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Know which way to miRoam: Red means ‘GO’, Green ‘STOP’!

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Know which way to miRoam: Red means ‘GO’, Green ‘STOP’!

By Carla B. Mellough

Induced pluripotent stem cells (iPSC) are shown to have many of the characteristics of human embryonic stem cells (hESC) including prolonged proliferative capacity and differentiation across all germ layers. The therapeutic potential of human iPSC (hiPSC), however, remains controversial. Whilst the use of hiPSC would overcome some of the ethical issues surrounding hESC, their suitability and usefulness as a replacement cell type has recently fallen into question. For example, concerns have been raised over the retention of epigenetic memory from the iPSC cell of origin (see iPSC don’t forget their origins) which questions their level of reprogramming, and may be the cause of their apparently biased pluripotent potential. However, conversely, it has been suggested that in fact iPSC and hESC are very similar (see Similar Human iPSC and ESC Chromatin States Suggests Usefulness in Regenerative Medicine). Various hiPSC clones differ with regards to their level of reprogramming and differentiation potential and being able to identify those of greatest potential therapeutic value would no doubt help the advancement of hiPSC research.

A recent paper from Irvin Chen’s group at the University of California1 reports the results from their study in which they devised a simple yet elegant way in which to monitor the transition from fibroblasts to hiPSC and subsequent hiPSC differentiation. This was accomplished by taking advantage of the differential expression of microRNAs (miRNAs) across different cell types. MicroRNAs are one of the species of small non-coding RNAs which orchestrate key processes during development in a tissue-specific manner. Recently miRNA function has been shown to act by decreasing target mRNA levels, predominantly by mRNA destabilization2. The importance of miRNAs in multiple tissues and disease states is now being recognised, having wide ranging effects in every system studied to date and more recently, elucidation of the patterns of miRNA expression in a given cell type, or the ‘miRNome’, has received much research attention with numerous potential applications apparent3,4.

To achieve live cell monitoring, the authors constructed a bidirectional reporter vector conjugating EGFP in an anti-sense direction with miR-302a and d (miR-302a/d, preferentially expressed in embryonal carcinoma cells, hESC and iPSC) and mCherry with complementary miRNA target sites for miR-223, miR-15 and miR-142-3p (enriched in differentiated hematopoietic, fibroblast or malignant cells). Expression of miR-302a/d in pluripotent stem cells would thus result in a loss of EGFP expression but not mCherry, whilst expression of lineage-specific miRNAs would result in the loss of mCherry but not EGFP.

The authors confirmed the responsiveness of this reporter construct to specific miRNAs by demonstrating the loss of the reporter gene either upon ectopic miRNA expression by co-transduction with specific miRNA-expressing lentiviral vectors, or following bidirectional vector transduction in hESCs, hematopoietic cells, lymphoma cells and hematopoietic fetal liver progenitor cells. In all cases the vector acted as expected, by turning off either EGFP or mCherry. Transduction of human dermal fetal fibroblasts (HFF) with the reporter construct also gave robust EGFP expression.

Then equipped to monitor hiPSC formation, the authors co-transduced HFFs with the bidirectional reporter construct and vectors expressing OCT4, SOX2, KLF4 and c-MYC and monitored reporter expression over four weeks during hiPSC generation. They observed a decrease in EGFP and increase in mCherry indicating the induction of hESC-specific miR-302a/d. Interestingly, the authors note that although many cells expressed miR-302a/d, only a small proportion of these formed hESC-like colonies during the reprogramming period. Large granulated colonies were also observed, of which 90% were EGFP negative and mCherry positive, likely indicating partial reprogramming in these cells. Co-transduction with the reporter vector and reprogramming factors in their hands gave 0.03% generation efficiency for normal hiPSC colonies that expressed mCherry and lacked EGFP. Of 13 hiPSC clones generated, 3 were analysed which demonstrated stable expression of mCherry for over 20 passages, remained EGFP negative and displayed normal hESC characteristics and markers as assessed by immunostaining and RT-PCR analysis. Differentiation of these hiPSC into embryoid bodies induced a small reduction in mCherry and re-expression of EGFP, indicating the loss of stem cell-associated miR-302a/b and the emergence of differentiation-associated miRNAs. Further, the authors tested reporter expression in hiPSCs differentiated towards the neural lineage. They detected expression of both EGFP and mCherry in neural tube-like rosette structures indicating a lack of reporter vector target miRNAs, alongside downregulation of pluripotency and upregulation of neuroectodermal markers. Of note, cells within the neural-like tube were EGFP positive only, those surrounding expressed both EGFP and mCherry, whilst pigmented cells in differentiating hiPSC cultures expressed mCherry alone, indicating differential miRNA expression between differing neural cell types.

This study demonstrates the efficient use of a miRNA-dependent vector system to monitor iPSC generation and differentiation. The relative expression of miRNAs thus seems a useful diagnostic indicator in defining the developmental state of hESC or iPSC, or in the delineation of more mature lineages. In this experimental design, robust expression of EGFP in fibroblasts (reflecting the absence of miR-302a/d) was replaced by mCherry expression in fully reprogrammed hiPSC, reflecting expression of miR-302a/d and lack of miR-223/155/142-3p expression. This presents a novel way to pick the most ideal hESC-like iPSC colonies for expansion following the reprogramming process.

However, other considerations must be taken into account; of the three hiPSC clones tested, one exhibited reduced mCherry expression and showed lower levels of SSEA3 with authors noting flatter colony morphology and greater levels of spontaneous differentiation in this clone. Further, the authors reported the expression of hESC-specific miRNA in more than 50% of transduced HFFs in response to ectopic expression of reprogramming factors, yet only 0.03% of these continued to fully reprogram into hiPSCs. So although selecting cells on the basis of miR-302a/d is useful, it does not definitively mark hiPSCs and more work needs to be done in the elucidation of the stem cell-specific miRNome to allow more efficient monitoring of cellular reprogramming to pluripotency. As the authors highlight in their paper, this will also permit the selection and isolation of low frequency populations that are difficult to yield from heterogeneous differentiating populations, based on their expression or lack thereof of specific miRNAs.

In addition to the value of miRNAs in monitoring or driving the specification of various cell types and its benefits for hESC and hiPSC research, knowledge of the key players in a cell-specific miRNome context will no doubt also lead to new therapeutic strategies. For example, the targeting and knockdown of dominant mutant proteins causing cell dysfunction in various disease. A paper recently published by Robin Ali’s group at University College London provide the proof of principle for such therapy, demonstrating the use of a miRNA-based hairpin to silence peripherin, the dominant mutated allele of which is a major cause of blindness in humans5. This indicates we are already gaining important knowledge for the research potential and future therapeutic value of these small, but important non-coding RNAs.

 

References

1. Kamata et al. Live cell monitoring of hiPSC generation and differentiation using differential expression of endogenous microRNAs. PLoS One. 2010;5(7):e11834.

2. Guo et al. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 2010;466(7308):835-40.

3. Gangaraju VK & Lin H. MicroRNAs: key regulators of stem cells. Nat Rev Mol Cell Biol. 2009;10(2):116-25.

4. Slack FJ. Stem cells: Big roles for small RNAs. Nature. 2010;463(7281):616.

5. Georgiadis A et al. AAV-mediated knockdown of peripherin-2 in vivo using miRNA-based hairpins. Gene Ther. 2010;17(4):486-93.