Unveiling the functional heterogeneity of cytokine-primed human umbilical cord mesenchymal stem cells through single-cell RNA sequencing.

BACKGROUND: Mesenchymal stem cells (MSCs) hold immense promise for use in immunomodulation and regenerative medicine. However, their inherent heterogeneity makes it difficult to achieve optimal therapeutic outcomes for a specific clinical disease. Primed MSCs containing a certain cytokine can enhance their particular functions, thereby increasing their therapeutic potential for related diseases. Therefore, understanding the characteristic changes and underlying mechanisms of MSCs primed by various cytokines is highly important. RESULTS: In this study, we aimed to reveal the cellular heterogeneity, functional subpopulations, and molecular mechanisms of MSCs primed with IFN-γ, TNF-α, IL-4, IL-6, IL-15, and IL-17 using single-cell RNA sequencing (scRNA-seq). Our results demonstrated that cytokine priming minimized the heterogeneity of the MSC transcriptome, while the expression of MSC surface markers exhibited only slight changes. Notably, compared to IL-6, IL-15, and IL-17; IFN-γ, TNF-α, and IL-4 priming, which stimulated a significantly greater number of differentially expressed genes (DEGs). Functional analysis, which included Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, indicated that IFN-γ, TNF-α, and IL-4-primed hUC-MSCs are involved in interferon-mediated immune-related processes, leukocyte migration, chemotaxis potential, and extracellular matrix and cell adhesion, respectively. Moreover, an investigation of various biological function scores demonstrated that IFN-γ-primed hUC-MSCs exhibit strong immunomodulatory ability, TNF-α-primed hUC-MSCs exhibit high chemotaxis potential, and IL-4-primed hUC-MSCs express elevated amounts of collagen. Finally, we observed that cytokine priming alters the distribution of functional subpopulations of MSCs, and these subpopulations exhibit various potential biological functions. Taken together, our study revealed the distinct regulatory effects of cytokine priming on MSC heterogeneity, biological function, and functional subpopulations at the single-cell level. CONCLUSIONS: These findings contribute to a comprehensive understanding of the inflammatory priming of MSCs, paving the way for their precise treatment in clinical applications.

Protein post-translational modification by lysine succinylation: Biochemistry, biological implications, and therapeutic opportunities.

Lysine succinylation (Ksuc) is a novel protein post-translational modification (PTM) wherein a succinyl group modifies a lysine residue. Ksuc leads to significant chemical and structural changes to the modified protein. Recent studies have shown that Ksuc might play an important role in organism physiology and some pathophysiological processes, such as tumorigenesis and metabolic diseases. To provide an understanding of the molecular mechanism and functions of Ksuc in different organisms, we reviewed the current literature about Ksuc, mainly summarizing the research advances in eukaryotes and prokaryotes based on both traditional study methods and site prediction tools. We also discussed inhibitors or activators associated with Ksuc that may contribute to proteomic studies and could be useful in future clinical practice. A deeper understanding of Ksuc may shed new light on life science at the protein level and could lead to novel therapeutic strategies for various diseases.

Erythropoietin promotes myocardial infarction repair in mice by improving the function of Sca-1+ stem cells.

Myocardial infarction (MI) is one of the leading causes of death in the world. With the improvement of clinical therapy, the mortality of acute MI has been significantly reduced. However, as for the long-term impact of MI on cardiac remodeling and cardiac function, there is no effective prevention and treatment measures. Erythropoietin (EPO), a glycoprotein cytokine essential to hematopoiesis, has anti-apoptotic and pro-angiogenetic effects. Studies have shown that EPO plays a protective role in cardiomyocytes in cardiovascular diseases, such as cardiac ischemia injury and heart failure. EPO has been demonstrated to protect ischemic myocardium and improve MI repair by promoting the activation of cardiac progenitor cells (CPCs). This study aimed to investigate whether EPO can promote MI repair by enhancing the activity of stem cell antigen 1 positive stem cells (Sca-1+ SCs). Darbepoetin alpha (a long-acting EPO analog, EPOanlg) was injected into the border zone of MI in adult mice. Infarct size, cardiac remodeling and performance, cardiomyocyte apoptosis and microvessel density were measured. Lin- Sca-1+ SCs were isolated from neonatal and adult mouse hearts by magnetic sorting technology, and were used to identify the colony forming ability and the effect of EPO, respectively. The results showed that, compared to MI alone, EPOanlg reduced the infarct percentage, cardiomyocyte apoptosis ratio and left ventricular (LV) chamber dilatation, improved cardiac performance, and increased the numbers of coronary microvessels in vivo. In vitro, EPO increased the proliferation, migration and clone formation of Lin- Sca-1+ SCs likely via the EPO receptor and downstream STAT-5/p38 MAPK signaling pathways. These results suggest that EPO participates in the repair process of MI by activating Sca-1+ SCs.

