Osteogenic Differentiation Of Adipose-derived Stem Cells Research Articles

Bone Tissue Engineering with Adipose-Derived Stem Cells in Polycaprolactone/Graphene Oxide/Dexamethasone 3D-Printed Scaffolds.

We fabricated three-dimensional (3D)-printed polycaprolactone (PCL) and PCL/graphene oxide (GO) (PGO) scaffolds for bone tissue engineering. An anti-inflammatory and pro-osteogenesis drug dexamethasone (DEX) was adsorbed onto GO and a 3D-printed PGO/DEX (PGOD) scaffold successfully improved drug delivery with a sustained release of DEX from the scaffold up to 1 month. The physicochemical properties of the PCL, PGO, and PGOD scaffolds were characterized by various analytical techniques. The biological response of these scaffolds was studied for adherence, proliferation, and osteogenic differentiation of seeded rabbit adipose-derived stem cells (ASCs) from DNA assays, alkaline phosphatase (ALP) production, calcium quantification, osteogenic gene expression, and immunofluorescence staining of osteogenic marker proteins. The PGOD scaffold was demonstrated to be the best scaffold for maintaining cell viability, cell proliferation, and osteogenic differentiation of ASCs in vitro. In vivo biocompatibility of PGOD was confirmed from subcutaneous implantation in nude mice where ASC-seeded PGOD can form ectopic bones, demonstrated by microcomputed tomography (micro-CT) analysis and immunofluorescence staining. Furthermore, implantation of PGOD/ASCs constructs into critical-sized cranial bone defects in rabbits form tissue-engineered bones at the defect site, observed using micro-CT and histological analysis.

Toward the Development of a Tissue Engineered Gradient Utilizing CRISPR-Guided Gene Modulation.

Cellular, compositional, and mechanical gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have proven difficult due to the complex nature of these tissues. Current strategies for tissue engineering complex gradients often utilize stem cells; however, these multipotent cells require direction from environmental cues, which can be difficult to control both in vitro and in vivo. In this study, we utilize clustered regularly-interspaced short palindromic repeats (CRISPR)-guided gene modulation to direct the differentiation of multipotent adipose-derived stem cells (ASCs) to demonstrate the effectiveness of CRISPR-engineered cells in tissue engineering applications. Specifically, we screen CRISPR-interference (CRISPRi) constructs targeting the promotors of selected osteogenic inhibitors and demonstrate that ASC osteogenic differentiation and mineral deposition can be regulated with CRISPRi targeting of Noggin without the use of exogenous growth factors in tissue engineered constructs. As a proof of concept, we combine three technologies developed out of our laboratories to demonstrate the controlled deposition of these engineered cells in a gradient with CRISPR-activation multiplex-engineered aggrecan/collagen type-II-chondrogenic ASCs on a high density anisotropic type I collagen construct to create a cell and tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the enthesis. Our results display the promise of CRISPR-engineered ASCs to produce tissue gradients, similar to what is observed in native tissue.

Empagliflozin Ameliorates the Impaired Osteogenic Differentiation Ability of Adipose-Derived Stem Cells in Diabetic Osteoporosis by Activating Autophagy.

Adipose-derived stem cells (ASCs) from diabetic osteoporosis (DOP) mice showed impaired osteogenic differentiation capacity. Recent studies have shown that in addition to antidiabetic drugs, sodium-glucose co-transporter inhibitor-2 (SGLT-2), empagliflozin, can play multipotent roles through various mechanisms of action. In this study, we aimed to investigate the effects and underlying mechanisms of empagliflozin on osteogenic differentiation of ASCs in DOP mice. Our results showed that osteogenic differentiation potential and autophagy activity weakened in DOP-ASCs when compared to controls. However, empagliflozin enhanced autophagy flux by promoting the formation of autophagosomes and acidification of autophagic lysosomes, resulting in an increase in LC3-II expression and a decrease in SQSTM1 expression. Furthermore, empagliflozin contributed to the reversal of osteogenesis inhibition in DOP-ASCs induced by a diabetic microenvironment. When 3-methyladenine was used to block autophagy activity, empagliflozin could not exert its protective effect on DOP-ASCs. Nonetheless, this study demonstrated that the advent of cellular autophagy attributed to the administration of empagliflozin could ameliorate the impaired osteogenic differentiation potential of ASCs in DOP mice. This finding might be conducive to the application of ASCs transplantation for promoting bone fracture healing and bone regeneration in patients with DOP.

