Detection Of Cell Surface Markers Research Articles

Technique for Isolation and Culture of Rat Jaw Bone Marrow Mesenchymal Stem Cells.

Bone marrow mesenchymal stem cells (BMMSCs) are a type of stem cell with multi-directional differentiation potential. Compared with BMMSCs derived from appendicular bones, BMMSCs derived from the jaw have greater proliferative and osteogenic differentiation ability, gradually becoming important seed cells for jaw defect repair. However, the mandible has a complex bony structure and less cancellous content than appendicular bones. It is difficult to acquire a large number of high-quality jaw-derived marrow mesenchymal stem cells using traditional methods. This study presents a 'niche-based approach on stemness' for isolating and culturing rat jaw bone marrow mesenchymal stem cells (JBMMSCs). Primary rat JBMMSCs were isolated and cultured using the whole bone marrow adherent method combined with the bone slice digestion method. The isolated cells were identified as JBMMSCs through cell morphology observation, detection of cell surface markers, and multi-directional differentiation induction. The cells extracted by this method exhibit a 'fibroblast-like' spindle shape. The cells are long, spindle-shaped and fibroblast-like. The flow cytometry analysis shows these cells are positive for CD29, CD44, and CD90 but negative for CD11b/c, CD34, and CD45, which is congruent with BMMSCs characteristics. The cells show strong proliferation capacity and can undergo osteogenic, adipogenic, and chondrogenic differentiation. This study provides an effective and stable method for obtaining enough high-quality JBMMSCs with strong differentiation ability in a short time, which could facilitate further studies of the exploration of biological function, regenerative medicine, and related clinical applications.

Differentiation Potential Analysis of Bone Marrow Mesenchymal Stem Cells from Guizhou Miniature Pigs

In this study, different growth characteristics of hematopoietic stem cells and bone marrow mesenchymal stem cells were used to isolate and culture BMSCs by whole bone marrow adhesion method, and RT-PCR was used to detect cell surface markers CD29, CD31, CD34, CD44, CD71, CD73, CD90. The differentiation of osteocytes into adipocytes and osteoblasts was induced in vitro, and the differentiation ability was observed by staining with oil red O and rubinin. To investigate the isolation and culture methods of BMSCs from Guizhou miniature pigs (Sussa lvanius) in vitro, and to study their biological characteristics and potential for multi-differentiation. The results showed that a small number of adherent cells could be seen in the primary BMSCs cultured in vitro within 48 hours, and the confluent cells reached 90% in 5~6 days. The cell growth state was good, and the shape was spindle and polygonal. RT-PCR was used to detect the positive expression of CD29, CD44, CD71, CD73 and CD90 on the cell surface, and the negative expression of CD31 and CD34. Oil red O staining showed the formation of lipid droplets in the cells, and alizarine red staining showed calcium nodules in the cells, indicating that the cells isolated and cultured in this experiment had the ability of multidirection differentiation. It is consistent with the characteristics of BMSCs. The growth of BMSCs cultured in vitro was stable, and the expanded BMSCs had multidirectional differentiation potential, which was an ideal seed cell for tissue engineering.

DNA‐Mediated, On‐Membrane Sequential Assembly of Conjugated Polymer Nanoparticles for Sensitive Detection of Cell Surface Markers

AbstractFlow cytometry can provide detailed information about protein expression on cell surface and is, therefore, widely used in clinical testing. However, owing to the limited sensitivity of fluorescence signals, detection of low‐expression cell surface markers is challenging. The present report describes a DNA‐mediated, on‐membrane assembly of conjugated polymer nanoparticles (Pdots) that amplifies fluorescence signal from surface markers for sensitive detection via flow cytometry. Single‐stranded DNA (ssDNA)‐conjugated antibodies are first bound to cell surface markers, from which ssDNA‐modified Pdots are sequentially assembled using DNA hybridization. The use of DNA as a linker enables the distance‐controlled assembly of Pdots to prevent fluorescence quenching, whereas their on‐membrane sequential assembly allows amplification of the fluorescence signal without reducing binding ability of antibodies. Thus, two rounds of Pdot assembly achieve 31‐fold amplification of the fluorescence signal from CD19 on Nalm‐6 cells, which is 125‐fold brighter than that obtained using the conventional fluorescent dye‐based method. Moreover, the sequential assembly of 22 nm Pdots shows 24‐fold higher fluorescence than one‐step labeling with 81 nm Pdots, suggesting the advantage of the sequential assembly strategy in avoiding steric hindrance. The proposed method is expected to contribute to the sensitive detection of low‐expression surface markers for early and accurate diagnosis.

