Metformin modulates autophagic pathway in renal fibrosis induced by carbon tetrachloride in adult male albino rats.

BACKGROUNDS: Chronic kidney disease (CKD) is a global public health problem. All progressive chronic kidney disease (CKD) is characterized by tubulointerstitial fibrosis. Exposure to high concentrations of carbon tetrachloride (including vapor) can destroy the kidneys. Autophagy played an important role in maintaining the homeostasis of organs. Impaired autophagy was frequently associated with renal damage and fibrosis. Recent data suggests that metformin protects against a variety of kidney disorders. AIM: To investigate the protective role of metformin on carbon tetrachloride induced renal damage via autophagy pathway. MATERIALS AND METHODS: Forty adult male albino rats were divided into four equal groups (10 rats, each); Group 1: control group. Group 2: olive oil group received olive oil 1.5 mg/kg twice weekly S.C for 12 weeks. Group 3: The ccl4 group, the rats were received ccl4 1.5 mg/kg twice weekly S.C for 12 weeks. Group 4: CCL4 and Metformin group received concomitant treatment of CCL4, 1.5 mg/kg twice weekly S.C and 100 mg/kg/day Metformin orally for 12 weeks. After sacrifice, kidneys were taken from all animal groups and processed for light and electron microscopy, immunological studies and biochemical tests. Statistical analysis was done. RESULTS: Administration of ccl4 resulted in histopathological changes in the kidney tissue in the form of areas of tissue destruction, inflammatory cell infiltration, congestion and fibrosis. Ultrastructurally, irregular thickening of GBM was observed. Improvement was noticed with concomitant treatment of ccl4 with metformin. CONCLUSION: Metformin administration can modulate histological and biochemical effects in the renal tissue induced by of ccl4.

Acetylated oligopeptide and N-acetylcysteine protect against iron overload-induced dentate gyrus hippocampal degeneration through upregulation of Nestin and Nrf2/HO-1 and downregulation of MMP-9/TIMP-1 and GFAP.

Iron accumulation in the brain causes oxidative stress, blood-brain barrier (BBB) breakdown, and neurodegeneration. We examined the preventive effects of acetylated oligopeptides (AOP) from whey protein on iron-induced hippocampal damage compared to N-acetyl cysteine (NAC). This 5-week study used 40 male albino rats. At the start, all rats received 150 mg/kg/day of oral NAC for a week. The 40 animals were then randomly divided into four groups: Group I (control) received a normal diet; Group II (iron overload) received 60 mg/kg/day intraperitoneal iron dextran 5 days a week for 4 weeks; Group III (NAC group) received 150 mg/kg/day NAC and iron dextran; and Group IV (AOP group) received 150 mg/kg/day AOP and iron dextran. Enzyme-linked immunosorbent assay, spectrophotometry, and qRT-PCR were used to measure MMP-9, tissue inhibitor metalloproteinase-1 (TIMP-1), MDA, reduced glutathione (GSH) levels, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) gene expression. Histopathological and immunohistochemical detection of nestin, claudin, caspase, and GFAP was also done. MMP-9, TIMP-1, MDA, caspase, and GFAP rose in the iron overload group, while GSH, Nrf2, HO-1, nestin, and claudin decreased. The NAC and AOP administrations improved iron overload-induced biochemical and histological alterations. We found that AOP and NAC can protect the brain hippocampus from iron overload, improve BBB disruption, and provide neuroprotection with mostly no significant difference from healthy controls.

Antiapoptotic and chemotaxis-stimulating effects of poly (D, L-lactide-co-glycolide)-chitosan and whey proteins against aflatoxicosis-induced splenic and thymic atrophy.

