Rat Adipose Tissue-derived Mesenchymal Stem Research Articles

In vitro Study of Primary Isolation and Culture of Adipose-Derived Stem Cells and Induction of Chondrogenic Differentiation.

Articular cartilage has constrained potential to restore. The mesenchymal stem cellular remedy has presented new treatment possibilities for this circumstance. The experiment aimed to verify the chondrogenic differentiation capacity of rat adipose tissue-derived mesenchymal stem cells (AD-MSCs) in vitroinside the presence or absence of transforming growth factor-beta (TGF-β1). Rat's subcutaneous adipose tissue minced into a small piece (2-3 mm3) was aseptically collected from the subcutaneous fat under anaesthesia and then digested with collagenase type I (1 mg/mL). Spontaneous chondrogenesis occurred in both AD-MSCs pellet cultures and was similar in both TGF-β1 treated. The untreated pellet cultures were collected after 21 days. Histological assessment for evaluating the level of proteoglycan by alcin blue staining and immunohistochemistry for detecting the presence of collagen type II. A monoclonal antibody directed against collagen type II. Adipose-derived stem cells (AD-MSCs) isolated from rats were immunophenotyped for the expression of MSCs cell surface markers and was performed by Flow cytometer, which demonstrated AD-MSCs highly expressed CD73 (99.69±2.6%), CD90 (98.11±0.3%), and week expression CD44 (17.15±0.3%). The result of histological staining showed the presence of extracellular matrix (ECM) in the hyaline cartilage. This staining indicated a deposit of "acid mucopolysaccharides" in the proximity of the cells. Additionally, most cells are rounded cells stained positive for the presence of the cells encompassed by extracellular matrix (ECM), which were like chondrocytes as seen from the magnified view, lightly pink stained nuclei, and nuclear fast red stain. However, the immunohistochemistry method demonstrated that the presence of TGF-β1 decreased the levels of collagen type I and increased the levels of collagen type II. In conclusion, subcutaneous adipose tissue-derived stem cells can be used in cartilage tissue engineering.

MafA-positive insulin-producing cells derived from rat adipose stem cells are efficient in glucose hemostasis in vitro

Objective: MafA has been recognized as a specific pancreatic β-cells activator for Insulin expression. This study investigated the changes in glucose transport and consumption by MafA-positive Insulin-producing cells resulting from the differentiation of rat adipose tissue-derived mesenchymal stem cells. Methods: Adipose tissue-derived mesenchymal stem cells were differentiated into Insulin-producing cells in a 14-day protocol using nicotinamide and ITS. The ability to produce Insulin was confirmed by adipogenesis differentiation capacity and expression of Oct4, Nanog, and Sox2. Insulin and Glucagon concentrations in Insulin-producing cells were determined by ELISA. The ability of Insulin-producing cells for the presence of MafA, Glut2, ENO1, and IGF-I receptor genes was determined by Western blotting analysis. The amounts of Glucokinase and Pyruvate Carboxylase activities were measured by Fluorometric and Colorimetric methods, respectively. Results: The un-differentiated cells showed a promising capacity to differentiate toward adipocytes. The adipose tissue mesenchymal stem cells revealed significantly elevated levels of Oct4, Nanog, and Sox2 compared to differentiated adipocytes. The cells that differentiated into Insulin-producing cells secreted considerably more Insulin and Glucagon than undifferentiated cells.

Palmitic Acid Methyl Ester Enhances Adipogenic Differentiation in Rat Adipose Tissue-Derived Mesenchymal Stem Cells through a G Protein-Coupled Receptor-Mediated Pathway.

