Culture Of Adipose-derived Mesenchymal Stem Cells Research Articles

Purification and Characterization of Extracellular Vesicles from Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (ADSCs) can promote the regeneration and reconstruction of various tissues and organs. Recent research suggests that their regenerative function may be attributed to cell-cell contact and cell paracrine effects. The paracrine effect is an important way for cells to interact and transfer information over short distances, in which extracellular vesicles (EVs) play a functional role as carriers. There is significant potential for ADSC EVs in regenerative medicine. Multiple studies have reported on the effectiveness of these methods. Various methods for extracting and isolating EVs are currently described based on principles such as centrifugation, precipitation, molecular size, affinity, and microfluidics. Ultracentrifugation is regarded as the gold standard for isolating EVs. Nevertheless, a meticulous protocol to highlight precautions during ultracentrifugation is still absent. This study presents the methodology and crucial steps involved in ADSC culture, supernatant collection, and EV ultracentrifugation. However, even though ultracentrifugation is cost-effective and requires no further treatment, there are still some inevitable drawbacks, such as a low recovery rate and EV aggregation.

P141 The Roles and Therapeutic Implications of Macrophages in Inducing Calcinosis Via Activin-A Pathway and CD206 Expression in Patients with Systemic Sclerosis

Abstract Background/Aims Calcinosis may result from localised trans-differentiation of tissue resident stem cells in the subcutaneous layer of affected skin in systemic sclerosis (SSc), as a severe disabling manifestation linked to ischaemia and local trauma. Polarised macrophages synergistically promote osteogenesis: M1 macrophages recruit mesenchymal stem cells leading to osteogenic differentiation, while M2 are responsible for osteoblast formation. Previous studies have implicated two pathways-Activin-A signalling and CD206 expression by macrophages - in heterotopic osteogenesis. We investigated the pathogenesis of calcinosis through macrophages, using a novel tissue culture model created by stimulation of adipose derived mesenchymal stem cells (ASCs) with SSc macrophages and explored the relevance of the Activin-A pathway. Methods Activin-A was measured by ELISA in diffuse cutaneous systemic sclerosis (dcSSc) patients' (n = 72), and healthy control (HC) plasma samples (n = 42). Macrophages were derived from peripheral blood monocytes of dcSSc patients (n = 4) and cultured under basal and BzATP-stimulated conditions. dcSSc macrophages were maintained in co-culture with ASCs in osteogenic media ±anti-activin-A neutralising antibody (anti-AA). Osteogenesis was assessed using Alizarin Red. Results Activin-A was increased in dcSSc patients’ plasma when compared to HC, most notably in anti-centromere antibody (ACA) subgroup (plasma activin-A 521±199pg/ml vs 255 ± 170pg/ml [p = 0.000065]) (Figure 1A). Moreover, there was synthesis of Activin A by SSc macrophages, enhanced by stimulation with BzATP (0.1µM) (basal Activin A 3.3 (0-14.5), BzATP stimulated 21.7 (7.7-418) pg/ml p NS) (Figure 1B). In co-cultures, the addition of SSc macrophages to ASCs led to significantly enhanced synthesis of Activin A, which was reduced by the addition of anti-AA (ASC monoculture 987+/-87, SSc macrophages+AScs 1599+/- 122, p < 0.0065, SSc macrophages+ASCs+anti-AA 1034+/-108 pg/ml, p < 0.013) (Figure 1C). Moreover, adding SSc macrophages to ASC cultures significantly enhanced calcinosis, as measured by Alizarin red stain, but anti-AA did not fully supress the Alizarin staining (ASC: 0.5, 0.4-0.7; ASC+M: 13.0, 5.8-17.2 p = 0.0022; ASC+M+anti-AA p NS median, IQR, red pixels per field) (Figure1D). Conclusion Elevated plasma Activin-A, most notable in ACA subgroup, is consistent with systemic upregulation of the Activin-A pathway in SSc. The interaction of activated macrophages with ASCs represents a potential model for calcinosis. Targeting Activin-A might benefit in this severe complication. Disclosure K. Liang: None. S. Lopez Garces: None. T. Searle: None. H. Lopez: Corporate appointments; CEO of Murigenics. G. Martin: Corporate appointments; Senior Scientific Officer Riptide. C. Denton: None. D. Abraham: None. R.J. Stratton: None.