Feline umbilical cord-derived mesenchymal stem cells: isolation, identification, and antioxidative stress role through NF-κB signaling pathway.

At present, the differentiation potential and antioxidant activity of feline umbilical cord-derived mesenchymal stem cells (UC-MSCs) have not been clearly studied. In this study, feline UC-MSCs were isolated by tissue adhesion method, identified by flow cytometry detection of cell surface markers (CD44, CD90, CD34, and CD45), and induced differentiation toward osteogenesis and adipogenesis in vitro. Furthermore, the oxidative stress model was established with hydrogen peroxide (H2O2) (100 μM, 300 μM, 500 μM, 700 μM, and 900 μM). The antioxidant properties of feline UC-MSCs and feline fibroblasts were compared by morphological observation, ROS detection, cell viability via CCK-8 assay, as well as oxidative and antioxidative parameters via ELISA. The mRNA expression of genes related to NF-κB pathway was detected via quantitative real-time polymerase chain reaction, while the levels of NF-κB signaling cascade-related proteins were determined via Western Blot. The results showed that feline UC-MSCs highly expressed CD44 and CD90, while negative for CD34 and CD45 expression. Feline UC-MSCs cultured under osteogenic and adipogenic conditions showed good differentiation capacity. After being exposed to different concentrations of H2O2 for eight hours, feline UC-MSCs exhibited the significantly higher survival rate than feline fibroblasts. A certain concentration of H2O2 could up-regulate the activities of SOD2 and GSH-Px in feline UC-MSCs. The expression levels of p50, MnSOD, and FHC mRNA in feline UC-MSCs stimulated by 300 μM and 500 μM H2O2 significantly increased compared with the control group. Furthermore, it was observed that 500 μM H2O2 significantly enhanced the protein levels of p-IκB, IκB, p-p50, p50, MnSOD, and FHC, which could be reversed by BAY 11-7,082, a NF-κB signaling pathway inhibitor. In conclusion, it was confirmed that feline UC-MSCs, with good osteogenesis and adipogenesis abilities, had better antioxidant property which might be related to NF-κB signaling pathway. This study lays a foundation for the further application of feline UC-MSCs in treating the various inflammatory and oxidative injury diseases of pets.

Immunological characteristics of human umbilical cord mesenchymal stem cells after hepatogenic differentiation.

Acute liver failure is one of the most intractable clinical problems. The use of bioartificial livers may solve donor shortage problems. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an excellent seed cell choice for artificial livers because they change their characteristics to resemble hepatocyte-like cells (HLCs) following artificial liver transplantation. This study aimed to determine whether the immunological characteristics of hUCMSCs are changed after being transformed into hepatocyte-like cells. HUCMSCs were isolated by the adherent method. The following hUCMSC surface markers were detected using flow cytometry: CD45, CD90, CD105, CD34, and octamer-binding transcription factor 4 (OCT-4). Functional detection of adipogenic differentiation was performed. The hUCMSCs were cultured in complete medium (control group) or induction medium (induction group), and flow cytometry was used to detect cell surface markers. Peritoneal lavage fluid was collected after intraperitoneal injection of 1 × 106 cells/mouse over 40minutes. The leukocyte count, labeled CD45, CD3, CD4 and CD8 antibodies, and flow detection of T lymphocyte subsets were determined using the peritoneal lavage fluid. Using phenotypic and functional identification, hUCMSCs were successfully isolated using a two-step induction method. The surface markers of the hUCMSCs cells changed after HLC induction. Invivo immune results showed that hUCMSCs and HLsC induced leukocyte production. Hepatic induction of hUCMSCs changes their cell surface markers. Both HLCs and hUCMSCs cause leukocytosis invivo, but the immune response induced by HLCs is slightly stronger.

