Human menstrual blood–derived stem cells promote the repair of impaired endometrial stromal cells by activating the p38 MAPK and AKT signaling pathways

Multiple studies have confirmed that human menstrual blood–derived stem cells (MenSCs) have potential applications in regenerative medicine or cell therapy. However, the contribution of MenSCs to endometrial repair is currently unknown. We evaluated the protective effects of MenSCs on impaired endometrial stromal cells (ESCs), as well as the signaling pathways involved in this process. Mifepristone was used to damage human ESCs, which were subsequently cocultured with MenSCs. The proliferation, apoptosis, and migration of ESCs were assessed, together with the expression of related signaling proteins including total p38 mitogen-activated protein kinase, P-p38, total protein kinase B (AKT), P-AKT, β-catenin, and vascular endothelial growth factor (VEGF). MenSCs significantly recovered the proliferation and migration ability of impaired ESCs, inhibited ESC apoptosis, and upregulated protein expression of P-AKT, P-p38, VEGF, and β-catenin. Our findings suggest that MenSC-based therapies could be promising strategies for the treatment of endometrial injury, and that AKT and p38 signaling pathways may be involved in this process.     

不同培养代次对经血源子宫内膜干细胞生物学活性的影响

该研究明确了不同培养代次经血源子宫内膜干细胞(menstrual blood derived endometrial stem cells,MenSCs)的生物学活性差异,为深入研究MenSCs生物学特性及其潜在临床应用提供理论支持。该研究使用钙黄绿素AM(Calcein-AM)染色检测体外培养至第三代(passage 3,P3)、P9和P15代MenSCs的形态;β-半乳糖苷酶染色检测不同培养代次MenSCs的衰老程度;活性氧试剂盒检测不同培养代次MenSCs中活性氧的变化;随后利用MTT、流式细胞术及细胞活死染色在接触式共培养条件下检测P3、P9和P15代MenSCs对小鼠脾淋巴细胞活性、细胞周期、死亡情况以及脾脏淋巴细胞中CD3^+和CD19^+淋巴细胞比例的影响。结果表明,随着培养代次的增加MenSCs细胞面积显著增大,在培养至P15代时MenSCs开始出现大量丝状伪足。衰老程度及活性氧的含量也随培养代次的增加而显著升高;随后与MenSCs接触式共培养体显著增加了小鼠脾淋巴细胞的活性,降低淋巴细胞死亡率,且随培养代次的增加,MenSCs对促进淋巴细胞存活、降低死亡的能力显著降低;进一步细胞周期检测发现,MenSCs无刺激淋巴细胞增殖分裂活性,但可显著降低淋巴细胞死亡及碎片化,且培养代次的增加可显著降低MenSCs维持淋巴细胞存活的能力;此外,不同培养代次MenSCs在体外均对小鼠脾淋巴细胞中CD3^+和CD19^+细胞亚群百分比无显著性影响。综上,MenSCs随着体外培养时间和代次的增加,出现明显的生物学活性降低等特征,且对淋巴细胞活性的调节能力显著降低,上述结果为临床应用中保障MenSCs质量、平衡细胞培养代次和细胞数量及保证稳定的MenSCs临床治疗效果提供理论支持。     

经血源子宫内膜干细胞培养、鉴定及体外分化潜能的研究

现阶段干细胞的来源常具有侵入性,该文旨在研究新来源于经血的经血源子宫内膜干细胞（menstrual blood-derived mesenchymal stem cells,MenSCs）的基本生物学特性及分化潜能。采用密度梯度法从女性经血中分离MenSCs,测定MenSCs群体倍增时间,流式细胞仪鉴定细胞表面抗原,免疫荧光法检测MenSCs nestin阳性表达情况,体外验证其成骨成脂分化潜能。结果表明,MenSCs具有典型的梭状结构,细胞倍增时间为32.2 h,均一地高表达CD29、CD90及CD105,不表达CD14、CD45、HLA-DR。免疫荧光表明,MenSCs为nestin阳性。MenSCs成脂诱导后,油红O染色为阳性。成骨诱导前期诱导组细胞胶原表达量升高,诱导两周后MenSCs形成钙结节,诱导组细胞ALP（alkaline phosphatase）活性连续3周呈上升趋势。以上证明,MenSCs具有来源广泛的优势,具有较高的增殖能力、较低免疫原性、nestin阳性及多向分化潜能等特性,可成为干细胞治疗的理想种子细胞。     

