Differentiation Of Human Mesenchymal Cells Research Articles

Effect of Atmospheric Pressure Plasma Jet Treatments on Magnesium Phosphate Cements: Performance, Characterization, and Applications.

Atmospheric pressure plasma treatments are nowadays gaining importance to improve the performance of biomaterials in the orthopedic field. Among those, magnesium phosphate-based cements (MPCs) have recently shown attractive features as bone repair materials. The effect of plasma treatments on such cements, which has not been investigated so far, could represent an innovative strategy to modify MPCs' physicochemical properties and to tune their interaction with cells. MPCs were prepared and treated for 5, 7.5, and 10 min with a cold atmospheric pressure plasma jet. The reactive nitrogen and oxygen species formed during the treatment were characterized. The surfaces of MPCs were studied in terms of the phase composition, morphology, and topography. After a preliminary test in simulated body fluid, the proliferation, adhesion, and osteogenic differentiation of human mesenchymal cells on MPCs were assessed. Plasma treatments induce modifications in the relative amounts of struvite, newberyite, and farringtonite on the surfaces on MPCs in a time-dependent fashion. Nonetheless, all investigated scaffolds show a good biocompatibility and cell adhesion, also supporting osteogenic differentiation of mesenchymal cells.

The Effect of Platelet-Rich Plasma on the Osteoblastic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

Mesenchymal stem cells (MSCs) are cell populations that have the potential to proliferate and differentiate. The process of stem cell differentiation from pluripotent cells to bone cells requires general changes in their pattern of gene expression, the most well-known of which are changes in miRNA-dependent settings. Platelet-enriched plasma (PRP) releases growth factors that are mitogenic to mesenchymal cells and can accelerate the process of osteogenic differentiation. The aim of this study was to investigate the effect of PRP on the expression changes of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a during osteogenic differentiation. MSCs were isolated from adipose tissue after abdominoplasty and evaluated by flow cytometry. The ef-fect of PRP (10%) on the process of osteogenic differentiation was determined by measuring the expression of Let-7a, mir-27a, mir-31, mir-30c, mir-21, and mir-106a using the real-time polymerase chain reaction (PCR) technique. The increase in Let-7a expression was significant on the 14th day compared to the 3rd day. mir-27a expression rose significantly on the 3rd day. The expression of mir-30 exhibited a significant increase on the 14th day. mir-21 expression was significantly enhanced on the 3rd day and was downregulated on the 14th day. mir-106a expression showed a significant decreasing tendency between days 3 and 14 in a time-dependent pattern. These findings indicate that PRP probably accelerates the process of differentiation into bone. PRP, as a biological catalyst, showed a clear and distinct impact on the miRNAs regulating bone differentiation of human mesenchymal cells.

Role of Stem Cells in Augmenting Dental Implant Osseointegration: A Systematic Review

Dental implants are a widely used treatment modality for oral rehabilitation. Implant failures can be a result of many factors, with poor osseointegration being the main culprit. The present systematic review aimed to assess the effect of stem cells on the osseointegration of dental implants. An electronic search of the MEDLINE, LILACS, and EMBASE databases was conducted. We examined quantitative preclinical studies that reported on the effect of mesenchymal stem cells on bone healing after implant insertion. Eighteen studies that fulfilled the inclusion criteria were included. Various surface modification strategies, sites of placement, and cell origins were analyzed. The majority of the selected studies showed a high risk of bias, indicating that caution must be exercised in their interpretation. All the included studies reported that the stem cells used with graft material and scaffolds promoted osseointegration with higher levels of new bone formation. The mesenchymal cells attached to the implant surface facilitated the expression of bio-functionalized biomaterial surfaces, to boost bone formation and osseointegration at the bone–implant interfaces. There was a promotion of osteogenic differentiation of human mesenchymal cells and osseointegration of biomaterial implants, both in vitro and in vivo. These results highlight the significance of biomodified implant surfaces that can enhance osseointegration. These innovations can improve the stability and success rate of the implants used for oral rehabilitation.