Isolation, culture and identification of SD rat bone marrow mesenchymal stem cells in tissue engineering

Seed cells are an important part of bone tissue engineering. In this study, we investigated methods for the isolation, culture and identification of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs from SD rats were isolated and cultured using the whole bone marrow adherent method. Morphological observation and flow cytometry were used for the detection of cell surface markers. The osteogenic and adipogenic differentiation of cultured cells was induced and evaluated. BMSCs could be effectively isolated and purified using the whole bone marrow adherent method. The primary BMSCs cultured in vitro formed spindle-shaped, spiral and vigorous cell colonies. The flow cytometry results showed that CD90 was highly expressed on the cell surface, but CD34 and CD45 were not expressed. After culture in osteogenic and adipogenic induction medium, positive Alizarin Red and Oil Red O staining was observed, indicating the potential for multidirectional differentiation. The whole bone marrow adherent method is simple and practical, which makes the isolation, culture and amplification of BMSCs in vitro easy, and the method has little effect on cell activity and is an effective method for obtaining high-purity BMSCs.

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines.

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

Ricolinostat promotes the generation of megakaryocyte progenitors from human hematopoietic stem and progenitor cells

BackgroundEx vivo production of induced megakaryocytes (MKs) and platelets from stem cells is an alternative approach for supplying transfusible platelets. However, it is difficult to generate large numbers of MKs and platelets from hematopoietic stem cells and progenitor cells (HSPCs).MethodsTo optimize the differentiation efficiency of megakaryocytic cells from HSPCs, we first employed a platelet factor 4 (PF4)-promoter reporter and high-throughput screening strategy to screen for small molecules. We also investigated the effects and possible mechanisms of candidate small molecules on megakaryocytic differentiation of human HSPCs.ResultsThe small molecule Ricolinostat remarkably promoted the expression of PF4-promoter reporter in the megakaryocytic cell line. Notably, Ricolinostat significantly enhanced the cell fate commitment of MK progenitors (MkPs) from cord blood HSPCs and promoted the proliferation of MkPs based on cell surface marker detection, colony-forming unit-MK assay, and quantitative real-time PCR analyses. MkPs generated from Ricolinostat-induced HSPCs differentiated into mature MKs and platelets. Mechanistically, we found that Ricolinostat enhanced MkP fate mainly by inhibiting the secretion of IL-8 and decreasing the expression of the IL-8 receptor CXCR2.ConclusionThe addition of Ricolinostat to the culture medium promoted MkP differentiation from HSPCs and enhanced the proliferation of MkPs mainly by suppressing the IL-8/CXCR2 pathway. Our results can help the development of manufacturing protocols for the efficient generation of MKs and platelets from stem cells in vitro.

Comparative Analysis of Human Adipose-Derived Stromal/Stem Cells and Dermal Fibroblasts.

Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.

Sublethal doxorubicin promotes migration and invasion of breast cancer cells: role of Src Family non-receptor tyrosine kinases

BackgroundDoxorubicin (Dox) is a widely used chemotherapy, but its effectiveness is limited by dose-dependent side effects. Although lower Dox doses reduce this risk, studies have reported higher recurrence of local disease with no improvement in survival rate in patients receiving low doses of Dox. To effectively mitigate this, a better understanding of the adverse effects of suboptimal Dox doses is needed.MethodsEffects of sublethal dose of Dox on phenotypic changes were assessed with light and confocal microscopy. Migratory and invasive behavior were assessed by wound healing and transwell migration assays. MTT and LDH release assays were used to analyze cell growth and cytotoxicity. Flow cytometry was employed to detect cell surface markers of cancer stem cell population. Expression and activity of matrix metalloproteinases were probed with qRT-PCR and zymogen assay. To identify pathways affected by sublethal dose of Dox, exploratory RNAseq was performed and results were verified by qRT-PCR in multiple cell lines (MCF7, ZR75-1 and U-2OS). Regulation of Src Family kinases (SFK) by key players in DNA damage response was assessed by siRNA knockdown along with western blot and qRT-PCR. Dasatinib and siRNA for Fyn and Yes was employed to inhibit SFKs and verify their role in increased migration and invasion in MCF7 cells treated with sublethal doses of Dox.ResultsThe results show that sublethal Dox treatment leads to increased migration and invasion in otherwise non-invasive MCF7 breast cancer cells. Mechanistically, these effects were independent of the epithelial mesenchymal transition, were not due to increased cancer stem cell population, and were not observed with other chemotherapies. Instead, sublethal Dox induces expression of multiple SFK—including Fyn, Yes, and Src—partly in a p53 and ATR-dependent manner. These effects were validated in multiple cell lines. Functionally, inhibiting SFKs with Dasatinib and specific downregulation of Fyn suppressed Dox-induced migration and invasion of MCF7 cells.ConclusionsOverall, this study demonstrates that sublethal doses of Dox activate a pro-invasive, pro-migration program in cancer cells. Furthermore, by identifying SFKs as key mediators of these effects, our results define a potential therapeutic strategy to mitigate local invasion through co-treatment with Dasatinib.