MSC-derived exosomes enhance the anticancer activity of drugs in 3D spheroid of breast cancer cells

The unique properties of the MSC-derived exosome (MSC-Exos) make it an interesting drug-delivery tool for tumor cells. Drug delivery via exosomes helps overcome challenges to access the core of solid tumors. Here, we investigated exosomal chemotherapy drug formulations on the 3D multicellular spheroid models of breast cancer cell lines. The multicellular 3D spheroid of BT-474 and MDA-MB-231 breast cancer cell lines were made by liquid overlay technique. MSC-Exos were isolated from adipose-derived mesenchymal stem cells (ADMSCs) culture medium and characterized by their biological characteristics. Then paclitaxel (PTX) and cisplatin (CIS) were loaded into MSC-Exos. The cellular uptake and cytotoxic effect of these nanocarriers were evaluated by MTT and flow cytometry. MSC-Exos could efficiently facilitate cellular uptake in multicellular spheroids of breast cancer cell lines. EXO-PTX significantly inhibited the growth of spheroid in both MDA-MB-231 and BT-474 multicellular spheroids (P < 0.001). Moreover, apoptosis was more prominently inducted by EXO-PTX compared to the free PTX (P < 0.03). For cisplatin, although the exosomal formulation contained lower drug concentration compared to the free drug, the potency was increased. Based on our findings, the exosomal formulation of drugs induced higher toxicity and apoptosis in the 3D multicellular spheroids model with considerably lower drug concentrations. Drug-loaded MSC-Exos could be a potentially suitable alternative for cancer chemotherapy practices.

Mesenchymal Stem Cell Extracellular Vesicles from Tissue-Mimetic System Enhance Epidermal Regeneration via Formation of Migratory Cell Sheets.

The secretome of adipose-derived mesenchymal stem cells (ASCs) offers a unique approach to understanding and treating wounds, including the critical process of epidermal regeneration orchestrated by keratinocytes. However, 2D culture techniques drastically alter the secretory dynamics of ASCs, which has led to ambiguity in understanding which secreted compounds (e.g., growth factors, exosomes, reactive oxygen species) may be driving epithelialization. A novel tissue-mimetic 3D hydrogel system was utilized to enhance the retainment of a moreregenerative ASC phenotype and highlight the functional secretome differences between 2D and 3D. Subsequently, the ASC-secretome was stratified by molecular weight and the presence/absence of extracellular vesicles (EVs). The ASC-secretome fractions were then evaluated to assess for the capacity to augment specific keratinocyte activities. Culture of ASCs within the tissue-mimetic system enhanced protein secretion ~ 50%, exclusively coming from the > 100kDa fraction. The ASC-secretome ability to modulate epithelialization functions, including migration, proliferation, differentiation, and morphology, resided within the "> 100kDa" fraction, with the 3D ASC-secretome providing the greatest improvement. 3D ASCEV secretion was enhanced two-fold and exhibited dose-dependent effects on epidermal regeneration. Notably, ASC-EVs induced morphological changes in keratinocytes reminiscent of native regeneration, including formation of stratified cell sheets. However, only 3D-EVs promoted collective cell sheet migration and an epithelial-to-mesenchymal-like transition in keratinocytes, whereas 2D-EVs contained an anti-migratory stimulus. This study demonstrates how critical the culture environment is on influencing ASC-secretome regenerative capabilities. Additionally, the critical role of EVs in modulating epidermal regeneration is revealed and their translatability for future clinical therapies is discussed.