The feasibility of human adipose-derived mesenchymal stem cells induced to endothelial-like cells and the safety of induced cells intraocular application in a short period

Objective To evaluate the feasibility of differentiation of human adipose mesenchymal stem cells (hADSCs) into endothelial-like cells and the safety of induced cells intraocular application. Methods HADSCs were induced to endothelial-like cells in vitro.The experimental group was added with streptomycin, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and the control group was added with the same amount of phosphate buffered saline (PBS), the expression of von Willebrand factor (vWF), a marker of endothelial cells, was observed in the two groups.Six C57BL/6J mice were randomly divided into experimental group and control group by using the random number method, 3 mice for each group, the induced cells were injected intravitreally into C57BL/6J mice as experimental group, and the same amount of PBS buffer was injected intravitreally into C57BL/6J mice as control group.The changes of retinal cells were observed by electron microscopy.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results After induction of hADSCs in vitro, the endothelial cell marker was expressed.VWF immunofluorescence of the experimental group showed strong green fluorescence, and the color rendering rate was 100%.The control group showed no coloration of vWF immunofluorescence, and the color positive rate was 0.After 1 month of intravitreal injection, the retinal ganglion cells and rod cells were not degraded and necrotic. Conclusions HADSCs can differentiate into endothelial-like cells in vitro, and there is no retinal toxicity in a short-term. Key words: Adipose derived mesenchymal stem cells; Endothelium-like cells; Induced differentiation; Toxicity

The ability of H2O2 to induce differentiation of human mesenchymal stem cells into chondrocytes

Background and Aim: Cartilage disorders may deteriorate following oxidative stress injuries affecting mature chondrocytes. Meantime, mesenchymal stem cells (MSCs) can differentiate into chondrocytes in the presence of oxidative conditions and act as a source of compensation for injured chondrocytes. The present study aimed to investigate the effect of H2O2 on MSCs differentiation into chondrocytes in order to cast light on the dual roles of oxidative stress in the pathogenesis of diseases. Materials and Methods: Human mesenchymal stem cells were isolated from abdominal adipose tissue of three different donors and cultured in the presence of 50 μM H2O2 in order to differentiate into chondrocytes. We determined cell viability by tetrazolium assay and measured reactive oxygen species (ROS) level by flow cytometry. Presence of glycoseaminoglycans was confirmed by safranin staining. Results: The percentage of cells containing ROS was significantly higher in the cells treated with hydrogen peroxide (29.2 ± 1) compared to that in the untreated control cells (7.7 ± 1.4). A significant increase in glycoseaminoglycan content was observed in H2O2 treated cells compared to that in the control cells both on the 9th day (treated: 1.57×104 ± 0.1 vs control: 0.91×104 ± 0.09) and 21st day (treated: 2.87×104 ± 0.2 vs control: 0.96×104 ± 0.07). In addition, comparison of glycoseaminoglycan content on the 9th and 21st days showed a significantly higher content in both treated and control cells on the 21st day (p<0.05). Conclusion: Hydrogen peroxide resulted in increased differentiation of adipose tissue-derived MSCs into chondrocytes. Therefore, we concluded that, oxidative stress had positive role in the induction of chondrocyte differentiation. © 2018, Kurdistan University of Medical Sciences. All rights reserved.

2D imprinted substrates and 3D electrospun scaffolds revolutionize biomedicine.

