Human Adipose-derived Mesenchymal Cells Research Articles

Decellularized Placental Sponge Seeded with Human Mesenchymal Stem Cells Improves Deep Skin Wound Healing in the Animal Model.

Skin injuries lead to a large burden of morbidity. Although numerous clinical and scientific strategies have been investigated to repair injured skin, optimal regeneration therapy still poses a considerable obstacle. To address this challenge, decellularized extracellular matrix-based scaffolds recellularized with stem cells offer significant advancements in skin regeneration and wound healing. Herein, a decellularized human placental sponge (DPS) was fabricated using the decellularization and freeze-drying technique and then recellularized with human adipose-derived mesenchymal cells (MSCs). The biological and biomechanical properties and skin full-thickness wound healing capacity of the stem cells-DPS constructs were investigated in vitro and in vivo. The DPS exhibited a uniform 3D microstructure with an interconnected pore network, 89.21% porosity, a low degradation rate, and good mechanical properties. The DPS and MSCs-DPS constructs were implanted in skin full-thickness wound models in mice. An accelerated wound healing was observed in the wounds implanted with the MSCs-DPS construct when compared to DPS and control (wounds with no treatment) during 7 and 21 days postimplantation follow-up. In the MSCs-DPS group, the wound was completely re-epithelialized, the epidermis layer was properly organized, and the dermis and epidermis' bilayer structures were restored after 7 days. Our findings suggest that DPS is an excellent carrier for MSC culture and delivery to skin wounds and now promises to proceed with clinical evaluations.

Effects of green light-emitting diode irradiation on hepatic differentiation of hepatocyte-like cells generated from human adipose-derived mesenchymal cells

Light-emitting diode (LED) irradiation has been used in the differentiation of mesenchymal stem cells into a variety of cell types. This study investigated the effect of green LED (GLED) irradiation on the differentiation of adipocyte-derived mesenchymal cells into hepatocyte-like cells (HLCs) and the mechanism of its action. HLCs in the hepatocyte maturation phase were irradiated with GLED (520 nm, 21 W/m2, 5 min/day for 10 days). The cells were then assessed for expression of hepatocyte maturity genes and opsin 3 (OPN3), hepatocyte function, viability, apoptosis, and levels of reactive oxygen species (ROS), intracellular adenosine triphosphate (ATP) and calcium ions (Ca2+). GLED irradiation increased Alpha-1 antitrypsin and Ornithine transcarbamylase gene expression, promoted Cytochrome P450 3A4 activity and urea synthesis, and elevated intracellular ROS, ATP and Ca2+ levels. OPN3 expression was significantly more upregulated in GLED-irradiated HLCs than in the non-irradiated HLCs. No significant difference in cell viability or apoptosis was observed between GLED-irradiated and non-irradiated HLCs. GLED irradiation can promote hepatocyte maturation and functions through OPN3. GLED irradiation also stimulated mitochondrial function via Ca2+/ATP/ROS activation. GLED irradiation has potential to support cell-based transplantation in patients.

Towards Regenerative Audiology: Immune Modulation of Adipose-Derived Mesenchymal Cells Preconditioned with Citric Acid-Coated Antioxidant-Functionalized Magnetic Nanoparticles.

