Differentiation Of Adipose-derived Stromal Cells Research Articles

Characteristics of the Dynamic Evolutionary Pathway of ADSCs Induced Differentiation into Astrocytes Based on scRNA-Seq Analysis.

We employed single-cell transcriptome sequencing to reveal the dynamic gene expression changes during the differentiation of adipose-derived stromal cells (ADSCs) into astrocytes. Single-cell RNA sequencing was conducted on cells from the ADSCs group and the induced groups at 2, 7, 14, and 21days using the 10 × Chromium platform. Data underwent quality control and dimensionality reduction. Cell differentiation trajectories were constructed using Monocle2, and differentially expressed genes (DEGs) in each cell cluster were identified using differential selection algorithms. DEGs at each time point were annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and regulatory intensities of transcription factors were analyzed using SCENIC. Integrating all groups, a total of five samples were divided into 13 cell clusters (0-12 clusters). DEGs between clusters and those compared with ADSCs at various induced time points showed distinct specificities. Monocle2 constructed cell differentiation trajectories; ADSCs can differentiate into mature astrocytes not only through the direct pathway from the 1 branch to the 3 branch but also through an indirect pathway, involving the 1 branch to the 2 branch before progressing to the 3 branch. SCENIC analysis highlighted the critical regulatory roles of STAT1, MYEF2, and SOX6 transcription factors during the differentiation of ADSCs into astrocytes. ADSCs can differentiate into mature astrocytes through two distinct pathways: direct and indirect. By the 14th day of induction, mature astrocytes have formed, characterized by a cell cycle arrest in mitosis. Further induction leads to degenerative senescence changes in differentiated cells.

Genomic characteristics of adipose-derived stromal cells induced into neurons based on single-cell RNA sequencing

Adipose-derived stromal cells (ADSCs) can be induced to differentiate into neurons, representing the most promising avenue for cell therapy. However, the molecular mechanism and genomic characteristics of the differentiation of ADSCs into neurons remain poorly understood. In this study, cells from the adult ADSCs group, induction 1h, 3h, 5h, 6h, and 8h groups were selected for single-cell RNA sequencing (scRNA-Seq). Samples from these seven-time points were sequenced and analyzed. The expression of neuron marker genes, including NES, MAP2, TMEM59L, PTK2B, CHN1, DNM1, NRSN2, FBLN2, SCAMP1, SLC1A1, DLG4, CDK5, and ENO2, was found to be low in the ADSCs group, but highly expressed in differentiated cell clusters. The expression of stem cell marker genes, including CCND1, IL1B, MMP1, MMP3, MYO10, and BMP2, was the highest in the ADSCs cluster. This expression decreased significantly with the extension of induction time. Gene ontology (GO) enrichment analysis of upregulated genes in the induced samples showed that the biological processes related to neuronal differentiation and development, such as neuronal differentiation, projection, and apoptosis, were significantly upregulated with a longer induction time during cell cluster differentiation. The results of the cell communication analysis demonstrated the gradual formation of complex neural network connections between ADSC-derived neurons through receptor and ligand pairs at 5h after the induction of differentiation.

Endoplasmic reticulum stress and death receptor-mediated apoptosis in the neuronal differentiation of adult adipose-derived stromal cells

Apoptosis is the primary cause of cell death in the differentiation of Adipose-derived stromal cells (ADSCs) into neurons. However, the relationship between endoplasmic reticulum stress (ERS) and death receptor-mediated apoptosis in ADSC-induced neuronal differentiation is not clear. ADSCs were isolated and induced to differentiate into neurons using β-mercaptoethanol. The expression of neuron-specific enolase (NSE), GRP94, CHOP, Fas/FasL, TNFR1/TNF-α, DR5/TRAIL, Caspase8, and Caspase3 in ADSCs was examined using immunocytochemistry and Western blotting before induction, during pre-induction, and after induction. Transmission electron microscopy (TEM) was used to observe changes in the morphology of the endoplasmic reticulum (ER), and the MTT assay was employed to measure cell viability in the uninduced and induced groups. Additionally, the number of apoptotic cells during the induction process was measured using flow cytometry with Annexin V/PI. With increasing induction time, the positive expression rates of CHOP, Fas/FasL, Caspase8, Caspase-3, and NSE gradually increased, while the positive expression rate of GRP94 decreased. TNFR1/TNF-α and DR5/TRAIL peaked at 5 h post-induction and then decreased at 8 h. TEM revealed swelling and expansion of the ER, vacuolar changes, and degranulation in cells. The MTT assay showed a gradual decrease in the absorbance of surviving cells in all groups. Flow cytometry indicated an increasing rate of apoptosis in cells. Therefore, ERS in the normal culture and growth of ADSCs, manifesting as enhanced unfolded protein response (UPR), maintains the normal survival of ADSCs. However, in the process of ADSC-induced differentiation into neurons, ERS and death receptor-mediated apoptosis are significant causes of cell death.

