How mesenchymal stem cells transform into adipocytes:Overview of the current understanding of adipogenic differentiation

Mesenchymal stem cells(MSCs)are stem/progenitor cells capable of self-renewal and differentiation into osteoblasts,chondrocytes and adipocytes.The transformation of multipotent MSCs to adipocytes mainly involves two subsequent steps from MSCs to preadipocytes and further preadipocytes into adipocytes,in which the process MSCs are precisely controlled to commit to the adipogenic lineage and then mature into adipocytes.Previous studies have shown that the master transcription factors C/enhancer-binding protein alpha and peroxisome proliferation activator receptor gamma play vital roles in adipogenesis.However,the mechanism underlying the adipogenic differentiation of MSCs is not fully understood.Here,the current knowledge of adipogenic differentiation in MSCs is reviewed,focusing on signaling pathways,noncoding RNAs and epigenetic effects on DNA methylation and acetylation during MSC differentiation.Finally,the relationship between maladipogenic differentiation and diseases is briefly discussed.We hope that this review can broaden and deepen our understanding of how MSCs turn into adipocytes.

Acetylated-PPARγexpression is regulated by different P53 genotypes associated with the adipogenic differentiation of polyploid giant cancer cells with daughter cells

Objective:Polyploid giant cancer cells(PGCCs)with daughter cells express epithelial–mesenchymal transition(EMT)-associated proteins.Highly malignant tumor cells with EMT properties can transdifferentiate into mature tumor cells.In this study,we elucidated the potential for,and underlying mechanism of,adipogenic differentiation of PGCCs with daughter cells(PDCs).Methods:Cobalt chloride was used to induce PGCC formation in HEY(wild-type P53)and MDA-MB-231(mutant P53)cells;these cells were then cultured in adipogenic differentiation medium.Oil red O staining was used to confirm adipogenic differentiation,and the cell cycle was detected with flow cytometry.The expression of adipogenic differentiation-associated proteins and P300 histone acetyltransferase activity were compared before and after adipogenic differentiation.Animal xenograft models were used to confirm the adipogenic differentiation of PDCs.Results:PDCs transdifferentiated into functional adipocytes.Two different cell cycle distributions were observed in PDCs after adipogenic differentiation.The expression levels of PPARγ,Ace-PPARγ,and Ace-P53 were higher in PDCs after adipogenic differentiation than in cells before adipogenic differentiation.Ace-PPARγand FABP4 expression increased in HEY cells and decreased in MDA-MB-231 PDCs after p53 knockdown.A485 treatment increased Ace-P53,Ace-PPARγ,and FABP4 expression in HEY PDCs by inhibiting SUMOylation of P53.In MDA-MB-231 PDCs,A485 treatment decreased Ace-P53,Ace-PPARγ,and FABP4 expression.Animal experiments also confirmed the adipogenic differentiation of PDCs.Conclusions:Acetylation of P53 and PPARγplays an important role in the adipogenic differentiation of PDCs.

YAP promotes osteogenesis and suppresses adipogenic differentiation by regulatingβ-catenin signaling

YAP（yes-associated protein） is a transcriptional factor that is negatively regulated by Hippo pathway, a conserved pathway for the development and size control of multiple organs. The exact function of YAP in bone homeostasis remains controversial. Here we provide evidence for YAP＇s function in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis.YAP is selectively expressed in osteoblast（OB）-lineage cells. Conditionally knocking out Yap in the OB lineage in mice reduces cell proliferation and OB differentiation and increases adipocyte formation, resulting in a trabecular bone loss. Mechanistically, YAP interacts with β-catenin and is necessary for maintenance of nuclear β-catenin level and Wnt/β-catenin signaling. Expression of β-catenin in YAP-deficient BMSCs（bone marrow stromal cells） diminishes the osteogenesis deficit. These results thus identify YAP-β-catenin as an important pathway for osteogenesis during adult bone remodeling and uncover a mechanism underlying YAP regulation of bone homeostasis.

