Proliferation Rate Of Mesenchymal Stem Cells Research Articles

Comparing the mesenchymal stem cells proliferation rate with different labeling assessments

Comparing the mesenchymal stem cells proliferation rate with different labeling assessments

THE EFFECT OF COMBINATIONS OF GROWTH FACTORS AND HYALURONIC ACID ON THE PROLIFERATION AND CHONDROGENIC DIFFERENTIATION OF HUMAN SYNOVIUM DERIVED MESENCHYMAL STEM CELLS

Growth factors and hyaluronic acid (HA) have been shown to stimulate functional activity of mesenchymal stem cells (MSCs) and thus may influence damaged tissue healing, including cartilage. In this study we investigated the effects of individual and combinatorial application of HA, platelet derived growth factor–BB (PDGF-BB), transforming growth factor β1 (TGF-β1), bone morphogenetic protein-4 (BMP-4) and fibroblast growth factor-2 (FGF-2) on the proliferation and chondrogenic differentiation of human synovium derived MSCs (SD MSCs). Isolated SD MSCs were expanded in α-MEM medium and characterized using immunocytochemistry, multilineage differentiation assay and CFU-assay. Proliferation activity of MSCs was examined with MTT-assay. Chondrogenic differentiation and glycosaminoglycan level in micro-pellet culture of MSCs were evaluated with histochemical and biochemical analysis. Our results showed that the individual use of FGF-2 and PDGF-BB significantly increased the rate of MSC proliferation compared to the control. At the same time, TGF-β1 did not affect cell growth. Combinations of growth factors: TGF-β1+FGF-2, TGF-β1+PDGF-BB, and FGF-2+PDGF-BB were able to significantly increase the proliferative activity of MSCs. The greatest synergistic effect was found with combination of all three factors TGF-β1, FGF-2, and PDGF-BB. Histochemical and biochemical analysis revealed that combination of TGF-β1 and BMP-4 have a significant effect on chondrogenic differentiation and the synthesis of glycosaminoglycans in micro-pellet culture of MSCs. The addition of HA to growth factors has minor effect on the proliferation and does not affect the chondrogenic differentiation of MSCs. This study demonstrated that combination of growth factors can promote proliferation and chondrogenic differentiation of SD MSCs and may be effective in enhancing cartilage healing.

Equine Platelet Lysate Gel: A Matrix for Mesenchymal Stem Cell Delivery.

A variety of bioscaffolds have been developed as carriers for the delivery of mesenchymal stem cells (MSCs), however, many of them are unable to provide direct cell nourishment, a critical factor for survival and retention of MSCs at the site of delivery. Platelet lysate is a plasma-derived product rich in growth factors that can be turned into a gel matrix following the addition of calcium chloride. Our objective was to characterize growth factor and cytokine release of equine platelet lysate gel (ePL gel) encapsulated with MSCs over time and to measure the viability and proliferation of ePL gel-encapsulated MSCs for up to 14 days. The release of interleukin-1β (IL-1β), interleukin-10 (IL-10), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor BB (PDGF-BB), as well as fibrinogen degradation, were measured from ePL gel with and without equine bone marrow-derived MSCs and compared with MSCs in monolayer. MSC proliferation and viability within the gel were assessed up to 14 days. Compared with monolayer MSC cultures, significantly higher concentrations of IL-1β, IL-10, and TGF-β were measured from supernatants collected from ePL gel containing MSCs at various time points. Significantly lower concentrations of PDGF-BB were measured in the supernatant when MSCs were incorporated in ePL gel while VEGF tended to be increased compared with MSCs in monolayer. Incorporation in ePL gel for up to 14 days did not appear to affect viability and proliferation rates of MSCs as these were found to be similar to those measured in monolayer cell culture. ePL gel may have the potential to serve as bioscaffold for MSC delivery since it appears to support the proliferation and viability of MSCs for up to 14 days.

