Smarca5-mediated epigenetic programming facilitates fetal HSPC development in vertebrates.

Nascent hematopoietic stem and progenitor cells (HSPCs) acquire definitive hematopoietic characteristics only when they develop into fetal HSPCs; however, the mechanisms underlying fetal HSPC development are poorly understood. Here, we profiled the chromatin accessibility and transcriptional features of zebrafish nascent and fetal HSPCs using ATAC-seq and RNA-seq and revealed dynamic changes during HSPC transition. Functional assays demonstrated that chromatin remodeler-mediated epigenetic programming facilitates fetal HSPC development in vertebrates. Systematical screening of chromatin remodeler-related genes identified that smarca5 is responsible for the maintenance of chromatin accessibility at promoters of hematopoiesis-related genes in fetal HSPCs. Mechanistically, Smarca5 interacts with nucleolin to promote chromatin remodeling, thereby facilitating genomic binding of transcription factors to regulate expression of hematopoietic regulators such as bcl11ab. Our results unravel a new role of epigenetic regulation and reveal that Smarca5-mediated epigenetic programming is responsible for fetal HSPC development, which will provide new insights into the generation of functional HSPCs both in vivo and in vitro.

Generation of Elf5-Cre knockin mouse strain for trophoblast-specific gene manipulation.

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5-Cre mice, we mated Elf5-Cre mice with Rosa26mT/mG reporter mice, and found that Elf5-Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5-Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5-Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.

Genistein-Chitosan Derivative Nanoparticles for Targeting and Enhancing the Anti-Breast Cancer Effect of Tamoxifen In Vitro.

Tamoxifen (TAM) is a classical anti-estrogenic drug that antagonizes estrogen by competitively binding to estrogen receptor α (ERα). However, drug resistance to TAM remains a significant challenge in breast cancer treatment. In this study, we aimed to design an actively targeted drug delivery system to enhance the proliferation inhibitory effects of TAM on ER positive breast cancer cells. Herein, chitosan (CS) was modified with genistein (GEN) to obtain the actively targeted GEN-CS. The TAM-loaded nanoparticles (TAM-GEN-CS-NPs) were constructed using an ionic-crosslinking method, with GEN-CS as the carrier material and sodium tripolyphosphate (TPP) as the crosslinking agent. As a result, TAM-GEN-CS-NPs exhibited a spherical morphology with an average size of 299.8 nm. The encapsulation efficiency and drug loading content were 85.77% and 14.13 µg/mg, respectively. Compared with free TAM, TAM-GEN-CS-NPs displayed obvious slow-release performance. In vitro cellular assays demonstrated that TAM-GEN-CS-NPs had active targeting and proliferation inhibitory effects on MCF-7 cells. The IC50 of TAM and TAM-GEN-CS-NPs were 10.25 µg/mL and 7.22 µg/mL, respectively. More importantly, the combination index (CI) value of TAM and GEN was less than 1, indicating synergistic effects. Therefore, TAM-GEN-CS-NPs hold the potential to enhance TAM therapy for breast cancer through active targeting and synergistic treatment strategies.

Long-term expansion of human hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are critical for the treatment of a variety of hematological diseases. However, the low number of HSCs lead to the clinical application difficult. To gain more functional human HSCs ex vivo, Sakurai et al. established a recombinant-cytokine-free and albumin-free culture system, i.e. PCL-PVAc-PEG-based culture, in combination with 740Y-P, butyzamide and UM171, to improve the long-term expansion of human cord blood HSCs.

Advances in Image-Based Artificial Intelligence in Otorhinolaryngology-Head and Neck Surgery: A Systematic Review.

OBJECTIVE: To update the literature and provide a systematic review of image-based artificial intelligence (AI) applications in otolaryngology, highlight its advances, and propose future challenges. DATA SOURCES: Web of Science, Embase, PubMed, and Cochrane Library. REVIEW METHODS: Studies written in English, published between January 2020 and December 2022. Two independent authors screened the search results, extracted data, and assessed studies. RESULTS: Overall, 686 studies were identified. After screening titles and abstracts, 325 full-text studies were assessed for eligibility, and 78 studies were included in this systematic review. The studies originated from 16 countries. Among these countries, the top 3 were China (n = 29), Korea (n = 8), the United States, and Japan (n = 7 each). The most common area was otology (n = 35), followed by rhinology (n = 20), pharyngology (n = 18), and head and neck surgery (n = 5). Most applications of AI in otology, rhinology, pharyngology, and head and neck surgery mainly included chronic otitis media (n = 9), nasal polyps (n = 4), laryngeal cancer (n = 12), and head and neck squamous cell carcinoma (n = 3), respectively. The overall performance of AI in accuracy, the area under the curve, sensitivity, and specificity were 88.39 ± 9.78%, 91.91 ± 6.70%, 86.93 ± 11.59%, and 88.62 ± 14.03%, respectively. CONCLUSION: This state-of-the-art review aimed to highlight the increasing applications of image-based AI in otorhinolaryngology head and neck surgery. The following steps will entail multicentre collaboration to ensure data reliability, ongoing optimization of AI algorithms, and integration into real-world clinical practice. Future studies should consider 3-dimensional (3D)-based AI, such as 3D surgical AI.

p16High senescence restricts cellular plasticity during somatic cell reprogramming.

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.

Response to Matters Arising: Characterization of placental fetal macrophages.

Chen et al.1 published a report that casts doubt on our main finding from a recent article.2 Although we acknowledge the importance of their observations, we are reserved about whether their observations would invalidate our conclusions that placental fetal macrophages are generated de novo via placental hemogenic endothelium. This Matters Arising response paper addresses the Chen et al.1 Matters Arising paper published concurrently in Developmental Cell.