Inhibition of peroxisome proliferator-activated receptor-γ in steroid-induced adipogenic differentiation of the bone marrow mesenchymal stem cells of rabbit using small interference RNA

Background Steroids inhibit osteogenic differentiation and decrease bone formation while concomitantly inducing adipose deposition in osteocytes.This leads to the fatty degeneration and necrosis of bone cells commonly seen in osteonecrosis of the femoral head.The peroxisome proliferator-activated receptor-γ （PPARγ） is an adipogenic transcription factor linked to the development of this disease and responsible for inducing adipogenesis over osteogenesis in bone marrow mesenchymal stem cells （BMSCs）.The aim of this study was to assess whether adipogenic differentiation could be suppressed,and thus osteogenic potential retained,by inhibiting PPARy expression in BMSCs.Methods Cells from the bone marrow of New Zealand rabbits were treated with 10-7 mol/L dexamethasone and infected with one of three small interference RNA （siRNA） adenovirus vectors （S1,S2,and S3） or non-targeting control siRNA （Con） and compared with dexamethasone-treated （model） and untreated （normal） cells.Cells were grown for 21 days and stained with Sudan Ⅲ for adipocyte formation.At various time points,cells were also assessed for changes in PPARγ,osteocalcin （OC）,Runx2,alkaline phosphatase （ALP） activity,and triglyceride （TG） content.Results Dexamethasone-treated model and control groups showed a significant increase in fatty acid-positive staining,which was inhibited in cells treated with PPARγ siRNA-treated,similar to normal untreated cells.All three siRNA groups significantly inhibited PPARγ mRNA and protein,adipocyte number,and TG content compared with the dexamethasonetreated model and control groups,matching that seen in normal cells.OC and Runx2 mRNA and protein,as well as ALP activity,were significantly higher in cells treated with siRNA against PPARγ,similar to that seen in the normal cells.These osteogenic markers were significantly lower in the dexamethasone-treated cell cultures.Conclusions The siRNA adenovirus vector targeting PPARγ can efficiently inhibit steroid-induced adipogenic differentiation in rabbit BMSCs and retain their osteogenic differentiation potential.

Effects of La^(3+) on osteogenic and adipogenic differentiation of primary mouse bone marrow stromal cells

In order to elucidate the action of La3＋ on bone metabolism,effects of La3＋ on the osteogenic and adipogenic differentiation of pri-mary mouse bone marrow stromal cells（BMSCs） were studied by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium bromide（MTT） test,alkaline phosphatase（ALP） activity measurement,mineralized function,oil red O stain and measurement.The results showed that La3＋ pro-moted the proliferation of BMSCs except at 1×10-10 and 1×10-6 mol/L.The effect of La3＋ on the osteogenic differentiation depended on con-centrations at the 7th day,but the osteogenic differentiation was inhibited at any concentration at the 14th day.La3＋ promoted the formation of mineralized matrix nodules except at 1×10-8 and 1×10-5 mol/L.La3＋ inhibited adipogenic differentiation except at 1×10-10 and 1×10-7 mol/L at the 10th day,and inhibited adipogenic differentiation except at 1×10-9 mol/L at the 16th day.These findings suggested that La3＋ might have protective effect on bone at appropriate dose and time.This would be valuable for better understanding the mechanism of the effect of La3＋ on bone metabolism.