Effects of nanofiber scaffolds coated with nanoparticulate and micro-particulate FDBA on the morphology, adhesion and proliferation of human mesenchymal stem cells.

Background:Freeze dried bone allograft nanoparticles on a nanofiber membrane may serve as an ideal scaffold for bone regeneration. This study aimed to assess the biological behavior of human MSCs in terms of proliferation and adhesion to nanoparticulate and microparticulate FDBA scaffolds on PLLA nanofiber membrane. Methods:In this experimental study, PLLA nanofiber scaffolds were synthesized by the electrospinning method. The FDBA nanoparticles were synthesized mechanically. The FDBA nanoparticles and microparticles were loaded on the surface of PLLA nanofiber membrane. A total of 64 scaffold samples in four groups of n-FDBA/PLLA, FDBA/PLLA, PLLA and control were placed in 24-well polystyrene tissue culture plates; 16 wells were allocated to each group. Data were analyzed using one-way ANOVA and Bonferroni test. Results:The proliferation rate of MSCs was significantly higher in the nanoparticulate group compared to the microparticulate group at five days (p = 0.034). Assessment of cell morphology at 24 hours revealed spindle-shaped cells with a higher number of appendages in the nanoparticulate group compared to other groups. Conclusion:MSCs on n-FDBA/PLLA scaffold were morphologically more active and flatter with a higher number of cellular appendages, as compared to FDBA/PLLA. It seems that the nanoparticulate scaffold is superior to the microparticulate scaffold in terms of proliferation, attachment, and morphology of MSCs in vitro.

Glutathione-related inflammatory signature in hepatocytes differentiated from the progenitor mesenchymal stem cells

N-acetylcysteine (NAC) as a glutathione inducer is known for its anti-inflammatory effects in inflammatory conditions. The aim of the present study was to know if supplementation of the culture medium with NAC can improve anti-inflammatory activities of hepatocytes during their differentiation from mesenchymal stem cells (MSCs). For this, in vitro hepatic differentiation of MSCs was performed in culture medium supplemented with NAC and selected pro- and anti-inflammatory factors were monitored for two weeks. Treatment of the MSCs undergoing hepatic differentiation with NAC (0.1 and 1.0 mM) caused a significant (~5-fold) increase in proliferation rate of MSCs, whereas the rate of hepatic differentiation was declined in NAC-treated cells as compared to those untreated with NAC. Under these circumstances, NAC caused a significant increase in total glutathione in cell lysate during 2 weeks of differentiation as compared to untreated group. NAC-related increase in glutathione was associated with significant alterations in tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8 and IL-10 levels secreted in the culture medium. A substantial decrease in the IL-6, IL-8 and TNF-α levels in the culture medium supplemented with NAC was obvious in hepatocytes recovered 14 days after differentiation. In contrast, the secretary IL-10 was significantly increased as a result of NAC treatments. These data suggest that NAC supplementation can improve anti-inflammatory activities of the hepatocytes derived from MSCs. NAC function mediated by glutathione synthesis can also help in modulation of proliferation of the stem cells and their differentiation into hepatocyte-like cells.

Association between Bone Marrow Mesenchymal Stem Cell Characterizations and the Administration of Antiarrhythmic Drugs in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery

