Systematic characterization of methods for inducing human naïve pluripotency [Registered Report Stage 1 Protocol]

ABSTRACT Since their derivation, human pluripotent stem cells (hPSCs) have been maintained in a “primed” pluripotent state in vitro, corresponding to embryonic cells of the post-implantation blastocyst. To mimic cells of the pre-implantation blastocyst, the field has devised protocols to revert primed hPSCs to an earlier embryonic, “naïve”, state that can be used to derive in vitro blastocyst-like structures, “blastoids”, providing an interesting model of human pre-implantation embryos. Despite their utility in understanding fundamental cell biology and early embryology, in vitro naïve hPSCs are prone to karyotypic abnormalities and the efficacy of their derivation varies across protocols and cell lines. To promote their reproducibility and reliability in the field, we here apply two widely adopted methods for naïve induction on several hPSC lines varying initial culture conditions while systematically assessing the quality of resulted cells by transcriptional profiling, karyotyping, and blastoid formation ability.</p

High resolution C1s XPS spectra.

(A) Surfaces analyzed after the protein G polypeptide graftin step. (B) Surfaces after the antibody immobilization step using anti-CD144 antibodies.</p

Fluorescence-based detection of immobilized IgG antibodies on conjugated protein G polypeptide spots.

(A) Schematic representation of antibody immobilization on protein G spots with (S-SMPB(+)) or without (S-SMPB(-)) covalent grafting of protein G polypeptide. (B) Spotted anti-CD31 or anti-CD144 antibodies detected through fluorophore-labelled anti-mouse antibodies as described in the last step of Fig 1. The protein G polypeptide concentration applied was 5.5 μM. (C) Successful immobilization of different primary antibodies on conjugated protein G polypeptide (S-SMPB (+)) based on the detection of fluorophore-labeled secondary antibodies added after protein G polypeptide and primary antibody immobilization. Surfaces without S-SMPB and/or without primary antibodies were used as negative controls. *P S3 Appendix.</p

Spreadsheet comprising the raw ELISA readouts used to generate Fig 3.

(XLSX)</p

Schematic representation of the antibody immobilization process followed by a fluorescence-based antibody detection step.

Schematic representation of the antibody immobilization process followed by a fluorescence-based antibody detection step.</p

ECFC capture from laminar flow conditions on functionalized surfaces.

Conditions tested include anti-C144 (present on ECFCs) on adsorbed or conjugated protein G polypeptide, anti-CD14 (not present on ECFCs) on conjugated protein G polypeptide (negative control), or collagen. (A) Fluorescence images of ECFC nuclei on modified surfaces after 1 h of exposure to cell suspension under flow conditions. (B) Quantification of number of cells per mm2 on the modified surfaces at the end of the 1 h of flow. Each symbol represents data collected using ECFCs from a separate donor. *P < 0.05 with N = 4.</p

Surface atomic composition assessed by XPS survey analyses^b'*'.

Surface atomic composition assessed by XPS survey analysesb'*'.</p