BACKGROUND: Aflatoxin B (AFB) induces toxicological effects on the liver and immune organs. The whey proteins can modulate the immune response during aflatoxicosis. Our work evaluates the novel polylactic acid-glycolic acid-chitosan-encapsulated bovine and camel whey proteins against AFB-induced thymic and splenic atrophy in rats. METHODS AND RESULTS: Seventy adult male Wister albino rats were divided into a control healthy group (G1) and six AFB1-intoxicated groups (G2-G7). One of the following supplements: distilled water, camel whey proteins (CWP), bovine whey proteins, poly (D, L-lactide-co-glycolide) (PLGA)- chitosan-loaded with camel whey protein microparticles (CMP), PLGA-chitosan loaded with bovine whey protein microparticles (BMP), and PLGA-chitosan nanoparticles were administered as prophylactic supplements to AFB1-intoxicated groups. The AFB-treated group showed significantly higher hepatic levels of oxidative stress and lower levels of antioxidants. In the aflatoxicated group, atrophy of the splenic lymphatic nodules and disfigurement in the organisation with an apparent decrease in the thickness of the cortex in the thymus were observed, as well as a decrease in splenic and thymic CD4+T and CD8+T lymphocytes. Moreover, CXCL12 levels were downregulated, whereas tumour necrosis factor-alpha, nuclear factor kappa B, and cleaved caspase-3 levels were upregulated. CWP, BMP, and CMP supplements markedly decreased oxidative stress, inflammation, and apoptosis, as well as significantly raised CXCL12, CD4+T, and CD8+T cells. CONCLUSIONS: The CWP, BMP, and CMP supplements rescue the liver and immune tissues from the toxic effects of AFB through their antioxidant, antiapoptotic, anti-inflammatory, and chemotaxis-enhancing roles.

Comparative Histological Study on the Effect of Tramadol Abuse on the Testis of Juvenile and Adult Male Albino Mice.

As a synthetic analog of codeine, tramadol is often prescribed to treat mild to moderate pains. This study was designed to estimate and compare the histological effect of tramadol on testes of both juvenile and adult male albino mice. A total number of 40 healthy male albino mice were classified into two main groups as follows: group I (juvenile group, includes 20 mice aged three weeks) subdivided equally into group Ia (control group received isotonic saline) and group Ib (tramadol-treated group received 40 mg/kg/d tramadol orally for 30 days); group II (adult group, includes 20 mice aged two months) subdivided equally into group IIa (control group received isotonic saline) and group IIb (tramadol-treated group). Juvenile and adult tramadol-treated groups showed numerous testicular changes, including blood vessels congestion, widening of intercellular spaces, vacuolization in interstitial tissues, luminal germ cells exfoliation, and increased expression of caspase-3 that indicated cellular apoptosis. In the ultrastructural examination, spermatogenic cells degenerated with the frequent appearance of apoptotic cells. Sertoli cells showed vacuolations, large lipid droplets, and disrupted intercellular cell junctions. These observed testicular changes were markedly observed in the juvenile group. Testicular abnormalities and apoptotic changes can be caused by tramadol administration. These abnormalities are more common in juvenile mice.

Therapeutic Potential of Mesenchymal Stem Cells versus Omega n - 3 Polyunsaturated Fatty Acids on Gentamicin-Induced Cardiac Degeneration.

This study compared the cardioprotective action of mesenchymal stem cells (MSCs) and PUFAs in a rat model of gentamicin (GM)-induced cardiac degeneration. Male Wistar albino rats were randomized into four groups of eight rats each: group I (control group), group II (gentamicin-treated rats receiving gentamicin intraperitoneally (IP) at dose of 100 mg/kg/day for 10 consecutive days), group III (gentamicin and PUFA group receiving gentamicin IP at dose of 100 mg/kg/day for 10 consecutive days followed by PUFAs at a dose of 100 mg/kg/day for 4 weeks), and group IV (gentamicin and MSC group receiving gentamicin IP at dose of 100 mg/kg/day followed by a single dose of MSCs (1 × 106)/rat IP). Cardiac histopathology was evaluated via light and electron microscopy. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), caspase-3 (apoptosis), Bcl2, and Bax expression was performed. Moreover, cardiac malonaldehyde (MDA) content, catalase activity, and oxidative stress parameters were biochemically evaluated. Light and electron microscopy showed that both MSCs and PUFAs had ameliorative effects. Their actions were mediated by upregulating PCNA expression, downregulating caspase-3 expression, mitigating cardiac MDA content, catalase activity, and oxidative stress parameters. MSCs and PUFAs had ameliorative effects against gentamicin-induced cardiac degeneration, with MSCs showing higher efficacy compared to PUFAs.