Background and Aim: Atrial fibrillation is the most common arrhythmia after coronary artery bypass grafting (CABG) and occurs in up to 30% of patients with heart failure. Mesenchymal stem cells (MSCs) can regenerate and improve cardiac function after tissue damage and are used in clinical trials. Due to the possible ability of MSCs to improve cardiac functions, in this work, we aimed to examine the probable association of the MSCs proliferation rate with the requirement for antiarrhythmic drugs in patients with severe left ventricular dysfunction after off-pump CABG. Methods: Bone marrow samples were obtained from the sternum of 30 patients who underwent off-pump CABG at Afshar Hospital and Seyed Al-Shohada Hospital. For calculating MSCs doubling time, the cells were counted after 4, 7, and 14 days using trypan-blue color and a hemocytometer. Results: There were no significant differences between MSCs' doubling time and the patient's age and gender. The percentage of women patients who require antiarrhythmic medicine was significantly higher than men after surgery. Also, we demonstrated that the BMSCs doubling time in female patients who received antiarrhythmic drugs was less than that of male patients who received antiarrhythmic drugs, but these differences were not significant. Conclusion: Based on this research, we concluded that women patients who received antiarrhythmic drugs were significantly higher than men, but there was no apparent relevancy between MSCs doubling time and antiarrhythmic drugs requirement in patients with severe left ventricular dysfunction. *Corresponding Author: Zakieh Sadat Sheikhalishahi; Email: z.sheikhalishahi@gmail.com Please cite this article as: Esmailidehaj M, Mirhosseini SJ, Sheikhalishahi ZS. Association between Bone Marrow Mesenchymal Stem Cell Characterizations and the Administration of Antiarrhythmic Drugs in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery. Arch Med Lab Sci. 2020;6:1-5 (e2). https://doi.org/10.22037/amls.v6.31297

Therapeutic role of bone marrow-derived stem cells and zinc sulfate for reduction of liver fibrosis

Introduction: Hepatic fibrosis is considered as the initial state of any chronic hepatic disease; progression causes accumulation of extracellular matrix (ECM) and develops into cirrhosis. In case of liver failure, transplantation was considered as the only suitable therapy but due to the hurdles in transplantation, therapy using stem cells was promoted. Mesenchymal stem cells (MSCs) are widely used for the treatment of a variety of diseases. Oxidative stress at the damage site causes poor MSC proliferation and engraftment. Thus, it is necessary to induce antioxidants with MSCs to enhance potency.
 Methods: To explore the therapeutic potential of zinc sulfate (ZnSO4) and MSCs on carbon tetra chloride (CCl4)-induced hepatic toxicity, 6-8 week-old female albino mice (BALB/c) were used. Initially, mice were infected with CCl4 and then intra-peritoneally injected with MSCs only, ZnSO4 only, or a combination of MSCs and ZnSO4. MSCs were isolated from femur and tibia of mice, and were cultured under control conditions.
 Results: The morphological results revealed that in contrast to MSC-only therapy, ZnSO4 enhanced the therapeutic potential of MSCs when administered to CCl4-injured mice. Biochemically, level of serum Alanine Transaminase (ALT) and total bilirubin was found to be significantly decreased in ZnSO4 + MSC transplanted mice. Histopathological examination also revealed that ZnSO4 + MSC transplantation induced a strong anti-apoptotic effect on CCl4-injured liver. Reverse transcription polymerase chain reaction (RT-PCR) detected a noteworthy anti-fibrotic effect of MSCs in the presence of ZnSO4, down-regulation of apoptotic marker (Bax), and up-regulation of anti-apoptotic (Bcl-xl) and hepatic (Albumin) markers.
 Conclusion: Thus, it was concluded that the presence of ZnSO4 reduced oxidative stress, enhanced the proliferation rate of MSCs, and significantly attenuated hepatic fibrosis.

Association between Characterizations of Bone Marrow Mesenchymal Stem Cells, Ejection Faction and Hospitalization Period in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery

Background and Aim: Severe left ventricular dysfunction patients with ejection fraction ≤ 30% are at a higher risk for complications and mortality than patients with ejection fraction >30%. The death of cardio-myocytes at injured regions leads to myocardium dysfunction. Bone marrow-derived mesenchymal stem cells are undifferentiated cells that have been used for the regeneration of damaged cardio-myocytes. Due to the inherent capability of mesenchymal stem cells to improve cardiac functions, in this research, our objective was to explore the possible association of the mesenchymal stem cells proliferation rate with Coronary Artery Bypass Grafting outcomes in patients with severe left ventricular dysfunction after off-pump Coronary Artery Bypass Grafting. Methods: For investigating the possible association of mesenchymal stem cells proliferation with Coronary Artery Bypass Grafting outcomes (ejection fraction, hypertension risk, and the time of hospital stay), we collected bone marrow samples from 30 patients (18 men and 12 women) who underwent off-pump Coronary Artery Bypass Grafting at Afshar Hospital and Seyed Al-Shohada Hospital (Yazd, Iran). Mesenchymal stem cells were isolated and cultured; then, cells were counted after 4, 7, and 14 days using trypan-blue color, and doubling times were calculated. Results: There was an association between doubling time and ejection fraction after surgery. Ejection fraction in postoperative patients increased, but this association was not significant. Also, our study showed that the risk of hypertension is equal in male and female patients. There were no significant differences in mesenchymal stem cells doubling time with hypertension and the time of hospital stay in the ICU. Conclusion: Based on this study, we concluded that there was no significant relationship between the rate of mesenchymal stem cell proliferation and Coronary Artery Bypass Grafting outcomes in patients with severe left ventricular dysfunction. *Corresponding Author: Zakieh Sadat Sheikhalishahi; Email: z.sheikhalishahi@gmail.com Please cite this article as: Esmailidehaj M, Mirhosseini SJ, Sheikhalishahi ZS. Association between Characterizations of Bone Marrow Mesenchymal Stem Cells, Ejection Faction and Hospitalization Period in Patients with Severe Left Ventricular Dysfunction after Off-Pump Bypass Surgery. Arch Med Lab Sci. 2019;5(4):30-36. https://doi.org/10.22037/amls.v5i4.31296

Enhanced osteogenic differentiation of mesenchymal stem cells on metal-organic framework based on copper, zinc, and imidazole coated poly-l-lactic acid nanofiber scaffolds.

The presence of inorganic bioactive minerals with polymers can accelerate and promote several processes including: bone cell joining, proliferation, differentiation, and expression of osteogenic proteins. In this study, zinc (Zn), copper (Cu), and imidazole metal-organic framework (MOF) nanoparticles were synthesized and coated over poly-l-lactic acid (PLLA) nanofibrous scaffolds for bone tissue engineering application. The surface and bioactive features of the scaffolds were characterized. The osteogenic potential of the scaffolds on human adipose tissue-derived mesenchymal stem cells (MSCs) was evaluated. Zn-Cu imidazole MOF coated PLLA scaffolds (PLLA@MOF) showed a comparable rate of MSC proliferation with the pure PLLA scaffolds and tissue culture plate (TCP). However, the PLLA@MOF potential of osteogenic differentiation was significantly greater than either pristine PLLA scaffolds or TCP. Hence, coating Zn-Cu imidazole MOF has a significant effect on the osteogenesis of MSC. Therefore, PLLA@MOF is novel scaffolds with bioactive components which are crucial for osteoconductivity and also able to provoke the osteogenesis and angiogenesis.

Occlusive dressing-induced secretomes influence the migration and proliferation of mesenchymal stem cells and fibroblasts differently

BackgroundFingertip injuries treated with occlusive dressings (ODs) lead to nearly scar-free, functionally, and aesthetically pleasing results. We hypothesized that paracrine factors in the wound fluid (secretome) may influence migration and proliferation of mesenchymal stem cells (MSCs) and fibroblasts and modulate the wound-healing process.MethodsWe could collect wound fluid samples from 4 fingertip injuries and 7 split skin donor sites at the 5th day during dressing change. Blood serum samples served as controls. The proliferation rate of MSCs and fibroblasts (HS27) was continuously measured through impedance analysis for 60 h and by Alamarblue analysis after 72 h. Cell migration was evaluated continuously for 15 h and confirmed by the in vitro wound-healing assay.ResultsMigration of MSCs under the influence of both wound fluids was significantly faster than controls from 4 to 6 h after incubation and reversed after 9 h. MSC proliferation in wound fluid groups showed a significant increase at 5 and 10 h and was significantly decreased after 45 h. Fibroblasts in wound fluid groups showed overall a significant increase in migration and a significant decrease in proliferation compared to controls.ConclusionOD-induced secretomes influence MSCs and fibroblasts and thereby possibly modulate wound healing and scar tissue formation.