Flexible Allyl-Modified Gelatin Photoclick Resin Tailored for Volumetric Bioprinting of Matrices for Soft Tissue Engineering.

Volumetric bioprinting (VBP) is a light-based 3D printing platform, which recently prompted a paradigm shift for additive manufacturing (AM) techniques considering its capability to enable the fabrication of complex cell-laden geometries in tens of seconds with high spatiotemporal control and pattern accuracy. A flexible allyl-modified gelatin (gelAGE)-based photoclick resin is developed in this study to fabricate matrices with exceptionally soft polymer networks (0.2-1.0kPa). The gelAGE-based resin formulations are designed to exploit the fast thiol-ene crosslinking in combination with a four-arm thiolated polyethylene glycol (PEG4SH) in the presence of a photoinitiator. The flexibility of the gelAGE biomaterial platform allows one to tailor its concentration spanning from 2.75% to 6% and to vary the allyl to thiol ratio without hampering the photocrosslinking efficiency. The thiol-ene crosslinking enables the production of viable cell-material constructs with a high throughput in tens of seconds. The suitability of the gelAGE-based resins is demonstrated by adipogenic differentiation of adipose-derived stromal cells (ASC) after VBP and by the printing of more fragile adipocytes as a proof-of-concept. Taken together, this study introduces a soft photoclick resin which paves the way for volumetric printing applications toward soft tissue engineering.

Chondrogenic Differentiation of Adipose-Derived Stromal Cells Induced by Decellularized Cartilage Matrix/Silk Fibroin Secondary Crosslinking Hydrogel Scaffolds with a Three-Dimensional Microstructure.

Finding an ideal scaffold is always an important issue in the field of cartilage tissue engineering. Both decellularized extracellular matrix and silk fibroin have been used as natural biomaterials for tissue regeneration. In this study, a secondary crosslinking method of γ irradiation and ethanol induction was used to prepare decellularized cartilage extracellular matrix and silk fibroin (dECM-SF) hydrogels with biological activity. Furthermore, the dECM-SF hydrogels were cast in custom-designed molds to produce a three-dimensional multi-channeled structure to improve internal connectivity. The adipose-derived stromal cells (ADSC) were seeded on the scaffolds, cultured in vitro for 2 weeks, and implanted in vivo for another 4 and 12 weeks. The double crosslinked dECM-SF hydrogels exhibited an excellent pore structure after lyophilization. The multi-channeled hydrogel scaffold presents higher water absorption ability, surface wettability, and no cytotoxicity. The addition of dECM and a channeled structure could promote chondrogenic differentiation of ADSC and engineered cartilage formation, confirmed by H&E, safranin O staining, type II collagen immunostaining, and qPCR assay. In conclusion, the hydrogel scaffold fabricated by the secondary crosslinking method has good plasticity and can be used as a scaffold for cartilage tissue engineering. The multi-channeled dECM-SF hydrogel scaffolds possess a chondrogenic induction activity that promotes engineered cartilage regeneration of ADSC in vivo.

GDF11 inhibits adipogenesis of human adipose-derived stromal cells through ALK5/KLF15/β-catenin/PPARγ cascade

Obesity is a metabolic disease characterized by excessive fat storage, and the adipogenic differentiation of adipose-derived stromal cells (ADSCs) is closely linked to its occurrence. Growth differentiation factor 11 (GDF11), a well-known molecule in the field of anti-aging, also has great potential in regulating stem cell differentiation. In this study, we found that GDF11 inhibited adipogenic differentiation of human ADSCs in vitro by activating the WNT/β-catenin and SMAD2/3 pathways while inhibiting the AKT pathway. Moreover, the transcription factor Kruppel-like factor 15 (KLF15) was discovered to be an important downstream factor for GDF11 in inhibiting adipogenesis via the WNT/β-catenin pathway. Furthermore, AlphaFold2 structure prediction and inhibitor-blocking experiments revealed that ALK5 is a functional receptor of GDF11. Collectively, we demonstrated that GDF11 is a potential target for inhibiting adipogenic differentiation and combating obesity.

