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.
