Introduction:Central nervous system (CNS) infiltration is a severe complication of acute myeloid leukemia (AML), often leading to relapse and poor prognosis. The underlying mechanisms remain poorly understood, limiting the development of effective targeted therapies. Conventional chemotherapy agents capable of crossing the blood-brain barrier (BBB) carry significant toxicity and fail to eliminate leukemia stem cells. Methods:Using multiple clinical cohorts, we established prognostic prediction model for AML. Using knockdown and overexpression experiments in a mouse leukemia CNS infiltration model, the role of risk factor gene in CNS infiltration was evaluated, along with the attenuating effect of its downstream proteins drug inhibition on leukemia CNS infiltration. Results:We identify Lactoferrin (LTF) as a risk factor associated with poor AML prognosis using a machine learning-based prognostic model. Through LTF knockdown and overexpression in a mouse leukemia model, we demonstrate its pivotal role in CNS invasion. Mechanistically, neutrophil serine proteases (NSPs) act as downstream effectors of LTF, and pharmacological inhibition with Brensocatib effectively blocks AML cell entry into the brain. Discussion:Our findings establish LTF as a mediator of CNS infiltration in AML and highlight NSP inhibition as a promising therapeutic strategy.

Multidrug resistance (MDR) remains a major obstacle in the clinical treatment of leukemia, severely limiting therapeutic efficacy and leading to poor patient outcomes. Drug efflux mediated by ATP-binding cassette (ABC) transporters represents a central mechanism driving cancer MDR. In this study, we identified ABCB6 as a critical mediator of MDR in leukemia through its role in promoting drug efflux. Using membrane-focused liquid chromatography‑tandem mass spectrometry (LC‑MS/MS) analysis of the leukemia cell line K562 and its MDR derivative K562/A02, we screened for differentially expressed transporters and identified ABCB6 as a candidate molecule. We further confirmed that ABCB6 is abundantly expressed on the plasma membrane of K562/A02 cells. Functional analyses demonstrated that altered ABCB6 expression did not significantly affect cell proliferation or apoptosis; however, targeted knockdown of ABCB6 markedly restored the sensitivity of K562/A02 cells to adriamycin (ADR) and cytosine arabinoside (Ara-C). Mechanistic studies revealed that ABCB6 contributes to ADR efflux from leukemia cells, thereby reducing intracellular drug accumulation and weakening its cytotoxic activity. Together, these findings establish ABCB6 as a previously unrecognized efflux transporter that contributes to MDR in leukemia and highlight ABCB6 as a potential therapeutic target for overcoming MDR.

In vivo genetic engineering of haematopoietic stem and progenitor cells (HSPCs) holds the potential to revolutionize the treatment landscape for numerous diseases. However, despite its transformative potential, it remains hindered by the difficulty in efficiently and specifically targeting quiescent human HSCs while maintaining their long-term functionality. Here, after screening 15 lipid nanoparticles (LNPs), we report an LNP that efficiently delivers reporter mRNA to human HSPCs both in ex vivo and in vivo settings when conjugated with the anti-CD34 antibody (CD34/LNPDP). Using CRISPR/Cas editing cargos, CD34/LNPDP achieves high editing efficiency in human HSPCs ex vivo. Intrafemoral administration of CD34/LNPDP in humanized mice results in efficient editing of the erythroid-specific BCL11A enhancer within human HSPCs, enabling the sustained long-term reactivation of fetal haemoglobin (HbF) expression in erythroid cells. In a humanized neutropaenia model harbouring an ELANE mutation, intrafemoral administration of CD34/LNPDP achieves robust editing, targeting exon 2 of ELANE in human HSPCs, partially restoring neutrophil development impairment under long-term observation. Collectively, CD34-targeted delivery enables in vivo HSPC modification without perturbing haematopoiesis, underscoring its suitability for clinical translation.

Irradiation with X-rays has been widely utilized in the clinical treatment of solid tumors and certain hematopoietic malignancies. However, this method fails to completely distinguish between malignant and normal cells. Prolonged or repeated exposure to radiation, whether due to occupational hazards or therapeutical interventions, can cause damage to normal tissues, particularly impacting the hematopoietic system. Therefore, it is important to investigate the effects of total body irradiation on the hematopoietic system of mice and to compare the inhibitory effects of various doses of irradiation on this system. In this study, we primarily employed flow cytometry to analyze mature lineage cells in the peripheral blood, as well as immature hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and spleen. Additionally, we evaluated the multilineage differentiation capacity of HSPCs through colony-forming cell assays. Our results indicated that peripheral B and T cells demonstrated increased sensitivity to irradiation, with significant cell death observed 1-day post-irradiation. Common lymphoid progenitor cells exhibited greater radiotolerance compared to other progenitor cell types, enabling them to maintain a certain population even at elevated doses. Moreover, notable differences were observed between intramedullary and extramedullary hematopoietic stem cells and common lymphoid progenitor cells regarding the extent of damage and recovery rate following irradiation. The multilineage differentiation capacity of HSPCs was also compromised during radiation exposure. In conclusion, different types of mature blood cells, along with immature HSPCs, exhibited varying degrees of sensitivity and tolerance to irradiation, resulting in distinct alterations in cell percentages and numbers.

