208 The effect of copper on the differentiation of adipose-derived stem cells into osteoblasts

2019 ◽  
Vol 31 (1) ◽  
pp. 229
Author(s):  
T. Bane ◽  
L. Siegel ◽  
J. Bertels ◽  
K. Ratz ◽  
M. Rubessa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Lysyl Oxidase ◽  
Collagen Matrix ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Significant Difference ◽  
Positive Effect

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate whether osteogenic medium infused with copper influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Copper is a key cofactor for lysyl oxidase, an enzyme involved in producing a collagen matrix through which bone can grow. Lysyl oxidase expression is up-regulated in bone marrow stromal cells (Khosravi et al. 2014 PLoS One 9, e100669). Our hypothesis was that the presence of copper in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 7 different treatments: 5 concentrations of copper in the osteogenic medium (0.1, 1, 10, 50, and 100 µM) plus 2 control treatments (osteogenic medium without copper and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa stains. In each well, we counted the total number of nodules that were either formed or forming. Data were analysed using the generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Armonk, NY). The least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that more nodules were formed in the 0.1 and 1 µM copper groups compared with the osteogenic control, but there was no statistical difference between those 2 treatments. Table 1 illustrates the total number of formed and forming nodules in addition to their standard deviation. There was a positive effect on nodule formation when copper concentrations of 0.1 and 1 µM were added to the osteogenic medium. In contrast, copper concentrations of 50 and 100 µM had a cytotoxic effect. These results confirm that low concentrations of copper have a positive effect on osteogenesis. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with copper incorporated into their matrix. Table 1.The average number (standard deviations in parentheses) of total formed and forming osteoblast nodules compared between treatment groups

189 The Effect of Vitamin K on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

2018 ◽  
Vol 30 (1) ◽  
pp. 234
Author(s):  
T. A. Bane ◽  
J. C. Bertels ◽  
K. M. Polkoff ◽  
M. Rubessa ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Vitamin K ◽  
Bone Mineralization ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Mineral Density ◽  
Alizarin Red ◽  
Significant Difference

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (osteogenic medium without vitamin K and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data were analysed by analysis of variance using the generalized linear model (GLM) procedure of SPSS (IBM/SPSS, Armonk, NY, USA); the least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison, and the α-level was set at 0.05. The results showed (in Table 1) that there was no positive effect on nodule formation when vitamin K was added to the medium; however, when 1000 nM vitamin K was added, nodule formation decreased. More experiments need to be conducted to determine if vitamin K can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin K incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS

2017 ◽  
Vol 29 (1) ◽  
pp. 207 ◽  
Author(s):  
J. C. Bertels ◽  
M. Rubessa ◽  
S.R. Schreiber ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
The Media ◽  
Positive Effect

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mM zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mM zinc, and 0.08 mM zinc, respectively. The largest nodule was found in the 0.08 mM zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix. Table 1. The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups

188 The Effect of Vitamin B12 on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

2018 ◽  
Vol 30 (1) ◽  
pp. 234
Author(s):  
T. A. Bane ◽  
J. C. Bertels ◽  
K. M. Polkoff ◽  
M. Rubessa ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Vitamin B12 ◽  
Difficult Problem ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Α Level

Large bone defects present a tremendous challenge to the treating surgeon. Tissue engineering using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules offer a potential solution to this difficult problem. The aim of this project was to evaluate if the osteogenic medium infused with vitamin B12 influences the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin B12 has been shown to have a stimulatory effect on osteoclastogenesis in vitro (Vaes et al. 2009 Calcified Tissue Int. 84, 413-422). Our hypothesis was that the presence of vitamin B12 in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). The ASC were divided in 8 different treatments: 8 concentrations of vitamin B12 in the osteogenic medium (0.1, 0.2, 1, 2, 10, and 20 μM) plus 2 control treatments (osteogenic medium without vitamin B12 and a negative control, DMEM). The medium was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red and Von Kossa stains. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data was analysed using the generalized linear model (GLM) procedure in SPSS (IBM/SPSS, Armonk, NY, USA). Bonferroni’s post hoc test was used to perform statistical multiple comparison. The α-level was set at 0.01. The results showed that the concentration of 20 μM vitamin B12 was detrimental for nodule formation. Table 1 illustrates the number of formed and forming nodules in addition to their standard deviation. There was no positive effect on nodule formation when different concentrations of vitamin B12 were added to the osteogenic medium. More experiments need to be conducted to determine if vitamin B12 can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin B12 incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

184 Effect of selenium on the differentiation of porcine adipose-derived stem cells into osteoblasts

2019 ◽  
Vol 31 (1) ◽  
pp. 216
Author(s):  
L. Siegel ◽  
T. Bane ◽  
J. Bertels ◽  
K. Ratz ◽  
M. Rubessa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Statistical Difference ◽  
Bone Growth ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Cell Therapies