Momentous advances in engineering in the 1980s and 1990s reduced significantly the manufacturing cost of equipment that can produce nano to low microscale materials. This was a key milestone in the field of biomedicine, as from the early 2000s, we entered the era of nanobiomedicine. Essentially, with very low capital cost, every laboratory in the world could develop cell culture substrates and/or implantable devices with morphological features down to the nanometer scale. The rationale of using such small dimensional features derives from the fact that the extracellular space is comprised of nanoto microscale supramolecular assemblies. Further, cells employ probing filopodia extensions and contractile intracellular machinery to gather topographical, spatial, mechanical and chemical information from the extracellular environment, which consequently determines cell functionality, lineage commitment and fate. Among the various fabrication technologies, lithography constitutes the most popular 2D top-down method, while electrospinning represents the most widely used 3D bottomup method [1–7]. The produced 2D and 3D constructs have enabled the study of cell and tissue responses at previously economical and technological prohibiting small scales and the rational design thereof for a specific application/clinical indication. Key to the design tenant is that topographical features and morphological structures should promote cellular polarization and organized extracellular matrix assembly, resembling the natural conformation of tissues. Using photolithography back in 1983, microscale V-shape grooves were created to study human gingival and porcine epithelial cell response [8]. By 1997, we were in a position to fabricate substrates with feature (square) accuracy down to 14 nm (depth) to study Xenopus neurites outgrowth using electron-beam lithography [9,10]. To date, we have successfully imprinted various gratings and geometric shapes along different scales onto thermoplastic polymers to create biologically relevant topographical interfaces. Data obtained from these and other studies clearly illustrate that topographical features are powerful modulators of cell morphology and differential function [11–15]. Further, nanoand microscale arrays have been shown to be potent tools in maintaining the phenotype fidelity of permanently differentiated cells and in differentiating stem cells toward specific lineages [16,17]. Multiple topographic arrays have also been used as high-throughput systems to elucidate the cell–surface interactions and enable identification of improved surfaces for optimal/required cell response [18,19]. Specifically, topographical features have been shown to directly modulate cell–material interaction and to affect the composition, orientation and conformation of adsorbed extracellular matrix components [20,21]. Further, nanoimprinted materials have been shown to either 2D imprinted substrates and 3D electrospun scaffolds revolutionize biomedicine

Involvement of Notch signaling in the osteogenic differentiation of human mesenchymal stem cells stimulated by pulsed electromagnetic fields

Biophysical stimulation with pulsed electromagnetic fields (PEMFs), used in clinics to promote bone repair, favour osteogenic differentiation in human mesenchymal stem cells (hMSCs), however their molecular mechanisms are not clarified. Notch is a pathway regulating cell fate decisions which play a role in skeletal development. Notch signaling is initiated by binding a Notch ligand to a cell surface Notch receptor, resulting in a cleavage of receptor and releasing Notch intracellular domain which translocates to the nucleus and activates transcription of nuclear Notch target genes, such as the Hes/Hey family.The aim of this study is to establish if the known PEMF-induced osteogenic effects may occur through the modulation of Notch pathway. Bone marrow hMSCs cultured in basal condition (control) and in osteoinductive medium (OM) for 28 days were unexposed or continuously exposed to PEMFs (75 Hz, 1.5 mT) (Igea, Carpi, Italy). To block Notch pathway, the Notch inhibitor DAPT was used to treat a series of hMSCs cultured in OM. At different time points (day 1,3,7,14,21,28), osteogenic markers (alkaline phosphatase activity, osteocalcin and matrix mineralization), mRNA expression of osteogenic transcription factors (Runx2, Dlx5, Osterix) as well as of Notch receptors (Notch1-4), their ligands (Jagged1, Dll1 and Dll4) and nuclear target genes (Hey1, Hey2, Hes1, Hes5) were analysed.Our results showed that osteogenic markers and transcription factors increased in OM compared to control and they were further stimulated by PEMFs. Notably, PEMFs significantly increased the expression of Notch4, Dll4, Hey1, Hes1 and Hes5 in the middle phase of differentiation in OM compared to control. In the presence of DAPT, osteogenic markers as well Hes1 and Hes5 expression were significantly inhibited, in unexposed and PEMF-exposed hMSCs. Hey1 was not inhibited by DAPT suggesting a possible regulation by other signaling pathway.These new findings show that PEMFs favor osteogenic differentiation acting through Notch pathway, adding important knowledge concerning the molecular mechanisms by which PEMFs can modulate osteogenesis. This work was supported by grants from IGEA, Carpi, Italy.

Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation.

Endogenous bioelectric signaling via changes in cellular resting potential (V mem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of V mem has been functionally implicated in dedifferentiation, tumorigenesis, anatomical re‐specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome‐wide transcriptional responses to V mem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to V mem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both subnetwork enrichment and PANTHER analyses identified a number of key genetic modules as targets of V mem change, and also revealed important (well‐conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm, and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that V mem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

Gene expression and protein secretion during human mesenchymal cell differentiation into adipogenic cells.