Adipogenic differentiation from stem cells has become a research target due to the increasing interest in obesity. It has been indicated that adipocytes can secrete palmitic acid methyl ester (PAME), which is able to regulate stem cell proliferation. However, the effects of PAME on adipogenic differentiation in stem cell remain unclear. Here, we present that the adipogenic differentiation medium supplemented with PAME induced the differentiation of rat adipose tissue-derived mesenchymal stem cells (rAD-MSCs) into adipocyte. rAD-MSCs were treated with PAME for 12 days and then subjected to various analyses. The results from the present study show that PAME significantly increased the levels of adipogenic differentiation markers, PPARγ and Gpd1, and enhanced adipogenic differentiation in rAD-MSCs. Furthermore, the level of GPR40/120 protein increased during induction of adipocyte differentiation in rAD-MSCs. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced adipogenic differentiation. Moreover, PAME significantly increased phosphorylation of extracellular signal-regulated kinases (ERK), but not AKT and mTOR. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced ERK phosphorylation and adipogenic differentiation. PAME also increased the intracellular Ca2+ levels. Cotreatment with PAME and a Ca2+ chelator or a phospholipase C (PLC) inhibitor prevented the PAME-enhanced ERK phosphorylation and adipogenic differentiation. Our data suggest that PAME activated the GPR40/120/PLC-mediated pathway, which in turn increased the intracellular Ca2+ levels, thereby activating the ERK, and eventually enhanced adipogenic differentiation in rAD-MSCs. The findings from the present study might help get insight into the physiological roles and molecular mechanism of PAME in regulating stem cell differentiation.

Low-level laser therapy alleviates the deleterious effect of doxorubicin on rat adipose tissue-derived mesenchymal stem cells

Cancer is a leading cause of death worldwide, and doxorubicin (DOX) has become one of the most commonly prescribed drugs. Stem cell (SC) therapy is proving to be a promising strategy to alleviate DOX adverse effects on non-cancerous cells. However, the drug also has a toxic action on SCs, reducing the efficiency of cell therapy from a preventive view. The present study shows that the DOX toxicity in mesenchymal SCs (MSCs) can be partially overcome by low-level laser irradiation (LLLI). To achieve this, we applied the low-level red laser (wavelength: 660 nm; output power: 30 mW; laser beam: 0.028 cm2; irradiation: 1.07 mW/cm2; Ga-Al-As Photon Laser III, DMC, São Paulo, Brazil) in rat adipose tissue-derived MSCs before their exposure to different DOX concentrations. Results revealed that the DOX reduced the viability and adenosine triphosphate level of MSCs. These findings were followed by significantly increased apoptosis as well as oxidative stress in the MSCs. Interestingly, LLLI at the dose of 0.2 J alleviated the effects of DOX on cell viability and apoptosis, and inhibited oxidative stress in the MSCs. In summary, this study provides a crucial step toward the future application of LLLI as a protective approach against DOX-induced toxicity in MSCs, particularly cell death. This study also lays the groundwork for further investigation into the role of oxidative stress and inflammation as an instructive milieu for cell protection.

Improved Efficiency of Cardiomyocyte-Like Cell Differentiation from Rat Adipose Tissue-Derived Mesenchymal Stem Cells with a Directed Differentiation Protocol.

Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and “rosette-like structures” from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

The neuroprotective effect of rat adipose tissue-derived mesenchymal stem cell-conditioned medium on cortical neurons using an in vitro model of SCI inflammation

ObjectivesIn this study, a new approach was used with an in vitro model in which neural cells were exposed to conditioned media from the injured spinal cord (SCI-CM) mimicking a local inflammatory microenvironment . Subsequently, the neuroprotective effect of rat adipose tissue-derived msesenchymal stem cell-conditioned media (ATMSC-CM) was investigated through a cell-free based therapy, which was used to treat cortical neurons and astrocytes under inflammation.MethodsPrimary cell cultures isolated from postnatal day (P6) Wistar rat brain cortex were exposed to SCI-CM derived from the central lesion, rostral and caudal segments of injured spinal cord. After 48 h incubation, the SCI-CM was replaced and primary cultures were cultivated either in DMEM media alone or in ATMSC-CM for 72 h. The impact of ATMSC-CM on the viability of neurons and astrocytes was assessed using a CyQUANT® Direct Cell Proliferation Assay Kit as well as immunocytochemistry analysis.ResultsImmunocytochemical analysis revealed significant decrease in the number of MAP2 positive neurons exposed to SCI-CM compared to Control. Protection by ATMSC-CM was associated with increased survival of neurons compared to primary culture cultivated in DMEM media alone. The ATMSC-CM effect on astrocytes was more variable and without any significant impact.ConclusionThe results demonstrate that SCI-CM mimicking inflammation can reduce cortical neuron survival, and subsequent exposure to ATMSC-CM can stabilize the neuronal population most likely via released neuroprotective and trophic factors. In addition, astrogliosis was not affected by ATMSC-CM.

