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The Molecular Mechanisms of Cell Fate Reprogramming in Vertebrate Eggs

Periodic Reporting for period 1 - ReproMech (The Molecular Mechanisms of Cell Fate Reprogramming in Vertebrate Eggs)

Reporting period: 2019-08-01 to 2021-07-31

During embryonic development, cells become increasingly committed to a certain cell fate. They rarely, if ever, change to another type. For example, as skin cell do not naturally change to, or give rise to a brain cell. However, this stable commitment of a cell can be reverted by certain experimental procedures, as for example when the nucleus of a specialised cell is transplanted to an enucleated egg. During this reprogramming, the gene expression pattern of the differentiated cell can be changed to that of an embryonic cell. This process is of interest because identifying how reprogramming takes place can help us to understand how cells maintain their identity. This is important because many pathological conditions arise from loss of cell identity. Second, the embryonic stem cells that can be obtained by reprogramming a specialised cell of one kind, e.g. a skin cell, can then be made to produce healthy specialized cells of an other kind, e.g. brain cells. This has potential application in cell-replacement therapy, where these cells can compensate the loss of irreversibly damaged or defective cells. It might be possible to derive replacement heart, neuronal or pancreatic cells from another cell type of the same individual, thereby avoiding the need for immunosuppression treatments.
Despite its enormous potential, the usefulness of this approach is limited by its low efficiency, as only a small number of cells can be fully reprogrammed. The reason why adult body cells like skin do not respond to nuclear reprogramming is likely related to an inherent resistance of a specialized kind of cell to change to a totipotent kind.
The goal of our project was to elucidate the factors responsible for inherent resistance of specialized cells, as well as the molecules present in vertebrate eggs that can in some cases overcome these barriers to reprogramming. Once this is understood on a molecular level, we aimed at lowering the resistance factors and increasing the reprogramming molecules, so that reprogramming can become more efficient. We used nuclear transplantation of specialised cell nucleus to Xenopus eggs as a model system to understand this process.Outcomes of this study will in the future help to identify treatments that can improve the generation of high quality embryonic cells useful for cell replacement. Furthermore, our results will help to gain a better insight into the mechanisms important for cellular memory and the stability of cell differentiation during normal development and disease.
The project consisted of three stages:

Stage 1: We successfully investigated how postulated resistance factors change around genes of differentiated nuclei upon reprogramming treatment. We compared changes on sperm nuclei, which are specialised nuclei whose reprogramming always succeeds in order to produce offspring upon fertilisation, to gut cells, which are specialised cells that normally are not efficiently reprogrammed and instead maintain some of their specialised cell characteristics. We discovered that in gut cells, postulated resistance factors are maintained around genes important for their specialised function as gut cells. Surprisingly, also sperm cells contain genes that maintain these resistance factors around genes important for embryonic development. We proposed that these resistance factors are important to memorise which genes are important for the specialised function of the cell, and that the same factors ensure that this knowledge is passed on to the next generation of cells, even when challenged with the reprogramming activities of the egg.

Stage 2: We addressed molecules that are recruited to differentiated nuclei upon reprogramming in vertebrate eggs to identify candidate factors that could either maintain the resistant state of nuclei (reprogramming inhibitors) or that remove the resistance factors (reprogramming facilitators) associated with cellular memory genes.

Stage 3: We continued with validating the identified candidate facilitators and inhibitors of reprogramming in somatic cell nuclear transplantation to vertebrate eggs: On the one hand, we were lowering the concentration of reprogramming inhibitors to decrease the resistance of specialised cells to reprogramming. On the other hand, we were increasing the concentration of reprogramming facilitators during nuclear transfer to eggs in order to aid efficient transition to totipotency.

We presented the results of our work at international scientific meetings, both in person and online. We communicated our results to the general public during an artist in residence program at our institute and by using our home page and social networking platforms such as twitter.
We expect that the direct beneficiaries of our work will be academic and clinical scientists working in the field of regenerative medicine. In the longer term, the wider public will benefit from our work, as our scientific advances could improve medical treatments and hence enhance quality of life.
As humans age, the function of their cells and tissues very commonly begins to deteriorate, and an ideal way of alleviating the discomfort and malfunction of ageing cells and tissues is to be able to provide replacement cells of the same genetic constitution as the recipient, thereby eliminating the need for immunosuppression if cells from other individuals are used. The provision of whole organs or complex tissues from accessible cells, such as skin, would be a formidable task, but the derivation of individual cell-types seems realistic. It is now possible to derive functional photoreceptors, neurons, cardiac cells, etc. from adult skin, and these can be transplanted to recipient hosts. There is still much to be discovered about the integration of such replacement cells into a recipient’s tissue and their continued function after transfer to an individual, but it is likely that improvements in this area will be forthcoming. A current disadvantage of the suggested production of specialized cells of one kind from another unrelated cell-type is the low efficiency of the process by transcription factor overexpression and hence the need for extensive multiplication of the derived cells, during which time genetic and other changes in these cells may arise. Therefore, an improved efficiency of person-specific replacement cell derivation would be highly beneficial.
We point out that the short-term outcome of our proposed project is to elucidate how vertebrate eggs reprogram differentiated nuclei at high efficiencies and within hours. We successfully identified changes of proposed reprogramming barriers during the conversion of a specialised cell to a totipotent kind after nuclear transplantation to vertebrate eggs. We revealed candidate factors that prevent and facilitate these changes, which were validated until the end of this project. In the long run, we anticipate that our results will contribute to scientific developments that (1) improve the efficiency of deriving person-specific stem cells from accessible adult tissues and (2) improve the quality of the reprogrammed cells by ensuring a complete switch in cell identity. Lastly, results from this project will be of great help for scientists in the broad area of research dealing with the stability of the differentiated state and its dysregulation (embryonic development, in vitro differentiation, reprogramming, ageing, and cancer).
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