Effect assessment of a type of xeno-free and serum-free human adipose-derived mesenchymal stem cells culture medium by proliferation and differentiation capacities.

Human mesenchymal stem cells (hMSCs) possess broad prospects in pre-clinical research. In vitro amplification of hMSCs requires appropriate medium to reach the number of seed cells with clinical significance. However, the uncertainty of the heterologous components of the traditional fetal bovine serum (FBS) culture medium has great safety risks. Moreover, existing commercial hMSCs medium is very expensive, therefore a safer and more optimal hMSCs medium is urgently needed. Accordingly, we developed five components adipose-derived hMSCs (hADMSCs) medium without xenogenic components, named E5 SFM. which is mainly composed of knockout serum replacement (KSR), and additionally four components such as fibroblast growth factor and transferrin. Here, we mainly compared the E5 SFM with traditional FBS-containing medium and a commercial medium by surface markers testing, proliferation assay as well as osteogenic, adipogenicand chondrogenic differentiation assessment. We demonstrated that hADMSCs cultured in the E5 SFM showed similar morphological characteristics and immunophenotypes to those in other media. Notably, cell proliferative capability was similar to that in the commercial medium, but higher than that in the FBS-containing medium and other media. Additionally, their capabilities of adipogenic and osteogenic differentiation were significantly higher than those of other media. Consequently, we concluded that the E5 SFM medium can not only effectively promote cell proliferation of hMSCs, but also has optimal differentiative capacity and clear and simple ingredients.

Tissue-mimetic culture enhances mesenchymal stem cell secretome capacity to improve regenerative activity of keratinocytes and fibroblasts in vitro.

Mesenchymal stem/stromal cells (MSCs) are a heterogenous population of multipotent and highly secretory cells currently being investigated in the field of wound healing for their ability to augment tissue responses. The adaptive response of MSC populations to the rigid substrate of current 2D culture systems has been considered to result in a deterioration of regenerative 'stem-like' properties. In this study, we characterise how the improved culture of adipose-derived mesenchymal stem cells (ASCs) within a tissue-mimetic 3D hydrogel system, that is mechanically similar to native adipose tissue, enhances their regenerative capabilities. Notably, the hydrogel system contains a porous microarchitecture that permits mass transport, enabling efficient collection of secreted cellular compounds. By utilising this 3D system, ASCs retained a significantly higher expression of ASC 'stem-like' markers while demonstrating a significant reduction in senescent populations, relative to 2D. Additionally, culture of ASCs within the 3D system resulted in enhanced secretory activity with significant increases in the secretion of proteinaceous factors, antioxidants and extracellular vesicles (EVs) within the conditioned media (CM) fraction. Lastly, treatment of wound healing cells, keratinocytes (KCs) and fibroblasts (FBs), with ASC-CM from the 2D and 3D systems resulted in augmented functional regenerative activity, with ASC-CM from the 3D system significantly increasing KC and FB metabolic, proliferative and migratory activity. This study demonstrates the potential beneficial role of MSC culture within a tissue-mimetic 3D hydrogel system that more closely mimics native tissue mechanics, and subsequently how the improved phenotype augments secretory activity and potential wound healing capabilities of the MSC secretome.

Adipose-Derived Mesenchymal Stem Cells Differentiation Toward Cardiomyocyte-Like Cells on the PCL/PANI Nanofibrous Scaffold: An Experimental Study.

