Adipogenic Induction Medium Research Articles

Biological characteristics of and protein expressions of p27kip1/PTEN in bone marrow mesenchymal stem cells from patients with systemic lupus erythematosus

Objective To evaluate biological behaviors of bone marrow mesenchymal stem cells(BMSCs)from patients with systemic lupus erythematosus(SLE), to confirm that the BMSCs are aging stems cells, and to explore mechanisms underlying their aging. Methods BMSCs were isolated from bone marrow of 6 patients with SLE(patient group)and 8 healthy human controls(control group)by density-gradient centrifugation and plastic adherence, and cultured in vitro. Optical microscopy was conducted to observe morphological changes and growth of BMSCs, and growth curves were drawn. The differentiation ability of BMSCs was evaluated through culture of them with adipogenic and osteogenic induction medium. Flow cytometry was performed to identify cellular surface markers and to analyze cell cycle and apoptosis, and scratch assay to assess the migration ability of BMSCs. Immunofluorescence assay and Western blot analysis were carried out to analyze the distribution and expression of p27kip1/PTEN in BMSCs respectively. Results Patient-derived BMSCs, which had a broad, flat and polygonal shape, showed decreased growth rate, migration activity as well as adipogenic and osteogenic ability compared with those from the controls. There was a significant increase in the proportion of BMSCs in early stage(17.98% ± 3.26% vs. 8.23% ± 3.25%, t = 3.91, P = 0.011)as well as in middle to late stages(16.80% ± 9.63% vs. 3.33% ± 2.21%, t = 2.99, P = 0.048)of apoptosis in the patient group compared with the control group. Moreover, compared with the control group, the patient group showed a significantly higher proportion of BMSCs arrested in the G0/G1 phase(92.34% ± 5.80% vs. 78.65% ± 3.22%, t = 3.635, P = 0.015), but a lower proportion in the S phase(0.86% ± 1.72% vs. 5.06% ± 1.874%, t = 3.084, P = 0.027). The protein expressions of p27 and PTEN were significantly higher in the patient group than in the control group(p27/β-actin: t = 2.784, P = 0.039; PTEN/β-actin: t = 4.812, P = 0.041). Conclusion The BMSCs from SLE patients exhibit senescence-related features, which may be associated with elevated expression levels of p27kip1/PTEN. Key words: Lupus erythematosus, systemic; Mesenchymal stem cells; Aging; Apoptosis; p27kip1/PTEN

Effect of Vitamin D on Adipogenesis under Hypoxia

BackgroundObesity and pandemic vitamin D (vitD) deficiency both share similar pathological associations with adipose tissue chronic inflammation leading to well‐known conditions such as insulin resistance and hypertension. Understanding adipogenesis is of major relevance for human health, as increased adiposity leads to cellular hypoxia followed by inflammation, oxidative damage and adipocyte dysfunction resulting in metabolic diseases. We have shown earlier that vitD supplementation has significant beneficial effects on adipocyte inflammation and improved insulin sensitivity in vitD deficient mice. The modulation of adipogenesis under these circumstances might be beneficial in improving its metabolic functioning. Therefore, we hypothesize that vitD modulates adipogenesis in pathological conditions (hypoxia).MethodsHuman mesenchymal stem cells (hMSCs) were differentiated into adipocytes ± vitD [10−7 M] containing adipogenic induction and maintenance medium under either hypoxic (1% O2) or normoxic (21% O2) conditions. At the end of differentiation cycles the process was assessed by the oil red O staining of the adipocytes. Fully differentiated adipocytes were stimulated with lipopolysaccharide (LPS 10 ng/ml) ± vitD [10−7 M] for 24 hrs and cells and cell culture supernatants were collected from both hypoxic and normoxic basal ± vitD [10−7 M] as well LPS stimulated ± vitD [10−7 M]. The inflammatory marker interleukin‐6 (IL‐6) levels were measured by ELISA. RNA and cell lysate were prepared and analyzed for adipogenic, hypoxic and inflammation regulating proteins.ResultsThe adipogenic differentiation of hMSCs under hypoxia was significantly inhibited in comparison to the normoxia. VitD [10−9 M to 10−7 M] significantly enhanced adipogenic differentiations dose dependently under both hypoxic and normoxic culture conditions. VitD [10−7 M] inhibited basal IL‐6 secretion by 83% in hypoxic and 66% in normoxic hMSCs differentiated adipocytes in comparison to their respective controls (p<0.05). In addition, vitD dose dependently reduced LPS induced IL‐6 secretion from hMSCs differentiated adipocytes under both hypoxic (10−7M:10−8M:10−7M; 66%:40%:19%) and normoxic (10−7M:10−8M:10−7M; 56%:44%:2%) condition in comparison with the LPS 10 ng/ml alone.ConclusionOur data demonstrates that vitD enhances the adipogensis of hMSCs under hypoxic culture condition; it inhibits basal IL‐6 release as well LPS induced IL‐6 release under both hypoxia and normoxia. This suggests that sufficient vitD levels would be able to modulate the hypoxia induced inflammation and adipogenic differentiation potential in obese subjects. These vitD actions might therefore partially mediate the beneficial effect on insulin sensitivity of adipose tissue and reduce the risk of known co‐morbidities of obesity.Support or Funding InformationSupported in part by the Academy of Finland and DynaHEALTH EU No.633595

Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc.

Modulating stem cell-substrate interactions and differentiation by controlling substrate topography via microphase separation

Event Abstract Back to Event Modulating stem cell-substrate interactions and differentiation by controlling substrate topography via microphase separation Varun Arvind1, Sebastian L. Vega1, 2, Lucas Mccabe3, Prabhas V. Moghe1, N Sanjeeva Murthy4 and Joachim Kohn4 1 Rutgers, The State University of New Jersey, Department of Biomedical Engineering, United States 2 University of Pennsylvania, Department of Bioengineering, United States 3 Rutgers, The State University of New Jersey, Deaprtment of Mathematics, United States 4 Rutgers, The State University of New Jersey, New Jersey Center for Biomaterials, United States Introduction: Biomaterials used in regenerative medicine are required to direct the differentiation of human mesenchymal stem cells (hMSCs)[1]. Equally important is the maintenance of multipotency of hMSCs for clinical and Industrial use. In this work, we generated a range of surface textures by phase-separation (polymer demixing) for modulating stem cell-substrate interactions and hMSC differentiation[2]. In combination with 3D printing, this process can be a powerful tool for fabricating scaffolds for tissue engineering. Materials and Methods: Surface patterns were fabricated by varying the ratio of two immiscible polymers poly(DTE carbonate) (PDTEC) and polystyrene (PS). Polymers were spin-coated onto coverslips to obtain phase-separated films. PS was selectively removed to obtain patterned PDTEC surfaces. Substrates were cultured with hMSCs in a 1:1 ratio of osteogenic and adipogenic induction media so as not to restrict lineage commitment. Cytoskeletal F-actin was fluorescently stained with Alexa conjugated Phalloidin, and phosphorylated focal adhesions (pFAK) were immunolabeled. High-resolution confocal images were used to analyze the morphological features and cytoskeletal anisotropy. Results and Discussion: Reproducible surface patterns (Fig. 1A) were obtained at each of the PDTEC:PS compositions. There were three categories of surface patterns: flat (D0, D100), continuous (D40, D60), and discontinuous (D20, D80). The x in Dx refers to the wt% PDTEC. D0 is the uncoated substrate, D100 is the uniform PDTEC film, D20 had islands and D80 had pits.The height of the features were ~7µm, their widths were ~ 16, 48, 68 and 151 µm in D20, D40, D60, and D80, respectively. Figure 1. (A) Bright field optical micrographs of the surface patterns with uniform chemistry, but varying topography. (B) Differentiation commitment of hMSCs on varying surface topographies exposed to mixed differentiation media for 14 days. (C) Quantification of cytoskeletal anisotropy of hMSCs at 72hrs in differentiation media. (D) Quantification of focal adhesion major-axis length. Results are given as mean ± s.e.; statistics by Tukey's ANOVA; * is p < 0.05. hMSCs cultured in differentiation induction media were assayed at 14 days to assess lineage commitment. Continuous topographies favored hMSCs differentiation, whereas discontinuous topographies supported hMSC multipotency (Fig. 1B). There were no significant differences between D0 and D100, indicating that observed changes in lineage commitment are due to topographic features, and not to surface chemistry. Examination of the hMSCs 72 hours post-seeding showed no significant changes in area or aspect ratio, indicating that cell morphology was not constrained by surface topography. Continuous features promoted cytoskeletal anisotropy (Fig. 1C). Cytoskeletal major-axis length measurements[3] showed that continuous features promote the development of mature pFAK, and discontinuous features do not (Fig. 1D). These data indicate that the observed long-term differentiation profiles can be predicted from early changes in cytoskeletal and pFAK organization. Conclusions: Tunable substrate textures can be easily and reproducibly produced on surfaces of devices with complex shapes such as bone screws, plates and scaffolds via microphase separation. Discontinuous patterns promote multipotency in hMSC. Actin organization and pFAK maturity can be used as reliable predictors of stemness. The National Resource for Polymeric Biomaterials funded by the National Institutes of Health (NIH grant EB001046)

