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Modelling in vivo lineage reprogramming of human astrocytes into induced neurons in the adult mouse brain

Periodic Reporting for period 1 - ReproXimera (Modelling in vivo lineage reprogramming of human astrocytes into induced neurons in the adult mouse brain)

Reporting period: 2020-02-01 to 2022-01-31

The emergence of reprogramming technologies has revolutionized the classical view of cellular identity as an unchangeable entity. Direct lineage reprogramming enables the fate conversion of one terminally differentiated cell into another demonstrating that changes associated with cellular differentiation are not irreversible. This approach has opened an unprecedented perspective for lost neurons’ replacement following injury and neurodegenerative diseases.

In the last decade, successful conversion of murine glial cells into induced neurons has been achieved by transduction with viruses encoding transcription factors that play key role in neurogenesis. More recently, the achievement of in vivo reprogramming of diverse brain cells has opened an unprecedented perspective. However, in vivo reprogramming studies have been restricted to murine glia. Human astroglia reprogramming represents a challenge in the field since it remains unknown whether such lineage reprogramming can also be achieved in vivo in the case of human glia which differs markedly from their rodent counterparts in size and complexity.

In this project I have tackled few fundamental questions in the field: First, I have investigated whether human astrocytes can be reprogrammed into induced neurons. Second, I have studied how reprogramming competence of human astrocytes is affected by their maturation stage. Although preliminary, the results obtained in this project show the feasibility of mature human astrocytes reprogramming in vitro.
During the period of this action, I have been working to tackle the question if human astrocytes can be reprogrammed into induced neurons. Towards this aim, I have carried out differentiation of human-induced pluripotent stem cells (hiPSCs) into astrocytes. Following a protocol based in the overexpression of gliogenic factors, hiPSC are efficiently reprogrammed into astrocytes. I have characterized the differentiated cells for their expression profile of astrocytic markers. The resulting astrocytes convincingly express the characteristic markers of mature astrocytes. Additionally, I have analysed if the generated astrocytes develop functional properties using Ca2+ imaging experiments. The experiments showed that astrocytes exhibit spontaneous calcium transients. Interestingly, astrocytes are connected between them propagating the calcium signals generating calcium waves. All together, these results are showing that we can generate mature and functional human astrocytes from hiPSCs.

In parallel, I have generated a genome-edited hiPSCs line using CRISPR-Cas9 technology. The modification allows the expression of an inducible Cre recombinase and a red fluorescent protein as a reporter in astrocytes. The subsequent analysis of the genome-edited line showed that the cassette has been inserted in the correct location in the locus and it is transcribed. Analysis of astrocytes derived from this line, showed that the inserted genes are translated into functional proteins, so the cells can be visualized due to the presence of the reporter protein. Similarly, the inducible form of Cre recombinase expressed in the astrocytes, turns active and functional upon tamoxifen administration, activating Cre dependent reporters. I have differentiated human astrocytes derived from the genome-edited line and, taking advantage of the inducible system, I am testing if these astrocytes are able to reprogram. Using Cre inducible vectors encoding for different reprogramming factors known to be capable to reprogram murine astrocytes into induced neurons, I am characterizing in vitro the reprogramming of hiPSCs-derived human astrocytes into induced neurons.
In this project, we have focused on human astroglia because of its translational importance of understanding reprogramming competence of human cells for regenerative medicine. The results obtained from this work will help to gain insight in the direct lineage conversion process in human cells. This remarkable knowledge is critical to understand the biology of the process itself for future application and design of strategies for repair of the central nervous system.

During the reported period we have achieved important deliverables. We have extended our expertise in human astrocyte differentiation from hiPSCs. This is a valuable tool which makes possible the study of human cell biology and function. Furthermore, we have generated a genome-edited hiPSCs line which allows for the inducible and controllable expression of any desire gene specifically in human astrocytes. This is a new tool, not available till now, that can be useful for the scientific community working in a broad range of different fields related with human astrocytes. Our experiments using this line to induce human astrocyte reprogramming indicate that the system is working properly. Additionally. preliminary data have shown cells with neuronal morphology and expressing neuronal markers can be found after few weeks under reprogramming conditions. In summary, the results obtained in this period of the action, suggest that human astrocytes reprogramming is feasible. More research in this direction needs to be done, but our findings pave the way for further exploration of human cells reprogramming.
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