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Retinal Differentiation of mRNA-Reprogrammed iPSCs – Safe and Efficient!

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Review of “Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells toward a Retinal Lineage” from Stem Cells Translational Medicine by Stuart P. Atkinson

The application of synthetic mRNAs to reprogramming represents a safe and efficient strategy to generate induced pluripotent stem cells (iPSCs) [1, 2]. This method lacks the risks related to genomic integration and the overexpression of potentially oncogenic factors although we understand less about their differentiation capacity.

With the hope of changing this, a new study from researchers at the laboratory of Jason S. Meyer (Indiana University-Purdue University, USA) aimed to assess the abilities of mRNA-induced iPSCs to differentiate into cells of the retina in the hope that this strategy can become an effective cell replacement therapy [3].

The authors first used the same human fibroblasts to generate iPSC lines using both mRNA (miPSC) and retroviral (riPSC) methods of transduction with reprogramming factors. Both iPSCs types expressed similar levels of pluripotency markers, exhibited an ESC-like morphology, and underwent embryoid body-mediated differentiation in a similar manner.

Using previously described protocols, the study then followed miPSC and riPSC differentiation through known retinal developmental stages. Encouragingly, they demonstrated similar proportions of both primitive neuroretinal cells expressing appropriate markers (PAX6, SOX1, OTX2, and LHX2) at day 10 and optical vesicle (OV)-like structures containing retinal progenitors expressing CHX10 and PAX6, but not SOX1, at day 30 (See attached figure).

But how about terminally differentiated retinal cell types? The authors noted that pigmented retinal epithelial (RPE) cells arose in both cultures within the first 30 days of differentiation, eventually forming expandable monolayers of hexagonal RPE-like cells over the following 4-8 weeks. Additionally, differentiation of CHX10-positive retinal progenitor cells derived from riPSCs and miPSCs for 70 days generated similar proportions of cells expressing genes associated with developing retinal ganglion cells, photoreceptors, and interneurons of the retina. 

However, the authors did observe significant differences in the levels of BRN3-positive retinal ganglion cells between cultures, although this did not correlate to the type of reprogramming strategy used, but showed variation between experimental repeats in both miPSC and riPSC cultures. 

So, all-in-all, this study proposes the differentiation of mRNA-induced iPSCs to be a safe and efficient means to produce retinal cells for translational and therapeutic applications such as disease modeling, pharmacological screening and cell replacement. The authors note that miRNA-based reprogramming is also compatible with the removal of xenogeneic components from media commonly used to maintain and differentiate hiPSCs, and so may be the most applicable strategy moving on towards clinical application.

References

  1. Schlaeger TM, Daheron L, Brickler TR, et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol 2015;33:58-63.
  2. Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 2010;7:618-630.
  3. Sridhar A, Ohlemacher SK, Langer KB, et al. Robust Differentiation of mRNA-Reprogrammed Human Induced Pluripotent Stem Cells Toward a Retinal Lineage. Stem Cells Transl Med 2016;5:417-426.