Zinc Sulphate Mediates the Stimulation of Cell Proliferation of Rat Adipose Tissue-Derived Mesenchymal Stem Cells Under High Intensity of EMF Exposure.

Unlike the role of mesenchymal stem cells (MSCs) in regenerative medicine, their application in cell therapy can be complicated by factors such as a reduction in the proliferation potential, senescent tendency of MSCs to expand, and an age-dependent decline in their number and functions. It was shown that there is an association between exposure to electromagnetic fields (EMFs) and response to stress, cell proliferation, aging, and cell death. Furthermore, the zinc ion, as an essential trace element, was reported to be involved in the regulation of the growth and cell proliferation. In this report, ratadipose tissue-derived mesenchymal stem cells (rADSCs) were randomly divided into four groups-group I (control without any ZnSO4 and EMF); group II (ZnSO4 treatment without EMF exposed); group III (EMF exposed without ZnSO4); and group ІV (EMF exposed with ZnSO4)-to evaluate whether 0.14μg/ml Zinc sulfate (ZnSO4) could affect cell proliferation of rADSCs under extremely low frequency-electromagnetic field (ELF-EMF). The methyl thiazoltetrazolium (MTT) method was used to determine the cell proliferation of rADSCs following exposure to ELF-EMF in the presence of ZnSO4. The immunocytochemistry method as well as flow-cytometry was used to identify the cell surface markers. Next, oil red O, alizarin red, toluidine blue, and cresyl violet staining was done to evaluate the adipogenic, osteogenic, chondrogenic, and neurogenic differentiation of rADSCs as the pluripotent capacity of rADSCs, respectively. The results showed that an exposure to ELF-EMF caused a decrease in the proliferation of rADSCs. However, the ZnSO4 supplementation significantly increased the cell proliferation of ELF-EMF-exposed rADSCs. In addition, in the presence of 0.14μg/ml ZnSO4, rADSCs appeared to be growing faster than the control group and ZnSO4 significantly decreased the doubling time of ELF-EMF-exposed rADSCs. It seems that ZnSO4 would be a good element to induce the cell proliferation of ELF-EMF-exposed rADSCs.

Enhancement of osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells by zinc sulphate under electromagnetic field via the PKA, ERK1/2 and Wnt/β-catenin signaling pathways

Zinc ion as an essential trace element and electromagnetic fields (EMFs) has been reported to be involved in the regulation of bone metabolism. The aim of this study was to elucidate the effects of zinc sulphate (ZnSO4) on the osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ADSCs) in the presence of EMF as a strategy in osteoporosis therapy. Alkaline phophatase (ALP) activity measurement, calcium assay and expression of several osteoblastic marker genes were examined to assess the effect of ZnSO4 on the osteogenic differentiation of ADSCs under EMF. The expression of cAMP and PKA was evaluated by ELISA. The expression of β-catenin, Wnt1, Wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5) and reduced dickkopf1 (DKK1) genes were used to detect the Wnt/β-catenin pathway. It was found that ZnSO4, in the presence of EMF, resulted in an increase in the expression of osteogenic genes, ALP activity and calcium levels. EMF, in the presence of ZnSO4, increased the cAMP level and protein kinase A (PKA) activity. Treatment of ADSCs with (MAPK)/ERK kinase 1/2 inhibitor, or PKA inhibitor, significantly inhibited the promotion of osteogenic markers, indicating that the induction of osteogenesis was dependent on the ERK and PKA signaling pathways. Real-time PCR analysis showed that ZnSO4, in the presence of EMF, increased the mRNA expressions of β-catenin, Wnt1, Wnt3a, LRP5 and DKK1. In this study, it was shown that 0.432 μg/ml ZnSO4, in the presence of 50 Hz, 20 mT EMF, induced the osteogenic differentiation of ADSCs via PKA, ERK1/2 and Wnt/β-catenin signaling pathways.