25-Hydroxyvitamin D Increases Insulin-Stimulated Glucose Uptake by Enhancing Adipocyte Differentiation.

Vitamin D and its receptor (vitamin D receptor; VDR) regulate calcium homeostasis in mammals. Recently, studies have shown that serum concentrations of 25-hydroxyvitamin D (25VD) are negatively associated with insulin resistance and the incidence of type 2 diabetes. In adipose tissues, glucose transporter 4 (GLUT4) contributes to insulin-stimulated glucose uptake; however, the effect of 25VD on glucose uptake in adipocytes remains unclear. We examined the role of 25VD in glucose uptake and the differentiation of adipose-derived stromal cells. Insulin-stimulated glucose uptake in adipocytes was increased by treatment with 25VD and decreased by VDR knockdown. The expression levels of GLUT4 were upregulated by 25VD treatment. 25VD exposure increased the expression of adipocyte differentiation-related genes including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins through VDR, thereby enhancing the formation of mature adipocytes. Moreover, 25VD increased the expression levels of 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), which catalyzes the conversion of cortisone to cortisol in a concentration-dependent manner. 25VD-stimulated adipocyte differentiation was suppressed by HSD11B1 knockdown. Cortisone together with 25VD enhanced adipocyte differentiation, whereas synthesized glucocorticoid dexamethasone-induced adipocyte differentiation is not promoted by 25VD. Overall, these results indicate that 25VD stimulates adipocyte differentiation through the induction of HSD11B1 expression, leading to increased insulin-induced glucose uptake in adipocytes.

High-fat diet decreases H3K27ac in mice adipose-derived stromal cells.

The study goal was to analyze the effects of a high-fat diet (HFD) on the histone 3 lysine 27 (H3K27) posttranscriptional modifications and the expression of histone-modifying enzymes in adipose-derived stromal cells (ASCs) from white adipose tissue (WAT). Male C57BL/6J mice received control or HFD for 12 weeks. The ASCs were isolated from subcutaneous and visceral (epididymal) WAT, cultivated, and evaluated for expression of H3K27trimethylation (H3K27me3) and H3K27 acetylation (H3K27ac) by Western blot. The transcription of histone-modifying enzymes was analyzed by real-time polymerase chain reaction. When compared with control, HFD ASCs showed a decrease in H3K27ac enrichment in subcutaneous and visceral WAT and ATP-citrate lyase expression in subcutaneous WAT. Curiously, the expression of CREB-binding proteinwas increased in visceral ASCs from HFD-fed mice. These results show that an HFD significantly reduces acetylation of H3K27 in ASCs and the expression of ATP-citrate lyase in subcutaneous ASCs, suggesting that, in this fat depot, the H3K27ac reduction could be partly due to lower acetyl-coenzyme A availability. H3K27ac is an epigenetic mark responsible for increasing the transcription rate and its reduction can have an important impact on ASC proliferation and differentiation potential.

Specific features of ex-obese patients significantly influence the functional cell properties of adipose-derived stromal cells.

Adipose‐derived stromal cells (ADSC) are increasingly used in clinical applications due to their regenerative capabilities. However, ADSC therapies show variable results. This study analysed the effects of specific factors of ex‐obese patients on ADSC functions. ADSC were harvested from abdominal tissues (N = 20) after massive weight loss. Patients were grouped according to age, sex, current and maximum body mass index (BMI), BMI difference, weight loss method, smoking and infection at the surgical site. ADSC surface markers, viability, migration, transmigration, sprouting, differentiation potential, cytokine secretion, telomere length and mtDNA copy number were analysed. All ADSC expressed CD73, CD90, CD105, while functional properties differed significantly among patients. A high BMI difference due to massive weight loss was negatively correlated with ADSC proliferation, migration and transmigration, while age, sex or weight loss method had a smaller effect. ADSC from female and younger donors and individuals after weight loss by increase of exercise and diet change had a higher activity. Telomere length, mtDNA copy number, differentiation potential and the secretome did not correlate with patient factors or cell function. Therefore, we suggest that factors such as age, sex, increase of exercise and especially weight loss should be considered for patient selection and planning of regenerative therapies.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4’-Dihydroxyflavone, U0126, and N-Acetyl Cysteine