HIF-1α increases the osteogenic capacity of ADSCs by coupling angiogenesis and osteogenesis via the HIF-1α/VEGF/AKT/mTOR signaling pathway

BackgroundStabilization and increased activity of hypoxia-inducible factor 1-α (HIF-1α) can directly increase cancellous bone formation and play an essential role in bone modeling and remodeling. However, whether an increased HIF-1α expression in adipose-derived stem cells (ADSCs) increases osteogenic capacity and promotes bone regeneration is not known.ResultsIn this study, ADSCs transfected with small interfering RNA and HIF-1α overexpression plasmid were established to investigate the proliferation, migration, adhesion, and osteogenic capacity of ADSCs and the angiogenic ability of human umbilical vein endothelial cells (HUVECs). Overexpression of HIF-1α could promote the biological functions of ADSCs, and the angiogenic ability of HUVECs. Western blotting showed that the protein levels of osteogenesis-related factors were increased when HIF-1α was overexpressed. Furthermore, the influence of upregulation of HIF-1α in ADSC sheets on osseointegration was evaluated using a Sprague–Dawley (SD) rats implant model, in which the bone mass and osteoid mineralization speed were evaluated by radiological and histological analysis. The overexpression of HIF-1α in ADSCs enhanced bone remodeling and osseointegration around titanium implants. However, transfecting the small interfering RNA (siRNA) of HIF-1α in ADSCs attenuated their osteogenic and angiogenic capacity. Finally, it was confirmed in vitro that HIF-1α promotes osteogenic differentiation and the biological functions in ADSCs via the VEGF/AKT/mTOR pathway.ConclusionsThis study demonstrates that HIF-1α has a critical ability to promote osteogenic differentiation in ADSCs by coupling osteogenesis and angiogenesis via the VEGF/AKT/mTOR signaling pathway, which in turn increases osteointegration and bone formation around titanium implants.Graphical

Methyltransferase-like 3 modulates osteogenic differentiation of adipose-derived stem cells in osteoporotic rats.

Osteoporosis (OP) is a metabolic bone disease involving reduced bone mass. Adipose-derived stem cells (ASCs) play an important role in bone regeneration. Emerging evidence suggests that methyltransferase-like 3 (METTL3) plays a significant role in bone development and metabolism. Therefore, this study investigates changes to METTL3 in the osteogenic differentiation of adipose stem cells in osteoporotic rats (OP-ASCs) and explores ways to enhance their osteogenic ability. An animal model of osteoporosis was established by removing both ovaries in rats. Real-time PCR and western blotting were performed to detect the expression of METTL3 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Furthermore, alkaline phosphatase staining was used to confirm the osteogenic potential of stem cells. Mettl3 small interfering RNA and Mettl3 overexpression lentivirus were used to assess the role of METTL3 in osteogenic differentiation of ASCs and OP-ASCs. The osteogenic differentiation capacity and Mettl3 expression significantly decreased in OP-ASCs. Moreover, Mettl3 silencing down-regulated the osteogenic ability of ASCs, and overexpression of Mettl3 recovered the impaired osteogenic capacity in OP-ASCs in vitro. The Mettl3 expression levels and osteogenic potential of OP-ASCs decreased. However, overexpression of METTL3 rescued the osteogenic ability of OP-ASCs, providing a new target for treatment of osteoporotic bone defects.

Comprehensive Analysis of Novel Genes and Pathways Associated with Osteogenic Differentiation of Adipose Stem Cells.

Background Adipose-derived stem cells (ADSCs) are an important alternative source of mesenchymal stem cells (MSCs) and show great promise in tissue engineering and regenerative medicine applications. However, identifying the novel genes and pathways and finding the underlying mechanisms regulating ADSCs osteogenic differentiation remain urgent. Methods We downloaded the gene expression profiles of GSE63754 and GSE37329 from the Gene Expression Omnibus (GEO) Database. We derived differentially expressed genes (DEGs) before and after ADSC osteogenic differentiation, followed by Gene Ontology (GO) functional and KEGG pathway analysis and protein-protein interaction (PPI) network analysis. 211 differentially expressed genes (142 upregulated genes and 69 downregulated genes) were aberrantly expressed. GO analysis revealed that these DEGs were associated with extracellular matrix organization, protein extracellular matrix, and semaphorin receptor binding. Conclusions Our study provides novel genes and pathways that play important roles in regulating ADSC osteogenic differentiation, which may have potential therapeutic targets for clinic.