L-carnitine contributes to enhancement of neurogenesis from mesenchymal stem cells through Wnt/β-catenin and PKA pathway.

The identification of factors capable of enhancing neurogenesis has great potential for cellular therapies in neurodegenerative diseases. Multiple studies have shown the neuroprotective effects of L-carnitine (LC). This study determined whether neuronal differentiation of rat adipose tissue-derived mesenchymal stem cells (ADSCs) can be activated by LC. In this study, protein kinase A (PKA) and Wnt/β-catenin pathways were detected to show if this activation was due to these pathways. The expression of LC-induced neurogenesis markers in ADSCs was characterized using real-time PCR. ELISA was conducted to assess the expression of cyclic adenosine monophosphate (cAMP) and PKA. The expression of β-catenin, reduced dickkopf1 (DKK1), low-density lipoprotein receptor-related protein 5 (LRP5), Wnt1, and Wnt3a genes as Wnt/β-catenin signaling members were used to detect the Wnt/β-catenin pathway. It was observed that LC could promote neurogenesis in ADSCs as well as expression of some neurogenic markers. Moreover, LC causes to increase the cAMP levels and PKA activity. Treatment of ADSCs with H-89 (dihydrochloride hydrate) as PKA inhibitor significantly inhibited the promotion of neurogenic markers, indicating that the PKA signaling pathway could be involved in neurogenesis induction. Analyses of real-time PCR data showed that the mRNA expressions of β-catenin, DKK1, LRP5c-myc, Wnt1, and Wnt3a were increased in the presence of LC. Therefore, the present study showed that LC promotes ADSCs neurogenesis and the LC-induced neurogenic markers could be due to both the PKA and Wnt/β-catenin signaling pathway. Impact statement Neural tissue has long been believed as incapable of regeneration and the identification of cell types and factors capable of neuronal differentiation has generated intense interest. Mesenchymal stem cells (MSCs) are considered as potential targets for stem cell-based therapy. L-carnitin (LC) as an antioxidant may have neuroprotective effects in oxidative damage and possibly in neurodegenerative disorders. We have tried to evaluate the effect of LC as an antioxidant on the neurogenic differentiation of ADSCs in order to further elucidate the simultaneous effects on the capability of the neural regeneration. In this study, PKA and Wnt/β-catenin signaling pathways were detected to see if LC could also activate these pathways. The results of this study showed that 200 µM LC promoted ADSCs neurogenic differentiation, and that it was correlated with the PKA and Wnt/β-catenin signaling pathways.

The effects of mesenchymal stem cells on the structure and contractile force of the carotid artery in a rat aneurysm model

Background/aim: An aneurysm is a pathological enlargement of an artery characterized by the thinning of the elastic fiber layer in the tunica media. Because the aneuritic artery wall is weakened, these vessels can rupture and cause serious bleeding. Surgical methods are often used for the treatment of aneurysms. However, cell-based therapies are less invasive and potentially safer alternatives. In this study, the therapeutic efficacy of rat adipose tissue-derived mesenchymal stem cells (MSCs) was investigated in a new carotid artery aneurysm model.Materials and methods: Arteries were pretreated with elastase to create aneurysms. Gelatin matrices containing MSCs were applied to the outer surface of the elastase-treated carotid artery sections.Results: Healing of the aneuritic arteries for which MSC applications were performed was significantly better than in the aneuritic group. The histological structure of the vessels was largely reconstituted, and the contractile force of the MSC-treated group was similar to the untreated healthy group.Conclusions: Application of MSCs facilitates the healing of aneurysms. Hereby, MSC application could be a promising approach for clinical applications after further validation processes are concluded.

L-carnitine significantly decreased aging of rat adipose tissue-derived mesenchymal stem cells.