The Hippo pathway is an evolutionarily conserved pathway that governs organ growth and homeostasis. The core of the Hippo pathway is a kinase cascade, the key components MST1/2, LATS1/2 and YAP/TAZ are thought to serve as tumor suppressors, but studies have found that YAP/TAZ are generally absent in hematopoietic system. Notably, MST1/2 expressions are high in HSPCs and LSCs, and present study indicated MST has a role in promoting the development of leukemia. These findings suggest that there is a non-canonical Hippo pathway in leukemia, and the role of MST1/2 in leukemogenesis is still ambiguous. In this study, using mouse model of acute myeloid leukemia (AML) driven by MLL-AF9, we investigated the impact of MST1/2 on leukemogenesis. Though with similar prolonged survival time, mice of the two groups exhibited totally different modes of remission: the onset time of AML in Mst1-/- group was delayed, while mice of Mst2-/- group could continue to survive with a high leukemic burden. Besides, loss of Mst1 or Mst2 could both promote the differentiation of AML, with only the knockout of Mst1 could induce AML cells apoptosis significantly. Given the dual regulatory features of MST1, we performed total RNA-seq, protein phosphorylation by MS and Crispr screening to search for downstream effectors. The results indicate that MEIS1 is a vital mediator of mechanical cues. Knockout of Mst1 declines the phosphorylation of MEIS1. Overexpression of MEIS1 phosphorylation-activated type in Mst1 deletion cells will accelerate leukemia progression rather than inactivation type. These findings suggest that MST1 regulates LSCs through MEIS1-dependent phosphorylation mechanism. We extend these findings to leukemia cell lines and other AML mouse model showed conserved pathways regulated by MST1-MEIS1. Overall, these results provide a rationale to target MST1-MEIS1 for AML treatment.

Conventional chemotherapy for killing cancer cells using cytotoxic drugs suffers from low selectivity, significant toxicity, and a narrow therapeutic index. Hyper-specific targeted drugs achieve precise destruction of tumors by inhibiting molecular pathways that are critical to tumor growth. Myeloid cell leukemia 1 (MCL-1), an important pro-survival protein in the BCL-2 family, is a promising antitumor target. In this study, we chose to investigate the effects of S63845, a small-molecule inhibitor that targets MCL-1, on the normal hematopoietic system. A mouse model of hematopoietic injury was constructed, and the effects of the inhibitor on the hematopoietic system of mice were evaluated via routine blood tests and flow cytometry. The results showed that S63845 affected the hematopoiesis of various lineages in the early stage of action, causing extramedullary compensatory hematopoiesis in the myeloid and megakaryocytic lineages. The maturation of the erythroid lineage in the intramedullary and extramedullary segments was blocked to varying degrees, and both the intramedullary and extramedullary lymphoid lineages were inhibited. This study provides a complete description of the effects of MCL-1 inhibitor on the intramedullary and extramedullary hematopoietic lineages, which is important for the selection of combinations of antitumor drugs and the prevention of adverse hematopoiesis-related effects.

Limited numbers of available hematopoietic stem cells (HSCs) limit the widespread use of HSC-based therapies. Expansion systems for functional heterogenous HSCs remain to be optimized. Here, we present a convenient strategy for human HSC expansion based on a biomimetic Microniche. After demonstrating the expansion of HSC from different sources, we find that our Microniche-based system expands the therapeutically attractive megakaryocyte-biased HSC. We demonstrate scalable HSC expansion by applying this strategy in a stirred bioreactor. Moreover, we identify that the functional human megakaryocyte-biased HSCs are enriched in the CD34 + CD38 - CD45RA-CD90 + CD49f low CD62L - CD133 + subpopulation. Specifically, the expansion of megakaryocyte-biased HSCs is supported by a biomimetic niche-like microenvironment, which generates a suitable cytokine milieu and supplies the appropriate physical scaffolding. Thus, beyond clarifying the existence and immuno-phenotype of human megakaryocyte-biased HSC, our study demonstrates a flexible human HSC expansion strategy that could help realize the strong clinical promise of HSC-based therapies.