Owing to the fact that the heart tissue is not able to repair itself. Biomaterial-based scaffolds are important cues in tissue engineering (TE) applications. Recent advances in TE have led to the development of suitable scaffold architecture for various tissue defects. Given the importance of cellular therapy, it was the aim of the present study to differentiate cardio myocyte cells from human adipose-derived mesenchymal stem cells (Ad-MSCs) using suitable induction reagents (namely, 5-azacytidine and transforming growth factor beta (TGF-β)) on poly-caprolactone (PCL)/Poly aniline (PANI) Nano fibrous scaffolds prepared by electrospinning. For this purpose, the adipose-derived mesenchymal stem cells (Ad-MSCs) were initially isolated and characterized before cultivation on the PCL/PANI Nano fibrous scaffold to be treated for 21 days with 5-azacytidine either singly or in combination with TGF-β in medium. The scaffold's morphological and cell attachment properties were investigated using electron microscopy (SEM). Finally, the cardio myocyte differentiation of Ad-MSCs on the scaffold was studied using both quantitative Real-time PCR (qPCR) and flow-cytometry while the expression rates of the cardio myocytes' specific genes (Gata4, NKX2.5, MYH-7, and Troponin I) were also determined. The results of Ad-MSCs culture, MTT assay, and SEM indicated that the cells had well proliferated on the PCL/PANI scaffolds, showing the biocompatibility of the nanofibers for cellular growth and adhesion. After 21 days of induced cardio myocyte differentiation by both agents, Real-time PCR revealed increases in the expressions of Gata4, Troponin I, MYH-7, and NKX2.5 genes in the cells cultured on the PCL/PANI scaffolds while the flow-cytometry test approved the expression of troponin I. The data obtained showed that the PCL/PANI Nano fibrous scaffolds were able to promote and support mesenchymal stem cell transformation to cardio myocyte cells. Generally speaking, the results of the study might be exploited in future in vitro and in vivo experimental model studies of cardio myocyte differentiation using co-polymer scaffolds.

Melatonin Promotes the Therapeutic Effect of Mesenchymal Stem Cells on Type 2 Diabetes Mellitus by Regulating TGF-β Pathway.

Abundant evidence proves the therapeutic effect of adipose-derived mesenchymal stem cells (ADMSCs) in the treatment of diabetes mellitus. However, the problems have not been solved that viability of ADMSCs were inconsistent and the cells quickly undergo senescence after in vitro cell culture. In addition, the therapeutic effect of ADMSCs is still not satisfactory. In this study, melatonin (MLT) was added to canine ADMSC culture medium, and the treated cells were used to treat type 2 diabetes mellitus (T2DM). Our research reveals that adding MLT to ADMSC culture medium can promote the viability of ADMSCs. This effect depends on the binding of MLT and MLT receptors, which activates the transforming growth factor β (TGF-β) pathway and then changes the cell cycle of ADMSCs and improves the viability of ADMSCs. Since ADMSCs were found to be used to treat T2DM by anti-inflammatory and anti-endoplasmic reticulum (ER) stress capabilities, our data demonstrate that MLT augment several effects of ADMSCs in remission hyperglycemia, insulin resistance, and liver glycogen metabolism in T2DM patients. This suggest that ADMSCs and MLT-ADMSCs is safe and vabulable for pet clinic.

Integration of a fiber-based cell culture and biosensing system for monitoring of multiple protein markers secreted from stem cells

We propose a new platform that can integrate three-dimensional cell culture scaffold and a surface-enhanced Raman spectroscopy (SERS)-based biosensor by stacking them to form a multilayer system, which would allow monitoring of the protein markers secreted from cultured stem cells without periodic cell and/or media collection. The cell culture scaffold supported the proliferation and osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). The SERS capture substrate detected protein markers in combination with SERS tag made with Au–Ag alloy nanoboxes. Incorporating the different Raman reporters into the SERS tag allowed easy identification of target proteins for multiplex assays. The resultant SERS-based immunoassay could detect the pg/mL levels of protein markers without crosstalk and interference. When one ADSC culture scaffold and multiple SERS capture substrates were integrated and incubated in differentiation culture media, our system was sufficiently sensitive to monitor time-dependent secretion of three different osteogenic protein markers from ADSCs during their osteogenic differentiation. Since the sensor and cell culture scaffold can be manipulated independently, various cell and biomarker combinations are possible to obtain relevant information regarding the actual state of the different types of cells.

SIRT5 deficiency enhances the proliferative and therapeutic capacities of adipose-derived mesenchymal stem cells via metabolic switching.

