Breakthrough! Peking University Du Peng Team Realized Stable Culture of Totipotent Stem Cells in Vitro

Since the establishment of the first embryonic stem cell (ESC), the culture of totipotent cells in vitro is equivalent in function and molecule to blastomeres in vivo with embryonic and extra-embryonic development potential.

On May 14, 2021, the Du Peng team of Peking University was in Cell   Published online entitled " Mouse totipotential stem cells captured and maintained through spliceosomal expression "The research paper, which Report mouse ESCs spliceosome inhibition drives the transition from pluripotent to totipotent. Using splice inhibitor Pladienolide B, this study can achieve stable in vitro culture on totipotent ESC at the molecular level compared with 2-cell and 4-cell blastomeres (called totipotent oocyte-like cells (TBLC)).

The combination of mouse chimeric detection and single cell RNA sequencing (scRNA-seq) shows that TBLC has strong bidirectional development ability and can produce a variety of embryonic and extracellular cell lineages . Mechanically, spliceosome repression leads to extensive splicing inhibition of pluripotent genes, while totipotent genes with almost no short introns are effectively spliced and transcribed and activated. This study provides a method to capture and maintain totipotent stem cells.

In addition, on May 5, 2021, Du Peng of Peking University and Richard I. Gregory of Harvard Medical School jointly communicated in Nature   Published online entitled " Global miRNA dosage control of embryonic germ layer specification This study identified the miRNA dose control mechanism of developmental regulation, involving variable transcription initiation (ATI) of DGCR8. ATI occurs downstream of the stem loop of DGCR8 mRNA and bypasses the self-regulating feedback loop during mouse embryonic stem (mES) cell differentiation. Removing the stem ring results in DGCR8: The stoichiometric imbalance of DROSHA protein leads to irreversible microprocessor aggregation, reduced primary miRNA processing, reduced mature miRNA abundance, and extensive inhibition of lipid metabolism mRNA targets. Although global miRNA dose control is not essential for mES cells to withdraw from pluripotency, its imbalance will change lipid metabolism pathway and interfere with embryo development by destroying germ layer specifications in vitro and in vivo. This miRNA dose control mechanism is conserved in humans. The results of this study identify a promoter switch that can balance the automatic adjustment and aggregation of microprocessors to accurately control the global miRNA dose and control the fate of stem cells during early embryonic development (click to read).

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Stem cell research is widely related to regenerative medicine, assisted reproduction, cancer, metabolic disorder and aging. Embryonic stem cells (ESCs) with high differentiation potential in vitro are basic and important. Totipotent cells have the highest development potential, which usually refer to mammalian fertilized eggs and 2-cell and 4-cell blastomeres. During early embryonic development, totipotent blastomeres specialize into inner cell mass (ICM) and trophoectoderm (TE), while ICM produces primitive endoderm (PrE) and epidermal cells (EPI) of blastocysts. After embryo implantation, EPI produces embryonic tissue containing three germ layers, while PrE produces visceral endoderm (VE) and parietal endoderm (PE), and finally develops into yolk sac tissue. TE produces extrauterine tissues, such as the extrauterine placental cone (EPC) and the extrauterine ectoderm (ExE), and ultimately forms the placenta.

ESC cultured in vitro is usually derived from ICM and can differentiate into any cell type of adult organisms, called pluripotent cells, which are characterized by the expression of transcription factors, such as POU5F1 (OCT4), ZFP42, NANOG and SOX2. Recently, it has been reported that a small proportion (0.1%-1%) of cultured ESC activate the expression of MERVL and MT2 transposons and other totipotent genes (such as ZSCAN4s). Two studies have reported a new type of expanded pluripotent stem cells (EPSC), which can realize embryonic and extrembryo differentiation and can be established and maintained in media with different chemical mixtures. However, a recent study questioned and doubted the totipotency of EPSC at molecular and functional levels. Up to now, it is impossible to capture and maintain totipotent stem cells which are similar in molecule and function to totipotent blastomeres in vivo in vitro.

Splicosome is a macromolecular ribonucleoprotein (RNP) complex with five core subunits and several cofactors, and it is a dynamic molecular machine for splicing and maturation of messenger RNA (mRNA). Recent studies have shown that spliceosome can also directly control the origin, extension and termination of transcription, highlighting their non-splicing independent function. Interestingly, in many different cancers, it has been determined that mutations of several key splicing factors play a vital role in tumorigenesis. However, the possible physiological correlation between spliceosome in the fate transformation of stem cells and early embryo development is still unclear.  

This study reports that mouse ESCs spliceosome inhibition drives the transition from pluripotent to totipotent. Using splice inhibitor Pladienolide B, this study can achieve stable in vitro culture on totipotent ESC compared with 2-cell and 4-cell blastomeres (called totipotent oocyte-like cells (TBLC)) at the molecular level.

The combination of mouse chimeric detection and single cell RNA sequencing (scRNA-seq) shows that TBLC has strong bidirectional development ability and can produce a variety of embryonic and extraembryonic cell lineages. Mechanically, spliceosome repression leads to extensive splicing inhibition of pluripotent genes, while totipotent genes with almost no short introns are effectively spliced and transcribed and activated. This study provides a method to capture and maintain totipotent stem cells.

Reference message:

Https://www.cell.com/cell/fulltext/S0092-8674 (21) 00500-6

Source: iNature

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