Several approaches to expand human hematopoietic stem cells (hHSCs) clinically along with retainable capability of multipotential differentiation have been reported, but only a few have advanced to evaluation in clinical trials, which limits the application of HSC-based therapy. Here we show a phthalide derivative, Levistilide A (LA), can serve as a promising molecule to expand functional human umbilical cord blood (UCB) HSCs ex vivo. An in-house screen identified LA out of nine natural products as an outstanding candidate for hHSCs expansion. Additionally, our data indicated that LA treatment not only increased the numbers of phenotype-defined HSCs, but also enhanced their colony formation ability. Xenotransplantation assays showed that LA treatment could maintain unaffected engraftment of hHSCs with multilineage differentiation capacity. Further experiments revealed that LA enhanced the antioxidant activity of hHSCs by reducing intracellular and mitochondrial reactive oxygen species (ROS) levels. The identification of LA provides a new strategy in solving the clinical issue of limited numbers of UCB HSCs.

The use of umbilical cord blood transplant has been substantially limited by the finite number of hematopoietic stem and progenitor cells in a single umbilical cord blood unit. Small molecules that not only quantitatively but also qualitatively stimulate enhancement of hematopoietic stem cell (HSC) self-renewal ex vivo should facilitate the clinical use of HSC transplantation and gene therapy. Recent evidence has suggested that the cyclin-dependent kinase inhibitor, p18(INK4C) (p18), is a critical regulator of mice HSC self-renewal. The role of p18 in human HSCs and the effect of p18 inhibitor on human HSC expansion ex vivo need further studies. Here we report that knockdown of p18 allowed for an increase in long-term colony-forming cells in vitro. We then identified an optimized small molecule inhibitor of p18, 005A, to induce ex vivo expansion of HSCs that was capable of reconstituting human hematopoiesis for at least 4 months in immunocompromised mice, and hence, similarly reconstituted secondary recipients for at least 4 more months, indicating that cells exposed to 005A were still competent in secondary recipients. Mechanistic studies showed that 005A might delay cell division and activate both the Notch signaling pathway and expression of transcription factor HoxB4, leading to enhancement of the self-renewal of long-term engrafting HSCs and the pool of progenitor cells. Taken together, these observations support a role for p18 in human HSC maintenance and that the p18 inhibitor 005A can enhance the self-renewal of long-term HSCs.

Summary: Attempts to generate functional blood cells from human pluripotent stem cells (hPSCs) remain largely unsuccessful, mainly due to the lack of understanding of the regulatory network of human hematopoiesis. In this study, we identified leucine-rich-repeat-containing G-protein-coupled receptor 4 (LGR4) as an essential regulator of early hematopoietic differentiation of hPSCs. The deletion of LGR4 severely impairs mesoderm development, thereby limiting hematopoietic differentiation both in vitro and in vivo. In contrast, LGR5 is dispensable for hPSC hematopoiesis. The four R-spondin proteins show differential activities and dependencies on LGR4 in hematopoietic differentiation. The deletion of LGR4 almost entirely abolishes the enhancement induced by R-spondin1 and R-spondin3, but not R-spondin2. In addition, ZNRF3 is required for the response of R-spondin1–R-spondin3. At the mechanistic level, LGR4 regulates transforming growth factor beta (TGF-beta) signaling to control hematopoietic differentiation. Together, our results reveal vital roles of LGR4 in hematopoietic development and uncover distinct functions and underlying mechanisms for R-spondins.

Background Ding Chuan Tang (DCT), a traditional Chinese herbal formula, has been consistently prescribed for the therapeutic management of wheezing and asthma-related indications since the Song Dynasty (960–1279 AD). This study aimed to identify molecular network pharmacology connections to understand the biological asthma-linked mechanisms of action of DCT and potentially identify novel avenues for asthma drug development. Methods Employing molecular docking (AutoDock Vina) and computational analysis (Cytoscape 3.6.0) strategies for DCT compounds permitted examination of docking connections for proteins that were targets of DCT compounds and asthma genes. These identified protein targets were further analyzed to establish and interpret network connections associated with asthma disease pathways. Results A total of 396 DCT compounds and 234 asthma genes were identified through database search. Computational molecular docking of DCT compounds identified five proteins (ESR1, KDR, LTA4H, PDE4D and PPARG) mutually targeted by asthma genes and DCT compounds and 155 docking connections associated with cellular pathways involved in the biological mechanisms of asthma. Conclusions DCT compounds directly target biological pathways connected with the pathogenesis of asthma including inflammatory and metabolic signaling pathways.