Tailoring of Novel Azithromycin-Loaded Zinc Oxide Nanoparticles for Wound Healing.

Skin is the largest mechanical barrier against invading pathogens. Following skin injury, the healing process immediately starts to regenerate the damaged tissues and to avoid complications that usually include colonization by pathogenic bacteria, leading to fever and sepsis, which further impairs and complicates the healing process. So, there is an urgent need to develop a novel pharmaceutical material that promotes the healing of infected wounds. The present work aimed to prepare and evaluate the efficacy of novel azithromycin-loaded zinc oxide nanoparticles (AZM-ZnONPs) in the treatment of infected wounds. The Box-Behnken design and response surface methodology were used to evaluate loading efficiency and release characteristics of the prepared NPs. The minimum inhibitory concentration (MIC) of the formulations was determined against Staphylococcus aureus and Escherichia coli. Moreover, the anti-bacterial and wound-healing activities of the AZM-loaded ZnONPs impregnated into hydroxyl propyl methylcellulose (HPMC) gel were evaluated in an excisional wound model in rats. The prepared ZnONPs were loaded with AZM by adsorption. The prepared ZnONPs were fully characterized by XRD, EDAX, SEM, TEM, and FT-IR analysis. Particle size distribution for the prepared ZnO and AZM-ZnONPs were determined and found to be 34 and 39 nm, respectively. The mechanism by which AZM adsorbed on the surface of ZnONPs was the best fit by the Freundlich model with a maximum load capacity of 160.4 mg/g. Anti-microbial studies showed that AZM-ZnONPs were more effective than other controls. Using an experimental infection model in rats, AZM-ZnONPs impregnated into HPMC gel enhanced bacterial clearance and epidermal regeneration, and stimulated tissue formation. In conclusion, AZM -loaded ZnONPs are a promising platform for effective and rapid healing of infected wounds.

Adipose tissue-derived mesenchymal stem cells reduce endometriosis cellular proliferation through their anti-inflammatory effects.

OBJECTIVE: Endometriosis is a chronic debilitating inflammatory condition characterized by the presence of endometrial tissues outside the uterine cavity. Pelvic soreness and infertility are the usual association. Due to the poor effectiveness of the hormone therapy and the high incidence of recurrence following surgical excision, there is no single effective option for management of endometriosis. Mesenchymal stem cells (MSCs) are multipotent stromal cells studied for their broad immunoregulatory and anti-inflammatory properties; however, their efficiency in endometriosis cases is still a controversial issue. Our study aim was to evaluate whether adipose tissue-derived MSCs (AD-MSCs) could help with endometriosis through their studied anti-inflammatory role. METHODS: Female Wistar rats weighting 180 to 250 g were randomly divided into two groups: group 1, endometriosis group; established by transplanting autologous uterine tissue into rats' peritoneal cavities and group 2, stem cell treated group; treated with AD-MSCs on the 5th day after induction of endometriosis. The proliferative activity of the endometriosis lesions was evaluated through Ki67 staining. Quantitative estimation of interferon γ, tumor necrosis factor-α, interleukin (IL)-6, IL-1ß, IL-10, and transforming growth factor ß expression, as well as immunohistochemical detection of CD68 positive macrophages, were used to assess the inflammatory status. RESULTS: The size and proliferative activity of endometriosis lesions were significantly reduced in the stem cell treated group. Stem cells efficiently mitigated endometriosis associated chronic inflammatory reactions estimated through reduction of CD68 positive macrophages and the expression of the proinflammatory cytokines. CONCLUSION: Stem cell therapy can be considered a novel remedy in endometriosis possibly through its anti-inflammatory and antiproliferative properties.

Therapeutic efficiency of adipose-derived mesenchymal stem cells in healing of experimentally induced gastric ulcers in rats.

