Complex haploinsufficiency in pluripotent cells yields somatic cells with DNA methylation abnormalities and pluripotency induction defects.

A complete knockout of a single key pluripotency gene may drastically affect embryonic stem cell function and epigenetic reprogramming. In contrast, elimination of only one allele of a single pluripotency gene is mostly considered harmless to the cell. To understand whether complex haploinsufficiency exists in pluripotent cells, we simultaneously eliminated a single allele in different combinations of two pluripotency genes (i.e., Nanog+/-;Sall4+/-, Nanog+/-;Utf1+/-, Nanog+/-;Esrrb+/- and Sox2+/-;Sall4+/-). Although these double heterozygous mutant lines similarly contribute to chimeras, fibroblasts derived from these systems show a significant decrease in their ability to induce pluripotency. Tracing the stochastic expression of Sall4 and Nanog at early phases of reprogramming could not explain the seen delay or blockage. Further exploration identifies abnormal methylation around pluripotent and developmental genes in the double heterozygous mutant fibroblasts, which could be rescued by hypomethylating agent or high OSKM levels. This study emphasizes the importance of maintaining two intact alleles for pluripotency induction.

Extracellular vesicles over adeno-associated viruses: Advantages and limitations as drug delivery platforms in precision medicine.

Tissue-specific uptake and sufficient biodistribution are central goals in drug development. Crossing the blood-brain barrier (BBB) represents a major challenge in delivering therapeutics to the central nervous system (CNS). Since its discovery in the late 19th century, considerable efforts have been invested in an attempt to decipher the BBB structure complexity and plasticity. In parallel, another prevalent approach is to improve a delivery system by harnessing the biological machinery in an attempt to enhance therapeutic-agent permeability. Here, we review the advantages and limitations of using extracellular vesicles over AAV systems as a delivery system for therapy, focusing on neurodevelopmental disorders.

Extensive Nuclear Reprogramming Underlies Lineage Conversion into Functional Trophoblast Stem-like Cells.

Induced pluripotent stem cells (iPSCs) undergo extensive nuclear reprogramming and are generally indistinguishable from embryonic stem cells (ESCs) in their functional capacity and transcriptome and DNA methylation profiles. However, direct conversion of cells from one lineage to another often yields incompletely reprogrammed, functionally compromised cells, raising the question of whether pluripotency is required to achieve a high degree of nuclear reprogramming. Here, we show that transient expression of Gata3, Eomes, and Tfap2c in mouse fibroblasts induces stable, transgene-independent trophoblast stem-like cells (iTSCs). iTSCs possess transcriptional profiles highly similar to blastocyst-derived TSCs, with comparable methylation and H3K27ac patterns and genome-wide H2A.X deposition. iTSCs generate trophoectodermal lineages upon differentiation, form hemorrhagic lesions, and contribute to developing placentas in chimera assays, indicating a high degree of nuclear reprogramming, with no evidence of passage through a transient pluripotent state. Together, these data demonstrate that extensive nuclear reprogramming can be achieved independently of pluripotency.