Generation of adult human induced pluripotent stem cells using nonviral minicircle DNA vectors

Abstract

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques utilize integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. Here we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thereby capable of high expression in mammalian cells. Their repeated transfection into hASCs, an abundant somatic cell source that is amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ~4 weeks.

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

“…Their use results in sustained transgene expression due to lower activation of nuclear transgene silencing mechanisms, and reduced immunogenic responses [30]; the latter resulting in higher safety. The clinical potential of mCs has been exploited for non-viral DNA reprogramming to generate human induced pluripotent stem cells [31]. An analysis of mC DNA transfection in a murine stem cell line CGR8-NS using microporation reported up to 60% transfection with high cell survival (~90%) [32].…”

CD117+amniotic fluid stem cells

“…First trimester AFS cells, otherwise, can be reprogrammed with high efficiency and with a stable maintenance of the pluripotent phenotype only with the employment of a chemical substance (i.e., valproic acid, an FDA-approved drug for the treatment of epilepsy), thus reducing the risks associated with the random integration of the reprogramming transgenes into the host genome. [103][104][105][106][107][108] Many questions concerning AFS cell biological properties remain to be solved including origin, biological function in vivo, epigenetic state, and immunological reactivity. Answers to all of these issues are necessary before AFS cell employment in therapy.…”

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells

“…[5][6][7][8] Recent efforts have thus focused on nonviral methods as a potentially safer alternative. iPSCs have been nonvirally generated using electroporation of nonintegrating episomal plasmids, 4,9,10 small molecule modulators, 11 minicircle DNA vectors, 12 recombinant proteins, 13,14 and synthetic messenger (m)RNA technology. 15 Although promising, these approaches report low reprogramming efficacy, and protocols involving animal-origin feeder cells and conditioned media containing serum may raise safety concerns for translation.…”