Introduction and Background: Based on stem cells, bioactive molecules and supportive structures, regenerative medicine (RM) is promising for its potential impact on field of hearing loss by offering innovative solutions for hair cell rescue. Nanotechnology has recently been regarded as a powerful tool for accelerating the efficiency of RM therapeutic solutions. Adipose-derived mesenchymal cells (ADSCs) have already been tested in clinical trials for their regenerative and immunomodulatory potential in various medical fields; however, the advancement to bedside treatment has proven to be tedious. Innovative solutions are expected to circumvent regulatory and manufacturing issues related to living cell-based therapies. The objectives of the study were to test if human primary ADSCs preconditioned with magnetic nanoparticles coated with citric acid and functionalized with antioxidant protocatechuic acid (MNP-CA-PCA) retain their phenotypic features and if conditioned media elicit immune responses in vitro. MNP-CA-PCA was synthesized and characterized regarding size, colloidal stability as well as antioxidant release profile. Human primary ADSCs preconditioned with MNP-CA-PCA were tested for viability, surface marker expression and mesenchymal lineage differentiation potential. Conditioned media (CM) from ADSCs treated with MNP-CA-PCA were tested for Il-6 and IL-8 cytokine release using ELISA and inhibition of lectin-stimulated peripheral blood monocyte proliferation. Results: MNP-CA-PCA-preconditioned ADSCs display good viability and retain their specific mesenchymal stem cell phenotype. CM from ADSCs conditioned with MNP-CA-PCA do not display increased inflammatory cytokine release and do not induce proliferation of allergen-stimulated allogeneic peripheral blood monocytes in vitro. Conclusions: While further in vitro and in vivo tests are needed to validate these findings, the present results indicated that CM from ADSCs preconditioned with MNP-CA-PCA could be developed as possible cell-free therapies for rescuing auditory hair cells.

A 3D Bioprinted Human Meniscus Shape Enriched with Mesenchymal Cells

Background and objectives: Regenerative medicine, with its massive development over the years, has the potential to solve some of the most problematic medical issues, such as functional organ transplantation. The aim of this study was to create a human meniscal shape 3D-printed enriched with human adipose-derived mesenchymal cells. Materials and Methods: Human infrapatellar fat pad was harvested, and mesenchymal cells were isolated. The mesenchymal stem cells were differentiated to the chondrocite lineage and a hydrogel (a nanofibrillar cellulose, sodium alginate, D-mannitol, and Hepes buffer solution combination) cell mixture was bioprinted to create three human-size meniscus structures. The obtained structures were evaluated regarding the cell viability, appropriate size in relation to a native meniscus, and some mechanical characteristics. Results: The human meniscal shape created respected the anatomic characteristic of a native structure. Cell viability of approximately 97% and extracellular matrix formation after the printing process were observed. The mean maximum force for the meniscus with mesenchymal cells was 6.5 N (+/−0.5 N) compared to the mean maximum force for the native meniscus of 10.32 N (+/−0.7 N), which is statistically relevant (p < 0.01). Conclusion: This paper presents the potential of bioprinting viable cell structures that could in the future present enough mechanical strength to replace a human organ, such as a meniscus. There are still limitations regarding the ink and the printing process, but we are confident that these problems will soon be solvable.

The feasibility of human adipose-derived mesenchymal stem cells induced to endothelial-like cells and the safety of induced cells intraocular application in a short period

Objective To evaluate the feasibility of differentiation of human adipose mesenchymal stem cells (hADSCs) into endothelial-like cells and the safety of induced cells intraocular application. Methods HADSCs were induced to endothelial-like cells in vitro.The experimental group was added with streptomycin, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and the control group was added with the same amount of phosphate buffered saline (PBS), the expression of von Willebrand factor (vWF), a marker of endothelial cells, was observed in the two groups.Six C57BL/6J mice were randomly divided into experimental group and control group by using the random number method, 3 mice for each group, the induced cells were injected intravitreally into C57BL/6J mice as experimental group, and the same amount of PBS buffer was injected intravitreally into C57BL/6J mice as control group.The changes of retinal cells were observed by electron microscopy.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results After induction of hADSCs in vitro, the endothelial cell marker was expressed.VWF immunofluorescence of the experimental group showed strong green fluorescence, and the color rendering rate was 100%.The control group showed no coloration of vWF immunofluorescence, and the color positive rate was 0.After 1 month of intravitreal injection, the retinal ganglion cells and rod cells were not degraded and necrotic. Conclusions HADSCs can differentiate into endothelial-like cells in vitro, and there is no retinal toxicity in a short-term. Key words: Adipose derived mesenchymal stem cells; Endothelium-like cells; Induced differentiation; Toxicity

Paracrine of human adipose-derived mesenchymal stem cells in three-dimensional culture containing functionized self assembling peptide nanofiber hydrogel

