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Reprogramming of elite B cells to induced hematopoietic stem cell with microRNAs and transcription factors

Periodic Reporting for period 1 - miRStem (Reprogramming of elite B cells to induced hematopoietic stem cell with microRNAs and transcription factors)

Reporting period: 2017-09-01 to 2019-08-31

In medicine, transplantation of HSCs is widely used to treat blood cell disorders and cancer patients treated with high dose chemotherapy. However, HSCs are highly limited both by the number of cells that can be obtained for transplantations and the identification of immunologically matched donors. A promising strategy to overcome these limitations would be to generate HSCs by reprogramming patient derived cells in vitro. The proposed “miRStem” project aimed to generate transplantable HSCs from B cells transiently exposed to C/EBPA at high efficiencies, by combining microRNA and TF based reprogramming strategies.
The overall objectives of the project were:
- to identify microRNA candidates for HSC induction
- to construct a new traceable vector allowing the inducible expression of specific transcription factors and microRNAs
- to test the efficiency of microRNAs in combination with transcription factors to reprogram somatic B cells into induced hematopoietic stem cells.
This work aimed to provide a new method for the direct transdifferentiation into HSCs and novel insights into HSC biology.
We used public dataset and performed bioinformatic analysis to identify microRNA candidates for HSC induction. We identified a set of microRNA specific to HSC. Among them, a cluster of three microRNAs (miR-125a, miR99b and miR-let-7e) is evolutionary conserved and we decided to test the ability of this cluster to induce HSC reprogramming.
We designed and built a new retroviral vector allowing the inducible expression of 3 TF in combination with up to 5 microRNAs. In addition this vector carry a fluorescent protein (2 version of the vector were constructed with GFP and RFP) allowing to track cells after transduction. We therefore constructed a retroviral vector expressing under the control of a Tet-inducible promoter a polycistronic RNA of Lmo2, Gata2 and Scl and expressing GFP (or RFP) under the control of an independent promoter. We then used this vector to clone in the 3’UTR of the polycistronic RNA the miR-125 or a cluster of 3 miRNAs (miR-125a, miR99b and miR-let-7e). The functionality of this vector was tested in mouse embryonic fibroblast and we validated the inducible expression of the 3 TFs and the microRNAs by qPCR and western-Blot.
At the time of the beginning of the project, a transgenic mouse strain was generated by the laboratory of Georges Lacaud in Manchester. This mice has a cassette of expression for Scl and Gata2 under the control of a Tet-inducible expression. It allows the generation of hematopoietic progenitors from mouse embryonic fibroblast. We decided to use these MEF derived from this mice as a positive control for induction of HSC in vitro. We tested the ability of miR-125 and miR-125 cluster to induce, in combination with Lmo2, Gata2 and Scl, HSC reprogramming of B cells after a short pulse of C/EBPA. We found that unfortunately the induction of HSC from B cells was very low using our cocktail of TFs and microRNAs and that the short exposure to C/EBPA did not significantly increased the reprogramming efficiency. In contrast, the control protocol of HSC induction from MEF converted as expected the MEF into hematopoietic progenitors. We also tested the ability of our TF and miRNA cocktail to reprogram B cells in vivo but again the reprogramming efficiency was very low or null.
Once we obtained our main result, we concluded that our protocol unfortunately did not increase the efficiency of HSC reprogramming. At the same time in the laboratory, we had exciting finding about the role of genome topology in the gene regulation and the cell fate decision. We decided to then focused on deciphering the role of genome architecture during different process of cell fate conversion. I set up in the lab a new technique called umi-4C allowing to detect and quantify chromatin interactions with a region of interest. This technique has been used in a study that has been published in 2019 (Tian et al., NCB, 2019) in which I am an co-author and the MSCIF is acknowledged. A method paper is also in preparation and will be submit for publication in the next months. I also leaded a study describing the role of genome topology and the specific architectural protein CTCF during cell fate conversion. I am actually writing a manuscript that will be submitted for publication before the end of the 2019 and the MSCIF will be acknowledged again.
I have attended to the Keystone meeting «Chromatin Architecture and Chromosome Organization (X5)», from the 23th to the 27th October 2018 in Whistler in Canada and the EMBO Workshop “The genome in three dimension” in Kyllini, Greece, from the 20th to the 24th of May 2019 where I presented posters of my work.
The research proposed here provide new insight in the field of the HSC engineering. Even if we showed that our microRNA cocktail was not enough to induce HSC reprogramming our analysis could be used in order to screen for the role of other microRNA in different contexts.
Moreover, we developed a new retroviral vector allowing the tet-inducible expression of 3 TF with up to 5 microRNAs. This vector also contain a fluorescent protein allowing to track infected cells either in vivo or in vitro. We also generated chromosome conformation capture data during exit of pluripotency of stem cells that are already available in the Gene Expression Omnibus under accession number GSE126618. Finally, also generated chromosome conformation capture data and other genomic data during cell fate conversion of human B cell into macrophage that will be published by the end of the year.