BackgroundMesenchymal stromal cells (MSC) can be obtained from potentially any tissue from the human body, but cells purified from different sources are undoubtedly different, and for each medical application, the MSC with the best regenerative potential should be chosen.ResultsBone marrow-derived mesenchymal stromal cells (BM-MSC), adipose tissue-derived mesenchymal stromal cells (AT-MSC) and Wharton’s Jelly-derived mesenchymal stromal cells (WJ-MSC) were isolated from human tissues and were cultured under differentiation media supplemented with fetal bovine serum. We quantified the expression of stem cell and adipocyte genetic markers using quantitative real time PCR, as well as the secretion of cytokines, extracellular matrix components and growth factors using Luminex and ELISA. All three MSC differentiated into adipogenic cells. AT-MSC showed the highest shift in ADIPOQ, CEBPA and PPARG mRNA expression. BM-MSC kept high expression levels of stem-cell markers SOX2 and POU5F1. WJ-MSC showed the lowest increase in mRNA expression when cells were induced to differentiate into adipocytes. Regarding protein secretion, adipocyte-like cells generated from WJ-MSC secreted the highest chemokine levels. AT-MSC-derived adipocyte-like cells secreted the lowest cytokine amounts and the highest quantity of collagen types I and III. Adipocyte-like cells obtained from BM-MSC secreted high amounts of most angiogenic factors, growth factors TGF-β1 and TGF-β2, collagens type II and IV, heparan sulfate, laminin and aggrecan.ConclusionMesenchymal stromal cells purified from different tissues have a different behavior when induced to differentiate into adipocyte-like cells.

A pilot study on differentiation of human amnion mesenchymal stem cells into ocular surface epithelial cells by co-culture in vitro

Background Recent studies indicated that human amnion mesenchymal stem cells (hAMSCs) can be induced to differentiate into multiple types of cells in vitro,but whether the hAMSCs can differentiate into ocular surface cells has not been reported yet.Objective This study was to investigate the feasibility of inducing differentiation of hAMSCs into ocular surface cells by co-culturing with human bulbar conjunctiva fibroblasts (hBCFs).Methods This study was approved by Ethic Committee of Affiliated Second Hospital of Guangzhou Medical University.HAMSCs were isolated from placenta under the informed consent of healthy delivery women.hAMSCs were cultured,passaged and identified by detecting the expressions of CD44,CD45,CD73,CD90 in the cells with flow cytometer,osteogenesis and adipogenic differentiation experiments.Human conjunctival tissue was obtained during the eye operation under the informed consent of patients and hBCFs were isolated and cultured with explant culture.The cells were divided into the hAMSCs culture group and the hAMSCs and hBCFs co-culture group and cultivated in Transwell chambers for 7 days.The expressions of cytokeratin 19 (CK19) and α-smooth muscle actin (α-SMA) in the cells were assayed by immnofluorescence technique.Results Cultured hAMSCs showed the slender shape and cell body enlarged with passage.CD44,CD73 and CD90 were expressed in the cells,and the expression of CD45 was absent.After 3-4 weeks of osteogenesis and adipogenic induce,the cells showed red staining for alizarin and oil red O.In the co-culture group of hAMSCs and hBCFs,hAMSCs presented the epithelioid cell-like in shape and showed the positive response for CK19 and weaker response for α-SMA.However,in the hAMSCs culture group,the cells showed the positive response for α-SMA and absent response for CK19.Conclusions The hAMSCs can differentiate into ocular surface cells after being induced by hBCFs.And the differentiation mechanism is possibly relevant to mesenchymal cells epithelium. Key words: Human amnion mesenchymal stem cell; Co-culture; Induce; Differentiation; Ocular surface cell

In vitro induction of differentiation of human adipose-derived mesenchymal stem cells into melanocytes: an experimental study