Gastric (peptic) ulcer is a major gastrointestinal disorder with high morbidity and mortality. While several drugs have been used to treat gastric ulcers, such as proton pump inhibitor-based triple therapy for Helicobacter pylori eradication, but hey result in adverse side effects. Therefore, development of new alternative therapies is desirable. Many recent studies have shown that mesenchymal stem cells (MSCs) might have an enhancing effect on the ulcerated gastric mucosa. The aim of this study is to evaluate the efficacy of MSCs in the treatment of indomethacin-induced gastric ulcer, and to compare it with the normal ulcer autohealing. This work was performed on 36 adult male albino rats, divided into four groups: Group I (control group), Group II (ulcer group), Group III (autohealing group), and Group IV (stem cells-treated group). The histological changes of gastric mucosa were examined in sections stained with H&E using light microscope for expression of vascular endothelial growth factors (VEGF) and proliferating cell nuclear antigen (PCNA) in immunohistochemical stained sections using image analyzer. The results from MSCs-treated group revealed restoration of the normal architecture of the gastric mucosa with comparison to the autohealing group which showed excessive granulation tissue and heavy cellular infiltration with disorganized architecture of the fundic mucosa. Immunohistochemical examination showed strong expression of both VEGF and PCNA in the MSCs-treated group. So it was concluded that MSCs accelerate gastric ulcer healing when injected intraperitoneally, compared to autohealing process which showed delayed healing.

The Possible Protective Role of Barley Seeds on the Spleen after Administration of Glucocorticoids in Adult Albino Rats: A Histological and Immunohistochemical Study.

BACKGROUND: Glucocorticoids (GCs) are the main treatment strategy in many autoimmune disease and inflammatory diseases; however, they have immunosuppressive effect on many organs. The barley seeds contain many antioxidant compounds, which may improve the antioxidant status and related physiological functions. Our aim in this work is to evaluate the possible protective role of barley seeds on some immune cells in the spleen against immunosuppressive effect of GCs in adult albino rats. MATERIALS AND METHODS: Forty-five adult albino rats were equally divided into 3 groups. Group I: normal vehicle control (n = 15), Group II: steroid-treated animals (n = 15), and Group III: steroid/barley-treated group (n = 15). Specimens from spleen were processed for light and electron microscopy. RESULTS: In steroid-treated group, the histological changes in white and red pulp were in the form of loss of architecture and wide empty spaces among the cells. Most of the cells showed degenerative change, dilatation of blood sinusoids, and deposition of fibrinoid material among the cells of the RP. However, multiple lysosomal bodies were observed in both dendritic and macrophage cells. These changes are improved in steroid/barley-treated group in the form of increasing the number and size of the lymphatic follicles. Most of the splenic cells regained normal structure. Dendritic cell marker CD86 and macrophage marker CD68 expression are increased. CONCLUSION: Barley protects the spleen tissues from steroid-induced structural changes; this could be mediated through its antioxidant effects, so barely is recommended as a healthy diet in patients consuming steroids.

Testing alternatives: the use of adipose-derived mesenchymal stem cells to slow neurodegeneration in a rat model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease. Unfortunately, the effectiveness of anti-Parkinson treatments gradually diminishes owing to the progressive degeneration of the dopaminergic terminals. The research described here investigated the effect of adipose-derived mesenchymal stem cells (AD-MSC) versus that of an anti-Parkinson drug in a rat model of Parkinsonism. Forty adult rats were divided into four equal groups, each group receiving a different treatment: vehicle, rotenone, rotenone + AD-MSC, or rotenone + carbidopa/levodopa. Behavioral tests were carried out before and at the end of the treatment and specimens harvested from the midbrain were processed for light and electron microscopy. Genetic expression of glial fibrillary acidic protein (GFAP) and Nestin mRNA was assessed. Expression of the Lamin-B1 and Vimentin genes was measured, along with plasma levels of Angiopoietin-2 and dopamine. Treatment with rotenone induced pronounced motor deficits, as well as neuronal and glial alterations. The AD-MSC group showed improvements in motor function in the live animals and in the microscopic picture presented by their tissues. The fold change of both genes (GFAP and Nestin) decreased significantly in the AD-MSC and carbidopa/levodopa groups compared to the group with Parkinson's disease. Plasma levels of Angiopoietin-2 and dopamine were significantly increased after treatment (P < 0.001) compared to levels in the rats with Parkinson's disease. AD-MSC reduced neuronal degeneration more efficiently than did the anti-Parkinson drug in a rat model of Parkinsonism.

