Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA

Abstract

The therapeutic promise of induced pluripotent stem cells (iPSCs) has spurred efforts to circumvent genome alteration when reprogramming somatic cells to pluripotency. Approaches based on episomal DNA, Sendai virus, and messenger RNA (mRNA) can generate “footprint-free” iPSCs with efficiencies equaling or surpassing those attained with integrating viral vectors. The mRNA method uniquely affords unprecedented control over reprogramming factor (RF) expression while obviating a cleanup phase to purge residual traces of vector. Currently, mRNA-based reprogramming is relatively laborious due to the need to transfect daily for ~2 weeks to induce pluripotency, and requires the use of feeder cells that add complexity and variability to the procedure while introducing a route for contamination with non-human-derived biological material. We accelerated the mRNA reprogramming process through stepwise optimization of the RF cocktail and leveraged these kinetic gains to establish a feeder-free, xeno-free protocol which slashes the time, cost and effort involved in iPSC derivation.

Bioengineering of Articular Cartilage: Past, Present and Future

“…There is always a risk of tumourigenicity associated with the use of stem cells and in particular the use of viral vectors. Newer methods that generate iPSCs without viral vectors have been developed to reduce the risk of tumourigenicity [61][62][63][64]. Overall chondrogenic differentiation of iPSCs is still in its formative stages of development and further work is required to evaluate its full potential in the field of osteochondral regeneration.…”

Expanding Horizons of Cellular Plasticity in Regenerative Medicine

“…37 However, limitations of such approach include inherent complexities related to cellular procedures and purification of reprogrammed cells. 37 In addition, the expression of reprogramming factors is robust for approximately 24 hours after mRNA transfection. Unfortunately, there is a long two-to three-week lag between expression of reprogramming factor proteins and induction of pluripotency in human cells.…”

“…Finally, repeated transfections that are needed to generate induced pluripotent stem cells is time intensive. 37 In this theme issue, we have reviewed the emergent significance of miRNA in tissue repair and regeneration. 38 The observation that miRNA does not integrate into the genome makes miRNA-based therapeutic strategies translationally valuable.…”

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.…”