Knockdown of PDX1 enhances the osteogenic differentiation of ADSCs partly via activation of the PI3K/Akt signaling pathway

BackgroundOsteoporosis (OP) is a systemic bone disease manifested as low bone mass, destruction of bone microstructure, increased bone fragility and fracture risk. The purpose of this study was to explore the role and mechanism of PDX1 for osteogenic differentiation of adipose derived stem cells (ADSCs).MethodsGSE37329 dataset was retrieved from NCBI Gene Expression Omnibus (GEO) database and performed bioinformatic analyses. ADSCs were incubated with normal medium, osteogenic induction medium (OIM) and OIM+si-PDX1. Then, alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS) were performed to assess the role of PDX1 for osteogenesis of ADSCs. PI3K inhibitor, LY294002 was then added to further explore the mechanism of PDX1 for osteogenic differentiation of ADSCs. Western blot assay was used to assess the osteogenic-related markers. Graphpad software was used to perform statistically analysis.ResultsA total of 285 DEGs were obtained from analysis of the dataset GSE37329, of which 145 were upregulated and 140 were downregulated genes. These differentially expressed genes mainly enriched in cell differentiation and PI3K/Akt signaling pathway. Moreover, PDX1 was decreased in osteogenic induced ADSCs. Knockdown of PDX1 significantly increased osteogenic differentiation capacity and p-PI3K and p-Akt protein levels. Administration with LY294002 could partially reversed the promotion effects of si-PDX1.ConclusionIn conclusion, knockdown of PDX1 promotes osteogenic differentiation of ADSCs through the PI3K/Akt signaling pathway.

LINC00370 modulates miR-222-3p-RGS4 axis to protect against osteoporosis progression

BackgroundWe aimed to determine the role of the LINC00370/miR-222-3p/RGS4 axis in modulating the process of adipose-derived stem cell (ADSC) osteogenic differentiation. MethodsWe first evaluated the differential expression of LINC00370, miR-222-3p and RGS4 between normal and osteogenically induced ADSCs. Moreover, we transfected ADSCs with LINC00370 siRNA and an miR-222-3p inhibitor to determine the role of LINC00370 in modulating the process of ADSC osteogenic differentiation. Finally, we analyzed the dual-luciferase reporter gene to identify the relationship between LINC00370 and miR-222-3p. We first created osteoporotic rat models by ovariectomy (OVX) and treated with pcDNA-LINC00370. HE and immunohistochemical staining of OCN were performed to assess the changes in bone microarchitecture. ResultsLINC00370 and RGS4 expression was remarkably upregulated in the osteogenic ADSC group compared with the normal medium group. On the other hand, miR-222-3p expression was remarkably decreased in the osteogenic group compared with the normal medium group. Knockdown of LINC00370 reduced the osteogenic differentiation of ADSCs. Moreover, the inhibitor of miR-222-3p partially reversed the reduction of osteogenic differentiation by LINC00370 knockdown. Knockdown of LINC00370 reduced the expression of p-Akt and p-PI3K. The inhibitor of miR-222-3p partially reversed the reduction of the expression of p-Akt and p-PI3K by LINC00370 knockdown. A dual luciferase reporter assay indicated that LINC00370 can directly bind miR-222-3p. LINC00370 suppressed OP progression in OVX and partially upregulated OCN protein expression. ConclusionCollectively, the above results confirm that LINC00370 promotes the process of ADSC osteogenic differentiation via the miR-222-3p/RGS4 axis. Moreover, LINC00370 could protect against OVX-induced OP.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment.

