The present study identified the miRNA expressed in human H7/H9 ESC, as well as their changes when they are differentiated into RGC using well established techniques. We looked at both abundance and log2 fold changes to understand the miRNA profile of these cells.
It has become apparent that miRNA play a significant role in both the healthy functioning of a cell (including RGC) as well as mediating the degenerative processes of an unhealthy one11. There is also evidence that miRNA can be exploited as powerful therapeutic strategies for degenerating RGC. Rather than having acute and substantial effects on cell biology, evidence suggests that miRNA are instead a buffering system, modulating gene expression to protect against large changes often caused by environmental and stressful challenges8,9. By understanding the miRNA profile of a cell type, a greater understanding of the role the miRNA play can be explored. We have previously profiled the miRNA of rodent RGC12, and thus build on this literature with a profile of human RGC, generated from ESC.
Our ESC remained relatively stable in culture, with only a small number of miRNA changing, and only by a small amount, with three showing statistical significance. All three of these, miR-106a22,23, miR-92a24, and miR-42425 were downregulated over time and have been linked to cell proliferation and migration and likely reflect the changing culture conditions as more cells occupy the flask. The miR-302 family of miRNA were the most abundant miRNA found in the ESC, and their role as a specific marker of ESC has already been established26, yet served as useful positive control of both the stemness of our cells, and its reduction, a marker of successful differentiation.
After differentiation, expectedly, there were substantial changes in the miRNA profile of the cells. As mentioned previously, the miR-302 family were all reduced substantially to undetectable levels, given their specificity for ESC. Other ESC specific miRNA include miR-372 and miR-367, and these were also reduced to undetectable levels27.
miR-204 expression is dramatically increased, which has been implicated as the master regulator of retinal development. Over one third of the ~800 genes that miR-204 targets are expressed in the retina, and its targets have been implicated in a variety of diseases including the RGC pathology, glaucoma28,29. miR-125b was also found to be differentially abundant and has been implicated in non-neuronal retinal cells such as retinal pigment epithelium (RPE) differentiated from human ESC30. Interestingly, their study suggested that miR-125b and let-7 drove differentiation down a non-neuronal retinal lineage. Let-7a was another that was over-abundant in our ESC-RGC, and therefore our study demonstrates that these two (miR-125b and let-7a) are not RPE specific, but are instead also found in RGC. miR-125b is further upregulated in retinal cell death31. Finally, miR-125b has been implicated as a biomarker for glaucoma due to its differential expression in the aqueous humor of glaucoma patients compared to controls32.
The increase in expression of miR-181a corroborates literature on this miRNA being strongly expressed in the retina. Based on hybridization studies, the highest expression was in amacrine cells, but was also detectable in the ganglion cell layer33,34. Equally, in fish, miR-181a is required for the growth of amacrine processes and RGC axons35.
miR-125a was also overexpressed, but the literature has few details of its role in the eye, with some suggestion it is involved in RPE loss in cell lines after exposure to exosomes from dry AMD patients36 as well as retinoblastoma in mice37. miR-216b was overexpressed, and has been seen in the developing retina, driving differentiation of retinal neurons (and away from the retinal glia lineage) through a reduction in notch and Foxn3 signalling38,39. Finally, it’s been suggested to play a protective role in preventing diabetic retinopathy40, similar to miR-15b, another overexpressed miRNA41.
Recently, we published a miRNA sequencing analysis of purified RGC from rodents12. While many studies have sequenced the total retinal cell population, this is to our knowledge the only study that purified RGC. As such, comparisons with this study provide a unique assessment of the congruence in miRNA expression between rodent and human (ESC-derived) RGC. Of the 13 most abundant miRNA identified in our human ESC-RGC, 7 of these were also the most abundant miRNA identified in rat purified RGC, whereas 6 were only found in the human ESC-RGC (Table. 1). It is however unknown whether those miRNA unique to the human RGC are a consequence of species differences, or the cell culture environment in which the ESC-derived RGC were generated. Interestingly, miR-191 was flagged as a miRNA unique to (rat) RGC, not identified in other miRNA assays of other retinal cell populations12, and is also found to be abundant in the present study.
Following differentiation and purification, it is unclear if the cells are stable or still undergoing changes. Comparing miRNA expression between 6 and 48 h in culture identifies several changes. While most of these changes are in low abundant miRNA, miR-135, -23a, and -107 all increased dramatically, whereas let-7b, -7d, -7i, -7 g, and -7 l (but not let-7a) all decreased dramatically. Let-7 is involved in neural progenitor development42, as well as late differentiation of both retinal neurons and glia43. It is thus tempting to speculate that the downregulation of let-7 is a consequence of maturation of the RGC. This is further corroborated by miR-135 being detectable in whole huma retina44,45. There are many subtypes of RGC within both animal and human retina, subtypes which so far have been relatively explored within rodent and human retinal organoids46,47,48. It would be of great use to further explore the subtype profile of these RGC, to determine if they belong to a distinct class of RGC or make up several expected subtypes.
In conclusion, the present study identifies the miRNA profile of both ESC and ESC-derived RGC, characterizing the differences. ESC-derived RGCs appear to express a variety of miRNA identified to be important in retinal development and function, while downregulating ESC-specific miRNA. Several of these miRNA have also been identified in purified rat RGC, however, six miRNA appeared to be unique to the human RGC. Further understanding of the biology of human RGC can be achieved by taking into consideration their unique miRNA signature.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- Source: https://www.nature.com/articles/s41598-024-83381-9