FGF2-induced PI3K/Akt signaling evokes greater proliferation and adipogenic differentiation of human adipose stem cells from breast than from abdomen or thigh.

In this study, human adipose stem cells were isolated from subcutaneous fat in the thigh (htASCs), abdomen (haASCs) and breast (hbASCs). Flow cytometry was used to detect cell surface markers, and an enzyme-linked immunosorbent assay was used to detect paracrine activity. Paracrine gene expression in the three cell types was examined using real-time qPCR, and adipogenic ability was assessed using Oil Red O staining. RNA from third-passage haASCs and hbASCs was sequenced. The results showed that the differentiation potential marker markers CD49d and CD54 were similar across hbASCs from 10 subjects. The hbASCs showed higher colony forming ability and expression of fibroblast growth factor-2, tissue inhibitor of metalloproteinase-1 and stromal cell derived factor-1 than htASCs and haASCs. Stimulating hbASCs with FGF2 promoted adipogenic differentiation, while treating the cells with the PI3K inhibitor LY294002 inhibited differentiation. These results suggest that the PI3K/Akt signaling pathway can promote proliferation and adipogenic differentiation of adipose stem cells, and that activation of this pathway by FGF2 may explain why hbASCs show greater proliferation and adipogenic differentiation than haASCs and htASCs.

Therapeutic effect of bone marrow mesenchymal stem cells in a rat model of carbon tetrachloride induced liver fibrosis

BackgroundLiver fibrosis is a major medical problem with high mortality and morbidity rates where the formation of regenerative nodules and cirrhosis leads to loss of liver function and may result in the development of hepatocellular carcinoma. bone marrow mesenchymal stem cells (BM-MSCs) have drawn attention as a novel approach for treatment of liver fibrosis. This study aimed to evaluate the therapeutic effect of BM-MSCs on the liver structure in carbon tetrachloride (CCl4) induced liver fibrosis in male rats relative to resveratrol and Silybum marianum as standard drugs derived from herbal plants. MethodsFifty adult male albino rats (Sprague Dawley strain; 180–220 g mean body weight) were purchased from the Laboratory Animal Unit in the Nile Center of Experimental Research, Mansoura, Egypt. Liver function were determined, isolation and preparation of BM- MSCs and detection of cell-surface markers by flow cytometry. ResultsAnimals exposed to CCl4 developed liver injury characterized by significant increase of liver enzymes, malondialdehyde (MDA), tumor necrosis factor alpha (TNFα), and CYP450, inhibition of antioxidant enzymes, and decreased albumin. Treatment with stem cells enhanced liver state more effectively than resveratrol and S. marianum. It significantly decreased AST, ALT, ALP, MDA, TNF-α, and CYP450 and increased albumin, SOD, GSH, GST, and CAT. Histopathological study and atomic force microscope results confirmed the therapeutic effects of MSCs. ConclusionsBM-MSCs could restore liver structure and function in CCL4 induced liver fibrosis rat model, ameliorating the toxicity of CCl4 and improving liver function tests.

CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation

Summary CRISPR-Cas9 engineered CYBBko THP-1 cell lines display an inflammatory profile with increased IL-1β, IL-6 and TNF-α secretion as consequence of NADPH-induced ROS deficiency. This phenotype is reminiscent of the one observed in phagocytes from CGD patients.

CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BackgroundChimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects.MethodsCD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs.ResultsAll retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing.ConclusionBoth CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

Biology and prognostic impact of clonal plasmacytoid dendritic cells in chronic myelomonocytic leukemia.