Objective To study the paracrine of human adipose-derived mesenchymal stem cells (ADMSCs) in three-dimensional culture containing functionized self assembling peptide nanofiber hydrogel and the influencing factors. Methods ADMSCs were isolated and extracted. Three kinds of peptides (RADAI-16, RGD, KLT) were prepared and functionized self assembling peptide nanofiber hydrogel was made by mixing RADAI-16, RGD and KLT with a volume ratio of 2∶1∶1. Under AFM, the morphology of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was observed (n=4). ADMSCs were cultured under the three-dimensional condition containing peptide nanofiber hydrogel. ADMSCs were subjected to in situ three-dimensional cultivation for 1 day, then the concentrations of hepatocyte growth factor (HGF, n=3) and vascular endothelial growth factor (VEGF, n=3) were determined in the supernatatn by enzyme linked immunosorbent assay (ELISA). The cells were extracted in peptide nanofiber hydrogel and the expression of heme oxygenase-1 (HO-1, n=3) was detected by Western blotting. The cultured ADMSCs in hypoxic condition and normal circumstance served as controls. Results ADMSCs showed long spindle shape and grew in a whirlpool shape. The diameter of RADAI-16, RGD, KLT and functionized self assembling peptide nanofiber hydrogel was (17.34±1.82), (15.50±1.41), (13.77±1.18) and (20.26±1.25) nm, respectively. The concentration of HGF under three conditions was (47.31±6.75), (247.86±17.59), and (297.25±17.95) ng/L, respectively, and that of VEGF was (218.30±3.03), (267.13±4.27) and (289.14±3.11) ng/L, respectively. Both HGF and VEGF were paracrined more in condition of functionized self assembling peptide nanofiber hydrogel (P=0.000). Western blotting results showed that the expression of HO-1 in the three-dimensional culture group was about 2 times (P=0.000) higher than that in the ordinary culture group, and was 1.2 times higher than that in the hypoxia culture group (P=0.033). Conclusion The three-dimensional culture mediated by the functionized self assembling peptide nanofiber hydrogel can promote the paracrine of ADMSCs, such as HGF and VEGF, and hypoxia is one of the important elements. Key words: Adipose derived Mesenchymal stem cells; Three-dimensional culture; Functionized self assembling peptide nanofiber hydrogel; Paracrine; Hypoxia

Role of RNA-binding protein HuR in human adipocyte differentiation

Objective To investigate the expressions of RNA-binding protein human antigen R(HuR), fatty acid binding protein type 4(FABP4), fatty acid synthetase(FASN), and lipoprotein lipase(LPL)during the differentiation of human adipocytes, and to explore their possible roles. Methods Human adipose-derived mesenchymal stem cells were induced by adipogenic differentiation, and the adipogenesis of cells was observed by oil red O staining. The expressions of HuR, FABP4, FASN, and LPL mRNA and protein were detected by real-time PCR and Western blotting. After HuR was silenced by siRNA, the change of adipogenesis for human adipose-derived mesenchymal stem cells was observed and the expressions of adipogenic genes were detected. Results The expressions of HuR, FABP4, FASN, and LPL mRNA and protein were significantly increased after human adipose-derived mesenchymal stem cells were induced to differentiate into adipocytes(all P<0.01). After HuR expression was down-regulated by siRNA, the adipogenic level of human adipose-derived mesenchymal stem cells was reduced, with decreased protein levels of FABP4, FASN, and LPL(all P<0.05), which were without changes for their mRNA levels. Conclusion HuR promotes the differentiation of human adipocytes mainly via regulating the changes of FABP4, FASN, and LPL protein levels. (Chin J Endocrinol Metab, 2017, 33: 854-860) Key words: Adipocyte differentiation; RNA binding protein; Human antigen R; Adipocyte fatty acid binding protein; Fatty acid synthase; Lipoprotein lipase

Effect of over-expressed miR-148a on secretion and expression of cytokines in human adipose-derived mesenchymal stem cell differentiation process

