Search Results (42)

(1) Background: Vascular mural cells reside in the media and outer layers of the vessel wall. Their ability to proliferate and migrate or to change phenotype in response to external cues is a central feature of the vascular response to injury. Genetically engineered mice are used for loss- or gain-of-function analyses or lineage tracing in vivo, their primary cells for mechanistic studies in vitro. Whether and how cultivation conditions affect their phenotype and function is often overlooked. (2) Methods: Here, we systematically studied how the cultivation of primary mural cells isolated from the aorta of adult wild-type mice in either basal medium (DMEM) or special media formulated for the cultivation of fibroblasts or pericytes affects their phenotype and function. (3) Results: Medium composition did not alter cell viability, but the mRNA levels of differentiated smooth muscle cell markers were highest in vascular mural cells expanded in DMEM. Conversely, significantly higher numbers of proliferating and migrating cells were observed in cells expanded in Pericyte medium, and cytoskeletal rearrangements supported increased migratory capacities. Significantly reduced telomere lengths and metabolic reprogramming was observed in aortic mural cells cultured in Fibroblast medium. (4) Conclusions: Our findings underline the plasticity of primary aortic mural cells and highlight the importance of the culture media composition during their expansion, which could be exploited to interrogate their responsiveness to external stimuli or conditions observed in vivo or in patients. Full article

Donkeys (Equus asinus) hold significant agricultural value in China, particularly for their hides and meat, which possess notable medicinal and dietary importance. However, their reproductive efficiency remains suboptimal compared with other livestock. Ovarian function is a key determinant of fertility, yet the molecular mechanisms underlying donkey ovarian biology remain largely unexplored. To address this gap, we performed single-cell RNA sequencing of donkey ovaries, generating a high-resolution transcriptomic atlas comprising 17,423 cells. Cross-species comparative analysis revealed a high degree of evolutionary conservation in core ovarian cell types, including endothelial, epithelial, immune, and smooth muscle cells, among vertebrates. In contrast, granulosa and theca cells exhibited distinct transcriptional profiles across species, reflecting lineage-specific adaptations. Notably, we identified key genes with donkey-specific expression patterns, including NR3C1 in endothelial cells, LIPE in granulosa cells, and DHRS9 in theca interna cells. Furthermore, an in vitro cumulus–oocyte complex model demonstrated the critical role of GATM in mammalian oocyte maturation. Collectively, these findings provide a comprehensive characterization of ovarian cell-type conservation and species-specific adaptations, offering key molecular insights into the mechanisms underlying cross-species differences in reproductive efficiency. Full article

Papillary thyroid cancer (PTC) contains mesenchymal stem/stromal cells (MSCs), but their contribution to PTC progression is not clear. In this study, we compared the transcriptional signatures of normal thyroid (NT) and PTC-derived MSCs with the aim of determining if these have distinct transcriptomes that might influence PTC progression. We used flow cytometry in combination with a panel of MSC clusters of differentiation (CD) markers and showed that both thyroid MSC populations expressed MSC markers and lacked expression of markers not normally expressed by MSCs. In addition, we determined that both MSC populations could differentiate to adipocytes and osteocytes. Analysis of single cell RNA sequencing data from both MSC populations revealed, regardless of tissue of origin, that both contained similar numbers of subpopulations. Cluster analysis revealed similarity in expression of both MSC populations for stromal markers, the vascular marker VEGFA and the smooth muscle marker CALD1, while smaller subpopulations expressed markers of more lineage-committed thyroid cells. PTC MSCs also showed upregulated expression of 28 genes, many of which are known to be involved in epithelial–mesenchymal transition (EMT) and/or disease progression in several types of cancers, including but not limited to breast cancer, gastric cancer, cervical carcinoma, bladder cancer and thyroid cancer. This included several members of the S100 and IGFBP gene families. Taken together, these data support a role for PTC MSCs in PTC progression. Full article

