A fast 2C-induction method reveals a barrier role of SP2 for totipotency

mRNA translational dynamics mediates totipotent-like reprogramming

It is demonstrated that coordinated regulation of both cytosolic and mitochondrial translation during totipotent-like reprogramming is offered, offering a new perspective for understanding cell fate determination.

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.

Rapid and efficient generation of human 8-cell-like cells for embryo modelling

Pcgf5 controls the exit from totipotency in mouse embryonic stem cells.

Mouse embryonic stem cell (ESC) cultures contain a rare subpopulation of two-cell-like cells (2CLCs) that transiently reactivate a two-cell embryo-like transcriptional program characteristic of zygotic genome activation (ZGA), including the endogenous retrovirus MERVL and Zscan4, and thereby regain a totipotent-like state. Polycomb repressive complex 1 (PRC1)-mediated H2AK119ub1 has been implicated in restraining entry into the 2C-like state through Pcgf6, yet the factors governing exit from this state and loss of totipotency remain poorly defined. Here, we show that among the six Pcgf paralogs, Pcgf5, which is most prominently upregulated in 2CLCs and forms an MERVL-driven chimeric transcript (Pcgf5MT2C_Mm) during ZGA in 2-cell embryos, controls exit from the 2C-like state in mouse ESCs. Using a reporter ESC line carrying MERVL-tdTomato and Zscan4c-EGFP (MtZG), we manipulated Pcgf5 dosage bidirectionally. Doxycycline (Dox)-inducible overexpression (OE) of Pcgf5 reduced the double-positive (DP) 2C-like population. Conversely, CRISPR-mediated knockout (KO) of Pcgf5 by targeting a common exon shared by all Pcgf5 variants (hereafter, total Pcgf5) increased the DP population. Time-lapse imaging directly confirmed that these changes reflected genuine differences in duration of the 2C-like state: OE shortened, whereas KO prolonged, the time cells spent in this state. These findings reveal that a Polycomb group factor controls not only entry into but also exit from the 2C-like state.

The Centriole as a Structural Ratchet That Restricts Cellular Reprogramming to Totipotency

Somatic cell nuclear transfer succeeds (Wilmut et al., 1997) but depends on oocyte cytoplasm that has undergone natural centriole elimination. No method using transcription factors, small molecules, or culture conditions alone has produced sustained totipotency from a fully differentiated somatic cell—though transient totipotent-like states can be induced from pluripotent stem cells, and stable totipotent-like stem cells (TLSCs) have been derived from mouse embryonic stem cells through chemical chromatin remodeling (Yang et al., 2022). Here we propose the centriole functions as a structural stabilizer of the differentiated state. Through conservative replication, asymmetric inheritance, and active regulatory roles, the mother centriole physically maintains the somatic gene regulatory network. Oocytes eliminate centrioles before totipotency in every metazoan examined; the germline resets the hardware at each generation, but the soma cannot. We outline a three-phase protocol—Eliminate (PLK4 PROTAC-mediated centriole removal), Reprogram (Tet-On DUX4 plus TPRX1), and Rebuild (de novo centriole assembly)—and predict that centriole elimination combined with totipotency factors will yield stable, self-renewing totipotent cells, distinct from transient 8C-like cells. We specify six quantitative falsification criteria, compare four alternative models, and propose a two-phase experimental design with a composite totipotency index as the primary endpoint.

Eliminate, Reprogram, and Rebuild

Background. Transient totipotent-like states (8CLCs, 2CLCs) can now be induced from pluripotent stem cells through transcription factor expression (DUX4) or chemical chromatin remodeling (TLSCs) - without centriole manipulation. Stable, self-renewing totipotent cells have been achieved from ESCs. Yet no method has produced sustained totipotency from a fully differentiated somatic cell. Why? Hypothesis. We propose that the centriole - through active regulatory mechanisms (DID-RNA, CAMC remodeling, NANOG sequestration, cilium-dependent signaling) - actively maintains the differentiated state and thus constitutes a somatic barrier to sustained totipotency. It is not the only barrier: TLSCs prove that the chromatin barrier can be overcome chemically. But somatic cells carry the burden of having traversed the differentiation ratchet - old centrioles accumulated through asymmetric inheritance - that ESCs do not. Entropy is not the barrier. Entropy accumulates passively in all structures (Second Law of Thermodynamics) and DEGRADES the centriole’s active regulatory function over time. When that function fails, the cell does not revert to totipotency - it becomes malignant. The barrier to totipotency is the centriole’s active maintenance of the differentiated state; entropy is what breaks the barrier, not what creates it. Irreversible differentiation means the irreversible shutdown of some gene regulatory networks and the activation of others. In naive cells (ESCs, iPSCs), gene networks are open - all programs remain accessible. TLSCs succeed without centriole manipulation because they start from this open state. Somatic cells have closed networks: genes required for totipotency are silenced, often at the chromatin level, but also - we propose - physically, through the centriolar ratchet. This additional hardware burden, a consequence of differentiation history rather than chronological age, may explain why somatic reprogramming arrests at the 8CLC stage - cells touch totipotency but cannot hold it. The centriole as a differentiation ratchet. The centriole is a material structure that ages passively with time in all cells - dividing and post-mitotic alike. Multicellular animals accumulate old centrioles in stem cells through asymmetric inheritance rather than eliminating them. This accumulation is the physical basis of irreversible differentiation: the ratchet permits forward movement along differentiation trajectories but forbids spontaneous reversal. Aging is the price of true differentiation. Plants, which lack centrioles in somatic cells, employ modulation (reversible differentiation). Prediction. Centriole elimination combined with totipotency factors (DUX4 + TPRX1) will convert non-totipotent 8CLCs into stable, self-renewing totipotent cells - defined as >50% MERVL+ after 10 passages, with competence for trophectoderm differentiation. The centriole is not a lock on totipotency per se - it is a lock on the STABILITY of totipotency in differentiated cells that have passed through the ratchet. Evidence. We present a meta-analysis of four convergent evidence streams: (1) centriole elimination during oogenesis across five model organisms, (2) the molecular distinction between pluripotency and totipotency programs, (3) the 2CLC/8CLC/TLSC literature establishing that totipotency can be accessed - but not sustained - from somatic cells, and (4) the centriole’s role as a conditional entropy accumulator in the MCARA framework. Experimental Design. Three-phase protocol: Phase 1 - Eliminate (PLK4 PROTAC/RP-1664-mediated centriole removal), Phase 2 - Reprogram (Tet-On DUX4 + TPRX1), Phase 3 - Rebuild (re-expression of de novo centriole biogenesis factors: PLK4, SAS-6, STIL, CPAP). Two species: Phase 1-2 in human fibroblasts (~$137K), Phase 2-3 with tetraploid complementation in mouse cells (~$126K). Critical controls: OSKM + p53/p38 inhibitors without elimination; elimination + neural factors (lock vs sensor discrimination); elimination + TLSC protocol. Significance. If confirmed, this would demonstrate that the centriole is a somatic barrier to sustained totipotency - not the only barrier, but one that must be addressed when starting from aged, differentiated cells. The implications span regenerative medicine, aging reversal, and the understanding of why somatic cells cannot spontaneously dedifferentiate.