BackgroundMesenchymal stem cells (MSCs) have therapeutic potential for multiple ischemic diseases. However, in vitro expansion of MSCs before clinical application leads to metabolic reprogramming from glycolysis to oxidative phosphorylation, drastically impairing their proliferative and therapeutic capacities. This study aimed to define the regulatory effects of Sirtuin 5 (SIRT5) on the proliferative and therapeutic functions of adipose‐derived MSCs (ADMSCs) during in vitro expansion.MethodsADMSCs were isolated from wild‐type (WT) and Sirt5‐knockout (Sirt5−/−) mice. Cell counting assay was used to investigate the proliferative capacities of the ADMSCs. Dihydroethidium and senescence‐associated β‐galactosidase stainings were used to measure intracellular ROS and senescence levels. Mass spectrometry was used to analyze protein succinylation. Oxygen consumption rates and extra cellular acidification rates were measured as indicators of mitochondrial respiration and glycolysis. Metabolic‐related genes expression were verified by quantitative PCR and western blot. Hind limb ischemia mouse model was used to evaluate the therapeutic potentials of WT and Sirt5−/− ADSMCs.ResultsSIRT5 protein levels were upregulated in ADMCs during in vitro expansion. Sirt5−/− ADMSCs exhibited a higher proliferation rate, delayed senescence, and reduced ROS accumulation. Furthermore, elevated protein succinylation levels were observed in Sirt5−/− ADMSCs, leading to the reduced activity of tricarboxylic acid cycle‐related enzymes and attenuated mitochondrial respiration. Glucose uptake, glycolysis, and pentose phosphate pathway were elevated in Sirt5−/− ADMSCs. Inhibition of succinylation by glycine or re‐expression of Sirt5 reversed the metabolic alterations in Sirt5−/‐ ADMSCs, thus abolishing their enhanced proliferative capacities. In the hind limb ischemia mouse model, SIRT5−/− ADMSCs transplantation enhanced blood flow recovery and angiogenesis compared with WT ADMSCs.ConclusionsOur results indicate that SIRT5 deficiency during ADMSC culture expansion leads to reversed metabolic pattern, enhanced proliferative capacities, and improved therapeutic outcomes. These data suggest SIRT5 as a potential target to enhance the functional properties of MSCs for clinical application.

Effect of cAMP Signaling Regulation in Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.

The impact of lactational status and days of gestation on insulin and glucose metabolism in pregnant mares

The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid–Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In both experiments, analysis of variance was used to compare averages through the Student–Newman–Keuls test. In the osteogenic study, no differences in any variable were detected between groups at Day 7. At Day 14, 0.01 nM T3 reduced cell density and the number of mineralized nodules despite the increase in ALP activity and mitochondrial metabolism (P < .05). ALP activity increased at 1,000 nM T3 concentration (P < .05). At Day 21, 0.01 nM T3 treatment increased ALP activity compared with control treatment (P < .05). At 1,000 nM concentration, T3 reduced mitochondrial metabolism and cell density (P < .05). In the chondrogenic study, the two T3 concentrations increased cell density compared with control treatment at Day 7. At Day 14, higher T3 concentration reduced mitochondrial metabolism, ALP activity, cell density, and PAS+ chondrogenic matrix percentage compared with control treatment (P < .05). Thus, T3 addition to equine AD-MSC cultures has no enhancement effect on osteogenic or chondrogenic differentiation and may, in fact, negatively affect cell density and matrix synthesis depending on hormone concentration and culture time.