Multipotent vascular stem cells (MVSCs) are found in the vascular wall and surrounding tissues and possess the ability to differentiate into mesenchymal lineages. Previous studies have shown that MVSCs can be activated in response to vascular injury and differentiate into vascular smooth muscle cells (SMCs), contributing to vascular remodeling and microvessel formation. However, it remains unclear as to whether and how microenvironmental changes in the extracellular matrix, such as substrate stiffness, modulates MVSC differentiation under pathological conditions. This study demonstrated that MVSCs cultured on stiff substrates exhibited increased cell spreading, stronger cell adhesion, and a higher expression of SMC markers, including myosin heavy chain (MHC), myocardin (MYCD), calponin 1 (CNN1), and smooth muscle α-actin (SMA). In contrast, MVSCs on soft substrates showed an elevated expression of the chondrogenic markers aggrecan 1 (AGC1) and collagen-II (COL2A1). The presence of TGF-β1 further increased the expression of SMC markers on stiff substrates and chondrogenic markers on the soft substrates. Collectively, these results establish substrate stiffness as a key regulator of MVSC lineage commitment through cytoskeletal reorganization, with TGF-β1 acting as a biochemical amplifier. Our findings highlight the substrate-stiffness-dependent differentiation of MVSCs and provide mechanistic insights into the role of MVSCs in vascular remodeling during atherosclerosis development and blood vessel regeneration. Full article

Mammary intraductal macrophages (foam cells) in humans are the most commonly encountered cells in spontaneous breast nipple discharge, nipple aspirate fluid, and ductal lavage, yet their origin remains unproven. These cells, in both humans and murine model systems, increase in pregnancy, pseudopregnancy, and other conditions like proliferative fibrocystic disease and intraductal neoplasia, ductal carcinoma in situ (DCIS), where there is intraductal ectasia and obstruction. Previous immunocytochemical studies with macrophage (CD68, lysozyme), epithelial (cytokeratin, estrogen receptor), and myoepithelial (smooth muscle actin, CALLA, maspin) markers have indicated that intraductal foam cells are of macrophage lineage. These foam cells engage in phagocytosis of both endogenous and exogenous substances present within the ducts and are not proliferative. Although it has been suggested that foam cells could derive from tissue-specific and niche-specific precursors or circulating monocytes, to date no experimental nor clinical studies have provided direct proof of their origin. In this study, we provide evidence in both human and murine bone marrow transplant studies that intraductal foam cells are bone marrow-derived. We first studied a registry of sex-mismatched bone marrow transplant recipients who later in life had undergone breast biopsies for either proliferative fibrocystic disease, DCIS, or gynecomastia, and studied these biopsies by XY chromosome fluorescence in situ hybridization (FISH) and informative microsatellite polymorphic markers. The intraductal foam cells were of bone marrow donor-origin. Then, in the experimental bone marrow transplant murine studies, donor marrow from female ROSA26 containing the lacZ reporter were transplanted into either irradiated female recipient transgenic mice carrying the highly penetrant MMTV-pymT or FVB/N background mice, where induced pluripotent stem (iPS) cells derived from tail vein fibroblasts of FVB/N-Tg(MMTV-PyVT)634Mul/J mice were subsequently injected into their mammary fat pads. In all of the transplanted recipient mice, the intraductal foam cells expressed the β-galactosidase (lacZ) reporter and also co-expressed markers of myeloid–macrophage lineage. The number of donor-derived intraductal foam cells increased in pseudopregnancy 5-fold and in intraductal neoplasia 10-fold. Although macrophages of different origins and lineages are undoubtedly present within both the murine and human breasts, those macrophages that qualify as phagocytic intraductal foam cells are bone marrow-derived. Full article

While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells (n = 393), or older cells (n = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity. Full article