Objective To establish a method for inducing human adipose-derived mesenchymal stem cells (hADSCs) to differentiate into melanocytes in vitro.Methods hADSCs were isolated from human subcutaneous fat tissue,and subjected to phenotypic analysis by flow cytometry.The differentiation potential of the hADSCs was evaluated by induction of osteogenic and adipogenic differentiation.Then,the fifth passage hADSCs were induced to differentiate into melanocytes by conditioned medium containing stem cell factor (SCF),endothelin 3,basic fibroblast growth factor (bFGF) for ten weeks.Those hADSCs receiving no induction treatment served as the normal control.Subsequently,real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expressions of melanin-associated genes including microophthalmia-associated transcription factor (MITF),ckit receptor tyrosine kinase (KIT),dopachrome tautomerase (DCT),tyrosinase,tyrosinase-related protein-1 (TYRP1),and sex determining region Y-box 10 (SOX10).Statistical analysis was done by one-factor analysis of variance and the least significant difference (LSD) test.Further more,the hADSC-derived melanocytes were identified by immunocytochemistry and immunofluorescence assay.Results As flow cytometry revealed,the isolated hADSCs showed high expressions of CD29,CD44,CD73,CD90,CD105 and CD166 with the positivity rates being more than 95％,but low or absent expressions of CD31,CD34,CD45 and human leukocyte antigen (HLA)-DR.Additionally,the osteogenic and adipogenic differentiation of hADSCs was successfully induced.The relative mRNA expression levels of MITF,KIT,DCT,tyrosinase,TYRP1 and SOX10 were 0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,and 0.225 ± 0.014 respectively in hADSCs induced for 10 weeks,which were significantly upregulated compared with the normal control hADSCs (all P ＜ 0.05).Moreover,the induced hADSCs showed positive immunocytochemical staining for MITF and HMB45,as well as positive cytoimmunofluorescence staining for TYRP1 and S100.Conclusion The hADSCs have been successfully induced to differentiate into cells with the biological characteristics of melanocytes. Key words: Human adipose-derived mesenchymal stem cells; Cell differentiation; Melanocytes

The mixed cultures and neuronal-like differentiation of human amnion-derived mesenchymal stem cells

Objective To observe growth of basic fibroblast growth factor (bFGF)-induced cultures of human amnion-derived mesenchymal stem cells (HAMs) and differentiation into neuronal-like cells.Comparative observation.Methods Amnia from full-term,uterine-incision delivery were donated by 12 healthy women.HAMs were obtained by cell separation and culture techniques,and were passaged and induced by bFGF.From the third passage,a total of 1 ml HAMs,at a density of 1.0 × 107/L,was separately harvested from six samples,which served as group A.A total of 1 ml HAMs,at a density of 1.0 × 107/L,was harvested separately from the remaining six samples,which served as group B.A total of 0.5 ml from the six samples of group A and 0.5 ml from the six samples of group B were combined to form group C.Differences in cell quantity among the three groups were compared by cell quantification and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis.Expressions of a glial cell marker,neuron-specific enolase,and Nestin were detected in the three groups by immunocytochemistry.Results Cell quantification and MTT analysis of live cells,as well as AMSC absorbance,were significantly greater in group C compared with groups A and B at 18 days of culture (P ＜ 0.05),and no significant difference was observed between groups A and B.Glial fibrillary acidic protein,neuron-specific enolase,and Nestin were expressed in all groups following bFGF induction.Conclusion Mixed AMSC cultures promoted proliferation,and bFGF-induced HAMs differentiated into neuronal-like cells. Key words: Amnion ; Mesenchymal stem cells ; Induction ; Mixed culture

Uric acid promotes neuronal differentiation of human placenta-derived mesenchymal stem cells in a time- and concentration-dependent manner.

Uric acid is an important, naturally occurring serum antioxidant. The present study investigates the use of uric acid for promoting proliferation and neuronal differentiation of mesenchymal stem cells derived from human placenta tissue. Human placenta-derived mesenchymal stem cells were pre-induced in the presence of either 0, 0.2, 0.4 or 0.8 mM uric acid in combination with 1 mM β-mercaptoethanol for 24 hours, followed by exposure to identical uric acid concentrations and 5 mM β-mercaptoethanol for 6 and 10 hours. Cells developed a neuronal-like morphology, with formation of interconnected process extensions, typical of neural cells. Immunocytochemistry and immunofluorescence staining showed neuron specific enolase positive cells were present in each group except the control group. A greater number of neuron specific enolase positive cells were observed in 0.8 mM uric acid in combination with 5 mM β-mercaptoethanol at 10 hours. After 24 hours of induction, Nissl bodies were detected in the cytoplasm of all differentiated cell groups except the control group and Nissl body numbers were greatest in human placenta-derived mesenchymal stem cells grown in the presence of 0.8 mM uric acid and 5 mM β-mercaptoethanol. These results suggest uric acid accelerates differentiation of human placenta-derived mesenchymal stem cells into neuronal-like cells in a time- and concentration-dependent manner.