Identification of HSC/MPP expansion units in fetal liver by single-cell spatiotemporal transcriptomics.

Limited knowledge of cellular and molecular mechanisms underlying hematopoietic stem cell and multipotent progenitor (HSC/MPP) expansion within their native niche has impeded the application of stem cell-based therapies for hematological malignancies. Here, we constructed a spatiotemporal transcriptome map of mouse fetal liver (FL) as a platform for hypothesis generation and subsequent experimental validation of novel regulatory mechanisms. Single-cell transcriptomics revealed three transcriptionally heterogeneous HSC/MPP subsets, among which a CD93-enriched subset exhibited enhanced stem cell properties. Moreover, by employing integrative analysis of single-cell and spatial transcriptomics, we identified novel HSC/MPP 'pocket-like' units (HSC PLUS), composed of niche cells (hepatoblasts, stromal cells, endothelial cells, and macrophages) and enriched with growth factors. Unexpectedly, macrophages showed an 11-fold enrichment in the HSC PLUS. Functionally, macrophage-HSC/MPP co-culture assay and candidate molecule testing, respectively, validated the supportive role of macrophages and growth factors (MDK, PTN, and IGFBP5) in HSC/MPP expansion. Finally, cross-species analysis and functional validation showed conserved cell-cell interactions and expansion mechanisms but divergent transcriptome signatures between mouse and human FL HSCs/MPPs. Taken together, these results provide an essential resource for understanding HSC/MPP development in FL, and novel insight into functional HSC/MPP expansion ex vivo.

Protocols for isolation and characterization of mouse placental hemogenic endothelial cells.

The murine mid-gestational placenta has been identified as a hematopoietic site during embryonic development. Here, we describe a protocol for isolation and characterization of the hemogenic endothelial (HE) cells from mouse placenta. We also describe techniques for dissection of placental tissues and for the optimization of tissue digestion and antibody conjugation conditions to identify HE cells via fluorescence-activated cell sorting. For details on the usage and application of this protocol, please refer to Liang et al. (2021).

De novo generation of macrophage from placenta-derived hemogenic endothelium.

Macrophages play pivotal roles in immunity, hematopoiesis, and tissue homeostasis. In mammals, macrophages have been shown to originate from yolk-sac-derived erythro-myeloid progenitors and aorta-gonad-mesonephros (AGM)-derived hematopoietic stem cells. However, whether macrophages can arise from other embryonic sites remains unclear. Here, using single-cell RNA sequencing, we profile the transcriptional landscape of mouse fetal placental hematopoiesis. We uncover and experimentally validate that a CD44+ subpopulation of placental endothelial cells (ECs) exhibits hemogenic potential. Importantly, lineage tracing using the newly generated Hoxa13 reporter line shows that Hoxa13-labeled ECs can produce placental macrophages, named Hofbauer cell (HBC)-like cells. Furthermore, we identify two subtypes of HBC-like cells, and cell-cell interaction analysis identifies their potential roles in angiogenesis and antigen presentation, separately. Our study provides a comprehensive understanding of placental hematopoiesis and highlights the placenta as a source of macrophages, which has important implications for both basic and translational research.

Generation of foxn1/Casper Mutant Zebrafish for Allograft and Xenograft of Normal and Malignant Cells.

Cell transplantation into immunodeficient recipients is a widely used approach to study stem cell and cancer biology; however, studying cell states post transplantation in vivo is inconvenient in mammals. Here, we generated a foxn1/Casper mutant zebrafish that is transparent and exhibits T cell deficiency. By employing the line for hematopoietic stem cell (HSC) transplantation (HSCT), we could achieve nonconditioned transplantation. Meanwhile, we found that fetal HSCs from 3 days post fertilization zebrafish embryos produce a better transplant outcome in foxn1/Casper mutants, compared with adult HSCs. In addition to HSCT, the foxn1/Casper mutant is feasible for allografts of myelodysplastic syndrome-like and muscle cells, as well as xenografts of medaka muscle cells. In summary, foxn1/Casper mutants permit the nonconditioned engraftment of multiple cell types and visualized characterization of transplanted cells in vivo.

pH-responsive thiolated chitosan nanoparticles for oral low-molecular weight heparin delivery: in vitro and in vivo evaluation.

The aim of present study was to investigate a pH-responsive and mucoadhesive nanoparticle system for oral bioavailability enhancement of low-molecular weight heparin (LMWH). The thioglycolic acid (TGA) was first covalently attached to chitosan (CS) with 396.97 ± 54.54 µmol thiol groups per gram of polymer and then the nanoparticles were prepared with thiolated chitosan (TCS) and pH-sensitive polymer hydroxypropyl methylcellulose phthalate (HPMCP) by ionic cross-linking method. The obtained nanoparticles were characterized for the shape, particle size, zeta potential, drug entrapment efficiency and loading capacity. In vitro results revealed the acid stability of pH-responsive nanoparticles, which had a significant control over LMWH release and could effectively protect entrapped drugs in simulated gastric conditions. By the attachment of the thiol ligand, an improvement of permeation-enhancing effect on freshly excised carp intestine (1.86-fold improvement) could be found. The mucoadhesive properties were evaluated using fluorescently labeled TCS or CS nanoparticles. As compared with the controls, a significant improvement of mucoadhesion on rat intestinal mucosa was observed in TCS/HPMCP nanoparticles via confocal laser scanning microscopy. The activated partial thromboplastin time (APTT) was significantly prolonged and an increase in the oral bioavailability of LMWH was turned out to be pronounced after oral delivered LMWH-loaded TCS/HPMCP nanoparticles in rats, which suggested enhanced anticoagulant effects and improved absorption of LMWH. In conclusion, pH-responsive TCS/HPMCP nanoparticles hold promise for oral delivery of LMWH.