Differentiation of amniotic fluid stem cells (AFSCs) into multiple lineages is controlled by epigenetic modifications, which include DNA methylation, modifications of histones, and the activity of small noncoding RNAs. The present study investigates the role of miRNAs in the differentiation of AFSCs and addresses how their unique signatures contribute to lineage-specific differentiation. The miRNA profile was assessed in AFSCs after 4 weeks of endothelial and muscular differentiation. Our results showed decreased expression of five miRNAs (miR-18a-5p, miR-125b-5p, miR-137, miR-21-5p, and let-7a) and increased expression of twelve miRNAs (miR-134-5p, miR-103a-3p, let-7i-5p, miR-214-3p, let-7c-5p, miR-129-5p, miR-210-3p, let-7d-5p, miR-375, miR-181-5p, miR-125a-5p, and hsa-let-7e-5p) in endothelial progenitor cells (EPCs) compared with undifferentiated AFSCs. AFSC differentiation into smooth muscle revealed notable changes in nine out of the 84 tested miRNAs. Among these, three miRNAs (miR-18a-5p, miR-137, and sa-miR-21-5p) were downregulated, while six miRNAs (miR-155-5p, miR-20a-5p, let-7i-5p, hsa-miR-134-5p, hsa-miR-214-3p, and hsa-miR-375) exhibited upregulation. Insights from miRNA networks promise future advancements in understanding and manipulating endothelial and muscle cell dynamics. This knowledge has the potential to drive innovation in areas like homeostasis, growth, differentiation, and vascular function, leading to breakthroughs in biomedical applications and therapies. Full article

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in the maintenance of aortic wall integrity. VSMCs have been suggested to have contractile and synthetic phenotypes and undergo phenotypic switching to contribute to the deteriorating aortic wall structure. Recently, the unprecedented heterogeneity and diversity of VSMCs and their complex relationship to aortic aneurysms (AAs) have been revealed by high-resolution research methods, such as lineage tracing and single-cell RNA sequencing. The aortic wall consists of VSMCs from different embryonic origins that respond unevenly to genetic defects that directly or indirectly regulate VSMC contractile phenotype. This difference predisposes to hereditary AAs in the aortic root and ascending aorta. Several VSMC phenotypes with different functions, for example, secreting VSMCs, proliferative VSMCs, mesenchymal stem cell-like VSMCs, immune-related VSMCs, proinflammatory VSMCs, senescent VSMCs, and stressed VSMCs are identified in non-hereditary AAs. The transformation of VSMCs into different phenotypes is an adaptive response to deleterious stimuli but can also trigger pathological remodeling that exacerbates the pathogenesis and development of AAs. This review is intended to contribute to the understanding of VSMC diversity in health and aneurysmal diseases. Papers that give an update on VSMC phenotype diversity in health and aneurysmal disease are summarized and recent insights on the role of VSMCs in AAs are discussed. Full article

Large animal experiments are important for preclinical studies of regenerative stem cell transplantation therapy. Therefore, we investigated the differentiation capacity of pig skeletal muscle-derived stem cells (Sk-MSCs) as an intermediate model between mice and humans for nerve muscle regenerative therapy. Enzymatically extracted cells were obtained from green-fluorescence transgenic micro-mini pigs (GFP-Tg MMP) and sorted as CD34+/45− (Sk-34) and CD34−/45−/29+ (Sk-DN) fractions. The ability to differentiate into skeletal muscle, peripheral nerve, and vascular cell lineages was examined via in vitro cell culture and in vivo cell transplantation into the damaged tibialis anterior muscle and sciatic nerves of nude mice and rats. Protein and mRNA levels were analyzed using RT-PCR, immunohistochemistry, and immunoelectron microscopy. The myogenic potential, which was tested by Pax7 and MyoD expression and the formation of muscle fibers, was higher in Sk-DN cells than in Sk-34 cells but remained weak in the latter. In contrast, the capacity to differentiate into peripheral nerve and vascular cell lineages was significantly stronger in Sk-34 cells. In particular, Sk-DN cells did not engraft to the damaged nerve, whereas Sk-34 cells showed active engraftment and differentiation into perineurial/endoneurial cells, endothelial cells, and vascular smooth muscle cells, similar to the human case, as previously reported. Therefore, we concluded that Sk-34 and Sk-DN cells in pigs are closer to those in humans than to those in mice. Full article