Effects of Cryopreservation on Canine Multipotent Stromal Cells from Subcutaneous and Infrapatellar Adipose Tissue.

Adipose derived multipotent stromal cells (ASCs) isolated from brown versus white adipose tissues, may have distinct in vitro properties, including response to cryopreservation, due to differences in tissue physiology. This study was designed to determine the ultrastructure, immunophenotype, in vitro expansion capabilities and multipotentiality of paired canine ASCs harvested from subcutaneous (SUB) and infrapatellar (IFP) adipose tissue up to cell passage (P) 3 before and after cryopreservation. Adipocyte and ASC ultrastructure from the same tissue were distinct, and morphologies of both differed between tissue sources and with cryopreservation. Cell expansion and colony forming unit frequencies were similar between ASCs from both tissue sources before and after cryopreservation. Most fresh cells were CD29+, CD44+, CD90+ and CD34− up to P3. Cryopreserved P1 and P3 cells had lower percentages of CD29+ and 44+ cells, respectively, compared to fresh. Peroxisome proliferator-activated receptor γ (PPAR-γ) gene expression and sex determining region Y-box 2 (SOX2), CD29 and CD44 protein expression was lower in cryopreserved versus fresh P3 ASCs. Both PPAR-γ and osteopontin (OPN) protein expression increased in fresh and cryopreserved P3 ASCs cultured in adipogenic and osteogenic induction medium, respectively, while SOX2 decreased. Based on the study findings, in vitro expansion and multipotentiality are not distinct among canine SUB and IFP ASCs before or after cryopreservation. However, cryopreservation alters ASC ultrastructure, immunophenotype and transcription factor expression from both tissue sources. Future studies are necessary to determine the impact of cryopreservation on cell potential for therapy and de novo tissue generation.

Isolation and characterization of mesenchymal stromal progenitors from the temporomandibular joint disc.

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John Wiley & Sons, Ltd.

Effects of different roughage sources and feeding levels on adipogenesis of ovine adipocytes.

The objective of the present study was to conduct an adipogenic evaluation of different roughage sources and feeding levels during ruminant adipocyte differentiation in vitro. Six wether sheep were divided into a timothy hay feeding group (TFG, n = 3) and an Italian ryegrass straw feeding group (IFG, n = 3). The sheep were fed high-roughage (HR), medium roughage (MR) and low-roughage (LR) diets in a one-way layout design each over a 6-day period. Sheep serum samples collected on the last day of each dietary treatment were added to an adipogenic induction medium for differentiation of preadipocytes derived from sheep subcutaneous adipose tissue. The cytoplasmic lipid accumulations in the TFG serum-treated preadipocytes were significantly higher than those of the IFG-serum treated preadipocytes on day 12. Messenger RNA expression of CCAAT/enhancer-binding protein (C/EBP)-α, C/EBP-β, C/EBP-δ, fatty-acid-binding protein (aP2) and stearoyl-coenzyme A desaturase (SCD) were regulated by each serum treatment. This study shows that different roughage source diets and roughage-to-concentrate ratio diets can regulate adipocyte differentiation via ruminant blood composition.

