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Investigating the extracellular microenvironment as a potential target to monitor and control large-scale haematopoietic differentiation

A Nair(1) M P Henry(1) C J O'Grady(1) V M Karels(1) S Menon(1) W Haerty(2) J Hasan(1)

1:Cell and Gene Therapy Catapult; 2:Earlham Institute, Norwich Research Park

Allogeneic stem cell-derived therapies are being investigated for several indications. In-vitro haematopoietic differentiation of pluripotent stem cells (PSCs) holds great promise for the development of novel therapies, however in many cases requires large cell numbers per dose, necessitating scalable manufacturing approaches like automated bioreactor systems over planar flask technologies. Despite the drive towards automation, cell therapies are inherently more prone to variability, with increased risk of batch failure and costs if processes are not sufficiently controlled. In-process analytics can help maintain, control, and reduce risks of failure; however, it is difficult to apply analytics that are used for monitoring expansion and differentiation in planar flasks to bioreactor systems.

To address these challenges, we explored the extracellular microenvironment during haematopoietic differentiation, analysing exosomal miRNAs. Several screening methods have been applied to the miRNA isolated from spent media to perform multi-parametric analysis for monitoring future large-scale differentiation processes.

Our initial proof of concept work has revealed potentially novel miRNA markers in the haematopoietic differentiation process for spent media via Illumina sequencing and NanoString Technologies. These markers have shown consistent differential expression across all the platforms assessed. In collaboration with Earlham Institute, we have successfully characterised a process for generating hematopoietic progenitor cells from iPSCs using miRNA sequencing and therefore, identified the extracellular microenvironment for monitoring large-scale haematopoietic differentiation.

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