Direct reprogramming of human astrocytes into functional neurons in cerebral organoids derived from genome edited hiPSCs

Project Information

Repro_organoid

Grant agreement ID: 750624

DOI

10.3030/750624

Project closed

EC signature date 24 February 2017

Start date 1 March 2017

End date 18 March 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 171 460,80

EU contribution

€ 171 460,80

Coordinated by

UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG-UNIVERSITAET MAINZ
Germany

Periodic Reporting for period 1 - Repro_organoid (Direct reprogramming of human astrocytes into functional neurons in cerebral organoids derived from genome edited hiPSCs)

Reporting period: 2017-03-01 to 2019-02-28

Summary of the context and overall objectives of the project

The classical dogma of the central nervous system as an unchangeable entity has been refuted by the advent of direct lineage reprogramming of non-neuronal cells into neurons. During the last decade many studies including from the host laboratory have shown that brain-resident cells such as astroglia, NG2 glia, and brain pericytes, can be converted into functional induced neurons (iNs) in vitro. Transduction of glia with viruses encoding transcription factors that play key roles in neurogenesis during development were sufficient to direct astroglia towards a glutamatergic or GABAergic neuron fate. Recently, several groups, including the host lab, have succeeded in going a step further by demonstrating the feasibility of reprogramming in vivo. In fact, the host laboratory has published that Sox2 (alone or in combination with Ascl1) is able to convert NG2 glia into iNs in the injured adult mouse cerebral cortex. However, it remains unknown whether such lineage reprogramming in vivo can also be achieved in the case of human astrocytes which differ markedly from their rodent counterparts in size and complexity. To study lineage conversion of human astrocytes in vivo has been hindered by the lack of experimental systems that allow for the growth and maturation of human astrocytes within a human in vivo-like tissue context. In this project, I took advantage of the recent advent of human cerebral organoids technology. Several groups have in parallel developed the technology of cerebral organoids derived from human induced pluripotent stem cells (hiPSCs). These 3D systems enable the generation of miniature organ-like structures that mimic in many aspects human neurodevelopment. As occurs in normal brain development, in these cerebral organoids astrogliogenesis follows upon neurogenesis of deep and superficial layer neurons. In this proposal I defined the following objectives (Figure 1):

1- Generation of hiPSC lines allowing for inducible and cell-type specific expression of reprogramming factors by genome-editing

2- Characterization of astrogliogenesis in human cerebral organoids

3- Induction of human astroglia reprogramming in a human in vivo-like tissue context using cerebral organoids

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the period covered by this grant I have made considerable progress in my project. Once settled at Prof. Berninger’s laboratory, I immediately started learning how to grow and maintain human induced pluripotent stem cells (hIPSCs), which are the starting point to grow cerebral organoids. Afterwards, I started working in the two first objectives of my proposal. For the first objective I used CRISPR/Cas9 technology to generate stable genome-edited hiPSC lines for the inducible and cell type-specific expression of reprogramming factors and for tracking cell-fate changes. Expression of reprogramming factors is specifically targeted to human astroglia and their potential cell fate changes are tracked with fluorescence markers. Direct reprogramming can be induced at different stages of astrogliogenesis within cerebral organoids derived from these genome-edited hiPSCs.

In the meantime, I also started with objective 2 of my proposal. Once I had the hIPSCs stock, I could start growing cerebral organoids. Towards this I have carefully characterized by immunofluorescence analyses, at what stage astrocytes emerge in these organoids and when their proliferation peaks. Further analyses of the cells will determine their maturation stage at different time points of cerebral organoids.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This proposal aimed at addressing one of the major challenges of the reprogramming field, namely the establishment of more physiological models to study human astrocyte direct lineage reprogramming in a human in vivo-like tissue context. This work will allow us to evaluate the influence of the different maturation stages and the ability of reprogrammed neurons to be recruited into functional neuronal circuits. A number of laboratories worldwide are focusing on direct reprogramming of human glial cells, however, due to the lack of appropriate systems, this process remains unexplored so far in human astrocytes in vivo.

Results obtained within this project has allowed me to perform a deep analysis of astrogliogenesis in this human in vivo-like tissue culture. Of special importance are results related to the maturation of astrocytes and proliferation within these cerebral organoids. This analysis is particularly relevant for future studies performed in human astrocytes integrated in cerebral organoids in which the maturation stage of the astrocytes is involved. CRISPR/Cas9 strategy has allowed the generation of a hIPSC line of special interest to study different processes related or involved in direct lineage reprogramming of human astrocytes into induced neurons, including mechanisms of reprogramming or signaling pathways involved in this process. Future experiments within this project will unveil if human astrocytes, even when mature, can undergo direct reprogramming and, whether maturation stage of these astrocytes influences this process and if so, how. This study will provide new important insights into the feasibility of human astrocyte-to-neuron conversion in an in vivo-like tissue context and the dependence of this process on the astrocyte maturation stage. Furthermore, results from this work will impact our current knowledge of human cell reprogramming and will establish new bases in the design of future strategies targeted towards neuron replacement in neurodegenerative disorders. This approach might lead to a breakthrough in the development of new therapies that might contribute to the cure of neurodegenerative diseases.

Figure 1