Triiodothyronine Has No Enhancement Effect on the Osteogenic or Chondrogenic Differentiation of Equine Adipose Tissue Stem Cells

The effects of two concentrations of triiodothyronine (T3; 0.01 and 1,000 nM) on the osteogenic and chondrogenic differentiation abilities of equine adipose-derived mesenchymal stem cells (AD-MSCs) were evaluated. The osteogenic study evaluated the effect of T3 using alkaline phosphatase activity (ALP) assay; cell viability and density; and formation of mineralized nodules at Days 7, 14, and 21 in culture. The chondrogenic study tested the effect of T3 through ALP assay, mitochondrial metabolism, cell density, and periodic acid–Schiff-positive (PAS+) matrix percentage at Days 7 and 14. In both experiments, analysis of variance was used to compare averages through the Student–Newman–Keuls test. In the osteogenic study, no differences in any variable were detected between groups at Day 7. At Day 14, 0.01 nM T3 reduced cell density and the number of mineralized nodules despite the increase in ALP activity and mitochondrial metabolism (P < .05). ALP activity increased at 1,000 nM T3 concentration (P < .05). At Day 21, 0.01 nM T3 treatment increased ALP activity compared with control treatment (P < .05). At 1,000 nM concentration, T3 reduced mitochondrial metabolism and cell density (P < .05). In the chondrogenic study, the two T3 concentrations increased cell density compared with control treatment at Day 7. At Day 14, higher T3 concentration reduced mitochondrial metabolism, ALP activity, cell density, and PAS+ chondrogenic matrix percentage compared with control treatment (P < .05). Thus, T3 addition to equine AD-MSC cultures has no enhancement effect on osteogenic or chondrogenic differentiation and may, in fact, negatively affect cell density and matrix synthesis depending on hormone concentration and culture time.

The influence of fibroblast growth factor 2 on the senescence of human adipose-derived mesenchymal stem cells during long-term culture.

Adipose‐derived mesenchymal stem cells (ASCs) exhibit great potential in regenerative medicine, and in vitro expansion is frequently necessary to obtain a sufficient number of ASCs for clinical use. Fibroblast growth factor 2 (FGF2) is a common supplement in the ASC culture medium to enhance cell proliferation. To achieve clinical applicability of ASC‐based products, prolonged culture of ASCs is sometimes required to obtain sufficient quantity of ASCs. However, the effect of FGF2 on ASCs during prolonged culture has not been previously determined. In this study, ASCs were subjected to prolonged in vitro culture with or without FGF2. FGF2 maintained the small cell morphology and expedited proliferation kinetics in early ASC passages. After prolonged in vitro expansion, FGF2‐treated ASCs exhibited increased cell size, arrested cell proliferation, and increased cellular senescence relative to the control ASCs. We observed an upregulation of FGFR1c and enhanced expression of downstream STAT3 in the initial passages of FGF2‐treated ASCs. The application of an FGFR1 or STAT3 inhibitor effectively blocked the enhanced proliferation of ASCs induced by FGF2 treatment. FGFR1c upregulation and enhanced STAT3 expression were lost in the later passages of FGF2‐treated ASCs, suggesting that the continuous stimulation of FGF2 becomes ineffective because of the refractory downstream FGFR1 and the STAT3 signaling pathway. In addition, no evidence of tumorigenicity was noted in vitro and in vivo after prolonged expansion of FGF2‐cultured ASCs. Our data indicate that ASCs have evolved a STAT3‐dependent response to continuous FGF2 stimulation which promotes the initial expansion but limits their long‐term proliferation.

Adipose tissue: A natural resource for multipotent mesenchymal stem cells with potential translation to trigerminal layers.

Background:The article reports basic science research that establishes that adipose tissue (AT)-derived mesenchymal stem cells (MSCs) have a potential to transgerminal translation.Study Design:MSC confirmation was obtained by phenotypic spindle-shaped cells as well as with four positive and three negative markers. The translineage translation of adipose-derived MSCs (ADMSCs) was established.Materials and Methods:The lipoaspirate was subjected to enzymatic digestion with collagenase. Stromal vascular factor (SVF) was isolated. With two passages, pure culture of ADMSCs was obtained. They were translated to all the three germinal layers.Results:AT-derived SVF contains ~30% MSCs. They are capable of being translated into endoderm, mesoderm and ectoderm.Conclusion:AT is a rich source for MSCs, with immense research possibilities for regeneration and rejuvenation.