5-(Hydroxymethyl)-2-furaldehyde Inhibits Adipogenic and Enhances Osteogenic Differentiation of Rat Bone Mesenchymal Stem Cells

Eucommiae Cortex (Eucommia ulmoides Oliver Bark) has been used for anti-osteoporosis usually as an ethnic drug for hundred years in China. In this study, a bioactive compound, 5-(hydroxymethyl)-2-furaldehyde (5-HMF), was isolated from Eucommiae Cortex. We found that after rat bone mesenchymal stem cells (bMSCs) were induced by 5-HMF at the concentration of 0.05, 0.10 and 0.20 microg/mL in the normal medium for 7 and 14 days, the mRNA expression of ALP, COL1alpha1 (7 days only), OCN and OPN increased. However, in the adipogenic induction medium (AIM), the mRNA expression of PPARgamma, FABP4, C/EBPalpha and LPL decreased with the 5-HMF treatment. Mineralized nodule formations were enhanced after bMSCs were induced by 5-HMF for 14 and 21 days in normal medium. In the AIM medium, 5-HMF not only inhibited the formation of adipose cells obviously, but also stimulated the mineralized nodule formation after induced for 21 days. These results indicated that 5-HMF was a powerful inhibitor of adipogenesis and enhancer of osteoblastogenesis. It may be one of the constituents contributing to anti-osteoporosis in Eucommiae Cortex.

COMP‐Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway

Mesenchymal stem cells (MSCs) are pleiotrophic cells that differentiate to chondrocytes, osteoblasts, or adipocytes, as a result of crosstalk by specific signaling pathways including MAPK pathway. Recently cartilage oligomeric matrix protein angiopoietin1 (COMP-Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms. Our results clearly demonstrated that MSCs cultured in each induction medium with COMP-Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5- and 2-fold, respectively) as well as up-regulate each gene, except for adipogenic differentiation. Accordingly, we found that phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts. Therefore, our findings suggest that COMP-Ang1 present a portal to promote MSCs differentiation to chondrocytes and osteoblasts through Ang1/Tie2 signaling pathway and provide insights into novel therapies for bone diseases.

Proton (1H) nuclear magnetic resonance spectroscopy to define metabolomic changes as a biomarker of adipogenic differentiation in human mesenchymal stem cells

The purpose of this study was to produce a metabolomic profile of a human mesenchymal stem cell (hMSC) pellet sample to identify biomarkers of adipogenic differentiation using nuclear magnetic resonance (NMR) spectroscopy. hMSC adipogenesis was monitored over four cycles of differentiation; one cycle consisted of treatment with adipogenic induction medium for 3 days followed by adipogenic maintenance medium for 1 day. Adipogenesis in control hMSCs was confirmed by Oil Red O staining. A NMR micro-imaging machine with a zqpr pulse (total volume) sequence was used to examine the metabolic changes in the cell pellet samples. Several lipid peaks were predominant among the various metabolite peaks in the NMR spectroscopy data. In particular, the lipid methylene (-[CH2]n-) signal at 1.3 ppm increased 3.6-fold during the first cycle, 15.7-fold during the second cycle, and 28.3-fold during the third cycle. Our findings indicate that NMR spectral peaks related to lipid metabolites can be used as a biomarker of hMSC adipogenesis.

