Parallel derivation of isogenic human primed and naive induced pluripotent stem cells

Abstract

Induced pluripotent stem cells (iPSCs) have considerably impacted human developmental biology and regenerative medicine, notably because they circumvent the use of cells of embryonic origin and offer the potential to generate patient-specific pluripotent stem cells. However, conventional reprogramming protocols produce developmentally advanced, or primed, human iPSCs (hiPSCs), restricting their use to post-implantation human development modeling. Hence, there is a need for hiPSCs resembling preimplantation naive epiblast. Here, we develop a method to generate naive hiPSCs directly from somatic cells, using OKMS overexpression and specific culture conditions, further enabling parallel generation of their isogenic primed counterparts. We benchmark naive hiPSCs against human preimplantation epiblast and reveal remarkable concordance in their transcriptome, dependency on mitochondrial respiration and X-chromosome status. Collectively, our results are essential for the understanding of pluripotency regulation throughout preimplantation development and generate new opportunities for disease modeling and regenerative medicine.

Dosage Sensing, Threshold Responses, and Epigenetic Memory: A Systems Biology Perspective on Random X‐Chromosome Inactivation

“…Recent developments in human embryo culture and new culture conditions for hESCs, will hopefully soon allow us to observe this transition to the XaXi state and investigate the functional roles of XACT and potentially TSIX. [ 98–101 ]…”

Identifying Human Naïve Pluripotent Stem Cells − Evaluating State‐Specific Reporter Lines and Cell‐Surface Markers

“…In the second culture formulation, naïve hPSCs are stabilised and propagated in a cocktail of low‐dose 2i, LIF and a PKC inhibitor (t2iL + PKCi) (Table ) . This set of factors can maintain naïve hPSCs that have been generated by NANOG and KLF2– induced , KLF4– induced, or OCT4 , KLF4 , SOX2 and MYC ‐induced reprogramming, by transgene‐free chemical resetting of primed hPSCs and captured directly from preimplantation human blastocysts . Naïve hPSCs maintained in t2iL + PKCi exhibit many of the similarities to 5i/L/A naïve hPSCs including a globally hypomethylated genome (Table ), a close transcriptional alignment with preimplantation human and primate epiblast cells, and, in a proportion of cells, XIST ‐positive active X‐chromosomes (Table ) .…”

“…Many of the drawbacks are also shared with the 5i/L/A cells . Curiously, a subset of naïve hPSC lines grown in RSeT media, a commercial derivative of ‘naive human stem cell medium’, showed transcriptional similarly to t2iL + PKCi cells, whereas other lines in the same RSeT conditions aligned more closely to primed hPSCs …”

FGF Signaling Pathway: A Key Regulator of Stem Cell Pluripotency

“…These similarities are defined in many aspects including dependence on activin A and fibroblast growth factor 2 (FGF2) signaling pathway (Li and Belmonte, 2017), their transcriptional and epigenetic profiles, random X-chromosome inactivation, culture requirements and morphology and their differentiation capacities (Brons et al, 2007;Tesar et al, 2007). Recently, numerous attempts have been made to generate human naïve PSCs by the resetting of primed PSCs (Hanna et al, 2010;Gafni et al, 2013;Takashima et al, 2014), as well as somatic cellular reprograming (Liu et al, 2017;Kilens et al, 2018;Giulitti et al, 2019). It has also been reported that hESCs isolated from preimplantation ICM could be cultured in vitro to acquire a naïve pluripotent state (Guo et al, 2016).…”