Background and ObjectivesFlavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4’-dihydroxyflavone (3, 4’-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs).Methods and ResultsTreatment of 3, 4’-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4’-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4’-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4’-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4’-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs.ConclusionsOur results showed that co-treatment of 3, 4’-DHF, U0126, and/or NAC cumulatively regulated osteogenesis in eADSCs, suggesting that 3, 4’-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Salidroside promoted osteogenic differentiation of adipose-derived stromal cells through Wnt/\u03b2-catenin signaling pathway

BackgroundBone disease causes short-term or long-term physical pain and disability. It is necessary to explore new drug for bone-related disease. This study aimed to explore the role and mechanism of Salidroside in promoting osteogenic differentiation of adipose-derived stromal cells (ADSCs).MethodsADSCs were isolated and treated with different dose of Salidroside. Cell count kit-8 (CCK-8) assay was performed to assess the cell viability of ADSCs. Then, ALP and ARS staining were conducted to assess the early and late osteogenic capacity of ADSCs, respectively.Then, differentially expressed genes were obtained by R software. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed genes were further analyzed. The expression of OCN, COL1A1, RUNX2, WNT3A, and β-catenin were measured by real-time PCR and Western blot analysis. Last, β-catenin was silenced by small interfering RNA.ResultsSalidroside significantly increased the ADSCs viability at a dose-response manner. Moreover, Salidroside enhanced osteogenic capacity of ADSCs, which are identified by enhanced ALP activity and calcium deposition. A total of 543 differentially expressed genes were identified between normal and Salidroside-treated ADSCs. Among these differentially expressed genes, 345 genes were upregulated and 198 genes were downregulated. Differentially expressed genes enriched in the Wnt/β-catenin signaling pathway. Western blot assay indicated that Salidroside enhanced the WNT3A and β-catenin expression. Silencing β-catenin partially reversed the promotion effects of Salidroside. PCR and Western blot results further confirmed these results.ConclusionSalidroside promoted osteogenic differentiation of ADSCs through Wnt/β-catenin signaling pathway.

Osteogenic differentiation of human mesenchymal stromal cells and fibroblasts differs depending on tissue origin and replicative senescence

The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-β signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-β signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-β signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

Osteogenic Differentiation of Adipose-Derived Stromal Cells: From Bench to Clinics.

In addition to mesenchymal stem cells, adipose-derived stem/stromal cells (ASCs) are an attractive source for a large variety of cell-based therapies. One of their most important potential applications is related to the regeneration of bone tissue thanks to their capacity to differentiate in bone cells. However, this requires a proper control of their osteogenic differentiation, which depends not only on the initial characteristics of harvested cells but also on the conditions used for their culture. In this review, we first briefly describe the preclinical and clinical trials using ASCs for bone regeneration and present the quantitative parameters used to characterize the osteogenic differentiation of ASCs. We then focus on the soluble factors influencing the osteogenic differentiation of ACS, including the steroid hormones and various growth factors, notably the most osteoinductive ones, the bone morphogenetic proteins (BMPs). Impact statement Adipose-derived stromal/stem cells are reviewed for their use in bone regeneration.

Tailored Melt Electrowritten Scaffolds for the Generation of Sheet-Like Tissue Constructs from Multicellular Spheroids.