Enhancement of adipogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells by supplementation with umbilical cord blood serum

Umbilical cord blood serum (UCBS) is a promising replacement for animal sera for the culture of human mesenchymal stem cells (hMSC), the unique serum composition of UCBS appearing to have variable effects on their proliferation and differentiation. Conditioning UCBS with methods such as charcoal stripping assists specific processes such as adipogenesis and osteogenesis in hMSCs. The charcoal stripping of serum removes lipophilic materials such as oestrogens, which are known inhibitors of adipogenesis. hMSC cultures supplemented with charcoal-stripped UCBS (CS-UCBS) show enhanced adipogenesis in adipogenic induction medium (AIM) containing indomethacin, 3-isobutyl-1-methylxanthine and dexamethasone. To obtain efficient adipogenesis without CS-UCBS, we have developed a modified protocol in which cells cultured separately with UCBS and CS-UCBS are constantly treated with minimal doses of insulin (1.1μg/ml) for 10days prior to the addition of AIM. hMSC cultures differentiated by using the modified protocol show improved adipogenesis under fetal bovine serum (FBS), UCBS and CS-UCBS conditions, with levels of adipogenesis being highest in UCBS, thereby eliminating the need for charcoal stripping. Furthermore, in each of the three sera, the insulin-pre-treated hMSCs accumulate lipid droplets faster and exhibit improved adipogenesis overall when compared with normal AIM-induced adipogenesis. We have also compared the levels of osteogenesis in hMSCs by using an induction medium devoid of dexamethasone. Maximum calcium deposition has been observed in hMSCs cultured with UCBS, as compared with those cultured with FBS or CS-UCBS. Our newly developed methods with a humanized serum supplement thus enhance the differentiation of cultured hMSCs.

218 DONOR-MATCHED FUNCTIONAL AND MOLECULAR CHARACTERIZATION OF CANINE MESENCHYMAL STEM CELLS DERIVED FROM DIFFERENT ORIGINS

Canine mesenchymal stem cells (cMSC) have been successfully isolated from several adult tissue sources. However, differences in the biological properties of MSC have been shown to be associated with donor variability. Further, the stem cell capacity of cMSC of various tissues isolated from a single donor is currently unclear. Therefore, this study investigated the functional and molecular characteristics of cMSC derived from bone marrow (cBM-MSC), adipose tissue (cA-MSC) and dermal skin (cDS-MSC) of a single donor. Three kinds of cMSC were isolated by following previously published protocols. AP activity was assessed with a chromogen kit (Abcam Inc., Cambridge, MA, USA). Expression of CD markers (CD45, 90 and 105) and stem cell transcription factors (Oct3/4, Nanog and Sox2) was analysed by immunocytochemical staining. All cells were induced into osteogenesis and adipogenesis by following protocols described earlier and confirmed by cytochemical staining and the detection of lineage specific genes by RT-PCR. Chromosomal stability was assessed by a method described earlier (Ock and Rho 2008 J. Vet. Med. Sci. 70, 1165–1172) and cell cycle status was determined by a flow cytometry. Telomere length was analysed by Telo TAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) and telomerase activity was evaluated by semiquantitative nested RT-PCR. Statistical analysis was performed by ANOVA using SPSS 12.0 and significance was tested when P &lt; 0.05. Expressions of AP activity and the transcription factors, such as Oct3/4, Nanog and Sox2 were absent in all cMSC. All 3 types of cMSC positively expressed the surface markers CD90 and 105 but not CD45. Exposure of all cell lines to osteogenic and adipogenic induction medium resulted in the calcium deposition evidenced by Alizarin red S staining and the accumulation of fat globules indicated by Oil red O staining, respectively. Differentiation was further confirmed by the detection of marker genes, such as Runx2 and Pparγ. However, the degree of osteogenic or adipogenic differentiation among the 3 kinds of cMSC was different and particularly, cA-MSC had enhanced cytochemical staining associated with expression of specific genes, Runx2 and Pparγ. Ploidy analysis showed that the diploid rate was high with over 90% in all cMSC and indicated no noticeable chromosomal abnormalities. Further, less than 52% of cells were found at G1 phase in all cMSC, with lowest percentage observed in cDS-MSC (33.3%). Regardless of varied tissue sources, cMSC from a single donor showed no differences in telomere lengths (∼18–19 kbp), but the telomerase activity was different with significantly higher levels found in cBM-MSC. In conclusion, the above results suggest that tissue specific cMSC derived from a single donor possess differences in stem cell capacity and support the consideration of tissue source before judging the suitability of cells for therapeutic applications. This work was supported by grant from Basic Science Research Program through NRF funded by the Ministry of Education, Science and Technology (2009-0064229).