Bone damage is a common occurrence and although traditional bone healing methods work well, they are not always able to fix all issues pertaining to the injury. However, recent research on stem cell therapies using adipose-derived stem cells (ASC) has shown that ASC that differentiate into osteoblasts can be used as a potentially better solution for bone injuries. Selenium is a nutritional trace element that has been found to be essential in the production of selenoproteins and bone growth. Selenium plays an integral role in osteoblast cell differentiation and proliferation. The aim of this experiment was to test whether adding selenium to osteogenic influences the differentiation potential of ASC into osteoblasts. Porcine ASC were isolated as described (Monaco et al. 2009, Open Tissue Eng. Regen. Med. J. 2, 20-33). Seven different treatments were given to the cells: a negative control of DMEM, a positive control of osteogenic medium, and 5 concentrations of selenium in the osteogenic medium (10, 5, 1, 0.5, and 0.1 µM). Medium was changed twice a week for 4 weeks, at the end of which the cells were stained with Alizarin Red S stain. The number of osteoblastic nodules in each well were counted and divided into the categories of “forming” and “formed.” A generalized linear model (GLM) procedure (SPSS Inc./IBM Corp., Chicago, IL, USA) was used to analyse the data. The least statistical difference (l.s.d.) post hoc test was used to perform statistical multiple comparison with an α-level of 0.05. The results showed (Table 1) that medium containing selenium concentrations of 0.1, 0.5, and 1.0 µM were able to develop more nodules than the osteogenic medium. There was no statistical difference in the total amount of forming and formed nodules in those 4 groups; however, there was a statistical tendency for a difference (P=0.06) between the medium with 1 µM selenium and the osteogenic medium. Medium containing 10 or 5 µM selenium had lower nodule counts (both forming and formed) than osteogenic medium. There was no statistical difference between 10 µM and DMEM. These results suggest that low concentrations of selenium have a positive effect on nodule formation, whereas higher concentrations are detrimental. Table 1.The average number (standard deviations in parentheses) of formed, forming, and total osteoblastic nodules in each of the different treatments

280 OSTEOGENIC ACTIVITY OF IN HOUSE-PRODUCED PORCINE BMP2 ON ADIPOSE-DERIVED STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 288 ◽  
Author(s):  
A. C. M. Ercolin ◽  
M. Mkrtschjan ◽  
M. Bionaz ◽  
T. Jensen ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Acetic Acid ◽  
Bone Regeneration ◽  
Treatment Time ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Accurate Analysis ◽  
Alizarin Red ◽  
Coding Sequence ◽  
In Cells

In our laboratory, we extensively study the possibility of using adipose-derived stem cells (ASC) for maxillofacial bone regeneration. This includes also the tissue repair of large critical-size osteotomies requiring the use of tridimensional scaffolds. Bone regeneration in scaffolds can be greatly enhanced by the use of specific growth factors such as BMP2. In the present study, we compared the activity of commercially available human BMP2 (hBMP2) with in house-produced porcine BMP2 (pBMP2). The latter was synthesised using the BMP2 coding sequence from mRNA obtained from porcine ASC cell cultures. The coding sequence of the mature protein was cloned into a pET-21 plasmid and produced in E. coli as inclusion bodies. The activity of pBMP2 and hBMP2 was tested on ASC isolated from male pigs at passage 4 and at approximately 80% confluence in 48-well plates. Cells were treated in triplicate with hBMP2 or pBMP2 at 0.5, 5, 50, 500, or 1000 ng mL–1, adipogenic medium (AM), osteogenic medium (OM), or normal DMEM medium supplemented with acetic acid (used to resuspend BMP2 as the control) for 5 or 17 days. Cells were harvested for Alizarin Red S (AR) quantification and expression of osteogenic genes. For the AR analysis, cells were fixed with formalin and treated with AR. The AR was then extracted by acetic acid and neutralized with ammonium hydroxide before spectrophotometer reading at an absorbance of 420 nm. Data were analysed using GLM of SAS (SAS Institute Inc., Cary, NC, USA) with treatment, time, concentration, and all interactions as main effects. Using an inverted robotic stage microscope, images of the entire well for each replicate were taken every 2 to 3 days. Images revealed formation of osteogenic nodules in OM and characteristic large cells filled with lipid droplets in AM. No evident nodule formation was observed in the other treated cells at any time point. The AR was higher than control in both hBMP2 and pBMP2 at 0.5, 50, and 1000 ng mL–1 but not at 5 and 500 ng mL–1. There was no overall difference between hBMP2 and pBMP2 but the former had the highest AR value at 5 days in cells treated with 0.5 ng mL–1 and pBMP2 at 17 days with 1000 ng mL–1. Interestingly, both had higher values compared to OM, particularly at 5 days. We also observed an increase of AR due to time in cells treated with acetic acid (control). Overall, the data appear to indicate an increase in calcium accumulation in cells treated with both hBMP2 and pBMP2, with an early increase in the former and a late and larger increase in the latter. This might indicate a larger but slower activity of pBMP2 compared with hBMP2. The lack of formation of osteogenic nodules by both BMP2 might indicate an insufficiency of BMP2 to induce osteogenesis in porcine ASC. This last observation, together with the lack of increased AR accumulation compared with control at the 5 and 50 ng mL–1 doses, suggests the need for a more accurate analysis of BMP2 activity by measuring expression of BMP2-related genes. Finally, the data provide preliminary support for the equivalency of activity of pBMP2 and hBMP2 for in vivo bone regeneration.