Biological characteristics of human menstrual blood‐derived endometrial stem cells

Successful isolation of human endometrial stem cells from menstrual blood, namely menstrual blood‐derived endometrial stem cells (MenSCs), has provided enticing alternative seed cells for stem cell‐based therapy. MenSCs are enriched in the self‐regenerative tissue, endometrium, which shed along the periodic menstrual blood and thus their acquisition involves no physical invasiveness. However, the impact of the storage duration of menstrual blood prior to stem cell isolation, the age of the donor, the number of passages on the self‐renewing of MenSCs, the paracrine production of biological factors in MenSCs and expression of adhesion molecules on MenSCs remain elusive. In this study, we confirmed that MenSCs reside in shedding endometrium, and documented that up to 3 days of storage at 4°C has little impact on MenSCs, while the age of the donor and the number of passages are negatively associated with proliferation capacity of MenSCs. Moreover, we found that MenSCs were actually immune‐privileged and projected no risk of tumour formation. Also, we documented a lung‐ and liver‐dominated, spleen‐ and kidney‐involved organic distribution profile of MenSC 3 days after intravenous transfer into mice. At last, we suggested that MenSCs may have potentially therapeutic effects on diseases through paracrine effect and immunomodulation.     

Promising effects of exosomes from menstrual blood-derived mesenchymal stem cells on endometriosis

Endometriosis as a non-malignant gynecological disease leads to dysregulation of numerous cellular functions including apoptosis, angiogenesis, migration, proliferation, and inflammation. Accumulating evidence has shed light on the importance of endometrial stem cells within the menstrual blood which are involved in the establishment and progression of endometriotic lesions in a retrograde manner. According to the fact that the therapeutic benefits of mesenchymal stem cells are provided through paracrine functions, we used exosomes from menstrual blood-derived stem cells (MenSCs) for treating endometriotic stem cells to inhibit their lesion formation tendency. Menstrual blood samples from healthy and endometriosis women were collected. Isolated MenSCs by the density-gradient centrifugation method were characterized by flow cytometry. Secreted exosomes were isolated from healthy MenSCs (NE-MenSCs) and used to treat endometriotic cells (E-MenSCs). 72 h after treatment, different mechanisms and pathways including inflammation, proliferation, apoptosis, migration, and angiogenesis were analyzed using Real-Time PCR, ELISA, immunocytochemistry, annexin V/PI, and scratching assay. Exosome treatment significantly reduce the expression level of markers related to inflammation, proliferation, migration, and angiogenesis in E-MenSCs which are aberrantly expressed in endometriosis. Moreover, apoptosis was induced in E-MenSCs after treatment which was evaluated in both gene and protein levels. In this study, we give preliminary evidence for the potential of MenSCs-Exo in ameliorating endometriosis. Regarding our results, we suggest that after relevant clinical trial, MenSCs-derived exosomes can be considered as a better treatment option to improve endometriosis compared to common and conventional treatments and show their potential as a cell-free product in endometriosis repair.     

Placenta Stem/Stromal Cell–Derived Extracellular Vesicles for Potential Use in Lung Repair

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next‐generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue‐repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.     

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.     

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixtyfold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G₀/G₁ cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling. © 1994 Wiley-Liss, Inc.     