Hypoxia Alters the Main Characteristics of the Hematopoietic Stem and Progenitor Cell Microenvironment in Vitro

Abstract 4803 Background:Hematopoietic stem and progenitor cells (HSPC) are located in a specialized microenvironment, called the stem cell niche, where their stem cell phenotype and differentiation are tightly regulated via interactions with the supporting mesenchymal stromal cells (MSC). These niches have been shown to be localized in regions with a lower oxygen tension which may also impact on the functional properties of MSC. For a better understanding to what extent hypoxia contributes to the establishment of an undifferentiated niche microenvironment that prevents inopportune differentiation of HSPC, we investigated MSC/HSPC co-cultures as well as MSC single cultures under low oxygen conditions. Design and Methods:Distribution, functional and phenotypical characteristics of CD34+ HSPC in hypoxic co-cultures (0.5% O2) were analyzed by flow cytometry. The effect of co-culture medium on the HSPC migration potential was tested in a transwell assay. The secretion of vascular endothelial growth factor A (VEGF-A), stromal-derived factor 1 (SDF-1), IL-6 and IL-8 by MSC was determined using ELISA whereas the expression of cell surface molecules was detected by flow cytometry. Moreover, the MSC proliferation as well as adipogenic and osteogenic differentiation was compared between hypoxic and normoxic culture conditions. Results:In the hypoxic co-culture, the adhesion of HSPC to the MSC layer was inhibited, whereas HSPC transmigration beneath the MSC layer was favoured. Increased VEGF-A secretion by MSC under hypoxic conditions, which enhanced the permeability of the MSC monolayer, was responsible for this effect. Furthermore, VEGF expression in hypoxic MSC was induced via hypoxia-inducible factor (HIF) signalling. Whereas IL-6 and IL-8 secretion were increased, SDF-1 expression by MSC was down-regulated under hypoxic conditions in a HIF-independent manner. The MSC immunophenotype which is characterized by expression of CD73, CD90, CD105, and CD166 was not significantly changed by hypoxia. Interestingly, a significant decrease of CD146 mRNA and protein expression levels was observed. The MSC proliferation was not significantly affected by lower oxygen tension. Culture of MSC in adipogenic induction medium for 14 days under hypoxia resulted in a reduced appearance of adipocyte-like cells containing lipid droplets and almost 50 % lower mRNA levels of fatty acid binding protein 2. The ALP activity as readout for osteogenic differentiation was decreased between 10% and 60% in hypoxic MSC. Conclusions:Low oxygen tension reduces the in vitro differentiation capacity and alters the cytokine secretion profile of primary human MSC. These functional changes may favour the homing and maintenance of quiescent HSC simulating the physiologically hypoxic niche conditions in vitro. Disclosures:No relevant conflicts of interest to declare.

Dynamic expression of DKK1 protein in the process whereby Epimedium‐derived flavonoids up‐regulate osteogenic and down‐regulate adipogenic differentiation of bone marrow stromal cells in ovariectomized rats

To observe the dynamic expression of DKK1 protein in the process whereby Epimedium-derived flavonoids (EFs) regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, and to provide experimental evidence for the mechanism of EFs in the treatment of postmenopausal osteoporosis. Bone marrow stromal cells from ovariectomized rats were separated and cultivated in osteoinductive or liquid medium for 15 days in vitro. EFs (10 µg/mL) were applied to both cultures. Alkaline phosphatase (ALP) staining, ALP activity determination, Oil Red O staining and fluorescence quantitative polymerase chain reaction were used to determine the influence of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats. Moreover, in order to explore the exact mechanism of EFs on osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats, enzyme linked immunosorbent assay was used to determine the dynamic expression of DKK1 protein in this process. EFs increased activity of ALP and mRNA expression of Runx2 (early osteoblast differentiation factor) and decreased mRNA expression of PPARγ-2 (key factor of fat generation). Importantly, EFs down-regulated expression of DKK1 protein in an osteogenic induction medium and inhibited up-regulation of DKK1 protein in an adipogenic induction medium. EFs regulate the balance between osteogenic and adipogenic differentiation of bone marrow stromal cells in ovariectomized rats by down-regulating expression of DKK1 protein. This may be an important molecular mechanism of EFs in the context of treatment of postmenopausal osteoporosis.