scholarly journals Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

2008 ◽  
Vol 41 (01) ◽  
pp. 08-14 ◽  
Author(s):  
Arash Zaminy ◽  
Iraj Ragerdi Kashani ◽  
Mohammad Barbarestani ◽  
Azim Hedayatpour ◽  
Reza Mahmoudi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Culture Media ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Adult Rats ◽  
Alizarin Red ◽  
Alp Activity ◽  
Proliferation And Differentiation

ABSTRACT Background: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

scholarly journals Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

2021 ◽  
Vol 22 (22) ◽  
pp. 12392
Author(s):  
Kiyofumi Takabatake ◽  
Masakazu Matsubara ◽  
Eiki Yamachika ◽  
Yuki Fujita ◽  
Yuki Arimura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Potential ◽  
Fat Cells ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Dedifferentiated Fat Cells ◽  
Dfat Cells

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.

186 The effects of different concentrations of MgSO4 in osteogenic differentiation

2019 ◽  
Vol 31 (1) ◽  
pp. 217
Author(s):  
L. R. Padoveze ◽  
M. Rubessa ◽  
C. E. Ambrosio ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
In Vitro Study ◽  
Control Group ◽  
Nodule Formation ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Physiological Processes

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.

scholarly journals A NOTCH1/LSD1/BMP2 co-regulatory network mediated by miR-137 negatively regulates osteogenesis of human adipose-derived stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Cong Fan ◽  
Xiaohan Ma ◽  
Yuejun Wang ◽  
Longwei Lv ◽  
Yuan Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteoblastic Differentiation ◽  
Negative Control ◽  
Bone Morphogenetic Protein 2 ◽  
Luciferase Reporter ◽  
Adipose Derived Stem Cells ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Morphogenetic Protein ◽  
Genes Expression

Abstract Background MicroRNAs have been recognized as critical regulators for the osteoblastic lineage differentiation of human adipose-derived stem cells (hASCs). Previously, we have displayed that silencing of miR-137 enhances the osteoblastic differentiation potential of hASCs partly through the coordination of lysine-specific histone demethylase 1 (LSD1), bone morphogenetic protein 2 (BMP2), and mothers against decapentaplegic homolog 4 (SMAD4). However, still numerous molecules involved in the osteogenic regulation of miR-137 remain unknown. This study aimed to further elucidate the epigenetic mechanisms of miR-137 on the osteogenic differentiation of hASCs. Methods Dual-luciferase reporter assay was performed to validate the binding to the 3′ untranslated region (3′ UTR) of NOTCH1 by miR-137. To further identify the role of NOTCH1 in miR-137-modulated osteogenesis, tangeretin (an inhibitor of NOTCH1) was applied to treat hASCs which were transfected with miR-137 knockdown lentiviruses, then together with negative control (NC), miR-137 overexpression and miR-137 knockdown groups, the osteogenic capacity and possible downstream signals were examined. Interrelationships between signaling pathways of NOTCH1-hairy and enhancer of split 1 (HES1), LSD1 and BMP2-SMADs were thoroughly investigated with separate knockdown of NOTCH1, LSD1, BMP2, and HES1. Results We confirmed that miR-137 directly targeted the 3′ UTR of NOTCH1 while positively regulated HES1. Tangeretin reversed the effects of miR-137 knockdown on osteogenic promotion and downstream genes expression. After knocking down NOTCH1 or BMP2 individually, we found that these two signals formed a positive feedback loop as well as activated LSD1 and HES1. In addition, LSD1 knockdown induced NOTCH1 expression while suppressed HES1. Conclusions Collectively, we proposed a NOTCH1/LSD1/BMP2 co-regulatory signaling network to elucidate the modulation of miR-137 on the osteoblastic differentiation of hASCs, thus providing mechanism-based rationale for miRNA-targeted therapy of bone defect.

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