CRMP2 derived from cancer associated fibroblasts facilitates progression of ovarian cancer via HIF-1α-glycolysis signaling pathway

As the predominant stroma cells of tumor microenvironment (TME), cancer associated fibroblasts (CAFs) are robust tumor player of different malignancies. However, less is known about the regulatory mechanism of CAFs on promoting progression of ovarian cancer (OvCA). In the present study, the conditioned medium of primary CAFs (CAF-CM) from OvCA was used to culture cell lines of epithelial ovarian cancer (EOC), and showed a potent role in promoting proliferation, migration and invasion of cancer cells. Mass spectrum (MS) analysis identified that Collapsin response mediator protein-2 (CRMP2), a microtubule-associated protein involved in diverse malignancies, derived from CAFs was a key regulator responsible for mediating these cell events of OvCA. In vitro study using recombinant CRMP2 (r-CRMP2) revealed that the protein promoted proliferation, invasion, and migration of OvCA cells through activation of hypoxia-inducible factor (HIF)-1α-glycolysis signaling pathway. The CRMP2 was abundantly expressed in OvCA, with a well correlation with metastasis and poor prognosis, as analyzed from 118 patients’ samples. Inhibition of the CRMP2 derived from CAFs by neutralizing antibodies significantly attenuated the tumor size, weights, and metastatic foci numbers of mice in vivo. Our finding has provided a novel therapeutic clue for OvCA based on TME.Subject terms: Cancer, Tumour biomarkers     

Engineering the migration and attachment behaviour of primary dermal fibroblasts

The availability of primary cells present in pathological conditions is often very limited due to stringent ethical regulation and patient consent. One such condition is chronic wounds, where dermal fibroblasts show a deficient migration. In vitro models with cellular tools that mimic the in vivo scenario would be advantageous to test new therapies for these challenging wounds. Since the availability of primary dermal fibroblasts present in chronic wounds is restricted and their “shelf-life” limited due to the increased senescence, our aim was to engineer human dermal fibroblasts with impaired migration using synthetic Arg-Gly-Asp (RGD) peptides. We studied fibroblast behaviour on three different two dimensional (2D) surfaces, representative of the dermal extracellular matrix and the materials used in the development of dermal scaffolds, in addition to commercially available, collagen-based 3D dermal scaffolds, demonstrating that the concentration of synthetic RGD peptides necessary to impair migration of dermal fibroblasts should be tailored to the particular surface/material and cell population used. The described technology could be translated to other cell types including established cell lines. A wide range of synthetic peptides exists, which differ in the amino acid sequence, thus increasing the possibilities of this technology.     

Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix

Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.     

microRNA-501-5p promotes cell proliferation and migration in gastric cancer by downregulating LPAR1

In spite of the achievement in treatment, the gastric cancer (GC) mortality still remains high. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play a crucial part in tumor progression. In this study, we explored the expression and function of microRNA-501-5p (miR-501-5p) in GC cell lines. Quantitative real-time polymerase chain reaction assay results suggested that miR-501-5p was significantly upregulated in GC tissues and cell lines. And, the Cell Counting Kit-8 colony formation and cell migration assay results showed that the downregulation of miR-501-5p decreased GC cell proliferation and migration. Besides that, we found that GC cell cycle was arrested in G2 phase and cell apoptosis rate was increased by silencing the expression of miR-501-5p in GC cell lines using the flow cytometry. We also found that miR-501-5p could directly target lysophosphatidic acid receptor 1 (LPAR1) and negatively regulate LPAR1 expression in GC cell lines by performing dual-luciferase reporter gene assay and Western blot analysis. And, LPAR1 was significantly downregulated in GC tissues and inversely correlated with miR-501-5p expression. Furthermore, LPAR1 downregulation promoted cell proliferation and migration, which were attenuated by cotransfection of miR-501-5p inhibitor in GC cells. In conclusion, miR-501-5p can promote GC cell proliferation and migration by targeting and downregulating LPAR1. miR-501-5p/LPAR1 may become a potential therapeutic target for GC treatment.     