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) was reported to promote both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis is initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM augments HAM-induced chondrogenesis but not osteogenesis of ADSCs. ADSCs were treated with SIM in a HAM (SIM plus HAM) by HA-coated wells or HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs was evaluated. SIM plus HAM increased chondrogenesis more than HAM or SIM alone, including cell aggregation, chondrogenic gene expression (collagen type II and aggrecan) and cartilaginous tissue formation (collagen type II and sulfated glycosaminoglycan). In contrast, SIM-induced osteogenesis in ADSCs was reduced in SIM plus HAM, including mRNA expression of osteogenic genes, osteocalcin and alkaline phosphatase (ALP), ALP activity and mineralization. SIM plus HAM also showed the most effective increases in the mRNA expression of BMP-2 and transcription factors of SOX-9 and RUNX-2 in ADSCs, while these effects were reversed by CD44 blockade. HAM suppressed the levels of JNK, p-JNK, P38 and p-P38 in ADSCs, and SIM plus HAM also decreased SIM-induced phosphorylated JNK and p38 levels. In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. The results from this study indicate that SIM may be an enhancer of HAM-initiated MSC-based chondrogenesis and avoid osteogenesis.

Effects of Black Jade on Osteogenic Differentiation of Adipose Derived Stem Cells under Benzopyrene

Jade, a popular gemstone symbolizing beauty, grace, and longevity, is known to improve blood circulation; however, scientific research evidence is still lacking. The effect of black jade extract on the expression levels of apoptotic and osteogenic genes was validated using qPCR and flow cytometry. In combination with the use of a fluorescence microscope, osteogenic differentiation and the stained osteocytes count were analyzed. Under the pressure of benzo(a)pyrene, dermal cell apoptosis was accelerated and the osteogenic differentiation of adipose-derived stem cells (ASCs) was suppressed; but black jade extract counteracted the effects. Through an anti-apoptotic mechanism, the extract suppressed the expression of apoptotic proteins Bax and cytochrome C to 9 and 4.8 times, respectively, compared to that in dermal cells exposed to benzo(a)pyrene. During osteogenic differentiation of ASCs, the extract enhanced their differentiation despite being exposed to benzo(a)pyrene, and the relative levels of the osteoblast differentiation markers osteoponin, osteocalcin, and sclerostin were 1.87, 2.54, and 3.9 times higher, respectively, than those in the conditioned medium by benzo(a)pyrene. These effects of the extract indicate that black jade extract is very useful when applied as a functional biomaterial.

Long noncoding RNA LINC00314 facilitates osteogenic differentiation of adipose-derived stem cells through the hsa-miR-129-5p/GRM5 axis via the Wnt signaling pathway

BackgroundMany studies have shown that long noncoding RNAs (lncRNAs) are closely related to the stimulation of osteogenic differentiation of adipose-derived stem cells (ADSCs) and the prevention of osteoporosis. Current research aimed to investigate the novel lncRNA and explored the function and molecular mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs.MethodsLncRNA and miRNA sequencing was performed in normal and osteogenic differentiation-induced ADSCs (osteogenic group). Abnormally expressed lncRNAs and miRNAs were obtained by the R software and the relative expression of LINC00314, miR-129-5p, and GRM5 during osteogenic induction was measured by RT-PCR. ADSCs were then transfected with pcDNA3.1-sh-LINC00314 and agomiR-129-5p. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were performed to identify the mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs.ResultsLINC00314 was significantly upregulated in the group of osteogenic-induced ADSCs. LINC00314 and GRM5 mimics increased the early and late markers of osteogenic differentiation, which manifest in not only the markedly increased ALP activity but also higher calcium deposition, while miR-129-5p mimic had the opposite effects. LINC00314 directly targeted miR-129-5p through luciferase reporter assay, and miR-129-5p suppressed GRM5 expression. Moreover, the LINC00314/miR-129-5p/GRM5 regulatory axis activated the Wnt/β-catenin signaling pathway.ConclusionsLINC00314 confers contributory function in the osteogenic differentiation of ADSCs and thus the LINC00314/miR-129-5p/GRM5 axis may be a novel mechanism for osteogenic-related disease.

Simvastatin Enhances the Chondrogenesis But Not the Osteogenesis of Adipose-Derived Stem Cells in a Hyaluronan Microenvironment