Inducing differentiation of human amnion-derived mesenchymal stem cells into insulin-secreting cells in vitro

Objective To investigate the potential of human amnion-derived mesenchymal stem cells to differentiate into insulin secreting cells in vitro.Methods The hAD-MSCs were isolated from human amnion by trypsin-collagenase digestion.The phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.The 3rd generation cells were inoculated at density of 2.5 × 106 unit/ml or 5 × 105 unit/ml in 6 well plates or preset coverslip 24 well plates.Induced groups were treated in serum-free HG-DMEM with 10 mmol/L nicotinamide and N2 supplement.The cells in the non-induced groups were incubated in LG-DMEM supplemented with 10％ fetal bovine serum.At days 7,14 and 21 after induction,insulin and β2 microglobulin was determined by immunocytochemical stain,the content of insulin in the culture supernatant was assayed by radioimmunoassay,and insulin mRNA and PDX-1 mRNA were detected by reverse transcriptase-polymerase chain reaction.Results ( 1 ) The hAD-MSCs highly expressed CD29,CD44,CD73,CD166,and vimentin.( 2 ) At 7,14,and 21 days,the percentages of insulin-positive cells in hAD-MSCs induced groups were 74.67％ ± 1.53％,75.00％:1.00％,and 74.33％ ±1.53％,respectively.Contents of insulin in the supematant of hAD-MSCs induced groups were ( 331.62 ± 1.76 ),( 330.50 ± 1.22 ),and ( 331.65 ± 0.48 ) μIU/ml,respectively,but non-induced groups were negative.(3) PDX-1 mRNA and PDX-1 protein were expressed before and after the induction of hADMSCs,but insulin mRNA was expressed only in the induced groups.( 4 ) Both hAD-MSCs induced groups and non-induced groups expressed β2 microglobulin ( all P ＞ 0.05 ).Conclusion The hAD-MSCs have a potential of differentiating into ISCs and thus may become a new cell source of therapy for type 1 diabetes. Key words: Human amnion-derived mesenchymal stem cells; Insulin secreting cells ; Differentiation

Experimental study on the induced differentiation of human amnion mesenchymal cells into osteoblasts

To investigate the feasibility of inducing differentiation of the human amniotic mesenchymal cells (hAMCs) into osteoblasts in vitro, so as to provide the seed cells for bone tissue engineering. The hAMCs were isolated from abandoned human amnion and cultured in osteogenic media to induce the osteogenic differentiation in vitro. After hAMCs were induced by osteogenic media for 15 days, morphological observation, immunocytochemistry and western blot were used to study the cellular morphology and expression of alkaline phosphatase (ALP), type I collagen, osteopontin and osteocalcin. The primary cultured hAMCs had long spindle shape or irregular shape, which were distributed evenly. The cells were usually suheultured in 5 or 7 days. After subculture, the cells became larger. After cultured by osteogenic media for 15 days, the hAMCs were detected to express ALP, osteocalcin and osteopontin, and secrete type I collagen. The hAMCs are isolated, cultured and amplified easily in vitro. The induced differentiated cells by osteogenic media have typical osteoblast morphological and functional characteristics, which can be used as seed cells for bone tissue engineering.

Effects of second‐generation tyrosine kinase inhibitors towards osteogenic differentiation of human mesenchymal cells of healthy donors

The BCR-ABL inhibitor imatinib is a standard first-line therapy for patients with chronic myeloid leukemia. However, it has been demonstrated that this long-term treatment is associated with altered bone metabolism. The mechanisms of this effect are not fully understood, but an inhibition of the platelet-derived growth factor receptor (PDGF-R) β axis has been suspected on the basis of some in vitro findings. We evaluated the osteoblastic differentiation of mesenchymal stem cells derived from bone marrow (hBM-MSCs) after in vitro treatment with dasatinib, nilotinib or bosutinib. Human bone marrow mesenchymal stem cells were induced to differentiate in osteoblastic cells by treatment with osteogenic medium with or without dasatinib, nilotinib or bosutinib. We found that the addition of dasatinib, and to a greater extend nilotinib, induced expression of osteogenic mRNA markers as compared with cultures with standard medium or osteogenic medium only. However, treatment with bosutinib did not induce an increase of osteogenic markers. In conclusion, we show that besides imatinib, other tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib, but not bosutinib, increase osteogenic markers in hBM-MSCs. Because bosutinib differs from the other TKIs because of its low affinity to other kinases such as PDGF-R, these experiments suggest that inhibition of PDGF-R may be involved in the induction of osteoblastogenesis by TKIs.