Mesenchymal stem cells are undifferentiated cells that have the ability to divide continuously and tissue regeneration potential during the transplantation. Aging and loss of cell survival, is one of the main problems in cell therapy. Since the production of free radicals in the aging process is effective, the use of antioxidant compounds can help in scavenging free radicals and prevent the aging of cells. The aim of this study is evaluate the effects of L-carnitine (LC) on proliferation and aging of rat adipose tissue-derived mesenchymal stem cells (rADSC). rADSCs were isolated from inguinal region of 5 male Rattus rats. Oil red-O, alizarin red-S and toluidine blue staining were performed to evaluate the adipogenic, osteogenic and chondrogenic differentiation of rADSCs, respectively. Flow cytometric analysis was done for investigating the cell surface markers. The methyl thiazol tetrazolium (MTT) method was used to determine the cell proliferation of rADSCs following exposure to different concentrations of LC. rADSCs aging was evaluated by beta-galactosidase staining. The results showed significant proliferation of rADSCs 48h after treatment with concentrations of 0.2mM LC. In addition, in the presence of 0.2mM LC, rADSCs appeared to be growing faster than control group and 0.2mM LC supplementation could significantly decrease the population doubling time and aging of rADSCs. It seems that LC would be a good antioxidant to improve lifespan of rADSCs due to the decrease in aging.

Influence of rat adipose tissue-derived mesenchymal stem cells on brain tissues following permanent unilateral common carotid artery occlusion in adult male albino rat

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In Vitro Toxic Effects of Zinc Oxide Nanoparticles on Rat Adipose Tissue-Derived Mesenchymal Stem Cells.

Zinc oxide nanoparticles (ZnO-NPs) are increasingly used in sunscreens, bio- sensors, food additives, pigments, manufacture of rubber products, and electronic materi- als. There are several studies about the effects of NPs on dermal fibroblast or keratino- cytes, but very little attention has been directed towards adipose-derived mesenchymal stem cells (ASCs). A previous study has revealed that ZnO-NPs restricted the migration capability of ASCs. However, the potential toxicity of these NPs on ASCs is not well un- derstood. This study intends to evaluate the effects of ZnO-NPs on subcutaneous ASCs. In this experimental study, In order to assess toxicity, we ex- posed rat ASCs to ZnO-NPs at concentrations of 10, 50, and 100 µg/ml for 48 hours. Tox- icity was evaluated by cell morphology changes, cell viability assay, as well as apoptosis and necrosis detection. ZnO-NPs concentration dependently reduced the survival rates of ASCs as re- vealed by the trypan blue exclusion and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium-bromide (MTT) tests. ZnO-NPs, at concentrations of 10 and 50 µg/ml, induced a significant increase in apoptotic indices as shown by the annexin V test. The concentration of 10 µg/ml of ZnO-NPs was more toxic. Lower concentrations of ZnO-NPs have toxic and apoptotic effects on subcutaneous ASCs. We recommend that ZnO-NPs be used with caution if there is a dermatological problem.

Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats.

Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect ofsplenectomy on stem cell infusion and elucidated its mechanism. Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the invivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation invitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.

259. Parathyroid Hormone Gene Modified Mesenchymal Stem Cells for Hypoparathyroidism

Hypoparathyroidism is a prominent complication of total thyroidectomy for thyroid cancer. Hypocalcemia significantly decrease the quality of life of patients with hypoparathyroidism. Medical management with calcium supplements and activated vitamin D analogues increase risk of renal complications. Hormone replacement is an alternative. In this study, we investigated the possibility of management hypoparathyroidism using parathyroid hormone gene modified mesenchymal stem cells. Rat model of hypoparathyroidism was established through parathyroid gland removing by operation. Human prepro-PTH gene was amplified by RT-PCR from parathyroid adenoma and cloned into lentiviral vector. In order to express functional PTH by non-parathyroid cells. Specific mutation was introduced into the prepro-PTH gene. PTH expression was determined after transfection of 293T cells with the vector. Rat adipose tissue-derived mesenchymal stem cells were cultured and infected with PTH gene-expressing lentiviral vectors. The therapeutic effect of PTH gene modified MSCs on experimental hypoparathyroidism will be presented.