Objective To investigate the effect of over-expressed miR-148a on secretion and expression of inflammatory and insulin sensitive protein, such as interleukin 6 (IL-6), leptin, resistin and tumor necrosis factor α (TNF-α), during human adipose-derived mesenchymal stem cells (hMSCs-Ad) differentiation. Methods hMSCs-Ad was infected with the lentivirus of hsa-miR-148a, then the secretion of inflammatory cytokines (IL-6, TNF-α) and insulin sensitive protein (leptin, resistin) during differentiation process.The cells were collected at 0, 4, 10 and 15 days for RNA extration.The expression levels of IL-6, leptin, resistin and TNF-α were measured by using enzyme-linked immunosorbent assay, IL-6 and leptin mRNA were examined by using real-time PCR. Results Compared with the control group, after hMSCs-Ad were infected with over-expressed miR-148a lentivirus, both secretion of IL-6 [day10: (50.92±14.93) ng/L vs (136.86±35.53) ng/L, t=3.86, P=0.018; day 15: (123.64±18.76) ng/L vs (178.59±7.54) ng/L, t=1.56, P=0.029]and leptin day 4: (12.28±0.56) ng/L vs (14.38±0.84) ng/L, t=3.06, P=0.038; day 15: (34.45±4.30) ng/L vs (115.85±28.81) ng/L, t=4, 79, P=0.009] were reduced, while the concentration of resistin was higher than that of the control group and the concentration of resistin was less 0.20 μg/L in both groups, and TNF-α did not express, in hMSCs-Ad differentiation.Similarly, IL-6(day 4: 0.041±0.018 vs 0.089±0.002, t=4.68, P=0.009; day 15: 0.138±0.015 vs 0.284±0.069, t=3.55, P=0.024) and leptin (day 10: 0.163±0.039 vs 0.497±0.042, t=10.13, P=0.001; day 15: 0.259±0.033 vs 1.146±0.346, t=4.41, P=0.012) mRNA expression levels were reduced in overexpressed miR-148a group. Conclusions It suggests that miR-148a can improve hMSCs-Ad inflammation status and improve insulin resistance through regulating the IL-6, leptin, resistin and TNF-α expressions, which indicates that miR-148a play a role in the process of obesity related inflammation and insulin sensitivity. Key words: miR-148a; Human adipose-derived mesenchymal stem cell; Adipokines; Inflammation; Obesity

Study on human adipose mesenchymal stem cells differentiating into retinal pigment epithelial-like cells and its in vivo application

Background Stem cell transplantation represents a promising treatment option for patients suffering from degenerative disorders. Accumulating evidences indicate that mesenchymal stem cells (MSCs) are able to differentiate into retinal pigment epithelial (RPE) -like cells. However, MSCs are difficult to obtain. Human adipose mesenchymal stem cells (ADSCs) are proved to have similar properties to MSCs, but relevant study is less. Objective This study was to assess the feasibility of human ADSCs differentiating into RPE-like cells and the safety of its application in vivo. Methods The third generation of human ADSCs were incubated into 6-well plate, and 100 ng/ml epithelial growth factor, 50 μmol/L taurine and 5×10-7 mol/L retinoic acid were added into the medium 12 hours after cultured to induce the cells, and conventional cultured cells were used as the control group. Induced cells were traced with PKH26, and Pan-cytoke ratin (Pan-CK) monoclonal antibody was used to identify the cells under the fluorescence microscope. Induced RPE-like cell suspension of 1 μl was intravetreally injected in the right eyes of 6 BALB/c mice, and equal volume of PBS was used in the same way in another 6 mice. The animals were sacrificed 1 month after injection, and the retinal morphology was examined by histopathology under the optical microscope. The ultrastructure of retinal ganglion cells (RGCs) was examined by the transmission electron microscope. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results Cultured human ADSCs grew well with the slender polygone shape. Cell membranes showed the red fluorescence for PKH26 after induced. In addition, Pan-CK was expressed in the cell membranes with the red fluorescence in the induced cells, but the response was absent in the control cells. One month after intravitreal injection, induced cells located on the retinal surface, and the retinal morphology was clear under the optical microscope. No abnormality in RGCs was seen under the transmission electron microscope. Conclusions Human ADSCs can differentiate into RPE-like cells after induction. PKH26 can mark induced cells well. There is no adverse effect of induced cells on retina after intravitreal injection in a short-term duration in mice. Key words: Cell differentiation; Adipose-derived mesenchymal stem cells; Pigment epithelium of eye/cytology; Stem cells transplantation; Retina/pathology; Ultrastructure; Mice, BALB/c