Islands of CD123high cells have been commonly described in the bone marrow of patients with chronic myelomonocytic leukemia (CMML). Using a multiparameter flow cytometry assay, we detected an excess of CD123+ mononucleated cells that are lineage-negative, CD45+, CD11c-, CD33-, HLA-DR+, BDCA-2+, BDCA-4+ in the bone marrow of 32/159 (20%) patients. Conventional and electron microscopy, flow cytometry detection of cell surface markers, gene expression analyses, and the ability to synthesize interferon alpha in response to Toll-like receptor agonists identified these cells as bona fide plasmacytoid dendritic cells (pDCs). Whole-exome sequencing of sorted monocytes and pDCs identified somatic mutations in genes of the oncogenic RAS pathway in the two cell types of every patient. CD34+ cells could generate high amount of pDCs in the absence of FMS-like tyrosine kinase 3-ligand(FLT3L). Finally, an excess of pDCs correlates with regulatory T cell accumulation and an increased risk of acute leukemia transformation. These results demonstrate the FLT3L-independent accumulation of clonal pDCs in the bone marrow of CMML patients with mutations affecting the RAS pathway, which is associated with a higher risk of disease progression.

SPR signals enhancement by gold nanorods for cell surface marker detection.

Introduction: The detection of micrometer-sized particles like cells is limited by surface plasmon resonance (SPR) biosensors because of having a depth of evanescent wave <500 nm. In this study, for the first time, we exhibited the use of streptavidin-functionalized gold nanorods (GNRs) as intensification labels for detection of cell surface markers in SPR-based biosensors. Methods: The GNRs (ʎ max: 735 nm) were modified with streptavidin using EDC/NHS coupling method and human umbilical vein endothelial cells (HUVECs) were selected as the cell model for detecting VE-cadherin on cell surface using real-time SPR device in the 785 nm wavelength of the laser source. Results: The investigations revealed that the plasmonic field extension produced from the gold layer and GNRs resulted in multiple enhancement of SPR signals when the wavelength of laser source in SPR instrument was matched with the wavelength of maximum absorbance in GNRs. Moreover, the results showed that the growth of ∆RU value in specific and non-specific bindings for various cell number injections were produced with increasing the cell number. Conclusion: The results displayed that cell detection can be performed in real- time form without any need to a time-consuming process used in conventional methods like immunocytochemistry, flow cytometry, and western blotting.

Human urine-derived stem cells play a novel role in the treatment of STZ-induced diabetic mice.

Human urine-derived stem cells (hUSCs) are a potential stem cell source for cell therapy. However, the effect of hUSCs on glucose metabolism regulation in type 1 diabetes was not clear. Therefore, the aim of the study was to evaluate whether hUSCs have protective effect on streptozotocin (STZ)-induced diabetes. hUSCs were extracted and cultivated with a special culture medium. Flow cytometry analysis was applied to detect cell surface markers. BALB/c male nude mice were either injected with high-dose STZ (HD-STZ) or multiple low-dose STZ (MLD-STZ). Serum and pancreatic insulin were measured, islet morphology and its vascularization were investigated. hUSCs highly expressed CD29, CD73, CD90 and CD146, and could differentiate into, at least, bone and fat in vitro. Transplantation of hUSCs into HD-STZ treated mice prolonged the median survival time and improved their blood glucose, and into those with MLD-STZ improved the glucose tolerance, islet morphology and increased the serum and pancreas insulin content. Furthermore, CD31 expression increased significantly in islets of BALB/c nude mice treated with hUSCs compared to those of un-transplanted MLD-STZ mice. hUSCs could improve the median survival time and glucose homeostasis in STZ-treated mice through promoting islet vascular regeneration and pancreatic beta-cell survival.