Effects of Type 2 Diabetic Serum on Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells.

Diabetic patients have an increased risk of osteoporosis-associated fractures. However, the results of most studies of the effects of diabetes on bone mass in patients with type 2 diabetes (T2DM) have been contradictory. To clarify these conflicting findings, we investigated the effects of diabetic serum on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). We used human sera from subjects with different levels of glycemic control to culture the MSCs and induce osteogenic differentiation. The rate of MSC proliferation differed when MSCs were cultured with sera from diabetic subjects with different levels of hyperglycemia. Hyperglycemic sera promoted MSC proliferation to some extent, but all the diabetic sera inhibited the differentiation of MSCs to osteoblasts. The effects of type 2 diabetic sera on the proliferation and osteogenic differentiation of MSCs are closely related to glycemic control. Our data demonstrate the importance of stratifying the study population according to glycemic control in clinical research into diabetic osteoporosis.

An insight into the role of magnesium in the immunomodulatory properties of mesenchymal stem cells

Magnesium (Mg2+) is a mineral with the ability to influence cell proliferation and to modulate inflammatory/immune responses, due to its anti-inflammatory properties. In addition, mesenchymal stem cells (MSCs) modulate the function of all major immune cell populations. Knowing that, the current work aimed to investigate the effects of Mg2+ enrichment, and its influence on the immunomodulatory capacity of MSCs. Murine C3H/10T1/2 MSCs were cultivated in media with different concentrations of Mg2+ (0, 1, 3 and 5 mM), in order to evaluate the effects of Mg2+ on MSC immunomodulatory properties, cell proliferation rates, expression of NFκB and STAT-3, production of IL-1β, IL-6, TGF-β, IL-10, PGE2 and NO, and TRPM7 expression. The results showed that TRPM7 is expressed in MSCs, but Mg2+, in the way that cells were cultivated, did not affect TRPM7 expression. Additionally, there was no difference in the intracellular concentration of Mg2+. Mg2+, especially at 5 mM, raised proliferation rates of MSCs, and modulated immune responses by decreasing levels of IL-1β and IL-6, and by increasing levels of IL-10 and PGE2 in cells stimulated with LPS or TNF-α. In addition, MSCs cultured in 5 mM Mg2+ expressed lower levels of pNFκB/NFκB and higher levels of pSTAT-3/STAT-3. Furthermore, conditioned media from MSCs reduced lymphocyte and macrophage proliferation, but Mg2+ did not affect this parameter. In addition, conditioned media from MSCs cultured at 5 mM of Mg2+ modulated the production profile of cytokines, especially of IL-1β and IL-6 in macrophages. In conclusion, Mg2+ is able to modulate some immunoregulatory properties of MSCs.

Effect of High Hydrostatic Pressure Applied Before Cryopreservation on the Survival Rate and Quality of Porcine Mesenchymal Stem Cells After Thawing

