Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34⁺ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34⁺ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34⁺ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca²⁺ release and extracellular Ca²⁺ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34⁺ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34⁺ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca²⁺ release from endoplasmic reticulum of CD34⁺ VW-SCs. Store-operated Ca²⁺ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca²⁺-free/Ca²⁺ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34⁺ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.
Mice ; Animals ; Endothelial Cells ; Cell Differentiation/physiology* ; Stem Cells ; Adventitia ; Fibroblasts ; Cells, Cultured ; Antigens, CD34/metabolism*

Periodontitis is a complex chronic inflammatory disease. The invasion of pathogens induces the inflammatory microenvironment in periodontitis. Cell behavior changes in response to changes in the microenvironment, which in turn alters the local inflammatory microenvironment of the periodontium through factors secreted by cells. It has been confirmed that periodontal ligament stem cells (PDLSCs) are vital in the development of periodontal disease. Moreover, PDLSCs are the most effective cell type to be used for periodontium regeneration. This review focuses on changes in PDLSCs, their basic biological behavior, osteogenic differentiation, and drug effects caused by the inflammatory microenvironment, to provide a better understanding of the influence of these factors on periodontal tissue homeostasis. In addition, we discuss the underlying mechanism in detail behind the reciprocal responses of PDLSCs that affect the microenvironment.
Humans ; Periodontal Ligament ; Osteogenesis ; Stem Cells ; Periodontitis/metabolism* ; Cell Differentiation/physiology* ; Cells, Cultured

Hepatic parenchymal cells are a type of liver cells that performs important functions such as metabolism and detoxification. The contribution of hepatic parenchymal cells, bile duct cells, and hepatic stem/progenitor cells to new hepatic parenchymal cells in the process of liver injury repair has become a controversial issue due to their strong proliferation ability. Lineage tracing technology, which has emerged in the past decade as a new method for exploring the origin of cells, can trace specific type of cells and their daughter cells by labeling cells that express the specific gene and their progeny. The article reviews the current literature on the origin and contribution of hepatic parenchymal cells by this technique. About 98% of new hepatic parenchymal cells originate from the existing hepatic parenchymal cells during liver homeostasis and after acute injury. However, under conditions of severe liver injury, such as inhibition of hepatic parenchymal cell proliferation, bile duct cells (mainly liver stem/progenitor cells) become the predominant source of hepatic parenchymal cells, contributing a steady increased hepatocyte regeneration with the extension of time.
Hepatocytes/metabolism* ; Liver/metabolism* ; Bile Ducts ; Stem Cells ; Liver Regeneration/physiology* ; Cell Differentiation

OBJECTIVE: To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs. METHODS: BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs. RESULTS: Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2. CONCLUSION The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.
Humans ; Adipogenesis ; Bone Diseases/metabolism* ; Bone Marrow Cells ; Cell Differentiation ; Cells, Cultured ; Mesenchymal Stem Cells/physiology* ; Multiple Myeloma/metabolism* ; Orlistat/pharmacology* ; Osteogenesis/genetics*

