In human embryos, naive pluripotent cells of the inner cell mass (ICM) generate epiblast, primitive endoderm and trophectoderm (TE) lineages, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and efficiently generate trophoblast stem cells (TSCs), while conventional PSCs form TSCs at low efficiency. Transient histone deacetylase and MEK inhibition combined with LIF stimulation is used to chemically reset conventional to naive PSCs. Here, we report that chemical resetting induces the expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allows for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a plastic intermediate state, characterised by co‐expression of naive and TSC markers, after which cells steer towards one of the two fates in response to the signalling environment. The efficiency and rapidity of our system will be useful to study cell fate transitions and to generate models of placental disorders.

Human induced pluripotent stem cells (iPSCs) are generated from somatic cells by the expression of a cocktail of transcription factors, and iPSCs have the capacity to generate in vitro all cell types of the human body. In addition to primed (conventional) iPSCs, several groups recently reported the generation of human naı ¨ve iPSCs, which are in a more primitive developmental state and have a broader developmental potential, as shown by their ability to form cells of the placenta. Human iPSCs have broad medical potential but their generation is often time-consuming, not scalable and requires viral vectors or stable genetic manipulations. To overcome such limitations, we developed protocols for high-efficiency generation of either conventional or naı ¨ve iPSCs by delivery of messenger RNAs (mRNAs) using a microfluidic system. In this protocol we describe how to produce microfluidic devices, and how to reprogram human somatic cells into naı ¨ve and primed iPSCs using these devices. We also describe how to transfer the iPSC colonies from the microfluidic devices over to standard multiwell plates for subsequent expansion of the cultures. Our approach does not require stable genetic modifications, is reproducible and cost-effective, allowing to produce patient-specific iPSCs for cell therapy, disease modeling, and in vitro developmental studies.

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In human embryos, naive pluripotent cells of the inner cell mass generate epiblast, primitive endoderm and Trophectoderm (TE) lineage, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and can generate trophoblast stem cells (TSCs), while conventional PSCs form amnion-like cells and lack the competence to generate TSCs. Transient histone deacetylase and MEK inhibitions with LIF stimulation can be used to chemically reset conventional to naive PSCs. Here we report that chemical resetting induced expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allowed for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a responsive intermediate state, in which conventional PSCs rapidly acquire competence to form TSCs without the need of stabilisation and expansion in a naive state. The efficiency and rapidity of our system will be useful for the study of cell fate transitions, and to generate models of placental disorders.