Cell and gene therapy for bone repair

Improving bone repair remains an important and challenging issue. Therapeutic approaches to amplify osteogenic cell pool or function include cell and gene therapies. We identified genes that promote human mesenchymal cell differentiation and bone formation. Targeting these or other genes may improve the efficacy of cell therapy for bone repair.

Chondrogenic differentiation of human bone mesenchymal stem cells treated with growth differentiation factor 5 under hypoxia

ObjectiveTo explore the feasibility and effectiveness of the self-assembly cartilage tissue engineered with chondrogenically differentiated human bone mesenchymal stem cells (hBMSCs) induced by growth differentiation factor-5 (GDF-5) under hypoxia.Methods After hBMSCs were isolated from ilium, flow cytometry was performed to detect the cell surface markers, hBMSCs were exposed to cartilage inducing medium containing 100 ng/mL of GDF-5 in hypoxic (2％ oxygen) and normoxic (21％ oxygen)conditions, respectively. RT-PCR was performed to detect the expressions of type Ⅱ collagen and aggrecan in the cells after 3 weeks. In hypoxic and normoxic conditions, the hBMSCs were induced in conditioned medium (CM) containing GDF-5 for 3 weeks. After digestion, the hBMSCs, according to a certain density, were inoculated on 24-well plates coated with 2％ of agarose. Induction in the CM continued for another 3 weeks.Examples of both groups were detected for types Ⅰ and l collagen with histology and immunohistochemistry.Toluidine blue staining was used to detect the expressions of cartilage-specific components.Results The hBMSCs grew in a manner of spindle-shaped whirlpool, with high expressions of CD44 and CD29 and a negative expression of CD45. The hBMSCs exposed to the CM containing GDF-5. in the hypoxic condition, expressed type Ⅱ collagen and aggrecan significantly more than those exposed to the chondrogenic growth factors in the nonnoxic condition ( P ＜ 0. 05). The hBMSCs, in the hypoxic condition, after induction by GDF-5,inoculation on agar surface and induction in the CM, were self-assembled to form cartilage-like tissue in proper shape and size. In the hypoxia group, the expression of type Ⅱ collagen was increased and the expression of type Ⅰ collagen was decreased. The toluidine blue staining in the hypoxia group was deeper than in the normoxia group.ConclusionsHypoxia may promote differentiation of hBMSCs into chondrocytes.The ” self-assembly” technology can make it possible that chondrogenically differentiated hBMSCs induced by GDF-5 under hypoxia can be built in vitro into successful tissue-engineered cartilage. Key words: Hypoxia, cellular; Chondrocytes; Bone marrow cells

Gene expression of markers of osteogenic differentiation of human mesenchymal cells on collagen I‐modified microrough titanium surfaces

Microrough, doubly acid etched titanium surfaces (Ti) were further modified by amination and covalent coupling of fibrillar collagen type I (ColTi). Human Mesenchymal Cells (HMC) adhesion and growth, and relevant osteogenic differentiation in nonosteogenic (basal) medium were evaluated by fluorescence microscopy, scanning electron microscopy, and RT-PCR for a three-week period. Results show strongly enhanced HMC adhesion and cell density at short experimental time on ColTi, together with complete spreading of the cell body over the microrough surface topography. RT-PCR analysis of several genes involved in osteogenesis indicate, since the first week of culturing, significant progression of HMC on ColTi along the osteogenic pathway. These results indicate that the adopted process of surface immobilization of collagen, mandatory to impart collagenase resistance in implant sites, does not impair biospecific interactions between HMC and collagen. Thus, it is possible to upgrade properties arising from the control of Ti surfaces topography by surface-chemistry driven enhanced recruitment of precursor osteogenic cells and pro-osteogenic stimula.