Current techniques for the detection of vasa vasorum (VV) in vascular pathology include staining for endothelial cell (EC) markers such as CD31 or VE-cadherin. However, this approach does not permit an objective assessment of vascular geometry upon vasospasm and the clinical relevance of endothelial specification markers found in developmental biology studies remains unclear. Here, we performed a combined immunostaining of rat abdominal aorta (rAA) and human saphenous vein (hSV) for various EC or vascular smooth muscle cell (VSMC) markers and found that the latter (e.g., alpha smooth muscle actin (α-SMA) or smooth muscle myosin heavy chain (SM-MHC)) ensure a several-fold higher signal-to-noise ratio irrespective of the primary antibody origin, fluorophore, or VV type (arterioles, venules, or capillaries). Further, α-SMA or SM-MHC staining allowed unbiased evaluation of the VV area under vasospasm. Screening of the molecular markers of endothelial heterogeneity (mechanosensitive transcription factors KLF2 and KLF4, arterial transcription factors HES1, HEY1, and ERG, venous transcription factor NR2F2, and venous/lymphatic markers PROX1, LYVE1, VEGFR3, and NRP2) have not revealed specific markers of any lineage in hSV (although KLF2 and PROX1 were restricted to venous endothelium in rAA), suggesting the need in high-throughput searches for the clinically relevant signatures of arterial, venous, lymphatic, or capillary differentiation. Full article

The objective of this study is to observe the effect of high selenium on the antioxidant and immune functions of growing goats based on transcriptome sequencing. Eighteen goats were randomly divided into three groups: (1) the control (CON) group was fed a basal diet, and (2) the treatment 1 group (LS) and treatment 2 group (HS) were fed a basal diet with 2.4 and 4.8 mg/kg selenium-yeast (SY), respectively. The results indicate that HS treatment significantly (p < 0.05) increased the apparent digestibility of either extract and significantly increased (p < 0.05) total antioxidant capacity, whereas it significantly (p < 0.05) decreased plasma aspartate aminotransferase and malondialdehyde relative to the control group. The LS treatment had significantly (p < 0.05) increased glutathione S-transferase and catalase compared to CON. A total of 532 differentially expressed genes (DEGs) between the CON and HS were obtained using transcriptome sequencing. Kyoto Encyclopedia of Genes and Genomes analysis identified upregulated (p < 0.05) DEGs mainly related to vascular smooth muscle contraction, alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, the VEGF signalling pathway, and proteoglycans in cancer; downregulated (p < 0.05) DEGs mainly related to the NOD-like receptor signalling pathway, influenza A, cytokine-cytokine receptor interaction, haematopoietic cell lineage, and African trypanosomiasis. Ontology analyses of the top genes show that the identified DEGs are mainly involved in the regulation of granulocyte macrophage colony-stimulating factor production for biological processes, the external side of the plasma membrane for cellular components, and carbohydrate derivative binding for molecular functions. Seven genes are considered potential candidate genes for regulating antioxidant activity, including selenoprotein W, 1, glutathione peroxidase 1, glutathione S-transferase A1, tumour necrosis factor, tumour necrosis factor superfamily member 10, tumour necrosis factor superfamily member 8, and tumour necrosis factor superfamily member 13b. The experimental observations indicate that dietary supplementation with 4.8 mg/kg SY can enhance antioxidant and immune functions by improving muscle immunity, reducing the concentrations of inflammatory molecules, and modulating antioxidant and inflammatory signalling pathways in growing goats. Full article