ABSTRACTThe aim of the present study was to examine the effects of varied high hydrostatic pressure (HHP) values on survival rate, proliferation rate, cell multipotency (transcript expression of SOX2, C-MYC, and REX1) and apoptosis (expression of phosphatidylserine (PS), SURVIVIN at the RNA level and BAX at the protein level) of porcine mesenchymal stem cells (MSCs). MSCs were isolated from porcine bone marrow and cultured in vitro. Before cryopreservation and storage in liquid nitrogen, MSCs were subjected to HHP at the varied pressures of 20, 30, 40, 50, or 60 MPa for 1 h at 24°C. Immediately after thawing and after 8 days of in vitro culture, cells were subjected to trypan blue staining, cell counting, real-time Polymerase Chain Reaction (PCR), western blotting, and fluorescence microscopy. BAX protein expression was only estimated immediately after HHP to exclusively examine the impact of HHP on apoptosis of MSCs. The viability of MSC subjected to 40, 50, and 60 MPa and estimated immediately after thawing increased significantly (P < 0.001 for 60 MPa and P < 0.05 for 40 and 50 MPa) in comparison to control. The proliferation rate of MSCs subjected to 40 MPa HHP was significantly higher than in the control group (P < 0.02) after 8 days of in vitro culture. After 8 days of in vitro culture, no significant differences were noted in the survival rates, PS exposure, or levels of SOX2, C-MYC, REX1, and SURVIVIN gene expression in all analyzed groups compared to control. In conclusion: 40–60 MPa HHP has a positive impact by improving cell viability in short term. 20–60 MPa HHP does not induce nor decrease apoptosis in MSCs. Fortunately, HHP does not induce differentiation of MSC. Our results calls for further analysis using HHP values higher than 60 MPa.

Autogenous Mesenchymal Stem Cells from the Vertebral Body Enhance Intervertebral Disc Regeneration via Paracrine Interaction: An in Vitro Pilot Study.

Several in vivo studies have found that transplanting mesenchymal stem cells (MSCs) into degenerative intervertebral discs (IVDs) leads to regeneration of disc cells. Since the exact underlying mechanisms are not understood, we investigated the mechanisms of action of MSCs in regeneration of degenerative IVDs via paracrine actions. Human MSCs and degenerative disc cells from the same donor vertebrae were directly or indirectly cocultured. The multidifferentiation potential, cell proliferation, collagen synthesis, and mRNA expression levels were assessed. The proliferation rates of MSCs and degenerative disc cells were higher in the coculture system than in the monolayer cultures or in the conditioned medium of each cell type. During coculturing with nucleus pulposus (NP) cells, mRNA expression of the extracellular matrix (ECM) components aggrecan, versican (VCAN), SOX9, and type II and type VI collagen was significantly increased in MSCs, whereas mRNA expression for type V collagen was increased in MSCs cocultured with annulus fibrosus (AF) cells. In addition, the accumulation of total ECM collagen was greater in cocultured degenerative disc cells than in monocultured cells. During coculturing, MSCs downregulated the expression levels of various proinflammatory cytokine genes in degenerative NP [interleukin-1α ( IL-1α), IL-1β, IL-6, and tumor necrosis factor-α ( TNF-α)] and AF cells ( IL-1α and IL-6), which are involved in the degradation of ECM molecules. In association with the trophic effect of MSCs on degenerative disc cells, upregulation of growth factor mRNA expression was shown in MSCs cocultured with degenerative NP cells [epidermal growth factor ( EGF), insulin-like growth factor-1 ( IGF-1), osteogenic protein-1 ( OP-1), growth and differentiation factor-7 ( GDF-7), and transforming growth factor-β ( TGF-β)] or degenerative AF cells ( IGF-1, OP-1, and GDF-7). In terms of MSC-based clinical approaches to IVD regeneration, implanting MSCs into a degenerative IVD may both stimulate MSC differentiation into an NP- or AF-like phenotype and stimulate the biological activation of degenerative disc cells for self-repair.

Stroke Serum Priming Modulates Characteristics of Mesenchymal Stromal Cells by Controlling the Expression miRNA-20a.