Directing adipose-derived stem cells (ADSCs) toward chondrogenesis is critical for ADSC-based articular cartilage regeneration. Simvastatin (SIM) is a lipid-lowering drug that also affects both chondrogenic and osteogenic differentiation of ADSCs by upregulating bone morphogenetic protein-2 (BMP-2). We previously found that ADSC chondrogenesis can be initiated and promoted in a hyaluronan (HA) microenvironment (HAM). Here, we further hypothesized that SIM can augment the HAM-induced chondrogenesis but not osteogenesis of ADSCs and that this synergism can be applied for articular cartilage regeneration. ADSCs were treated with SIM in a HAM in HA-coated wells or an HA-enriched fibrin (HA/Fibrin) hydrogel, and chondrogenic differentiation of ADSCs and formation of cartilage were evaluated. Treatment with SIM in a HAM (SIM plus HAM) induced the expression of BMP-2 and transcription factors for chondrogenesis (SOX-9) and osteogenesis (RUNX-2) in ADSCs, and these effects were reversed by CD44 blockade. Furthermore, ADSCs exposed to SIM plus HAM exhibited cell aggregation, chondrogenic gene (collagen type II and aggrecan) expression and sulfated glycosaminoglycan formation. By contrast, SIM-induced osteogenesis in ADSCs was reduced in a HAM, as determined by measuring osteogenic gene (osteocalcin and alkaline phosphatase) expression and calcium deposition. These inhibitory effects were not altered by changes in the HA molecular weight (80~2000 kDa). In addition, SIM enhanced articular cartilage regeneration, as demonstrated by implantation of an ADSCs/HA/Fibrin construct in an ex vivo porcine articular chondral defect model. These results indicate that a HAM may be used as a chondroinductive agent to enhance SIM-induced MSC chondrogenesis but prevent osteogenesis.

GREM1 inhibits osteogenic differentiation, senescence and BMP transcription of adipose-derived stem cells

ABSTRACT Purpose: Adipose-derived stem cells (ADSCs) are ideal for cell-based therapies to support bone regeneration. It is vital to understand the critical genes and molecular mechanisms involved in the functional regulation of ADSCs for enhancing bone regeneration. In the present study, we investigated the Gremlin 1 (GREM1) effect on ADSCs osteogenic differentiation and senescence. Materials and methods: The in vitro ADSCs osteogenic differentiation potential was evaluated by determining alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteogenic markers. Cell senescence is determined by SA-β-gal staining, telomerase assay, and the expression of aging markers. Results: GREM1 overexpression in ADSCs reduced ALP activity and mineralization, inhibited the expression of osteogenic related genes OCN, OPN, DSPP, DMP1, and BSP, and key transcription factors, RUNX2 and OSX. GREM1 knockdown in ADSCs enhanced ALP activity and mineralization, promoted the expression of OCN, OPN, DSPP, DMP1, BSP, RUNX2, and OSX. GREM1 overexpression in ADSCs reduced the percent SA-β-Gal positive cells, P16 and P53 expressions, and increased telomerase activity. GREM1 knockdown in ADSCs increased the percentage of SA-β-Gal positive cells, P16 and P53 expressions, and reduced telomerase activity. Furthermore, GREM1 reduced the mRNA expression levels of BMP2, BMP6, and BMP7. Conclusions: In summary, our findings suggested that GREM1 inhibited ADSCs senescence and osteogenic differentiation and antagonized BMP transcription.

Hypermethylation in Calca Promoter Inhibited ASC Osteogenic Differentiation in Rats with Type 2 Diabetic Mellitus.

The abnormal environment of type 2 diabetes mellitus (T2DM) leads to a substantial decrease in osteogenic function of stem cells. However, the gene sequence does not vary before and after disease for the patient. This phenomenon may be related to changes in osteogenesis-related gene expression caused by DNA methylation. In this study, we established T2DM models to extract adipose-derived stem cells (ASCs) for different gene identifications through DNA methylation sequencing. Specific fragments of methylation changes in the target gene (Calca) were identified by IGV analysis. CGRP was applied to compare the effects on ASCs-T2DM morphology via phalloidin staining, proliferation through CCK-8 assay, and osteogenic differentiation with osteogenic staining, qPCR, and repair of calvarial defect. Furthermore, 5-azacytidine (5-az) was used to intervene ASCs-T2DM to verify the relationship between the methylation level of the target fragment and expression of Calca. We found that the DNA methylation level of target fragment of Calca in ASCs-T2DM was higher than that in ASCs-C. CGRP intervention showed that it did not change the morphology of ASCs-T2DM but could improve proliferation within a certain range. Meanwhile, it could significantly enhance the formation of ALP and calcium nodules in ASCs-T2DM, increase the expression of osteogenesis-related genes in vitro, and promote the healing of calvarial defects of T2DM rat in a concentration-dependent manner. 5-az intervention indicated that the reduction of the methylation level in Calca target fragment of ASCs-T2DM indeed escalated the gene expression, which may be related to DNMT1. Taken together, the environment of T2DM could upregulate the methylation level in the promoter region of Calca and then decrease the Calca expression. The coding product of Calca revealed a promoting role for osteogenic differentiation of ASCs-T2DM. This result provides an implication for us to understand the mechanism of the decreased osteogenic ability of ASCs-T2DM and improve its osteogenic capacity.

