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1. EZH2 is a key component of hepatoblastoma tumor cell growth

   https://doi.org/10.1002/pbc.30774  

   Glaser, Kathryn ; Schepers, Emily J. ; Zwolshen, Harrison M. ; Lake, Charissa M. ; Timchenko, Nikolai A. ; Karns, Rebekah A. ; Cairo, Stefano ; Geller, James I. ; Tiao, Gregory M. ; Bondoc, Alexander J. ( November 2023 , Pediatric Blood & Cancer)

   Enhancer of zeste homolog 2 (EZH2) catalyzes the trimethylation of histone H3 at lysine 27 via the polycomb recessive complex 2 (PRC2) and plays a time‐specific role in normal fetal liver development. EZH2 is overexpressed in hepatoblastoma (HB), an embryonal tumor. EZH2 can also promote tumorigenesis via a noncanonical, PRC2‐independent mechanism via proto‐oncogenic, direct protein interaction, including β‐catenin. We hypothesize that the pathological activation of EZH2 contributes to HB propagation in a PRC2‐independent manner.

   We demonstrate that EZH2 promotes proliferation in HB tumor‐derived cell lines through interaction with β‐catenin. Although aberrant EZH2 expression occurs, we determine that both canonical and noncanonical EZH2 signaling occurs based on specific gene‐expression patterns and interaction with SUZ12, a PRC2 component, and β‐catenin. Silencing and inhibition of EZH2 reduce primary HB cell proliferation.

   EZH2 overexpression promotes HB cell proliferation, with both canonical and noncanonical function detected. However, because EZH2 directly interacts with β‐catenin in human tumors and EZH2 overexpression is not equal to SUZ12, it seems that a noncanonical mechanism is contributing to HB pathogenesis. Further mechanistic studies are necessary to elucidate potential pathogenic downstream mechanisms and translational potential of EZH2 inhibitors for the treatment of HB.

    

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2. Thermal inkjet bioprinting drastically alters cell phenotype

   https://doi.org/10.1088/1758-5090/acd3b3  

   Ablanedo Morales, Patricia ; Rodriguez, Brittany ; Furth, Michael E. ; Molina, Kayla B. ; Boland, Andrew J. ; Mohl, Jonaton E. ; Boland, Thomas ( May 2023 , Biofabrication)

   Since the first description of inkjet bioprinting of cells in 2003, quantifying the input and measuring the output of the printers has been the hallmark of the field of bioprinting, as it is virtually impossible to characterize cells that are inside the printing orifices or extrusion needles. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting. Primary and immortalized adult dermal fibroblasts were expanded for 2–3 passages upon receiving. The cells were harvested, resuspended in PBS, and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20µl drops were pipetted for hanging drop culture. IPC differentiation protocols were applied and the induction was begun approximately 45 min after printing. When differentiating aggregates, the initiation happened 45 min after the aggregates were transferred into the 96 wells. Standard immunostaining and RNA sequencing (RNA-Seq) were used to analyze the cell phenotypes. Preliminary results indicate that all cells expressed the three pluripotency markers oct-4, nanog, and sox-2. After applying a cardiomyocyte differentiation protocol, the cells stained positively for troponin-3. The cells also elongated and became more cardiomyocyte-like in their morphology. We analyzed bulk RNA seq data and our preliminary results show upregulation of some genes that have been implicated as stem cell markers: EPCAM, LEFTY1, ZFP42, and TEX19. In addition, differential expression of genes associated with pluripotency-relevant pathways shows some pathways are off like the MAPK/p38, MAPK/JNK1-3 which is expected for a pluripotent state. We also have data supporting the activation of the hippo pathway with transcriptional co-activator with PDZ binding motif (TAZ) highly upregulated and yes-associated protein staining the cell body. In addition, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. Bioprinted fibroblasts will at least temporarily adopt a more primitive or dedifferentiated state, reminiscent of pluripotency. While immunochemistry shows the classic transcription factors required for pluripotency, gene expression shows a more nuanced picture of the transformations that occur upon printing. Understanding these transformations, even if temporary will be crucial when trying to build tissues using bioprinting technologies.

    

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3. Selective Inhibition of mTORC1 Signaling Supports the Development and Maintenance of Pluripotency

   https://doi.org/10.1093/stmcls/sxad079  

   Kim, Jin Koo ; Villa-Diaz, Luis G. ; Saunders, Thomas L. ; Saul, Ruiz P. ; Timilsina, Suraj ; Liu, Fei ; Mishina, Yuji ; Krebsbach, Paul H. ( November 2023 , Stem Cells)

   Insight into the molecular mechanisms governing the development and maintenance of pluripotency is important for understanding early development and the use of stem cells in regenerative medicine. We demonstrate the selective inhibition of mTORC1 signaling is important for developing the inner cell mass (ICM) and the self-renewal of human embryonic stem cells. S6K suppressed the expression and function of pluripotency-related transcription factors (PTFs) OCT4, SOX2, and KLF4 through phosphorylation and ubiquitin proteasome-mediated protein degradation, indicating that S6K inhibition is required for pluripotency. PTFs inhibited mTOR signaling. The phosphorylation of S6 was decreased in PTF-positive cells of the ICM in embryos. Activation of mTORC1 signaling blocked ICM formation and the selective inhibition of S6K by rapamycin increased the ICM size in mouse blastocysts. Thus, selective inhibition of mTORC1 signaling supports the development and maintenance of pluripotency.

    

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4. Functional analyses of the polycomb‐group genes in sea lamprey embryos undergoing programmed DNA loss

   https://doi.org/10.1002/jez.b.23225  

   Saraceno, Cody ; Timoshevskiy, Vladimir A. ; Smith, Jeramiah J. ( October 2023 , Journal of Experimental Zoology Part B: Molecular and Developmental Evolution)

   During early development, sea lamprey embryos undergo programmatic elimination of DNA from somatic progenitor cells in a process termed programmed genome rearrangement (PGR). Eliminated DNA eventually becomes condensed into micronuclei, which are then physically degraded and permanently lost from the cell. Previous studies indicated that many of the genes eliminated during PGR have mammalian homologs that are bound by polycomb repressive complex (PRC) in embryonic stem cells. To test whether PRC components play a role in the faithful elimination of germline‐specific sequences, we used a combination of CRISPR/Cas9 and lightsheet microscopy to investigate the impact of gene knockouts on early development and the progression through stages of DNA elimination. Analysis of knockout embryos for the core PRC2 subunits EZH, SUZ12, and EED show that disruption of all three genes results in an increase in micronucleus number, altered distribution of micronuclei within embryos, and an increase in micronucleus volume in mutant embryos. While the upstream events of DNA elimination are not strongly impacted by loss of PRC2 components, this study suggests that PRC2 plays a role in the later stages of elimination related to micronucleus condensation and degradation. These findings also suggest that other genes/epigenetic pathways may work in parallel during DNA elimination to mediate chromatin structure, accessibility, and the ultimate loss of germline‐specific DNA.

    

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5. Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR

   https://doi.org/10.1038/s41467-022-34363-w  

   Sempou, Emily ; Kostiuk, Valentyna ; Zhu, Jie ; Cecilia Guerra, M. ; Tyan, Leonid ; Hwang, Woong ; Camacho-Aguilar, Elena ; Caplan, Michael J. ; Zenisek, David ; Warmflash, Aryeh ; et al ( November 2022 , Nature Communications)

   Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (V_m) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identifyKCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the V_mand thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion ofkcnh6leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating V_musing a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells.

    

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