Effects of scandium chloride on osteogenic and adipogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells(MSCs)are multi-potent cells that are able to differentiate and mature into various types of cells under a certain microenvironment for cell therapy and tissue regeneration.Scandium(Sc),an important rare earth element,recently has been intensively investigated in biomedical fields as well as industrial engineering,and chloride channels have been proven to be able to affect osteogenic differentiation.Thus,it is significant to investigate effects of ScCl_(3)on cell activities of MSCs.In this paper,rat bone MSCs(rBMSCs)were co-cultured with various concentrations of ScCl_(3)(1×10^(-8),1×10^(-6),and 1×10^(-4)mol/L)to evaluate their influence on cell proliferation as well as osteogenic and adipogenic differentiation in vitro.The results indicate that ScCl_(3)promotes the proliferation of rBMSCs initially,which is yet reduced upon ion accumulation.We used immunofluorescence staining,quantitative real time polymerase chain reactions,and assays measuring alkaline phosphatase activity,mineralized deposits,and intracytoplasmic lipids to reveal that rBMSCs treated with ScCl_(3)at concentrations of 1×10^(-8)-1×10^(-6)mol/L can enhance levels of osteogenic differentiation in a dosedependent manner and reduce adipogenic differentiation to a certain degree through Wnt/β-catenin signaling pathway.These results indicate that appropriate concentrations of ScCl_(3)can improve osteogenic differentiation in the lineage commitment of rBMSCs,and thus,promote bone remodeling.This study implies that ScCl_(3) possesses great potentials in the treatment of bone diseases and would provide new strategy of designing composites by SiCl3 doping for biomedical applications in the future.

Ligand density-dependent influence of arginine-glycine- aspartate functionalized gold nanoparticles on osteogenic and adipogenic differentiation of mesenchymal stem cells

Extracellular matrix （ECM） plays a very important role in regulating cell function and fate. It is highly desirable to fabricate biomimetic models to investigate the role of ECM in stem cell differentiation. In this study, arginine- glycine--aspartate （RGD）-modified gold nanoparticles （Au NPs） with tunable surface ligand density were prepared to mimic the ECM microenvironment. Their effect on osteogenic and adipogenic differentiation of human mesenchymal stem cells （MSCs） was investigated. The biomimetic Au NPs were taken up by MSCs in a ligand density-dependent manner. The biomimetic NPs with a high RGD density had an inhibitive effect on the alkaline phosphatase （ALP） activity, calcium deposition, and osteogenic marker gene expression of MSCs. Their effect on oil droplet formation and adipogenic marker gene expression was negative when RGD density was low, while their effect was promotive when RGD density was high. The biomimetic Au NPs regulated the osteogenic and adipogenic differentiation of MSCs mainly through affecting the focal adhesion and cytoskeleton. This study highlights the roles of biomimetic NPs on stem cell differentiation that could provide a meaningful strategy in fabricating functional biomaterials for tissue engineering and biomedical applications.

Imipramine Inhibits Adipogenic Differentiation in Both 3T3-L1 Preadioocvtes and Mouse Marrow Stromal Cells

Imipramine （IM） has been widely used clinically for the treatment of mental disorders. Its actions on tissues or organs other than the nervous system also need to be understood for its proper clinical use. In this study, the effects of IM on adipogenic differentiation in both 3T3-L1 preadipocytes and mouse marrow stromal cells （MSCs） were investigated. The results showed that fewer adipocytic cells were developed from 3T3-L1 preadipocytes in the presence of 0.001 to 1 μmol/L of IM as compared to control. Similar inhibitory effect was also observed in mouse MSCs. The decrease in the formation of adipocytes was accompanied with significant down-regulation at mRNA expression of the early adipogenic transcription factor, peroxisome proliferator-activated receptor γ2 （PPARγ2）. Western blot analysis further revealed that the protein expression of PPARγ2 was reduced markedly in ceils treated with IM at concentrations of 0.01, 0.1 and 1 μmol/L, suggesting that the suppression on PPAR72 was involved in IM＇s inhibition on MSCs adipogenesis. Moreover, IM at the above concentrations could stimulate the mRNA expression of β2-adrenergic receptor （AR） and β3-AR, which implicated that the effect of IM on adipogenic differentiation was partially mediated by β-ARs. Our results demonstrated for the first time that the conventional antidepressive imipramine exerts accompanied inhibitory effect on adipocyte formation, which may have possible clinical implications.

Molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rat bone marrow stromal stem cells(rBMSCs)

Objective：The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of r BMSCs.Methods：r BMSCs were harvested from SD rats,and determination of alkaline phosphatase（ALP）activity,quantification of mineralization by Alizarin Red S staining,and the m RNA expression of osteogenic differentiation markers（Runx2,BMP-2,and OSX）by RT-PCR after r BMSCs stimulated by osteogenic induction with（0.1–10）μg/m L of quercitrin,quantification of Lipid droplet by Oil Red O staining and the m RNA expression of adipogenic differentiation marker（,and a P2）by RT-PCR after r BMSCs stimulated by adipogenic induction with（0.1-10）μg/m L of quercitrin.Results：Quercitrin can up-regulate the m RNA expression of osteogenic differentiation markers（Runx2,BMP-2,and OSX）and increase ALP activity and mineralization after osteogenic induction,on the other hand quercitrin can suppress the m RNA expression of adipogenic differentiation markers（,and a P2）and decrease lipid droplet after adipogenic induction.Conclusion：This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of r BMSCs,which was associated with the up-regulation of Runx2,BMP-2,and OSX m RNA expression and the down-regulation of,and a P2 m RNA expression.

Proliferation and adipogenic differentiation of human adipose-derived stem cells isolated from middle-aged patients with prominent orbital fat in the lower eyelids

Aim:To evaluate the age-related effects on the adipogenic differentiation and proliferation potentials of human orbital adipose-derived stem cells(OASCs).Methods:Orbital adipose samples were harvested from the central fat compartment in the lower eyelids of 10 young and middle-aged patients during routine blepharoplasty surgery.After assessment of the morphological changes of adipocytes with aging,OASCs were isolated from the fat samples and expanded in vitro.Differences in the stem cell colony number(fibroblast colony-forming unit),growth rate and phenotype characterization(flow cytometry analysis)were evaluated.The ability of OASCs to differentiate into adipocytes was determined by oil red O staining and the mRNA expression level of peroxisome proliferator-activated receptorγ.Results:Fat cell size showed a decreasing trend with advancing age.Although no difference was found in the expression of cell surface markers,the colony number and proliferative rate of OASCs from middle-aged donors were significantly lower than those from the young donors.The adipogenic differentiation capacity of middle-aged OASCs was also reduced.These differences were statistically significant(P<0.001).Conclusion:The data showed that the progenitor cell number,proliferation capacity and adipogenic potential of OASCs decreased with aging,suggesting that using OASCs from elderly patients for therapeutic purposes might be restricted.

miR-103-3p regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome(MDS)may be related to the abnormal expression of microRNAs(miRNAs),which could influence the differentiation capacity of mesenchymal stem cells(MSCs)towards adipogenic and osteogenic lineages.In this study,exosomes from bone marrow plasma were successfully extracted and identified.Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its 0.52-fold downregulation in patients with MDS compared with controls(NOR)and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs.Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro,while inhibition of miR-103-3p showed the opposite results in NOR-MSCs.Thus,the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs,significantly impacting MDS-MSCs differentiation.The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation,thereby providing possible target for the treatment of MDS pathogenesis.

Unveiling the role of hypoxia-inducible factor 2alpha in osteoporosis:Implications for bone health