Signaling pathways regulating dose-dependent dual effects of TNF-α on primary cultured Schwann cells

After peripheral nerve injury, Schwann cells are rapidly activated to participate in the regenerative process and modulate local immune reactions. Tumor necrosis factor-α (TNF-α), one of the major initiators of the inflammatory cascade, has been known to exert pleiotropic functions during peripheral nerve injury and regeneration. In this study, we aimed to investigate the in vitro effects of TNF-α on peripheral neural cells. First, gene-microarray analysis was applied to the RNA samples extracted from injured peripheral nerves, providing the information of gene interactions post nerve injury. Then, after primary cultured Schwann cells were treated with increasing dosages (0–40 ng/ml) of TNF-α, cell proliferation and migration were examined by EdU incorporation and a transwell-based assay, and cell apoptosis was observed and quantified by electron microscopy and Annexin V-FITC assay, respectively. The results showed that lower dosages of TNF-α increased cell proliferation and migration, whereas higher dosages of TNF-α decreased cell proliferation and migration and enhanced cell apoptosis. The tests using a chemical inhibitor of TNF-α further confirmed the above effects of TNF-α. To understand how TNF-α produced the dose-dependent dual effects on primary cultured Schwann cells, we performed co-immunoprecipitation, Western blot analysis, and immunocytochemistry to decipher the complex network of biochemical pathways involving many signaling molecules, i.e., TNF receptor-associated death domain, Fas-associated death domain, receptor interacting protein, JNK, NF-κB p65, and caspases, thus assuming the mechanisms by which TNF-α activated the death and survival pathways and achieved a balance between the two opposite actions in primary cultured Schwann cells.     

hsa_circRNA_100533 regulates GNAS by sponging hsa_miR_933 to prevent oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the head and neck region. Circular RNA (circRNA), as one kind of noncoding RNA, involves in biological processes in diverse cancers. circRNA functions mainly as the microRNA (miRNA) sponge, competitively binding to miRNAs to regulate target gene expressions. However, the expression profiles and roles of circRNAs in OSCC are still unexplored. circRNA microarrays and quantitative real-time polymerase chain reaction was used to identify the hsa_circRNA_100533 downregulated in OSCC tissues and cell lines. Bioinformatics methods were used to predict the interactions among circRNAs, miRNA, and target genes. Based on the luciferase reporter assay and AGO2 RIP assay, we found that hsa_circRNA_100533 binds to miRNAs as a miRNA sponge. hsa_circRNA_100533 inhibited cell proliferation, migration, and promoted cell apoptosis in OSCC cell lines, which could be blocked by hsa-miR-933 overexpression. hsa_circRNA_100533 binds to hsa-miR-933 as a miRNA sponge to regulate GNAS expression, and to modulate cell proliferation, migration, and apoptosis. In summary, the hsa_circRNA_100533-miR-933-GNAS axis affect the proliferation and apoptosis of OSCC cells through the mechanism of competing endogenous RNAs. hsa_circRNA_100533 may function as promising diagnostic biomarkers and effective therapeutic targets for OSCC.     

Effects of the carbohydrate-binding protein galectin-3 on the invasiveness of human breast carcinoma cells

Galectin-3 is a Mr 30,000 protein with carbohydrate-binding specificity for type I and II ABH blood group epitopes and polylactosamine glycans expressed on cell surface and extracellular matrix glycoproteins such as laminin. Cell lines propagated from human normal mammary epithelia and from benign or infiltrating components of primary breast tumours express low levels of galectin-3 in the cytoplasm. However, galectin-3 when added exogenously in solution or when bound within a three-dimensional matrix markedly enhanced the migration of the primary tumour cell lines through a Matrigel barrier. Galectin-3 expression in the cytoplasm and intercellularly on surface membranes was greatly increased in cell lines propagated from malignant ascites and pleural effusions of late stage breast cancer. These cell lines were non-invasive in the Matrigel assay and exogenous galectin-3 had no enhancing effect on invasiveness. These results suggest that galectin-3 could play multiple roles in cell metastasis at an early invasive stage by acting in a paracrine manner to stimulate cell migration through an extracellular matrix, and in later stage cancers in synergy with other mediators of cell-cell aggregation. However, endogenous galectin-3 expression in human breast cancers is not correlated directly with their invasive potential in vitro. © 1996 Wiley-Liss, Inc.     

