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.

Genetic Diversity and Structure Revealed by Genomic Microsatellite Markers of Mytilus unguiculatus in the Coast of China Sea.

The hard-shelled mussel Mytilus unguiculatus plays an important role in mussel aquaculture in China due to its characteristic and nutritive value. In this study, 10 microsatellite loci are used to study the genetic diversity and genetic structure of seven location populations of M. unguiculatus in coastal areas of China. The results of amplification and genotyping show that the observed heterozygosity (Ho) and the expected heterozygosity (He) are 0.61~0.71 and 0.72~0.83, respectively. M. unguiculatus has high genetic diversity. The inbreeding index (FIS) of M. unguiculatus is significantly positive (FIS: 0.14~0.19), indicating that inbreeding might exist within populations. The genetic structure of M. unguiculatus is weak within populations from the East China Sea All results showed that genetic differences existed between the Qingdao population from the Yellow Sea and other populations from the East China Sea. It does not detect a population bottleneck event or expansion event in the populations. The results from this study can be used to provide important insights in genetic management units and sustainable utilization of M. unguiculatus resources and provide a better understand of genetic structure of marine bivalve with similar planktonic larval stage in the China Sea.

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.

Recovery and maintenance of NESTIN expression in umbilical cord-MSC using a novel culture medium.

Mesenchymal stem cells (MSC) are a popular candidate in cellular therapy for many diseases. MSCs are well known by their feature of self-renewal and their differentiation potential. NESTIN is a cytoskeletal protein expressed in MSC that functions directly in cell proliferation and differentiation. Here, we demonstrated that adding UltraGRO, a medium supplement, could maintain and partially recover the expression of NESTIN in human umbilical cord derived MSCs (UC-MSCs). Furthermore, the UC-MSCs cultured with UltraGRO showed a better immunomodulation ability in a colitis mouse model compared with those cultured in other types of media. This indicates that the use of novel culture medium benefits the maintenance of NESTIN expression and NESTIN may be one of the vital factors that regulates the performance of MSCs.

Strong genetic differentiation of the clam Meretrix lamarckii in the China Sea revealed by mitochondrial DNA marker.

The hard clam Meretrix lamarckii is ecologically and economically important in the coastal regions of China. In this study, we evaluated the genetic diversity and population structure of six M. lamarckii populations in the East China Sea and the South China Sea using mitochondrial cytochrome c oxidase subunit 1 (COI) and cytochrome b (Cytb) genes. We obtained 582 bp of partly sequences comprising 28 novel haplotypes of COI gene from 138 specimens and 1168 bp of partly sequences comprising 22 novel haplotypes of Cytb gene from 125 specimens. The haplotype diversity of COI and Cytb genes ranged from 0.606 to 0.862 and 0.562 to 0.863, respectively. The nucleotide diversity ranged from 0.0015 to 0.0038 in COI gene and ranged from 0.0007 to 0.0032 in Cytb gene. Thus, there is moderate-level genetic diversity in M. lamarckii in the China Sea. The F-statistics showed that the Zhoushan (ZS) and Xiangshan (XS) populations were significantly (P < 0.01) differed from the populations of Wenzhou (WZ), Zhangpu (ZP), Shantou (ST), and Zhanjiang (ZJ) in both COI and Cytb genes. Both haplotypes network and plot of STRUCTURE analysis suggested obviously genetic divergence between East China Sea and South China Sea regions. Knowledge on genetic variation and population structure of M. lamarckii populations along the Southeast China Sea obtained from this study will support the aquaculture management and conservation of M. lamarckii in China.

Pelagic larval dispersal habits influence the population genetic structure of clam Gomphina aequilatera in China.

Pelagic larval dispersal habits influence the population genetic structure of marine mollusk organisms via gene flow. The genetic information of the clam Gomphina aequilatera (short larval stage, 10 days) which is ecologically and economically important in the China coast is unknown. To determine the influence of planktonic larval duration on the genetic structure of G. aequilatera. Mitochondrial markers, cytochrome oxidase subunit i (COI) and 12S ribosomal RNA (12S rRNA), were used to investigate the population structure of wild G. aequilatera specimens from four China Sea coastal locations (Zhoushan, Nanji Island, Zhangpu and Beihai). Partial COI (685 bp) and 12S rRNA (350 bp) sequences were determined. High level and significant FST values were obtained among the different localities, based on either COI (FST = 0.100-0.444, P < 0.05) or 12S rRNA (FST = 0.193-0.742, P < 0.05), indicating a high degree of genetic differentiation among the populations. The pairwise Nm between Beihai and Zhoushan for COI was 0.626 and the other four pairwise Nm values were > 1, indicating extensive gene flow among them. The 12S rRNA showed the same pattern. AMOVA test results for COI and 12S rRNA indicated major genetic variation within the populations: 77.96% within and 22.04% among the populations for COI, 55.73% within and 44.27% among the populations for 12S rRNA. A median-joining network suggested obvious genetic differentiation between the Zhoushan and Beihai populations. This study revealed the extant population genetic structure of G. aequilatera and showed a strong population structure in a species with a short planktonic larval stage.