Effects of Adipose-derived Mesenchymal Stem Cell Exosomes on Corneal Stromal Fibroblast Viability and Extracellular Matrix Synthesis.

Background:Corneal stromal cells (CSCs) are components of the corneal endothelial microenvironment that can be induced to form a functional tissue-engineered corneal endothelium. Adipose-derived mesenchymal stem cells (ADSCs) have been reported as an important component of regenerative medicine and cell therapy for corneal stromal damage. We have demonstrated that the treatment with ADSCs leads to phenotypic changes in CSCs in vitro. However, the underlying mechanisms of such ADSC-induced changes in CSCs remain unclear.Methods:ADSCs and CSCs were isolated from New Zealand white rabbits and cultured in vitro. An Exosome Isolation Kit, Western blotting, and nanoparticle tracking analysis (NTA) were used to isolate and confirm the exosomes from ADSC culture medium. Meanwhile, the optimal exosome concentration and treatment time were selected. Cell Counting Kit-8 and annexin V-fluorescein isothiocyanate/propidium iodide assays were used to assess the effect of ADSC- derived exosomes on the proliferation and apoptosis of CSCs. To evaluate the effects of ADSC- derived exosomes on CSC invasion activity, Western blotting was used to detect the expression of matrix metalloproteinases (MMPs) and collagens.Results:ADSCs and CSCs were successfully isolated from New Zealand rabbits. The optimal concentration and treatment time of exosomes for the following study were 100 μg/ml and 96 h, respectively. NTA revealed that the ADSC-derived exosomes appeared as nanoparticles (40–200 nm), and Western blotting confirmed positive expression of CD9, CD81, flotillin-1, and HSP70 versus ADSC cytoplasmic proteins (all P < 0.01). ADSC-derived exosomes (50 μg/ml and 100 μg/ml) significantly promoted proliferation and inhibited apoptosis (mainly early apoptosis) of CSCs versus non-exosome-treated CSCs (all P < 0.05). Interestingly, MMPs were downregulated and extracellular matrix (ECM)-related proteins including collagens and fibronectin were upregulated in the exosome-treated CSCs versus non-exosome-treated CSCs (MMP1: t = 80.103, P < 0.01; MMP2: t = 114.778, P < 0.01; MMP3: t = 56.208, P < 0.01; and MMP9: t = 60.617, P < 0.01; collagen I: t = −82.742, P < 0.01; collagen II: t = −72.818, P < 0.01; collagen III: t = −104.452, P < 0.01; collagen IV: t = −133.426, P < 0.01, and collagen V: t = −294.019, P < 0.01; and fibronectin: t = −92.491, P < 0.01, respectively).Conclusion:The findings indicate that ADSCs might play an important role in CSC viability regulation and ECM remodeling, partially through the secretion of exosomes.

IL-10 and TGF-β1 evaluation in 3D human adipose-derived mesenchymal stem cells culture and hypoxic condition

IL-10 and TGF-β1 evaluation in 3D human adipose-derived mesenchymal stem cells culture and hypoxic condition

Adipose‑derived mesenchymal stem cells attenuate cisplatin‑induced apoptosis in epithelial ovarian cancer cells.

Environment-mediated drug resistance (EMDR) serves an important role in tumor chemotherapy resistance. Adipose‑derived mesenchymal stem cells (ADSC) are an important component of the tumor microenvironment. However, the role of ADSC in EMDR remains unclear. Therefore, in order to clarify whether ADSCs contribute to the cisplatin‑mediated apoptosis in epithelial ovarian cancer (EOC), the present study isolated ADSCs from the omentum of women with benign disease and collected the ADSC culture medium as conditioned medium. Subsequently, it was revealed that ADSCs decreased the sensitivity of EOC cells to cisplatin via an MTT assay. In addition, it was revealed that ADSCs may reduce cisplatin-induced apoptosis in EOC cells, as determined by Hoechst staining and flow cytometric analysis. Additionally, a lower level of cleaved caspase‑3 was observed, accompanied with decreased intracellular platinum accumulation in EOC cells indirectly co‑cultured with ADSCs. In conclusion, the findings of the present study suggested that ADSCs reduced cisplatin‑induced apoptosis and the intracellular level of platinum in EOC cells, which in turn resulted in cisplatin resistance. Therefore, ADSCs may serve as a therapeutic target for recurrent EOC.