BACKGROUND Osteoporosis(OP)has become a major public health problem worldwide.Most OP treatments are based on the inhibition of bone resorption,and it is necessary to identify additional treatments aimed at enhancing osteogenesis.In the bone marrow(BM)niche,bone mesenchymal stem cells(BMSCs)are exposed to a hypoxic environment.Recently,a few studies have demonstrated that hypoxiainducible factor 2alpha(HIF-2α)is involved in BMSC osteogenic differentiation,but the molecular mechanism involved has not been determined.AIM To investigate the effect of HIF-2αon the osteogenic and adipogenic differentiation of BMSCs and the hematopoietic function of hematopoietic stem cells(HSCs)in the BM niche on the progression of OP.METHODS Mice with BMSC-specific HIF-2αknockout(Prx1-Cre;Hif-2αfl/fl mice)were used for in vivo experiments.Bone quantification was performed on mice of two genotypes with three interventions:Bilateral ovariectomy,semilethal irradiation,and dexamethasone treatment.Moreover,the hematopoietic function of HSCs in the BM niche was compared between the two mouse genotypes.In vitro,the HIF-2αagonist roxadustat and the HIF-2αinhibitor PT2399 were used to investigate the function of HIF-2αin BMSC osteogenic and adipogenic differentiation.Finally,we investigated the effect of HIF-2αon BMSCs via treatment with the mechanistic target of rapamycin(mTOR)agonist MHY1485 and the mTOR inhibitor rapamycin.RESULTS The quantitative index determined by microcomputed tomography indicated that the femoral bone density of Prx1-Cre;Hif-2αfl/fl mice was lower than that of Hif-2αfl/fl mice under the three intervention conditions.In vitro,Hif-2αfl/fl mouse BMSCs were cultured and treated with the HIF-2αagonist roxadustat,and after 7 d of BMSC adipogenic differentiation,the oil red O staining intensity and mRNA expression levels of adipogenesis-related genes in BMSCs treated with roxadustat were decreased;in addition,after 14 d of osteogenic differentiation,BMSCs treated with roxadustat exhibited increased expression of osteogenesis-related genes.The opposite effects were shown for mouse BMSCs treated with the HIF-2αinhibitor PT2399.The mTOR inhibitor rapamycin was used to confirm that HIF-2αregulated BMSC osteogenic and adipogenic differentiation by inhibiting the mTOR pathway.Consequently,there was no significant difference in the hematopoietic function of HSCs between Prx1-Cre;Hif-2αfl/fl and Hif-2αfl/fl mice.CONCLUSION Our study showed that inhibition of HIF-2αdecreases bone mass by inhibiting the osteogenic differentiation and increasing the adipogenic differentiation of BMSCs through inhibition of mTOR signaling in the BM niche.

Maternal inappropriate calcium intake aggravates dietary-induced obesity in male offspring by affecting the differentiation potential of mesenchymal stem cells

BACKGROUND The effects of inappropriate dietary calcium intake in early life on later obesity have not been fully elucidated.AIM To raise the mechanism of maternal calcium intake on the multi-differentiation potential of mesenchymal stem cells among their male offspring.METHODS Four-week-old female C57BL/6N mice were fed by deficient,low,normal and excessive calcium reproductive diets throughout pregnancy and lactation.Bone MSCs(BMSCs)were obtained from 7-day-old male offspring to measure the adipogenic differentiation potential by the Wnt/β-catenin signaling pathway.The other weaning male pups were fed a high-fat diet for 16 wk,along with normalfat diet as the control.Then the serum was collected for the measurement of biochemical indicators.Meanwhile,the adipose tissues were excised to analyze the adipocyte sizes and inflammatory infiltration.And the target gene expressions on the adipogenic differentiation and Wnt/β-catenin signaling pathway in the adipose tissues and BMSCs were determined by real-time reverse transcription polymerase chain reaction.RESULTS Compared with the control group,maternal deficient,low and excessive calcium intake during pregnancy and lactation aggravated dietary-induced obesity,with larger adipocytes,more serious inflammatory infiltration and higher serum metabolism indicators by interfering with higher expressions of adipogenic differentiation(PPARγ,C/EBPα,Fabp4,LPL,Adiponectin,Resistin and/or Leptin)among their male offspring(P<0.05).And there were significantly different expression of similar specific genes in the BMSCs to successfully polarize adipogenic differentiation and suppress osteogenic differentiation in vivo and in vitro,respectively(P<0.05).Meanwhile,it was accompanied by more significant disorders on the expressions of Wnt/β-catenin signaling pathway both in BMSCs and adulthood adipose tissues among the offspring from maternal inappropriate dietary calcium intake groups.CONCLUSION Early-life abnormal dietary calcium intake might program the adipogenic differentiation potential of BMSCs from male offspring,with significant expressions on the Wnt/β-catenin signaling pathway to aggravate high-fat-diet-induced obesity in adulthood.

Is 1.28 parts per million biomarker specific for neural progenitor cells?