Melt electrowriting (MEW) is an additive manufacturing technology that is recently used to fabricate voluminous scaffolds for biomedical applications. In this study, MEW is adapted for the seeding of multicellular spheroids, which permits the easy handling as a single sheet-like tissue-scaffold construct. Spheroids are made from adipose-derived stromal cells (ASCs). Poly(ε-caprolactone) is processed via MEW into scaffolds with box-structured pores, readily tailorable to spheroid size, using 13-15 µm diameter fibers. Two 7-8 µm diameter "catching fibers" near the bottom of the scaffold are threaded through each pore (360 and 380 µm) to prevent loss of spheroids during seeding. Cell viability remains high during the two week culture period, while the differentiation of ASCs into the adipogenic lineage is induced. Subsequent sectioning and staining of the spheroid-scaffold construct can be readily performed and accumulated lipid droplets are observed, while upregulation of molecular markers associated with successful differentiation is demonstrated. Tailoring MEW scaffolds with pores allows the simultaneous seeding of high numbers of spheroids at a time into a construct that can be handled in culture and may be readily transferred to other sites for use as implants or tissue models.

Alterations of MEK1/2-ERK1/2, IFNγ and Smad2/3 associated Signalling pathways during cryopreservation of ASCs affect their differentiation towards VSMC-like cells

Vascular smooth muscle cells (VSMCs) play essential roles in regulating blood vessel form and function and they are required for vascular tissue regeneration. Multipotent adipose derived stromal cells (ASCs) can be differentiated into VSMC-like cells, which can be used as a potential VSMC source for the development of vascular tissue. However, the effects of cryopreservation on the differentiation of ASCs towards VSMCs are poorly studied to date. This study compared fresh ASCs (FA) vs. cryopreserved ASCs (CA) with respect to their differentiation potential towards VSMC-like cells. The expression of contractile VSMC markers (such as smoothelin) and cell contractility were investigated. It was found that VSMC-like cells derived from CA expressed smoothelin gene and protein at lower levels and showed compromised contractility in response to vasoconstrictors, when compared with those derived from FA. Moreover, it was demonstrated that this negative effect of cryopreservation could be mediated by MEK1/2-ERK1/2, IFNγ and Smad2/3 associated Signalling pathways. Treatment of CA with MEK1/2-ERK1/2 activator or IFNγ neutralizing antibodies enhanced Smad2/3 phosphorylation and showed a rescue of the negative effect of cryopreservation on the differentiation of ASCs towards VSMC-like cells. These findings are important for defining approaches that may use cryopreserved ASCs for vascular tissue regeneration.

Substrate elasticity regulates adipose-derived stromal cell differentiation towards osteogenesis and adipogenesis through β-catenin transduction

It is generally recognised that mesenchymal stem cells (MSCs) can differentiate into multiple lineages through guidance from the biophysical properties of the substrates. However, the precise biophysical mechanism that enables MSCs to respond to substrate properties remains unclear. In the current study, polydimethylsiloxane (PDMS) substrates with different stiffnesses were fabricated and the way in which the elastic modulus of the substrate regulated differentiation towards osteogenesis and adipogenesis in adipose-derived stromal cells (ASCs) was explored. Initially, a cell morphology change by SEM was observed between the stiff and soft substrates. The cytoskeleton stains including microfilament by F-actin and microtubule by α- and β-tubulin further showed a larger cell spreading area on the stiff substrate. Then the expression of vinculin, in charge for the linkage of adhesion molecules to the actin cytoskeleton, was enhanced on the stiff substrate. This change in focal adhesion plaque further triggered intracellular β-catenin signaling and promoted its nuclear translocation especially on the stiff substrate. The influence of β-catenin signaling on direct differentiation to osteogenic lineages was through direct binding between its downstream protein, Lef-1, and the osteogenic transcriptional factors, Runx2 and Osx, while on differentiation to adipogenic lineages was through modulating the expression of PPARγ. The imbalance of stiffness-induced β-catenin signaling finally induced a stronger osteogenesis and a weaker adipogenesis on the stiff substrate relative to those on the soft substrate. This study indicates the importance of stiffness on ASC differentiation and could help to increase understanding of the mechanism underlying molecular signal transduction from mechanosensing, mechanotransducing to stem cell differentiation. Statement of SignificanceMesenchymal stem cells can differentiate into multiple lineages, such as adipogenesis, myogenesis, neurogenesis, angiogenesis and osteogenesis, through influence of biophysical properties of the extracellular matrix. However, the precise bio-mechanism that triggers stem cell differentiation in response to matrix biophysical properties remains unclear. In the current study, we provide a series of experiments involving the characterization of cell morphology, microfilament, microtubule and adhesion capacity of adipose-derived stromal cells (ASCs) in response to substrate stiffness, and further elucidation of cytoplasmic β-catenin-dependent signal transduction, nuclear translocation and resultant promoter activation of transcriptional factors for osteogenesis and adipogenesis. This study provides an explanation on deeper understanding of bio-mechanism underlying substrate stiffness-triggered β-catenin signal transduction from active mechanosensing, mechanotransducing to stem cell differentiation.