In vitro induction of differentiation of human adipose-derived mesenchymal stem cells into melanocytes: an experimental study

Objective To establish a method for inducing human adipose-derived mesenchymal stem cells (hADSCs) to differentiate into melanocytes in vitro.Methods hADSCs were isolated from human subcutaneous fat tissue,and subjected to phenotypic analysis by flow cytometry.The differentiation potential of the hADSCs was evaluated by induction of osteogenic and adipogenic differentiation.Then,the fifth passage hADSCs were induced to differentiate into melanocytes by conditioned medium containing stem cell factor (SCF),endothelin 3,basic fibroblast growth factor (bFGF) for ten weeks.Those hADSCs receiving no induction treatment served as the normal control.Subsequently,real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expressions of melanin-associated genes including microophthalmia-associated transcription factor (MITF),ckit receptor tyrosine kinase (KIT),dopachrome tautomerase (DCT),tyrosinase,tyrosinase-related protein-1 (TYRP1),and sex determining region Y-box 10 (SOX10).Statistical analysis was done by one-factor analysis of variance and the least significant difference (LSD) test.Further more,the hADSC-derived melanocytes were identified by immunocytochemistry and immunofluorescence assay.Results As flow cytometry revealed,the isolated hADSCs showed high expressions of CD29,CD44,CD73,CD90,CD105 and CD166 with the positivity rates being more than 95％,but low or absent expressions of CD31,CD34,CD45 and human leukocyte antigen (HLA)-DR.Additionally,the osteogenic and adipogenic differentiation of hADSCs was successfully induced.The relative mRNA expression levels of MITF,KIT,DCT,tyrosinase,TYRP1 and SOX10 were 0.325 ± 0.012,0.042 ± 0.006,0.046 ± 0.013,0.036 ± 0.005,0.041 ± 0.003,and 0.225 ± 0.014 respectively in hADSCs induced for 10 weeks,which were significantly upregulated compared with the normal control hADSCs (all P ＜ 0.05).Moreover,the induced hADSCs showed positive immunocytochemical staining for MITF and HMB45,as well as positive cytoimmunofluorescence staining for TYRP1 and S100.Conclusion The hADSCs have been successfully induced to differentiate into cells with the biological characteristics of melanocytes. Key words: Human adipose-derived mesenchymal stem cells; Cell differentiation; Melanocytes

Adhesion, proliferation and differentiation of human adipose mesenchymal stem cells on PLGA

Objective To study the ability of adhesion, proliferation and differentiation of human adipose mesenchymal stern cells,which were cultured in PLGA,and thus to provide a basis for their in vivo implantation. Methods PLA was mixed with PGA in a ratio of 1 ： 1, and PLGA was obtained by insert moldling with organic solvents. In vitro, human adipose mesenchymal stem cells were adhered to PLGA. The attachment and the proliferation of human adipose mesenchymal stem cells in PLGA were analysed by scanning electron microscope, and the morphological change of the cells was investigated before and after induction. Results Human adipose mesenchymal stem cells were able to attach, proliferate and secrete extracellular matrix in porous PLGA （pore size： 100 -400 pm） with porosity of 85 % , and to differentiate into round lipoblast-like cells under the induction of adipose medium. After 14 days, the particulate of PLGA was overlaid by extracellular matrix. Conclusion Human adipose mesenchymal stem cells have the capacity to adhere to PLGA and proliferate, indicating that PLGA can be used as a carrier for human adipose mesenchymal stem cells in adipose tissue engineering. Key words: Human adipose mesenchymal stem cells; Scaffold material; Scanning electron microscope