Objective: To investigate the expression of programmed death protein-ligand 1 (PD-L1) in liver cancer stem-like cells (LCSLC) and its effect on the characteristics of tumor stem cells and tumor biological function, to explore the upstream signaling pathway regulating PD-L1 expression in LCSLC and the downstream molecular mechanism of PD-L1 regulating stem cell characteristics, also tumor biological functions. Methods: HepG2 was cultured by sphere-formating method to obtain LCSLC. The expressions of CD133 and other stemness markers were detected by flow cytometry, western blot and real-time quantitative polymerase chain reaction (RT-qPCR) were used to detect the expressions of stemness markers and PD-L1. The biological functions of the LCSLC were tested by cell function assays, to confirm that the LCSLC has the characteristics of tumor stem cells. LCSLC was treated with cell signaling pathway inhibitors to identify relevant upstream signaling pathways mediating PD-L1 expression changes. The expression of PD-L1 in LCSLC was down regulated by small interfering RNA (siRNA), the expression of stem cell markers, tumor biological functions of LCSLC, and the changes of cell signaling pathways were detected. Results: Compared with HepG2 cells, the expression rate of CD133 in LCSLC was upregulated [(92.78±6.91)% and (1.40±1.77)%, P<0.001], the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were also higher than those in HepG2 cells (P<0.05), the number of sphere-formating cells increased on day 7 [(395.30±54.05) and (124.70±19.30), P=0.001], cell migration rate increased [(35.41±6.78)% and (10.89±4.34)%, P=0.006], the number of transmembrane cells increased [(75.77±10.85) and (20.00±7.94), P=0.002], the number of cloned cells increased [(120.00±29.51) and (62.67±16.77), P=0.043]. Cell cycle experiments showed that LCSLC had significantly more cells in the G(0)/G(1) phase than those in HepG2 [(54.89±3.27) and (32.36±1.50), P<0.001]. The tumor formation experiment of mice showed that the weight of transplanted tumor in LCSLC group was (1.32±0.17)g, the volume is (1 779.0±200.2) mm(3), were higher than those of HepG2 cell [(0.31±0.06)g and (645.6±154.9)mm(3), P<0.001]. The expression level of PD-L1 protein in LCSLC was 1.88±0.52 and mRNA expression level was 2.53±0.62, both of which were higher than those of HepG2 cells (P<0.05). The expression levels of phosphorylation signal transduction and transcription activation factor 3 (p-STAT3) and p-Akt in LCSLC were higher than those in HepG2 cells (P<0.05). After the expression of p-STAT3 and p-Akt was down-regulated by inhibitor treatment, the expression of PD-L1 was also down-regulated (P<0.05). In contrast, the expression level of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in LCSLC was lower than that in HepG2 cells (P<0.01), there was no significant change in PD-L1 expression after down-regulated by inhibitor treatment (P>0.05). After the expression of PD-L1 was knockdown by siRNA, the expressions of CD133, Nanog, Oct4A and Snail in LCSLC were decreased compared with those of siRNA-negative control (NC) group (P<0.05). The number of sphere-formating cells decreased [(45.33±12.01) and (282.00±29.21), P<0.001], the cell migration rate was lower than that in siRNA-NC group [(20.86±2.74)% and (46.73±15.43)%, P=0.046], the number of transmembrane cells decreased [(39.67±1.53) and (102.70±11.59), P=0.001], the number of cloned cells decreased [(57.67±14.57) and (120.70±15.04), P=0.007], the number of cells in G(0)/G(1) phase decreased [(37.68±2.51) and (57.27±0.92), P<0.001], the number of cells in S phase was more than that in siRNA-NC group [(30.78±0.52) and (15.52±0.83), P<0.001]. Tumor formation in mice showed that the tumor weight of shRNA-PD-L1 group was (0.47±0.12)g, the volume is (761.3±221.4)mm(3), were lower than those of shRNA-NC group [(1.57±0.45)g and (1 829.0±218.3)mm(3), P<0.001]. Meanwhile, the expression levels of p-STAT3 and p-Akt in siRNA-PD-L1 group were decreased (P<0.05), while the expression levels of p-ERK1/2 and β-catenin did not change significantly (P>0.05). Conclusion: Elevated PD-L1 expression in CD133(+) LCSLC is crucial to maintain stemness and promotes the tumor biological function of LCSLC.
Humans ; Animals ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; B7-H1 Antigen/metabolism* ; Ligands ; Liver Neoplasms/pathology* ; RNA, Small Interfering/metabolism* ; Neoplastic Stem Cells/physiology* ; Cell Line, Tumor ; Cell Proliferation

Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders, the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown. Here, we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem cells (hMSCs). Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration, increases mitochondrial reactive oxygen species (ROS) production, and accelerates cellular senescence. Mechanistically, the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes, especially several key subunits of complex III including UQCRC2. Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs. These f indings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis, particularly for the mitochondrial respiration complex III, thus providing a new potential target to counteract human stem cell senescence.
Mesenchymal Stem Cells/physiology* ; Cellular Senescence ; Homeostasis ; Cell Cycle Proteins/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; Mitochondria/metabolism* ; Electron Transport Complex III/metabolism* ; Humans ; Cells, Cultured