Cardiovascular disease is a globally prevalent disease with far-reaching medical and socio-economic consequences. Although improvements in treatment pathways and revascularisation therapies have slowed disease progression, contemporary management fails to modulate the underlying atherosclerotic process and sustainably replace damaged arterial tissue. Direct cellular reprogramming is a rapidly evolving and innovative tissue regenerative approach that holds promise to restore functional vasculature and restore blood perfusion. The approach utilises cell plasticity to directly convert somatic cells to another cell fate without a pluripotent stage. In this narrative literature review, we comprehensively analyse and compare direct reprogramming protocols to generate endothelial cells, vascular smooth muscle cells and vascular progenitors. Specifically, we carefully examine the reprogramming factors, their molecular mechanisms, conversion efficacies and therapeutic benefits for each induced vascular cell. Attention is given to the application of these novel approaches with tissue engineered vascular grafts as a therapeutic and disease-modelling platform for cardiovascular diseases. We conclude with a discussion on the ethics of direct reprogramming, its current challenges, and future perspectives. Full article

In the capillary walls, vascular endothelial cells are covered with mural cells, such as smooth muscle cells and pericytes. Although pericytes had been thought to play simply a structural role, emerging evidence has highlighted their multiple functions in the embryonic, postnatal, and adult brain. As the central nervous system (CNS) develops, the brain’s vascular structure gradually matures into a hierarchical network, which is crucial for the proper development of neural lineage cells by providing oxygen and nutrients. Pericytes play an essential role in vascular formation and regulate blood‒brain barrier (BBB) integrity as a component of the neurovascular unit (NVU), in collaboration with other cells, such as vascular endothelial cells, astrocytes, neurons, and microglia. Microglia, the resident immune cells of the CNS, colonize the brain at embryonic day (E) 9.5 in mice. These cells not only support the development and maturation of neural lineage cells but also help in vascular formation through their extensive migration. Recent studies have demonstrated that pericytes directly contact microglia in the CNS, and their interactions have a profound effect on physiological and pathological aspects. This review summarizes the function of pericytes, focusing on the interplay between pericytes and microglia. Full article

Despite many decades of research, complications of atherosclerosis resulting from the rupture or erosion of unstable plaques remain the leading cause of death worldwide. Advances in cellular lineage tracing techniques have allowed researchers to begin investigating the role of individual cell types in the key processes regulating plaque stability, including maintenance of the fibrous cap, a protective collagen-rich structure that underlies the endothelium. This structure was previously thought to be entirely derived from smooth muscle cells (SMC), which migrated from the vessel wall. However, recent lineage tracing studies have identified endothelial cells (EC) as an essential component of this protective barrier through an endothelial-to-mesenchymal transition (EndoMT), a process that has previously been implicated in pulmonary, cardiac, and kidney fibrosis. Although the presence of EndoMT in atherosclerotic plaques has been shown by several laboratories using EC-lineage tracing mouse models, whether EndoMT is detrimental (i.e., worsening disease progression) or beneficial (i.e., an athero-protective response that prevents plaque instability) remains uncertain as there are data to support both possibilities, which will be further discussed in this review. Full article

Our laboratory reported the derivation of neural crest stem cell (NCSC)-like cells from the interfollicular epidermis of the neonatal and adult epidermis. These keratinocyte (KC)-derived Neural Crest (NC)-like cells (KC-NC) could differentiate into functional neurons, Schwann cells (SC), melanocytes, and smooth muscle cells in vitro. Most notably, KC-NC migrated along stereotypical pathways and gave rise to multiple NC derivatives upon transplantation into chicken embryos, corroborating their NC phenotype. Here, we present an innovative design concept for developing anisotropically aligned scaffolds with chemically immobilized biological cues to promote differentiation of the KC-NC towards the SC. Specifically, we designed electrospun nanofibers and examined the effect of bioactive cues in guiding KC-NC differentiation into SC. KC-NC attached to nanofibers and adopted a spindle-like morphology, similar to the native extracellular matrix (ECM) microarchitecture of the peripheral nerves. Immobilization of biological cues, especially Neuregulin1 (NRG1) promoted the differentiation of KC-NC into the SC lineage. This study suggests that poly-ε-caprolactone (PCL) nanofibers decorated with topographical and cell-instructive cues may be a potential platform for enhancing KC-NC differentiation toward SC. Full article