A Proposed Experimental Protocol for Testing Centriole-Mediated Somatic Totipotency Induction
Background. The centriole is proposed to function as a physical ratchet that locks somatic cells in a differentiated state: it duplicates conservatively, is never fully disassembled, and accumulates damage over time. Transient totipotent-like states (8C-like and 2C-like cells) revert within one to two divisions, and no method has produced sustained totipotency from a fully differentiated somatic cell. We hypothesise that centriole elimination, transient expression of totipotency factors, and de novo centriole assembly — “Eliminate, Reprogram, Rebuild” — recapitulate the germline cycle and may enable stable totipotency. The central experiment (centriole elimination combined with DUX4/TPRX1 expression in fibroblasts) has not been reported. Methods. Phase 0 establishes centriole proteome baselines (mass spectrometry, U-ExM, TRIM37 expression, karyotype). Centriole elimination is attempted with five independent methods (Plk4 siRNA, the PLK4 inhibitor RP-1664, CRISPR/Cas9 PLK4 knockout, PCM disruption, autophagy induction) across 17 experimental groups (n = 15 biological replicates per elimination arm; n = 10 for control arms). p53-dependent G1 arrest is bypassed with 53BP1/USP28 siRNA, TRIM37 siRNA, or inducible dominant-negative p53, with SKY/FISH karyotype surveillance at every passage. DUX4, TPRX1 and ZSCAN4 are delivered by doxycycline-inducible lentivirus; de novo centriole assembly restores PLK4/STIL/SAS-6/CPAP. Primary endpoints are the full zygotic genome activation panel (MERVL/HERVL, ZSCAN4, TPRX1, LEUTX, DPPA3, MLT2A1) maintained over ≥ 10 passages; secondary endpoints include trophectoderm differentiation, embryoid bodies, teratoma, chimera and tetraploid complementation assays, supported by RNA-seq, ATAC-seq, EPIC arrays, Hi-C and single-cell RNA-seq. Predicted outcomes. If the centriole is a true somatic barrier, elimination plus totipotency factors will produce a stable 8CLC-like state distinct from transient 8CLCs. Pre-registered contingency plans define falsification criteria for all four possible outcomes. Conclusions. The protocol provides a falsifiable test of the centriole-ratchet hypothesis and a route to sustained somatic totipotency, with implications for regenerative medicine.