Sustained Release of 17β-Estradiol Stimulates Osteogenic Differentiation of Adipose Tissue-Derived Mesenchymal Stem Cells on Chitosan-Hydroxyapatite Scaffolds

The aim of this study was to develop a 17β-estradiol (E2)-releasing scaffold-nanoparticle system in order to promote osteogenic differentiation of rat adipose tissue-derived mesenchymal stem cells (AdMSCs) for bone tissue regeneration. E2-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles with a diameter of ∼240 nm were produced via an emulsion-diffusion-evaporation method. Because of its higher encapsulation efficiency (54%), PLGA, which has a 65:35 composition, was chosen for the preparation of nanoparticles. Chitosan-hydroxyapatite (HA) scaffolds in macroporous structures with interconnected pores were prepared by combining microwave irradiation and gas-foaming techniques. PLGA nanoparticles were loaded onto scaffolds in 2 ways: via embedding after scaffold fabrication and during fabrication. While 100% of the loaded E2 was released during 55 days from scaffolds loaded by embedding, a controlled release behavior of E2 was observed over 135 days in scaffolds loaded during manufacture. The results of cell culture studies indicated that the controlled delivery of E2 from PLGA nanoparticles loaded on chitosan-HA scaffolds had a significant effect on the osteogenic differentiation of AdMSCs.

Isolation, culturing and characterization of rat adipose tissue-derived mesenchymal stem cells: a simple technique

In this study, our aim was to develop a new simple technique for isolation of mesenchymal stem cells from adipose tissue. For this purpose, mesenchymal stem cells were isolated from rat adipose tissue by using the primary explant culture technique. When the cells became confluent, they were passaged 4 times by using the standard trypsinization method with trypsin/EDTA solution. Cells at second passage were characterized by using immunofluorescence staining against CD13 and CD29 markers. The results showed that these cultured cells were positive for CD13 and CD29 markers. Flow cytometry analysis was also done against CD29, CD90, CD54, MHC Class I, CD45, CD106, and MHC Class II for characterization of mesenchymal stem cells. The results of flow cytometry analysis showed that these cells were mesenchymal stem cells. Half of the cells were cryopreserved at all passages for future applications. It is thought that these mesenchymal stem cells can be used in therapy of cardiovascular diseases as an alternative technique in the near future.

Differentiation of rat adipose tissue-derived mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro

To differentiate rat adipose tissue-derived mesenchymal stem cells (ADSCs) into cells with a nucleus pulposus-like phenotype in vitro, so as to lay a foundation for the cell-based transplantation therapy of degenerated intervertebral discs. Rat ADSCs were isolated only from the subcutaneous inguinal region and purified by limited dilution. ADSCs of the third passages were analyzed by fluorescence activated cell sorter (FACS) to detect the cell surface markers (Sca-1, CD44, CD45, CD11b). To induce ADSCs towards a nucleus pulposus-like phenotype, ADSCs were immobilized in 3-dimensional alginate hydrogels and cultured in an inducing medium containing transforming growth factor-beta1 (TGF-beta1) under hypoxia (2% O(2)), while control groups under normoxia (21% O(2)) in alginate beads in medium with or without the presence of TGF-beta1. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate phenotypic and biosynthetic activities in the process of differentiation. Meanwhile, Alcian blue staining were used to detect the formation of sulfated glycosaminoglycans (GAGs) in the differentiated cells. The purified ADSCs were fibroblast-like and proliferated rapidly in vitro. The flow cytometry showed that ADSCs were positive for Sca-1 and CD44, negative for CD45 and CD11b. The results of RT-PCR manifested that the gene expressions of Sox-9, aggrecan and collagen II, which were chondrocyte specific, were upregulated in medium containing TGF-beta1 under hypoxia (2% O(2)). Likewise, gene expression of HIF-1a, which was characteristics of intervertebral discs, was also upregulated. Simultaneously, Alcian blue staining exhibited the formation of many GAGs. The approach in our experiment is a simple and effective way to acquire a large quantity of homogenous ADSCs. Rat ADSCs can be differentiated into nucleus pulposus-like cells. ADSCs may replace bone marrow mesenchymal stem cells as a new kind of seed cells in regeneration of degenerated intervertebral discs using cell transplantation therapy.