Interleukin-18-primed human umbilical cord-mesenchymal stem cells achieve superior therapeutic efficacy for severe viral pneumonia via enhancing T-cell immunosuppression.

Coronavirus disease 2019 (COVID-19) treatments are still urgently needed for critically and severely ill patients. Human umbilical cord-mesenchymal stem cells (hUC-MSCs) infusion has therapeutic benefits in COVID-19 patients; however, uncertain therapeutic efficacy has been reported in severe patients. In this study, we selected an appropriate cytokine, IL-18, based on the special cytokine expression profile in severe pneumonia of mice induced by H1N1virus to prime hUC-MSCs in vitro and improve the therapeutic effect of hUC-MSCs in vivo. In vitro, we demonstrated that IL-18-primed hUC-MSCs (IL18-hUCMSC) have higher proliferative ability than non-primed hUC-MSCs (hUCMSCcon). In addition, VCAM-1, MMP-1, TGF-ß1, and some chemokines (CCL2 and CXCL12 cytokines) are more highly expressed in IL18-hUCMSCs. We found that IL18-hUCMSC significantly enhanced the immunosuppressive effect on CD3+ T-cells. In vivo, we demonstrated that IL18-hUCMSC infusion could reduce the body weight loss caused by a viral infection and significantly improve the survival rate. Of note, IL18-hUCMSC can also significantly attenuate certain clinical symptoms, including reduced activity, ruffled fur, hunched backs, and lung injuries. Pathologically, IL18-hUCMSC transplantation significantly enhanced the inhibition of inflammation, viral load, fibrosis, and cell apoptosis in acute lung injuries. Notably, IL18-hUCMSC treatment has a superior inhibitory effect on T-cell exudation and proinflammatory cytokine secretion in bronchoalveolar lavage fluid (BALF). Altogether, IL-18 is a promising cytokine that can prime hUC-MSCs to improve the efficacy of precision therapy against viral-induced pneumonia, such as COVID-19.

Human Platelet Lysate Maintains Stemness of Umbilical Cord-Derived Mesenchymal Stromal Cells and Promote Lung Repair in Rat Bronchopulmonary Dysplasia.

Mesenchymal stromal cells (MSCs) show potential for treating preclinical models of newborn bronchopulmonary dysplasia (BPD), but studies of their therapeutic effectiveness have had mixed results, in part due to the use of different media supplements for MSCs expansion in vitro. The current study sought to identify an optimal culture supplement of umbilical cord-derived MSCs (UC-MSCs) for BPD therapy. In this study, we found that UC-MSCs cultured with human platelet lysate (hPL-UCMSCs) were maintained a small size from Passage 1 (P1) to P10, while UC-MSCs cultured with fetal bovine serum (FBS-UCMSCs) became wide and flat. Furthermore, hPL was associated with lower levels of senescence in UC-MSCs during in vitro expansion compared with FBS, as indicated by the results of ß-galactosidase staining and measures of senescence-related genes (CDKN2A, CDKN1A, and mTOR). In addition, hPL enhanced the proliferation and cell viability of the UC-MSCs and reduced their doubling time in vitro. Compared with FBS-UCMSCs, hPL-UCMSCs have a greater potential to differentiate into osteocytes and chondrocytes. Moreover, using hPL resulted in greater expression of Nestin and specific paracrine factors (VEGF, TGF-ß1, FGF2, IL-8, and IL-6) in UC-MSCs compared to using FBS. Critically, we also found that hPL-UCMSCs are more effective than FBS-UCMSCs for the treatment of BPD in a rat model, with hPL leading to improvements in survival rate, lung architecture and fibrosis, and lung capillary density. Finally, qPCR of rat lung mRNA demonstrated that hPL-UCMSCs had lower expression levels of inflammatory factors (TNF-α and IL-1ß) and a key chemokine (MCP-1) at postnatal day 10, and there was significant reduction of CD68+ macrophages in lung tissue after hPL-UCMSCs transplantation. Altogether, our findings suggest that hPL is an optimal culture supplement for UC-MSCs expansion in vitro, and that hPL-UCMSCs promote lung repair in rat BPD disease.

Vaginal Microbiota and HPV Infection: Novel Mechanistic Insights and Therapeutic Strategies.