Nuclear magnetic resonance-visible mobile lipid, at 1.28 parts per million （ppm）, is thought to be due to mobile lipid droplets formed in cells and has been considered unique for neural progenitor cells. However, this idea remains controversial. The present study examined the 1.28 ppm biomarker in other stem cells and non-stem cells, and explored the relationship between 1.28 ppm biomarker and mobile lipid droplets. 1H nuclear magnetic resonance spectroscopy of EC109 cells, mesenchymal stem cells （MSCs） and adipogenic cells differentiated from MSCs was performed. Results show that 1.28 ppm biomarker was observed in human MSCs, but was absent from EC109 cells. Following adipogenic differentiation induced for 2 weeks, the 1.28 ppm biomarker climbed remarkably, with mobile lipid droplet generation, suggesting that the 1.28 ppm biomarker is not specific for neural progenitor cells because it is also observed in MSCs and adipogenic-induced differentiated cells. Moreover, it is possible to monitor MSCs differentiation following cell transplantation, using 1.28 ppm biomarker changes.

Nanoparticles and their effects on differentiation of mesenchymal stem cells

Over the past decades,advancements in nanoscience and nanotechnology have resulted in numerous nanomedicine platforms.Various nanoparticles,which exhibit many unique properties,play increasingly important roles in the field of biomedicine to realize the potential of nanomedicine.Due to the capacity of self-renewal and multilineage mesenchymal differentiation,mesenchymal stem cells(MSCs)have been widely used in the area of regenerative medicine and in clinical applications due to their potential to differentiate into various lineages.There are several factors that impact the differentiation of MSCs into different lineages.Many types of biomaterials such as polymers,ceramics,and metals are commonly applied in tissue engineering and regenerative therapies,and they are continuously refined over time.In recent years,along with the rapid development of nanotechnology and nanomedicine,nanoparticles have been playing more and more important roles in the fields of biomedicine and bioengineering.The combined use of nanoparticles and MSCs in biomedicine requires greater knowledge of the effects of nanoparticles on MSCs.This review focuses on the effects of four inorganic or metallic nanoparticles(hydroxyapatite,silica,silver,and calcium carbonate),which are widely used as biomaterials,on the osteogenic and adipogenic differentiation of MSCs.In this review,the cytotoxicity of these four nanoparticles,their effects on osteogenic/adipogenic differentiation of MSCs and the signalling pathways or transcription factors involved are summarized.In addition,the chemical composition,size,shape,surface area,surface charge and surface chemistry of nanoparticles,have been reported to impact cellular behaviours.In this review,we particularly emphasize the influence of their size on cellular responses.We envision our review will provide a theoretical basis for the combined application of MSCs and nanoparticles in biomedicine.

Transient expression of inactive RB in mesenchymal stem cells impairs their adipogenic potential and is associated with hypermethylation of the PPARγ2 promoter

The retinoblastoma gene product(pRb)is a chromatin-associated protein that can either suppress or promote activity of key regulators of tissue-specific differentiation.We found that twelve weeks after transfection of the exogenous active(ΔB/X andΔр34)or inactive(ΔS/N)forms of RB into the 10T1/2 mesenchymal stem cells and clonal selection not a single cell line did contain exogenous RB,despite being G-418 resistant.However,the consequences of the transient production of exogenous RB had different effects on the cell fate.TheΔB/X andΔр34 cells transfected with active form of RB showed elevated levels of inducible adipocyte differentiation(AD).On the contrary,theΔS/N cells transfected with inactive RB mutant were insensitive to induction of AD associated with abolishing of expression of the PPARγ2.Additionally,the PPARγ2 promoter in undifferentiatedΔS/N cells was hypermethylated,but all except−60 position CpG became mostly demethylated after cells exposure to AD.We conclude that while transient expression of inactive exogenous RB induces long term epigenetic alterations that prevent adipogenesis,production of active exogenous RBs results in an AD-promoting epigenetic state.These results indicate that pRb is involved in the establishment of hereditary epigenetic memory at least by creating a methylation pattern of PPARγ2.