A group of researchers has succeeded in directly reprogramming fibroblast cells into photoreceptors and transplanting them into mice, which resulted in partial restoration of vision [1]. This achievement can potentially lead to the development of cheap and effective treatments for retinopathies such as age-related macular degeneration (AMD), the leading cause of vision loss in older people.
Rod cells and cone cells are the two types of retinal photoreceptor cells, and their degeneration leads to the impairment or loss of vision. Currently, vision loss caused by photoreceptor degeneration is considered irreversible.
Methods to produce photoreceptor cells by reprogramming stem cells have previously been developed [2], but these methods are complicated and time-consuming, as they rely on induced pluripotent stem cells (iPSCs), which, themselves, have been produced from regular cells. This new method, however, entirely foregoes the stem cell stage and generates chemically induced photoreceptor-like cells (CiPCs) directly from non-pluripotent cells.
The authors chose fibroblasts as their source cells, as they are the most abundant cells in our connective tissue, charged with synthesizing the extracellular matrix and collagen. Three types of fibroblasts were used in the experiment: mouse embryonic fibroblasts (MEFs), human adult dermal fibroblasts (HADFs), and human fetal lung fibroblasts.
Chemically induced reprogramming
Chemically induced reprogramming of non-pluripotent cells has been around since 2006, when Dr. Shinya Yamanaka and his colleagues discovered that a cocktail of four chemicals, now collectively known as the Yamanaka factors and OSKM, could induce reprogramming into a pluripotent state by upregulating certain genes. After experimenting with different combinations of chemicals that were previously known to convert fibroblasts into neurons, the researchers discovered a formula that transformed them into CiPCs in only 10 days.
In order to confirm the success of the transformation, the MEFs were sourced from a transgenic Nrl-GFP mouse. Nrl is a transcription factor expressed in rod photoreceptors, while GFP stands for green fluorescent protein and is frequently used as a marker of expression [3]. Cells from these transgenic mice are programmed to produce GFP in the event of Nrl activation. Consequently, the presence of green fluorescence in the differentiated cells provided the necessary proof that the differentiation indeed went as planned. This result was backed by transcriptome analysis – a test that confirmed that the new cells expressed genes in a photoreceptor-like manner, while fibroblast-specific genes became downregulated.
The researchers then proceeded to investigate the mechanism of this chemically induced differentiation. One of their major findings was that this multi-step process was orchestrated by mitochondria. This was the first time mitochondria-to-nucleus signaling [4] was implicated in direct chemical reprogramming. Interestingly, one of the steps involved the increase in the production of mitochondrial reactive oxygen species (mROS). Until a few years ago, ROS were considered just a harmful byproduct of cellular metabolism (and one of longevity’s archenemies), but, since then, it has been shown to take part in numerous important cellular processes [5].
Transplantation successful in some cases
Fourteen transgenic mice of a different type, programmed to develop rapid retinal degeneration, were used as transplantation targets. Following the transplantation of CiPCs into the subretinal space, six of these mice demonstrated improved pupillary constriction (a marker of photoreceptor function) three to four weeks later. Predictably, none of the mice in the control group showed any improvement. To avoid the possibility that the improvement happened due to the recovery of the mice’s own photoreceptors, the researchers chose to transplant CiPCs relatively late: 31 days after the mice were born. At this point, due to the pace of the retinal degeneration, no native rod photoreceptors could exist.
The scientists then performed a light-avoidance test. Being nocturnal animals, mice naturally shun well-lit places and try to hide in the darkness. All CiPC mice who demonstrated pupillary constriction were also found to spend substantially more time in the dark than mice in the control group or CiPC mice that had not shown improvement in pupillary action.
To determine whether the transplanted photoreceptors continued to function and had connected to deeper neural cells, the researches followed up on the mice three months after the transplantation. They found that the mice that had exhibited improved visual function retained it. Moreover, CiPCs were found to develop synaptic terminals that apparently transmit light signals into the inner retinal neurons.
Conclusion
Retinopathies, often caused by the death of photoreceptors, inflict extreme suffering by inducing vision impairment and blindness. At least two of these diseases are age-related: AMD and diabetic retinopathy. Therefore, chemical reprogramming of non-pluripotent cells into transplantable rod photoreceptors represents an important milestone in treating some forms of age-related vision loss. The researchers contend that the low rate of conversion of fibroblasts into photoreceptors and the lack of proliferation of the transplanted cells may impede the translation of their discovery into clinical use, but they have high hopes for the future optimization of their technique.
Literature
[1] Mahato, B., Kaya, K. D., Fan, Y., Sumien, N., Shetty, R. A., Zhang, W., … & Mohanty, S. (2020). Pharmacologic fibroblast reprogramming into photoreceptors restores vision. Nature, 1-6.
[2] Gonzalez-Cordero, A., Kruczek, K., Naeem, A., Fernando, M., Kloc, M., Ribeiro, J., … & Sampson, R. D. (2017). Recapitulation of human retinal development from human pluripotent stem cells generates transplantable populations of cone photoreceptors. Stem cell reports, 9(3), 820-837.
[3] Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science, 263(5148), 802-805.
[4] Eisenberg‐Bord, M., & Schuldiner, M. (2017). Ground control to major TOM: mitochondria–nucleus communication. The FEBS journal, 284(2), 196-210.
[5] Shadel, G. S., & Horvath, T. L. (2015). Mitochondrial ROS signaling in organismal homeostasis. Cell, 163(3), 560-569.