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1×10(6)/cm(2) were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1×10(6)/cm(2) were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.

244 CHONDROGENIC PELLET CULTURES FOR CARTILAGE TISSUE ENGINEERING GROW BY DEPOSITION OF MATRIX AND NOT BY CELLULAR PROLIFERATION

Pellet cultures are commonly used to study chondrogenic differentiation in vitro. Our laboratory has demonstrated pellets made with chondrocytes grow in size during culture and produce cartilage matrix, but pellets made with adipose-derived mesenchymal stem cells (ASC) grow only slightly, producing little cartilage matrix. The objective of this study was to determine if differences in chondrocyte and ASC pellet growth result from differences in cell proliferation or in deposition of extracellular matrix. Primary chondrocytes and ASC from adult pigs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. To determine baseline proliferation rates in monolayer culture, cells were grown on coverslips in 10 µM bromodeoxyuridine (BrdU) for 24 h and immunostained for BrdU labelling. For pellet cultures, 5 × 105 cells were placed in 15 mL-conical tubes, pelleted by centrifugation in 1.0 mL of chondrogenic base media (CBM: DMEM + 40 µg mL–1 of proline, 50 µM ascorbic acid-2-phosphate, 100 nM dexamethasone, and 1× insulin-transferrin-selenium), and cultured in CBM for 1, and 4 weeks. To detect proliferation in pellets, 1- and 2-week cultured samples were labelled with 10 µM BrdU for 24 h before harvest. Pellets were fixed with 4% paraformaldehyde, embedded, and sectioned on a Leica CM1900 cryostat (Leica Microsystems, Wetzlar, Germany). To assess chondrogenic differentiation and matrix expression, sections were stained for collagen II, keratin sulfate, and chondroitin sulfate. Images were captured and distance between adjacent nuclei in 1- and 4-week pellets were measured using Zeiss imaging software. As expected, cells on coverslips showed BrdU labelling, with higher labelling in ASC cultures indicating faster proliferation (n = 5, 77.3 ± 3.74% chondrocyte v. 92.1 ± 2.88% ASC; α = 0.05; P &lt; 0.0001; Student’s t-test). However, BrdU labelling was not seen in sections from ASC or chondrocyte pellets (n = 5), at either 1 or 2 weeks. Absence of cellular proliferation in pellets was verified by negative staining for the mitotic marker Aurora KinaseB (AurKB). Cartilage matrix staining was strong in chondrocyte pellets at all time points and absent in ASC pellets. Cell nuclei were closely packed in both ASC and chondrocyte pellets at 1 week, but a significant increase in distance between adjacent nuclei with interspersed matrix staining was noted in chondrocyte pellets at 4 weeks (n = 4, 11.88 ± 0.67 µm at 1 week v. 26.85 ± 2.06 µm at 4 weeks; α = 0.05; P &lt; 0.0001; Student’s t-test). As TGFβ3 has been shown to induce chondrogenesis in ASC, ASC pellets were cultured in CBM + 10 ng of TGFβ3 for 1 and 2 weeks (n = 4). The TGFβ3 treatment did not induce cell proliferation in pellets, as sections were negative for BrdU. However, expression of cartilage markers keratan sulfate and chondroitin were noted. Based on our data, neither ASC nor chondrocytes proliferate in pellet culture, and chondrocyte pellet growth is due to extracellular matrix deposition.