Molecular mechanism of fatty degeneration in rotator cuff muscle with tendon rupture

Fatty degeneration often occurs in rotator cuff muscle with tendon rupture. However, the molecular mechanism underlying this change has not been fully clarified yet. We investigated the gene expression of Wnt10b and adipogenic marker gene, PPARγ and C/EBPα in C2C12 myogenic cell line under inhibition of Wnt10b by adipogenic induction medium, isobutylmethylxanthine, dexamethasone, and insulin (MDI). The role of Wnt-signal was confirmed by adding Lithium chloride (LiCl), which mimics Wnt signaling to the cultured cell with MDI. We also assessed the expression profiles of same genes in the rat rotator cuff tear model in vivo. MDI induced Oil red-O staining positive adipocytes and upregulated PPARγ and C/EBPα expression. LiCl inhibited adipogenic induction of MDI. Rotator cuff muscle with tendon rupture showed positive staining for Oil red-O. Real-time polymerase chain reaction analyses revealed decreased expression of Wnt10b followed by increased PPARγ and C/EBPα gene expression in the supraspinatus muscle. Fatty degeneration and its molecular events were remarkably seen in the distal one-third of the detached supraspinatus muscle versus control. Wnt signaling may regulate adipogenic differentiation both in the myoblasts in vitro and the muscle in vivo. Our results indicate that the reduction of Wnt10b in muscle with a rotator cuff tear is a key signal in fatty degeneration of the muscle.

Mannose-binding dietary lectins induce adipogenic differentiation of the marrow-derived mesenchymal cells via an active insulin-like signaling mechanism

We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis.

Stem Cells Derived from Human Amniotic Fluid Contribute to Acute Kidney Injury Recovery

Stem cells isolated from human amniotic fluid are gaining attention with regard to their therapeutic potential. In this work, we investigated whether these cells contribute to tubular regeneration after experimental acute kidney injury. Cells expressing stem cell markers with multidifferentiative potential were isolated from human amniotic fluid. The regenerative potential of human amniotic fluid stem cells was compared with that of bone marrow-derived human mesenchymal stem cells. We found that the intravenous injection of 3.5 × 10(5) human amniotic fluid stem cells into nonimmune-competent mice with glycerol-induced acute kidney injury was followed by rapid normalization of renal function compared with injection of mesenchymal stem cells. Both stem cell types showed enhanced tubular cell proliferation and reduced apoptosis. Mesenchymal stem cells were more efficient in inducing proliferation than amniotic fluid-derived stem cells, which, in contrast, were more antiapoptotic. Both cell types were found to accumulate within the peritubular capillaries and the interstitium, but amniotic fluid stem cells were more persistent than mesenchymal stem cells. In vitro experiments demonstrated that the two cell types produced different cytokines and growth factors, suggesting that a combination of different mediators is involved in their biological actions. These results suggest that the amniotic fluid-derived stem cells may improve renal regeneration in acute kidney injury, but they are not more effective than mesenchymal stem cells.

5-Aza-2′-deoxycytidine treatment induces skeletal myogenic differentiation of mouse dental pulp stem cells

