The naive inner cell mass of the human embryo is characterised by unlimited developmental plasticity as it gives rise to all embryonic and extraembryonic lineages. This state can be recapitulated in vitro using naive human pluripotent stem cells (hPSCs), a new prominent tool for basic and translational research with the potential for significant impact on health and economics. This state differs from the well-known and widely used conventional pluripotent state (primed hPSCs), which resembles a later stage of development primed toward the embryonic lineages. In vitro models are allowing significant progress in understanding the mechanistic roles of transcriptional factors and signalling pathways in pluripotency. However, we still do not know how naive hPSCs’ striking plasticity is regulated, also due to technical limitations in the manipulation of this recently discovered cell type.
Understanding naive hPSCs requirements has significant implications for biological and medical research, improving:
i) the awareness of similarities/differences with animal models;
ii) the implementation of optimised and standardised protocols for the generation and manipulation of this cell type and its derivatives, with consequent increase in a) stability of the cultures, b) efficiency in the generation of the desired cell types and c) comparison and integration of inter-laboratories/research institutions/companies’ results.
The JAK/STAT3 pathway has been demonstrated to play a fundamental role in maintaining murine naive pluripotency by i) expressional regulation and inhibition of early differentiation markers, and ii) metabolic reconfiguration. JAK/STAT3 activator LIF is routinely used in naive hPSCs culture and derivation media, but the molecular impact of JAK/STAT3 in human pluripotency was still not known. To understand if the pathway plays a role in naive pluripotency maintenance and/or acquisition, I unravelled the contribution of the JAK/STAT3 pathway to the transcriptional program of naive hPSCs in maintenance and during their derivation from primed hPSCs via chemical resetting, a non-genetic alterations-based protocol to produce naive hPSCs from the vast plethora of already available primed hPSC lines. Moreover, I am generating the first computational model of hPSCs metabolism to identify metabolic changes associated with the two states of human pluripotency, for further dissection of human developmental mechanisms and comparison with mouse data. This bioinformatic approach has been chosen and used as a deep metabolic investigation in naive hPSCs is at the moment prevented by technical limitations linked to the standard culture system of naive hPSCs, which relies on a feeder layer of cells on which naive hPSCs grow. This setting impacts different research and applied aspects in the use of naive hPSCs, such as confounding experimental results or non-implementable analyses, marked culture variability, high resource consumption, and limited scalability. Therefore, this project also aimed to establish and validate a new feeder-free culture system, and to propose it to the scientific community as a new standard for the cultivation of naive hPSCs to overcome all listed limitations.