Transplantation of mesenchymal stem cells (MSCs) expanded with fetal bovine serum (FBS) has some limitations, including the requirement of a long culture period to obtain a sufficient amount of stem cells. Priming of MSCs with serum from patients with ischemic stroke (stroke serum) increased the proliferation rate and the neurorestorative capacity of MSCs. We hypothesized that this novel priming method increases the proliferation rate of MSCs via the regulation of microRNAs (miRs). Thus, we investigated miR profiling in stroke serum-primed MSCs and tested whether the regulation of certain miRs may affect the proliferation rate of rat MSCs. The proliferation rate of MSCs cultured with stroke serum was higher than that of MSCs cultured with normal serum or FBS. Using miR microarray analysis, we compared the miR expression profiles between MSCs cultured in FBS and in stroke serum. Among miRs associated with cell proliferation, miR-20a was most significantly increased. Similarly, miR-20a was increased in MSCs obtained from the bone marrow of stroke rats compared with MSCs from normal rats. Furthermore, the deregulation of miR-20a by the transfection of MSCs with pre-miR-20a or anti-miR-20a was significantly correlated with the increased proliferation rate of MSCs. The overexpression of miR-20a in MSCs cultured in FBS improved the proliferation rate, while the knockdown of endogenous miR-20a decreased the proliferation rate. In addition, miR-20a promoted proliferation by suppressing the expression of p21 cyclin-dependent kinase inhibitor 1 (CDKN1A). A dual-luciferase reporter assay showed that CDKN1A is a target of miR-20a. Our findings indicate that stroke serum priming upregulated the expression of miR-20a, which promoted MSC proliferation by regulating the cell cycle inhibitor p21 CDKN1A, and suggest the possible roles of priming methods in modulating the characteristics of MSCs by controlling the expression of miR in MSCs.

The effect of magnetic field during freezing and thawing of rat bone marrow-derived mesenchymal stem cells

Previous studies showed that a programmed freezer with magnetic field can maintain a high survival rate of mesenchymal stem cells (MSCs). The purpose of this study was to evaluate the influences of magnetic field during freezing and thawing on the survival of MSCs isolated from rat bone marrow. The cells were frozen by a normal programmed freezer or a programmed freezer with magnetic field (CAS-LAB1) and cryopreserved for 7 days at −150 °C. Then, the cells were thawed in the presence or absence of magnetic field. Immediately after thawing, the number of surviving or viable cells was counted. The cell proliferation was examined after 1-week culture. Cryopreserved MSCs which were frozen by a normal freezer or a CAS freezer were transplanted into bone defects artificially made in calvaria of 4-week-old rats. Non-cryopreserved MSCs were used as a control. The rats were sacrificed at 8, 16, or 24 weeks after transplantation and the bone regeneration area was measured. Proliferation rates of MSCs after 1 week were significantly higher in the CAS-freezing-thawing group than in the CAS-freezing group. The extent of new bone formation in the CAS-freezing-thawing group tended to be larger than in CAS-freezing group 24 weeks after transplantation. These results suggest that a magnetic field enhances cell survival during thawing as well as freezing.

Effects of ultrasound in presence of microbubbles on cartilage tissue regeneration in three-dimensional printed scaffolds

Gas-filled microbubbles encapsulated with lipids and other surfactants are highly responsive to ultrasound, which has led to their effective role as ultrasound contrast agents (UCA). In this study, for the first time, we used lipid-coated microbubbles (MB) prepared in-house in order to better harness the beneficial effects of ultrasound stimulation on proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs) within a novel 3D printed poly (ethylene glycol) diacrylate (PEG-DA) hydrogel scaffolds. A significant increase in cell number (p <0.001) was observed with low intensity pulsed ultrasound (LIPUS) treatment in the presence of 0.5 % (v/v) MB after 1, 3, and 5 days of culture. MSC proliferation enhanced up to 40% after 5 days of culture in the presence of MB and LIPUS while this value was only 18% when excited with LIPUS alone. We investigated the effects of acoustic parameters such as excitation intensity, frequency, and pulse repetition period on MSC proliferation rate. Our 3-week chondrogenic differentiation results demonstrated that combining LIPUS with MB significantly enhanced both Glycosaminoglycan (GAG) and type II collagen production. Therefore, integrating LIPUS and MB appears to be a promising strategy for enhanced MSC growth and chondrogenic differentiation.

