Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA

Abstract

SUMMARY The generation of human induced Pluripotent Stem (iPS) cells holds great promise for development of regenerative medicine therapies to treat a wide range of human diseases. However, the generation of iPS cells in the absence of integrative DNA vectors remains problematic. Here we report a simple, highly reproducible RNA-based iPS generation approach that utilizes a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors, OCT4, KLF4, SOX2 with c-MYC or GLIS1 at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPS cells with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles and in vivo pluripotency to differentiate into all three germ layers. The VEE-RF RNA-based approach has broad applicability for the generation of iPS cells for ultimate use in human stem cell therapies in regenerative medicine.

THERAPY OF ENDOCRINE DISEASE: Islet transplantation for type 1 diabetes: so close and yet so far away

“…Epigenetic studies of produced iPSCs traced epigenetic memory or reminiscent marks, such as residual DNA methylation signatures of the starting cell types during early passages of iPSCs (34,35) which could explain the mechanisms behind such observations. Alternative strategies such as non-integrative vectors (36), adenoviruses (37), repeated mRNA transfection (38,39,40), protein transduction (41), and transposon-based transgene removal (42) have been successfully applied to develop transgene-free iPSC lines. This progression towards safe iPSC generation (reviewed in (43)) offers a promising technology for medical pertinence.…”

Vectorology and Factor Delivery in Induced Pluripotent Stem Cell Reprogramming

“…5). Such synthetic, noninfectious RNA replicons encoding the reprogramming factors were recently used to generate footprint-free iPSCs [16]. DNA-based alphaviral vectors are reported [123][124][125].…”

“…To achieve these goals, many distinct technologies are employed in current reprogramming protocols. These include nonintegrating adenoviral vectors [7], excisable PiggyBac (PB) transposon [8], excision of transgenes with the Cre-Lox system upon completion of reprogramming [9,10], repeated transfection with conventional plasmids [11], minicircle DNA [12], Epstein-Barr virus-based replicating episomal plasmids [4][5][6], protein transduction [13], mRNA transfection [14], negative-sense RNA vectors (Sendai viral vector) [15], positive-sense RNA vector/replicons [16], and the use of polycistrons mediated by 2A peptide [9,11], and/or Internal Ribosome Entry Site (IRES) [4]. This review summarizes information relative to vector designs and factor delivery systems used in current reprogramming protocols.…”

Direct reprogramming of somatic cells: an update

“…Therefore, iPSCs must be screened to select free cells for further applications (Gonzalez et al, 2009). To date, the safest technique of iPSC production is induction of pluripotency via mRNA (Warren et al, 2012;Yoshioka et al, 2013) or protein (Kim et al, 2009;Lee et al, 2012). These iPSCs are called "clean" iPSCs.…”