The invention relates to application of chrysin in in-vitro expansion of human hematopoietic stem cells. In the application, the concentration of chrysin in an amplification culture medium is 1-10 [mu]M. The invention also provides a human hematopoietic stem cell amplification culture medium and a method for amplifying the human hematopoietic stem cells. The chrysin has a good promotion effect onself-renewal of the human hematopoietic stem cells, and a new method can be provided for clinical treatment in the future.

Condyloma acuminate (CA) is a communicable disease caused by human papillomavirus (HPV). This study aimed to study the targeting relationship between miR-34a-5p and Jagged 1 (JAG1), as well as its regulatory effect in HPV-infected cells. Human keratinocyte HaCaT cells were infected with HPV16E6, and CA tissues were collected. The expression level of miR-34a-5p and JAG1 were detected in CA tissues and HPV-HaCaT cells. Cell proliferation, migration and invasion were respectively measured using 3-(4, 5)-dimethylthiahiazo-(−z-y1)-3, 5-diphenytetrazoliumromide (MTT), cell wound healing and Transwell assay. The potential binding sites of miR-34a-5p and JAG1 were predicted by website TargetScan, and confirmed using dual luciferase reporter gene assay. The proteins of Notch1 pathway-related were assessed using western blotting. The results showed that miR-34a-5p expression was decreased, and JAG1 expression was increased in CA tissues and HPV-HaCaT cells. Cell proliferation, migration and invasion were decreased when miR-34a-5p over-expression and JAG1 knock-down in HPV-HaCaT cells. Furthermore, miR-34a-5p had a targeting effect on JAG1. The expression level of Notch1, NICD, Hes1 and Hey1 were increased when miR-34a-5p knock-down. miR-34a-5p could inhibit cell development, and regulate the activity of Notch1 pathway through targeting JAG1 expression in HPV-infected keratinocytes.

The lack of efficient ex vivo expansion methods restricts clinical use of haematopoietic stem cells (HSC) for the treatment of haematological malignancies and degenerative diseases. Umbilical cord blood (UCB) serves as an alternative haematopoietic stem cell source. However, currently what limits the use of UCB-derived HSC is the very low numbers of haematopoietic stem and progenitor cells available for transplantation in a single umbilical cord blood unit. Here, we report that TNFSF15, a member of the tumour necrosis factor superfamily, promotes the expansion of human umbilical cord blood (UCB)-derived HSC. TNFSF15-treated UCB-HSC is capable of bone marrow engraftment as demonstrated with NOD/SCID or NOD/Shi-SCID/IL2Rgnull (NOG) mice in both primary and secondary transplantation. The frequency of repopulating cells occurring in the injected tibiae is markedly higher than that in vehicle-treated group. Additionally, signal proteins of the Notch pathway are highly up-regulated in TNFSF15-treated UCB-HSC. These findings indicate that TNFSF15 is useful for in vitro expansion of UCB-HSC for clinical applications. Furthermore, TNFSF15 may be a hopeful selection for further UCB-HSC application or study.

There is an increasing demand for the expansion of functional human hematopoietic stem cells (hHSCs) for various clinical applications. Based on our primary screening of antioxidant small molecule compounds library, a small molecule compound C2968 (chrysin) was identificated to expand cord blood CD34+ cells in vitro. Then we further verified the optimum concentration and explored its effect on hHSCs phenotype and biological function. C2968 could significantly increase the proportion and absolute number of CD34+CD38−CD49f+ and CD34+CD38−CD45RA−CD90+ cells under 2.5 μM. Furthermore, the total number of colony-forming units and the frequency of LT-HSCs in C2968-treated group were significantly higher than control, indicating the multipotency and long-term activity of hematopoietic stem and progenitor cells were sustained. Additionally, C2968 treatment could maintain transplantable HSCs that preserve balanced multilineage potential and promote rapid engraftment after transplantation in immunodeficient (NOG) mice. Mechanistically, the activity of chrysin might be mediated through multiple mechanisms namely delaying HSC differentiation, inhibiting ROS-activated apoptosis, and modulating of cyclin-dependent kinase inhibitors. Overall, chrysin showed good ex vivo expansion effect on hHSCs, which could maintain the self-renewal and multilineage differentiation potential of hHSCs. Through further research on its antioxidant mechanism, it may become a promising tool for further fundamental research and clinical umbilical cord blood transplantation of hHSCs.