Objective Tissue stem cells in dental pulp are assumed to possess differentiation potentials similar to mesenchymal stem cells (MSCs). The aim of this in vitro study is to examine the differentiation potentials of mouse dental pulp stem cells (DPSCs) and develop the appropriate differentiation assay systems for skeletal myogenic differentiation of these cells. Methods Dental pulps were extracted from mandible sections of C57/BL6 mice, and adherent dental pulp cells were isolated in culture. These cells were cultured in osteogenic or adipogenic induction medium to induce osteogenic and adipogenic differentiation. On the other hand, the skeletal myogenic differentiation potential of these cells was investigated using different conditions, such as serum-free medium, Myod1 overexpression, or 5-Aza-2′-deoxycytidine (5-Aza) treatment for DNA demethylation. Muscle-specific transcriptional factor expression was evaluated by RT-PCR, and myotube formation and myosin heavy chain expression were evaluated by phase-contrast microscopy and immunofluorescence staining, respectively. Results The adherent dental pulp cells exhibited a proliferative capacity and they showed osteogenic and adipogenic differentiation as seen in previous studies. Although the expression of Myod1 mRNA and myotube formation was not detected in serum-free conditions, the forced expression of Myod1 up-regulated the expression of Myogenin and Pax7 mRNA. However, myotube formation was not confirmed. Interestingly, myosin heavy chain expression and myotube formation were observed following 5-Aza treatment of these cells. Conclusions These results demonstrated that mouse DPSCs possess MSC-like differentiation potential. DNA demethylation induced by 5-Aza treatment resulted in the skeletal muscle differentiation in mouse DPSCs, suggesting that DNA demethylation might trigger this differential induction of mouse DPSCs.

Loss of Proliferation and Differentiation Capacity of Aged Human Periodontal Ligament Stem Cells and Rejuvenation by Exposure to the Young Extrinsic Environment

The application of periodontal ligament stem cells (PDLSCs) may be effective for periodontal regenerative therapy. As tissue regenerative potential may be negatively regulated by aging, whether aging and its microenvironment modify human PDLSCs remains a question. In this study, we compared the proliferation and differentiation capacity of PDLSCs obtained from young and aged donors. Then, we exposed aged PDLSCs to young periodontal ligament cell-conditioned medium (PLC-CM), and young PDLSCs were exposed to aged PLC-CM. Morphological appearance, colony-forming assay, cell cycle analysis, osteogenic and adipogenic induction media, gene expression of cementoblast phenotype, and in vivo differentiation capacities of PDLSCs were evaluated. PDLSCs obtained from aged donors exhibited decreased proliferation and differentiation capacity when compared with those from young donors. Young PLC-CM enhanced the proliferation and differentiation capacity of PDLSCs from aged donors. Aged PDLSCs induced by young PLC-CM showed enhanced tissue-regenerative capacity to produce cementum/periodontal ligament-like structures, whereas young PDLSCs induced by aged PLC-CM transplants mainly formed connective tissues. To our knowledge, this is the first study to mimic the developmental microenvironment of PDLSCs in vitro, and our data suggest that age influences the proliferation and differentiation potential of human PDLSCs, and that the activity of human PDLSCs can be modulated by the extrinsic microenvironment.

Therapeutic potential and related signal pathway of adipose‐derived stem cell transplantation for rat liver injury

Liver transplantation is the only currently effective therapy for end-stage chronic liver disease and severe acute liver failure, but its use is limited by high cost and a shortage of allografts. Here we explored the effectiveness of transplanting adipose-derived stem cells (ADSCs) into rats with experimentally induced liver injury. ADSCs obtained from rats were hepatogenic induced in vitro with MAPK pathways inhibitors preconditioning. In vivo, ADSCs were transplanted into rats via different routes and serum liver function markers from post-operative rats were tested. When grown in adipogenic induction medium, ADSCs were able to differentiate into adipocytes. In hepatogenic induction medium, ADSCs were able to differentiate into hepatocyte-like cells, with appropriate changes in morphology and appropriately elevated expression of hepatocyte-specific markers. ERK1/2 phosphorylation activity was also significantly upregulated during the hepatogenic differentiation process, and was blocked by the ERK/MAPK pathway-specific inhibitor PD98059. In a rat liver injury model, intravenously injected ADSCs successfully engrafted into recipient livers. We found that injection via the hepatic portal vein was more efficient than via the dorsal vein of the penis. ADSC transplantation into damaged livers significantly decreased the level of serum liver enzymes such as alanine aminotransferase and aspartate aminotransferase, and improved serum albumin level. Both the number of engrafted cells and the improvement of liver function reached a peak two weeks after transplantation. Transplanted ADSCs appear to be therapeutically effective in the rat liver injury model, which may ultimately provide a therapeutic alternative to liver transplantation in human patients.