Cervical cancer is a global public health concern. The complex interaction of genetic and environmental factors is critical for the progress of cervical cancer. Growing evidence suggests that microbes, human papillomavirus (HPV), and the immune system interact closely with each other to govern homeostasis of the vaginal environment and the health of the lower genital tract of females. Certain vaginal microbial strains may play either a protective or a pathogenic role in carcinogenesis of the cervix after HPV persistent infection. Probiotics can therefore present a putative therapeutic approach for cervical cancer. However, work in this field remains limited. Recent technological developments have allowed us to identify microbes and their products using culture-independent molecular detection techniques. In this review, we discuss the composition of the vaginal bacterial community, its commensal flora and the protective impact this has on the health of the female genital tract. This review will also describe critical immune factors in lower genital tract health and summarize the role of the vaginal microbiota in cervical carcinogenesis. Knowledge in this field has provided researchers with the clues and tools to propose the use of probiotics as a potential line of treatment for cervical cancer and has provided valuable insights into host-pathogen interaction dynamics within the female genital tract.

Multiplex precise base editing in cynomolgus monkeys.

Common polygenic diseases result from compounded risk contributed by multiple genetic variants, meaning that simultaneous correction or introduction of single nucleotide variants is required for disease modeling and gene therapy. Here, we show precise, efficient, and simultaneous multiplex base editing of up to three target sites across 11 genes/loci in cynomolgus monkey embryos using CRISPR-based cytidine- and adenine-base editors. Unbiased whole genome sequencing demonstrates high specificity of base editing in monkey embryos. Our data demonstrate feasibility of multiplex base editing for polygenic disease modeling in primate zygotes.

Knockdown of CHPF suppresses cell progression of non-small-cell lung cancer.

Purpose: The aim of the present study was to explore the role of CHPF in non-small-cell lung cancer (NSCLC) and to develop an shRNA vector-based therapy to repress the expression of CHPF gene in NSCLC cell lines. Methods: In this study, we used immunohistochemical staining to verify the expression of CHPF in NSCLC tissue. Then, we determined the expression of CHPF gene in different NSCLC cell lines with RT-PCR and Western blotting. Specific CHPF shRNA was used to knockdown the expression of CHPF. Celigo image cytometry, cell cycle analysis, and flow cytometry assay were performed. Results: The results showed that expression level of CHPF was higher in NSCLC tissues than normal lung tissues. Further, we established that CHPF expression knockdown in NSCLC cells could substantially restrain the cell proliferation, apoptosis, and cell cycle in vitro. Conclusion: On the basis of these results, we concluded that CHPF expression has an important role in the progression of human NSCLC cells. Therefore, its interference could possibly be used as a potential therapeutic target against NSCLC.

[Optimization of isolation and culture of Lin- Sca-1+ cardiac stem cells from newborn mice].

Cardiac stem cells (CSCs) transplantation has been recognized to be effective on the treatment of myocardial infarction (MI), but some techniques still need to be developed in the isolation and culture of CSCs, which is the key problem restricting the clinical application of CSCs. This study was focused on the isolation of Lin- (lineage-negative) Sca-1+ (stem cell antigen-1-positive) CSCs from newborn C57BL/6J mice (0-3 d) by mixed enzymatic-explant isolation in combination with immunomagnetic separation. The digesting time, digesting frequency, incubation temperature, stirring speed, centrifugation time and rotational speed were strictly controlled in the experiment. In order to increase the survival rate of CSCs, the medium changing time and manner were optimized in primary CSCs culture. The percentages of Sca-1+ cells in primary and passage cells were detected by flow cytometry and immunofluorescence staining. The results showed that: (1) the proportion of Lin- Sca-1+ cells within the collected cells could be as high as (85.03 ± 5.60)% after isolation and purification; (2) In vitro culture of Lin- Sca-1+ CSCs grew into spheres on the 5th day, and over the whole bottom of the dish on the 7th day. The growth curve showed that the cells were in logarithmic growth phase on the 3rd day; (3) Immunofluorescence staining data showed that the expression of Sca-1, the CSCs membrane-specific marker, was decreased after subculture, and flow cytometry data showed that the percentages of Sca-1+ cells were (71.82 ± 2.63)%, (58.38 ± 3.70)% and (46.19 ± 4.72)% in passage 1 (P1), P3, and P5 CSCs, respectively. The above results suggest that high purity of Lin- Sca-1+ CSCs can be obtained by enzymolysis combined with immunomagnetic separation method. Moreover, the CSCs culture system is stable. In our experiment, the Sca-1+ CSCs isolation and culture method has been successfully established, and it is simple, stable, effective and reliable. The method can provide a stable methodological basis for the treatment of MI by Lin- Sca-1+ CSCs transplantation.