Promotive effects of human induced pluripotent stem cell-conditioned medium on the proliferation and migration of dermal fibroblasts

Fibroblasts are a major cell type in the dermis. When skin is wounded in various ways such as by abrasions, cuts or diabetic ulcer, proliferation and migration of dermal fibroblasts is necessary for cutaneous wound healing. Numerous studies have shown that adult stem cells secrete paracrine factors and these are able to promote wound healing by activating migration and proliferation of effector cells such as dermal fibroblasts. However, the paracrine factors secreted from pluripotent stem cells and the effect of these on dermal fibroblast proliferation and migration have been poorly characterized. In this study we cultured human induced pluripotent stem cells without any animal-derived components including feeder cells, and investigated the effect of stem cell-conditioned medium (iPSC-CM) on dermal fibroblast proliferation and migration. Results showed that the proliferation of mouse embryonic fibroblasts (STO cells) and human dermal fibroblasts (HDFs) were significantly stimulated by iPSC-CM. We determined that the optimal concentration of iPSC-CM in promoting the proliferation of HDFs was a 75% dilution. Scratch wound assay and transwell migration assay also demonstrated the stimulatory effect of iPSC-CM on the migration of HDFs. iPSC-CM is believed to have advantages because of the unique capabilities of iPSCs, which include infinite self-renewal, pluripotency and variety of donor cells. Thus, iPSC-CM is anticipated to be a valuable source of paracrine factors which can potentially be used for wound healing applications.     

Mesenchymal stem cells from different sources and their derived exosomes: A pre-clinical perspective

Since the introduction of cell therapy as a strategy for the treatment of many diseases, mesenchymal stem cells have emerged as ideal candidates, yet the underlying mechanisms of their beneficial effects are only partially understood.At the start of the 21 st century, a paracrine effect was proposed as a mechanism of tissue repair by these cells. In addition, a role was suggested for a heterogeneous population of extracellular vesicles in cell-to-cell communication.Some of these vesicles including exosomes have been isolated from most fluids and cells, as well as from supernatants of in vitro cell cultures. Recent research in the field of regenerative medicine suggests that exosomes derived from mesenchymal stem cells could be a powerful new therapeutic tool. This review examines the therapeutic potential of these exosomes obtained from the sources most used in cell therapy: bone marrow, adipose tissue, and umbilical cord.     

Tricin inhibits proliferation of human hepatic stellate cells in vitro by blocking tyrosine phosphorylation of PDGF receptor and its signaling pathways

4',5,7-Trihydroxy-3',5'-dimethoxyflavone (Tricin), a naturally occurring flavone, has anti-inflammatory potential and exhibits diverse biological activities including antigrowth activity in several human cancer cell lines and cancer chemopreventive effects in the gastrointestinal tract of mice. The present study aimed to investigate the biological actions of tricin on hepatic stellate cells (HSCs) in vitro, exploring its potential as a treatment of liver fibrosis, since HSC proliferation is closely related to the progression of hepatic fibrogenesis in chronic liver diseases leading to irreversible liver cirrhosis and hepatocellular carcinoma. Tricin inhibited platelet-derived growth factor (PDGF)-BB-induced cell proliferation by blocking cell cycle progression and cell migration in the human HSC line LI90 and culture-activated HSCs. It also reduced the phosphorylation of PDGF receptor β and the downstream signaling molecules ERK1/2 and Akt, which might be due to its tyrosine kinase inhibitor properties rather than inhibition of the direct binding between PDGF-BB and its receptor. Our findings suggest that tricin might be beneficial in HSC-targeting therapeutic or chemopreventive applications for hepatic fibrosis.