Research progress and thinking of long non-coding RNAs regulating osteogenic differentiation of adipose derived stem cells

The potential of osteogenic differentiation of adipose-derived stem cells (ADSCs) is unquestionable, but the biological mechanism of it’s specific development is unknown. In the recent years, the characteristics and functions of long non-coding RNAs (lncRNAs) are closely related to the osteogenic differentiation of ADSCs. Some lncRNAs in the nucleus can promote osteogenic differentiation of ADSCs, and some cytoplasmic lncRNAs can also promote osteogenic differentiation of ADSCs. Part of the effect is the opposite. Therefore, the present article aims to review the recent advances in the molecular mechanisms of lncRNAs in osteogenic differentiation of adipose-derived stem cells. In this review, we have analyzed the research ideas of lncRNAs in osteogenic differentiation of ADSCs. Key words: Long chain non-coding RNA; Adipose-derived stem cells; Osteogenic differentiation

RETRACTED ARTICLE: Long non-coding RNA HIF1A-AS2 facilitates adipose-derived stem cells (ASCs) osteogenic differentiation through miR-665/IL6 axis via PI3K/Akt signaling pathway

BackgroundThis study was aimed to investigate the role and specific molecular mechanism of HIF1A-AS2/miR-665/IL6 axis in regulating osteogenic differentiation of adipose-derived stem cells (ASCs) via the PI3K/Akt signaling pathway.MethodsRNAs’ expression profile in normal/osteogenic differentiation-induced ASCs (osteogenic group) was from the Gene Expression Omnibus database. The analysis was carried out using Bioconductor of R. Gene Set Enrichment Analysis and Kyoto Encyclopedia of Genes and Genomes dataset were applied to identify up- and downregulated signaling pathways. Co-expression network of specific lncRNAs and mRNAs was structured by Cytoscape, while binding sites amongst lncRNA, mRNA, and miRNA were predicted by TargetScan and miRanda. ASCs were derived from human adipose tissue and were authenticated by flow cytometry. ASC cell function was surveyed by alizarin red and alkaline phosphatase (ALP) staining. Molecular mechanism of HIF1A-AS2/miR-665/IL6 axis was investigated by RNAi, cell transfection, western blot, and qRT-PCR. RNA target relationships were validated by dual-luciferase assay.ResultsHIF1A-AS2 and IL6 were highly expressed while miR-665 was lowly expressed in induced ASCs. HIF1A-AS2 and IL6 improved the expression level of osteoblast markers Runx2, Osterix, and Osteocalcin and also accelerated the formation of calcium nodule and ALP activity, yet miR-665 had opposite effects. HIF1A-AS2 directly targeted miR-665, whereas miR-665 repressed IL6 expression. Moreover, the HIF1A-AS2/miR-665/IL6 regulating axis activated the PI3K/Akt signaling pathway.ConclusionsLncRNA HIF1A-AS2 could sponge miR-665 and hence upregulate IL6, activate the PI3K/Akt signaling pathway, and ultimately promote ASC osteogenic differentiation.

Phospholipase C-β1 interacts with cyclin E in adipose- derived stem cells osteogenic differentiation.

Adipose-derived stem cells (ADSCs) are multipotent mesenchymal stem cells that have the ability to differentiate into several cell types, including chondrocytes, osteoblasts, adipocytes, and neural cells. Given their easy accessibility and abundance, they became an attractive source of mesenchymal stem cells, as well as candidates for developing new treatments for reconstructive medicine and tissue engineering. Our study identifies a new signaling pathway that promotes ADSCs osteogenic differentiation and links the lipid signaling enzyme phospholipase C (PLC)-β1 to the expression of the cell cycle protein cyclin E. During osteogenic differentiation, PLC-β1 expression varies concomitantly with cyclin E expression and the two proteins interact. These findings contribute to clarify the pathways involved in osteogenic differentiation and provide evidence to develop therapeutic strategies for bone regeneration.