BACKGROUND: Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study. METHODS: Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA for 24 hours followed by co-treatment with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway. RESULTS: The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 μmol/L, 100%; 2.5 μmol/L, 98.58%; 5.0 μmol/L, 95.18%; 10.0 μmol/L, 98.11%; 50.0 μmol/L, 99.38%, F   =  2.33, P   >  0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 μmol/L BA, 12.27% vs. Dex, 39.27%, t  = 20.62; flow cytometry: Dex + 50.0 μmol/L BA, 12.68% vs. Dex, 37.43%, t  = 11.56; Both P  < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 μmol/L BA, 1.09 vs. Dex, 2.76, t  = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 μmol/L BA, 0.38 vs . Dex, 0.73, t  = 10.62; SHH: Dex + 50.0 μmol/L BA, 0.50 vs . Dex, 0.12, t  = 34.01; SUFU: Dex+ 50.0 μmol/L BA, 0.75 vs . Dex, 1.19, t  = 10.78; GLI-1: Dex+ 50.0 μmol/L BA, 0.40 vs . Dex, 0.11, t  = 30.68. All P  < 0.05). CONCLUSIONS BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.
Humans ; Hedgehog Proteins/metabolism* ; bcl-2-Associated X Protein ; Caspase 3/metabolism* ; Signal Transduction/physiology* ; Apoptosis ; Apoptosis Regulatory Proteins/pharmacology* ; Dexamethasone/pharmacology* ; Mesenchymal Stem Cells/metabolism* ; Bone Marrow Cells

The study aims to explore the effect of mesenchymal stem cells-derived exosomes (MSCs-Exo) on staurosporine (STS)-induced chondrocyte apoptosis before and after exposure to pulsed electromagnetic field (PEMF) at different frequencies. The AMSCs were extracted from the epididymal fat of healthy rats before and after exposure to the PEMF at 1 mT amplitude and a frequency of 15, 45, and 75 Hz, respectively, in an incubator. MSCs-Exo was extracted and identified. Exosomes were labeled with DiO fluorescent dye, and then co-cultured with STS-induced chondrocytes for 24 h. Cellular uptake of MSC-Exo, apoptosis, and the protein and mRNA expression of aggrecan, caspase-3 and collagenⅡA in chondrocytes were observed. The study demonstrated that the exposure of 75 Hz PEMF was superior to 15 and 45 Hz PEMF in enhancing the effect of exosomes in alleviating chondrocyte apoptosis and promoting cell matrix synthesis. This study lays a foundation for the regulatory mechanism of PEMF stimulation on MSCs-Exo in inhibiting chondrocyte apoptosis, and opens up a new direction for the prevention and treatment of osteoarthritis.
Animals ; Rats ; Apoptosis ; Chondrocytes ; Electromagnetic Fields ; Exosomes/physiology* ; Mesenchymal Stem Cells/metabolism*

The homing and engraftment of hematopoietic stem cells (HSC) into bone marrow is the first critical step for successful clinical hematopoietic stem cell transplantation (HSCT). SDF-1 / CXCR4 is considered to be a very promising target to promote HSC homing. In recent years, with the in-depth research on the HSC homing, a variety of new strategies for promoting HSC homing and engraftment have been explored, such as nuclear hormone receptor, histone deacetylase inhibitor, prostaglandin and metabolic regulation, so as to increase the success rate of HSCT and improve the survival of patients. In this review, the recent research advances in the mechanism of HSC homing and strategies to promote HSC homing and engraftment were summarized and discussed.
Humans ; Hematopoietic Stem Cells/physiology* ; Bone Marrow ; Hematopoietic Stem Cell Transplantation ; Gene Expression Regulation ; Prostaglandins/metabolism*

Bone marrow macrophage is an important component of bone marrow microenvironment, which is closely related to hematopoietic regulation and hematopoietic stem cell transplantation(HSCT). Recent studies have shown that bone marrow macrophage is an important part of hematopoietic stem cell niche, which can help regulate the mobilization and function of hematopoietic stem/progenitor cells. After HSCT, the microenvironment of bone marrow is damaged and a large number of macrophages infiltrate into the bone marrow. Regulating the macrophage-related signal pathways can promote the recovery of hematopoiesis and the reconstruction of hematopoietic function. Co-culture of macrophages and hematopoietic stem cells (HSC) in vitro significantly increased the number of HSCs and their ability of clone formation, which suggests that macrophages play an important role in the regulation of hematopoiesis in the hematopoietic microenvironment of bone marrow. This paper reviews the recent research progress on the role of macrophages in bone marrow hematopoietic microenvironment.
Humans ; Bone Marrow/metabolism* ; Hematopoietic Stem Cells/physiology* ; Hematopoiesis/physiology* ; Stem Cell Niche ; Macrophages/metabolism*