary depending on the number of repeats present in the hexamer and VNTR domains. L1, Alu and SVA insertions are characterized by the hallmarks of L1-mediated retrotransposition such as flanking variable target site duplications (TSDs), polyA tails at their 3′ ends (A_n) and insertion at the consensus target sequence 5′-TTTT/AA-3′. 3’UTR, 3′ untranslated region

Fig. 3

Schematic of relative endogenous L1 expression levels and engineered retrotransposition frequencies in human ESCs and adult stem cells derived from mesoderm and ectoderm. Relative L1 mRNA and L1 ORF1p expression levels, and relative engineered L1 retrotransposition frequencies supported by the respective cell type are illustrated: ++++, very high; +++, high; ++, moderate; +/−, barely detectable and therefore very low. HSC, Hematopoietic Stem Cells; MSC, Mesenchymal Stem Cells; ESC, Embryonic Stem Cells; NPC, Neural Progenitor Cells; KER, Human Foreskin Keratinocytes

Fig. 4

Use of class II transposons as gene vectors. a Autonomous DNA transposons consist of a transposase-coding gene that is flanked by inverted terminal repeats (ITR; black arrows flanked by white arrows). b Bi-component transposon vector system for delivering plasmid-encoded transgenes. One component consists of a plasmid containing a gene of interest (GOI) flanked by transposon ITRs. The second component is a transposase expression plasmid. Black arrow, promoter driving expression of transposase gene. c The transposon carrying a GOI is excised from the donor plasmid and integrated at a chromosomal site by the transposase