Molecular pathways reflecting poor intrauterine growth are found in Wharton's jelly-derived mesenchymal stem cells.

Are molecular pathways reflecting the biology of small for gestational age (SGA) neonates preserved in umbilical cord-derived mesenchymal stem cells (MSCs)? MSCs from SGA newborns were found to express an altered EGR-1-dependent gene network involved in the regulation of cell proliferation and oxidative stress. Individuals with suboptimal intrauterine development are at greater risk of metabolic diseases such as type II diabetes, obesity and cardiovascular disease. Umbilical cords (n = 283) from the GUSTO (growing up in Singapore towards healthy outcomes) birth cohort study, and primary MSC isolates established from SGA and matched control cases (n = 6 per group), were subjected to gene expression analysis and candidate genes were studied for functional validation. Umbilical cord specimens were derived from babies born at the National University Hospital (NUH) in Singapore. Local ethical approval was obtained. MSC isolates were established in Wharton's jelly and molecular analysis was conducted by gene expression microarrays and RT-PCR. Cells from SGA and control groups were compared in the presence and absence of insulin and candidate gene function was studied via siRNA-mediated gene knockdown and over-expression experiments in MSCs. Using repeated measure ANOVAs, proliferation rates of MSCs isolated from SGA neonates were found to be significantly increased (P < 0.01). In the absence of insulin, EGR-1 levels were found to be significantly reduced in the group of SGA-derived MSCs, whereas EGR-1 expression was found to be up-regulated in the same group in the presence of insulin (P < 0.01). EGR-1 was found to induce expression of COX-2 in the SGA group (P < 0.01) and both, EGR-1 and COX-2 stimulated glucose uptake in MSCs (P < 0.01). EGR-1 and COX-2 levels were associated in whole umbilical cords (n = 283, P < 0.01) and EGR-1 positively correlated with abdominal circumference and birthweight (n = 91, P < 0.01 and n = 91, P < 0.01). Cell models may not entirely reflect the physiology of the host and patient follow-up studies will be necessary for further clinical validation. Our study suggests that Wharton's jelly-derived MSCs are useful in identifying pathways specific for fetal growth restriction. This work is supported by the Translational Clinical Research (TCR) Flagship Program on Developmental Pathways to Metabolic Disease funded by the National Research Foundation (NRF) and administered by the National Medical Research Council (NMRC), Singapore- NMRC/TCR/004-NUS/2008'. SICS Investigators are supported through the Agency for Science Technology and Research (A*STAR) funding. No potential conflicts of interest relevant to this article were reported.

The metabolic function of hepatocytes differentiated from human mesenchymal stem cells is inversely related to cellular glutathione levels

Differentiation of mesenchymal stem cells (MSCs) to hepatocytes-like cells is associated with alteration in the level of reactive oxygen species (ROS) and antioxidant defense system. Here, we report the role of glutathione in the functions of hepatocytes derived from MSCs. The stem cells undergoing differentiation were treated with glutathione modifiers [buthionine sulfoxide (BSO) or N-acetyl cysteine (NAC)], and hepatocytes were collected on day 14 of differentiation and analysed for their biological and metabolic functions. Differentiation process has been performed in presence of glutathione modifiers viz. BSO and NAC. Depending on the level of cellular glutathione, the proliferation rate of MSCs was affected. Glutathione depletion by BSO resulted in increased levels of albumin and ROS in hepatocytes. Whereas, albumin and ROS were inhibited in cells treated with glutathione precursor (NAC). The metabolic function of hepatocytes was elevated in BSO-treated cells as judged by increased urea, transferrin, albumin, alanine transaminase and aspartate transaminase secretions in the media. However, the metabolic activity of the hepatocytes was inhibited when glutathione was increased by NAC. We conclude that the efficiency of metabolic function of hepatocytes is inversely related to the levels of cellular glutathione. These data may suggest a novel role of glutathione in regulation of metabolic function of hepatocytes.

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7days at a temperature of −150°C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.