Regional Differentiation of Adipose-Derived Stem Cells Proves the Role of Constant Electric Potential in Enhancing Bone Healing

Mesenchymal stem cells (MSCs) have a great potential in the field of tissue engineering and regenerative medicine on account of their ability to self-renew and differentiate into various lineages. MSCs could be differentiated by a number of ways. Electric field is known to bring about differentiation, migration, proliferation, and reorientation of MSCs. Hence, we aim to create a bioreactor to attain osteodifferentiation of human-derived MSCs in the presence of osteoinduction medium (OIM) in combination with or without alternating current (AC) fields. A stimulation bioreactor was specially designed for the exposure of adipose-derived stem cells (ASCs) to an electric field of 20 mV/cm, 60 kHz. The electric field potential (E) within the chamber was simulated using COMSOL. The morphology, proliferation, and osteogenic differentiation of ASCs under the influence of electrical stimulation were studied. By week three, electrically stimulated ASCs exhibited their typical spindle-shaped morphology. Stimulated ASCs were more intensely stained with alkaline phosphatase and alizarin red, the markers of osteogenic differentiation, as compared to the unstimulated control groups. Darker stained regions correlated with the COMSOL simulation which showed constant electric potential at the same place. The results depicted a clear difference between the effect of constant and varying electric potential on osteodifferentiation of ASCs. Picogreen assay revealed lower DNA contents of electrically stimulated ASCs compared to the control group. In this study, we have additively enhanced the osteodifferentiation potential of ASCs by electrical stimulation and have proved that it is constant electric field potential which specifically augments osteogenic differentiation. We have successfully developed a bioreactor to improve the osteodifferentiation of ASCs by an electrical field, which could be applied in regenerative therapy strategies of bone fracture treatment.

Effects of concentrated growth factors on proliferation and osteogenic differentiation in Beagle adipose-derived stem cells

To investigate the effect of concentrated growth factor (CGF) on proliferation and differentiation in Beagle adipose-derived stem cells (ADSCs). Methods: ADSCs were isolated from adipose tissue of healthy Beagles and cultured. The multi-directional differentiation potential of ADSCs was identified. The ADSCs were assigned to a CGF group and a control group. The rate of proliferation was analyzed by CCK-8 assay. The osteogenic differentiation capability was detected by ALP staining after the osteoinduction. Bone formation-related gene expression was detected by RT-PCR. Results: CGF promoted the proliferation of ADSCs in vitro. ADSCs in the CGF group showed higher level of ALP activity than that in the control group (P<0.05). CGF stimulated the expression of the genes associated with osteogenesis, such as Col-I and Runx2. Conclusion: CGF can promote the proliferation and osteogenic differentiation in ADSCs in vitro.

Function of microRNAs in the Osteogenic Differentiation and Therapeutic Application of Adipose-Derived Stem Cells (ASCs).

Traumatic wounds with segmental bone defects represent substantial reconstructive challenges. Autologous bone grafting is considered the gold standard for surgical treatment in many cases, but donor site morbidity and associated post-operative complications remain a concern. Advances in regenerative techniques utilizing mesenchymal stem cell populations from bone and adipose tissue have opened the door to improving bone repair in the limbs, spine, and craniofacial skeleton. The widespread availability, ease of extraction, and lack of immunogenicity have made adipose-derived stem cells (ASCs) particularly attractive as a stem cell source for regenerative strategies. Recently it has been shown that small, non-coding miRNAs are involved in the osteogenic differentiation of ASCs. Specifically, microRNAs such as miR-17, miR-23a, and miR-31 are expressed during the osteogenic differentiation of ASCs, and appear to play a role in inhibiting various steps in bone morphogenetic protein-2 (BMP2) mediated osteogenesis. Importantly, a number of microRNAs including miR-17 and miR-31 that act to attenuate the osteogenic differentiation of ASCs are themselves stimulated by transforming growth factor β-1 (TGFβ-1). In addition, transforming growth factor β-1 is also known to suppress the expression of microRNAs involved in myogenic differentiation. These data suggest that preconditioning strategies to reduce TGFβ-1 activity in ASCs may improve the therapeutic potential of ASCs for musculoskeletal application. Moreover, these findings support the isolation of ASCs from subcutaneous fat depots that tend to have low endogenous levels of TGFβ-1 expression.