Therapeutic potential of BMSCs for premature ovarian failure in mice

To explore the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) on premature ovarian failure (POF) in mice induced by cyclophosphamide (CTX) and the possible mechanisms. Methods: Mouse BMSCs were identified through detection of cell surface markers by flow cytometry. The model of mouse POF was induced by intraperitoneal injection of CTX at a dose of 50 mg/kg, once daily for 15 days. BMSCs were transplanted into POF mice at 2×106 cells/mouse by tail veil. The ovarian tissues were collected for HE staining at 7 days after transplantation to observe the changes of ovarian structure and real-time PCR was performed to detect the folliculogenesis gene expression. Results: BMSCs showed positive expression of CD29 and CD90 while low expression for endothelial and hematopoietic cell markers CD31 and CD34. The numbers of primodial follicle, primary follicle, secondary follicle and antral follicle were significantly decreased, but the numbers of atretic follicle were significantly increased in CTX induced-POF mice (P<0.05). BMSCs transplantation effectively repaired the structure of damaged ovary. The significant reduction of atretic follicle and significant increase of antral follicle and secondary follicle were observed in ovaries of BMSCs-treated mouse(P<0.05). BMSCs-transplanted mouse ovaries showed the increased mRNA expression levels of Nano3, Nobox, and Lhx8 (P<0.05). Conclusion: BMSCs could effectively repair ovarian structure and promote follicle development in CTX-induced POF mouse.

Effect of hypoxia on the biological characteristics of human dental follicle cells

This study aimed to investigate the effects of hypoxia on the characteristics of human dental follicle cells (hDFCs). The tissue explant collagenase method was used to isolate hDFCs from young permanent teeth. The immunofluorescence technique was used to detect cell surface markers, and the multi-differentiation potential was detected by multilineage differentiation induction assay. Then, the hypoxic microenvironment was physically mimicked, and the cells were divided into the normoxia group (20%O₂) and the hypoxia group (2%O₂). The effects of hypoxia on cell migration and proliferation were examined by Transwell chamber test and CCK-8 assay, respectively. The gene and protein expression levels of stemness-related markers at both oxygen concentrations were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. After osteogenic induction of both groups, qRT-PCR was performed to evaluate the osteogenesis-related gene, and alizarin red staining was used to assess the formation of mineralized nodules. With the multi-differentiation capacity of osteogenic cells, adipogenic cells, and nerves, hDFCs demonstrate strong stem cell characteristics and possess the criteria of mesenchymal stem cells, which can meet the requirements of seed cells in dental tissue engineering. Hypoxia was conducive to the maintenance of hDFC stemness. Hypoxia promoted the migration and proliferation of hDFCs. The hDFCs were induced to osteogenic differentiation under hypoxic conditions, thereby enhancing osteogenesis. Hypoxic microenvironment plays an important role in maintaining the stemness and promoting the proliferation, migration, and differentiation of hDFCs. Thus, this microenvironment could also serve several important functions in future clinical applications.

Isolation and culture exploration of Anas platyrhynchos amniotic fluid stem cells in vitro

Objective: This research was designed to establish the system of isolation and culture of Anas platyrhynchos (duck) amniotic fluid stem cells (DAFSCs), and to explore its biological characteristics and differentiation ability in vitro. Material and methods: Main experimental reagents contained L-DMEM, fetal bovine serum, chicken serum, EGF, bFGF, L-glutamine, trypsin, rabbit anti-chicken CD44, CD73, CD105, nanog and SSEA-4 (Abcam, USA), FITC conjugated goat anti-rabbit secondary antibody IgG, DAPI, Trizol, inverse transcription kit, Propidium iodide, IBMX, INS, dexamethasone and indometacin. Cultivation system included L-DMEM with 10% FBS, 5% chicken serum, EGF 10 ng/mL, bFGF 10 ng/mL and 1% L-glutamine, and was cultured under 37°C, 5% CO2 and saturated humidity. Immunofluorescent detection is used to detect cell surface markers, while RT-PCR was used to detect related gene expression. Cell cycle was detected with Flow Cytometer and was analyzed by ModFitLT 2.0, induced differentiation, and Oil Red O staining. Results: More DAFSCs were gained via super-centrifugation and thermoelectric methods cost effectively. DAFSCs could go down to the future generation at passage 23(P23). CD44, CD73, CD105 and SSEA-4 were detected as positive with immunofluorescence histochemistry. GAPDH, GDNF, rex1 and JAG1 were detected as positive with RT-PCR. Cell cycle was detected on flow cytometer. Tentative exploration of differentiation ability that DAFSCs could be induced into adipocyte in vitro. Conclusion: DAFSCs can be isolated from matrix that have strong self-renewal capacity in vitro. DAFSCs can be induced into adipocyte in vitro. These testify that DAFSCs can be an ideal seeded cells having potentials for preservation and utilization of rare genetic resources. http://doi.org/10.5455/javar.2017.d200 XML PubReader