Stem Cell Reports最新文献

Heterogeneity among both primed and naive pluripotent stem cell lines remains a major unresolved problem. Here we show that expressing the maternal-specific linker histone H1FOO fused to a destabilizing domain (H1FOO-DD), together with OCT4, SOX2, KLF4, and LMYC, in human somatic cells improves the quality of reprogramming to both primed and naive pluripotency. H1FOO-DD expression was associated with altered chromatin accessibility around pluripotency genes and with suppression of the innate immune response. Notably, H1FOO-DD generates naive induced pluripotent stem cells with lower variation in transcriptome and methylome among clones and a more uniform and superior differentiation potency. Furthermore, we elucidated that upregulation of FKBP1A, driven by these five factors, plays a key role in H1FOO-DD-mediated reprogramming.

Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.

In Japan, the Act on Safety of Regenerative Medicine regulates unapproved regenerative medicine. Other nations market regenerative medicine products, bypassing regulatory approval. To identify unapproved orthopedic regenerative medicine, we have used data based on the Act. Platelet-rich plasma was often used. The common target was the knee. Prices averaged $2,490.

Here, we used single-cell RNA sequencing (scRNA-seq), single-cell ATAC sequencing (scATAC-seq), and single-cell spatial transcriptomics to characterize murine cortical OPCs throughout postnatal life. During development, we identified two groups of differentially localized PDGFRα⁺ OPCs that are transcriptionally and epigenetically distinct. One group (active, or actOPCs) is metabolically active and enriched in white matter. The second (homeostatic, or hOPCs) is less active, enriched in gray matter, and predicted to derive from actOPCs. In adulthood, these two groups are transcriptionally but not epigenetically distinct, and relative to developing OPCs are less active metabolically and have less open chromatin. When adult oligodendrogenesis is enhanced during experimentally induced remyelination, adult OPCs do not reacquire a developmental open chromatin state, and the oligodendrogenesis trajectory is distinct from that seen neonatally. These data suggest that there are two OPC groups subserving distinct postnatal functions and that neonatal and adult OPC-mediated oligodendrogenesis are fundamentally different.

Embryo size, specification, and homeostasis are regulated by a complex gene regulatory and signaling network. Here we used gene expression signatures of Wnt-activated mouse embryonic stem cell (mESC) clones to reverse engineer an mESC regulatory network. We identify NKX1-2 as a novel master regulator of preimplantation embryo development. We find that Nkx1-2 inhibition reduces nascent RNA synthesis, downregulates genes controlling ribosome biogenesis, RNA translation, and transport, and induces severe alteration of nucleolus structure, resulting in the exclusion of RNA polymerase I from nucleoli. In turn, NKX1-2 loss of function leads to chromosome missegregation in the 2- to 4-cell embryo stages, severe decrease in blastomere numbers, alterations of tight junctions (TJs), and impairment of microlumen coarsening. Overall, these changes impair the blastocoel expansion-collapse cycle and embryo cavitation, leading to altered lineage specification and developmental arrest.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Maintenance of mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC), but the underlying mechanisms remain ill defined. In this study, we monitored mitophagy in MuSCS under various myogenic states and examined the role of PINK1 in maintaining regenerative capacity. Results indicate that quiescent MuSCs actively express mitophagy genes and exhibit a measurable mitophagy flux and prominent mitochondrial localization to autophagolysosomes, which become rapidly decreased during activation. Genetic disruption of Pink1 in mice reduces PARKIN recruitment to mitochondria and mitophagy in quiescent MuSCs, which is accompanied by premature activation/commitment at the expense of self-renewal and progressive loss of muscle regeneration, but unhindered proliferation and differentiation capacity. Results also show that impaired fate decisions in PINK1-deficient MuSCs can be restored by scavenging excess mitochondrial ROS. These data shed light on the regulation of mitophagy in MuSCs and position PINK1 as an important regulator of their mitochondrial properties and fate decisions.

Embryonic stem cells (ESCs) are defined as stem cells with self-renewing and differentiation capabilities. These unique properties are tightly regulated and controlled by complex genetic and molecular mechanisms, whose understanding is essential for both basic and translational research. A large number of studies have mostly focused on understanding the molecular mechanisms governing pluripotency and differentiation of ESCs, while the regulation of proliferation has received comparably less attention. Here, we investigate the role of ZZZ3 (zinc finger ZZ-type containing 3) in human ESCs homeostasis. We found that knockdown of ZZZ3 negatively impacts ribosome biogenesis, translation, and mTOR signaling, leading to a significant reduction in cell proliferation. This process occurs without affecting pluripotency, suggesting that ZZZ3-depleted ESCs enter a "dormant-like" state and that proliferation and pluripotency can be uncoupled also in human ESCs.