Substrate elasticity regulates vascular endothelial growth factor A (VEGFA) expression in adipose-derived stromal cells: Implications for potential angiogenesis.

Adipose-derived stromal cells (ASCs) have potential in bioengineering angiogenesis due to their paracrine role in supporting endothelial tubulogenesis and vascular network formation. However, the precise mechanism of the inner angiogenic capacity of ASCs determined by the biophysical properties of the extracellular matrix needs to be further elucidated. In the current study, we fabricated two silicon-based elastomer polydimethylsiloxane (PDMS) substrates with different stiffnesses (stiff substrate, E = 195 kPa and soft substrate, E = 15 kPa) and found there were cytoskeletal changes in ASCs in response to different substrate stiffnesses. We then showed the expression of vinculin in focal adhesion plaques was enhanced and the nuclear translocation of β-catenin signaling was increased in ASCs on the stiff substrate relative to those on the soft substrate. We next used bioinformatics and found the downstream proteins of β-catenin signaling had binding sites in the promoter of vascular endothelial growth factor A (VEGFA), which is responsible for angiogenesis; then, we further confirmed the enhanced endogenous VEGFA expression in ASCs on the stiff substrate relative to that on the soft substrate. Finally, by using ectogenic VEGFA, we showed the stiff substrate could promote angiogenesis of ASCs in the form of more ring-like formations in 2D and vessel-like structure formations in 3D under VEGFA induction compared to that of the soft substrate. This study not only indicates the inner angiogenic capacity of ASCs but also elucidates the influence of substrate elasticity on ASC differentiation in bioengineering angiogenesis.

Inverse Opal Scaffolds with Gradations in Mineral Content for Spatial Control of Osteogenesis.

The design and fabrication of inverse opal scaffolds with gradations in mineral content to achieve spatial control of osteogenesis are described. The gradient in mineral content is established via the diffusion-limited transport of hydroxyapatite nanoparticles in a closely packed lattice of gelatin microbeads. The mineral-graded scaffold has an array of uniform pores and interconnected windows to facilitate efficient transport of nutrients and metabolic wastes, ensuring high cell viability. The graded distribution of mineral content can provide biochemical and mechanical cues for spatially regulating the osteogenic differentiation of adipose-derived stromal cells. This new class of scaffolds holds promise for engineering the interfaces between mineralized and unmineralized tissues.

Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles

BackgroundIncreasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs).ResultsHerein, we report that titanium dioxide (TiO2) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo.ConclusionsWe demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

Platelet-rich plasma respectively reduces and promotes adipogenic and myofibroblastic differentiation of human adipose-derived stromal cells via the TGF\u03b2 signalling pathway

Autologous fat grafting is a gold standard therapy for soft tissue defects, but is hampered by unpredictable postoperative outcomes. Fat graft enrichment with adipose-derived stromal cell (ASCs) was recently reported to enhance graft survival. Platelet-rich plasma (PRP) has also emerged as a biologic scaffold that promotes fat graft viability. Combined ASC/PRP fat grafting enrichment is thus a promising new regenerative medicine approach. The effects of PRP on ASC proliferation are well documented, but the impact of PRP on ASC differentiation has yet to be investigated in depth to further elucidate the PRP clinical effects. Here we analyzed the human ASC fate upon PRP treatment. PRP was found to sharply reduce the potential of ASCs to undergo differentiation into adipocytes. Interestingly, the PRP anti-adipogenic effect was accompanied by the generation of myofibroblast-like cells. Among the various factors released from PRP, TGFβ pathway activators played a critical role in both the anti-adipogenic and pro-myofibroblastic PRP effects. Overall, these data suggest that PRP participates in maintaining a pool of ASCs and in the repair process by promoting ASC differentiation into myofibroblast-like cells. TGFβ may provide an important target pathway to improve PRP clinical outcomes.