BACKGROUND: It has been reported that UM171 enables an ex vivo expansion of human hematopoietic stem cells. However, the intracellular regulatory mechanisms remain unclear. OBJECTIVE: To investigate the compound‐target interaction network of UM171 by High‐Throughput Docking (HTDocking) screening technology and TargetHunter, and to outline its potential mechanisms of action. METHODS: Using the online StemCellCKB interface, we searched the possible targets of UM171. Negative targets were excluded by validation experiments, and a surface plasmon resonance (SPR) assay was used to identify the interaction between UM171 and transforming growth factor &bgr;RI. Several important proteins which are known to regulate this pathway were investigated by western blot. RESULTS AND CONCLUSION: StemCellCKB program automatically calculated the docking scores, which indicated the possible targets of UM171. The KD50 values were calculated as 62.5 &mgr;mol/L by SPR, indicating the high affinity between UM171 and its target protein. Western blot analysis results revealed that the expression levels of main proteins in transforming growth factor &bgr; signaling pathway, including TGF‐&bgr;RI, Smad3, p‐Smad3, Smad4, were increased significantly in a concentration‐depend manner. Overall, UM171 can sustain the expansion of umbilical cord blood hematopoietic stem cells through the transforming growth factor &bgr; signaling pathway.

The thermal conductivity of cross-linked bulk polyethylene is studied using molecular dynamics simulation. The atomic structure of the cross-linked polyethylene (PEX) is generated through simulated bond formation using LAMMPS. The thermal conductivity of PEX is studied with different degrees of crosslinking, chain length, and tensile strain. Generally, the thermal conductivity increases with the increasing degree of crosslinking. When the length of the primitive chain increases, the thermal conductivity increases linearly. When the polymer is stretched along one direction, the thermal conductivity increases in the stretched direction and decreases in the direction perpendicular to it. However, the thermal conductivity varies slightly when the polymer is stretched in three directions simultaneously.

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of keratinocyte-derived skin tumor, which is regarded as the second most common skin cancer worldwide. Accumulating evidence has established that microRNAs (miRNAs) can play a critical role in tumor initiation, progression, and metastasis including cSCC. Abnormal expression of hsa-miR-142-5p has been elaborated in various tumors. Nevertheless, its expression and function in the development of cSCC remain unclear. In our study, we found that the expression of hsa-miR-142-5p in cSCC cells were greatly overexpressed compared to human benign epidermal keratinocyte cells. Moreover, inhibited hsa-miR-142-5p can repress cSCC cell growth and induce apoptosis while upregulated hsa-miR-142-5p exhibited a reverse phenomenon. Recently, cancer stem cells (CSCs) which possess the ability of self-renewal and proliferation and are able to produce cancer cells have been widely reported. However, the correlation between hsa-miR-142-5p and CSCs in cSCC is still unknown. Interestingly, we observed that overexpressing hsa-miR-142-5p can induce CSC-like properties in cSCC via activating Wnt signaling. In addition, the luciferase reporter assay data and bioinformatics analysis demonstrated that hsa-miR-142-5p can target the 3'UTR of PTEN mRNA. Taken these together, we draw a conclusion that hsa-miR-142-5p can trigger cancer stem cell-like properties of cSCC through inhibition of PTEN. Our findings may provide hsa-miR-142-5p as a new therapeutic target for cSCC.

Aging weakened innate and adaptive immunity both quantitatively and qualitatively. Some components in propolis could stimulate immune function in young animals or cultured immune cells in vitro. Few studies had been carried out in the aged. The present study was to evaluate the effects of Brazilian green propolis supplementation on the immunological parameters in aged mice. Eighty Kunming mice, aged 15–18 months, were randomly assigned to the control and three experimental groups supplemented with different doses (83.3, 157.4 and 352.9 mg/kg.bw respectively) of Brazilian green propolis. The experiment lasted for 4 weeks. Contents of total polyphenol, flavonoid, cinnamic acid and artepillin-C in Brazilian green propolis were analyzed. Splenic NK cytotoxic, T lymphocyte proliferation and antibody generation cells, as well as the phagocytosis of peritoneal macrophages, ear swelling, and serum contents of IgG, IgM, hemolysin and cytokines were measured. After 4 weeks of treatment, the phagocytosis of peritoneal macrophages was enhanced in 157.4 mg/kg and 352.9 mg/kg groups. Ear swelling increased in all propolis treatmented groups. Antibodies specific to sheep erythrocytes were higher in the groups receiving 157.4 and 352.9 mg/kg.bw than that of control group. IgG level dramatically increased in the groups receiving 83.3 and 157.4 mg/kg.bw in comparison to the control group. These results indicate that administration of Brazilian green propolis have a positive effect on innate and adaptive immunity in aged mice.