Therapeutic effect of adipose-derived mesenchymal stem cells on Cisplatin induced testicular damage in adult male albino rat.

Infertility represents a major medical, economic, and psychological problem. Stem cells therapy for infertility has a great interest nowadays especially for cancer survivors at pre-reproductive and reproductive age.  Thirty-two adult male albino rats were used, divided equally into four groups; Group I (Control group) received isotonic saline intraperitoneally (i.p.) as vehicle. Group II (Cisplatin-treated group) received Cisplatin (i.p.) at a single dose of 7 mg/kg, and then were sacrificed after 5 days. Group III (Stem-cell-treated group) received Cisplatin (i.p.) at a single dose of 7 mg/kg, then after 5 days received adipose-derived mesenchymal stem cells (ADMSCs) (1 × 106). Cells were injected in the rete testis, then after 60 days, the animals were sacrificed. Group IV (Auto healing group) received Cisplatin (i.p.) at a single dose of 7 mg/kg, and then left for 65 days then the animals were sacrificed. Cisplatin administration resulted in degenerative changes in the testicular architecture in the form of thickened irregular BM of seminiferous tubules. The germinal epithelium showed disorganization and marked reduction in the thickness, associated with Sertoli cells preservation. Features of apoptosis assured by elevated caspase-3 expression were noticed. The interstitium showed cellular infiltration and distorted Leydig cells. Injection of (ADMSCs) resulted in great improvement of testicular architecture and increase in the testosterone level associated with strong immune reaction of the CD-44. ADMSCs are recommended as a new treatment modality for male infertility. Abbreviation: i.p.: intraperitoneally; BM: basement membrane; ADMSCs: adipose-derived mesenchymal stem cells; WHO: World Health Organization; MSCs: mesenchymal stem cells; DMEM: Dulbecco modified eagles media; PBS: phosphate-buffered saline; FACS: fluorescence-activated cell sorting; ELISA: enzyme-linked immunosorbent assay; CP: Cisplatin; ROS: reactive oxygen species; CAT: catalase; SOD: superoxide dismutase; OS: oxidative stress; SSCs: spermatogonia stem cells; GCs: germ cells; UCMSCs: umblical cord mesenchymal stem cells; TGFb1: transforming growth factor beta-1; BMP4: Bone morphogenic protein 4; BMP8b: bone morphogenic protein 8b.

Calorie restriction enhances adult mouse lung stem cells function and reverses several ageing-induced changes.

Ageing is associated with decreased lung function and an increased incidence of lung infections. Several studies have suggested that long-term calorie restriction (CR) promotes health and longevity and results in the reduced risk of several diseases. The effect of CR is thought to be through improving the function of tissue stem cells. Stem cell function is known to decline with ageing. In this study, we examined the effects of ageing on lung epithelial and stem cells and the effect of CR on young and old lungs. We found that ageing results in a decrease in tracheal basal stem cells. CR induced an increase in basal stem cells in both young and old mice. In addition, ageing induced lung inflammation, and CR tended to reduce baseline lung inflammatory cell infiltration in young mice and significantly reduced ageing-induced lung inflammation. Furthermore, ageing reduced the number and function of mitochondria in lung and increased the level of mitochondrial reactive oxygen species. CR increased the number and function of mitochondria both in young and old mice. Moreover, ageing reduced lung stem cell colony-forming efficiency (CFE), and CR increased the CFE in both young and old mice. Finally, CR improved epithelial cell survival in injured lungs of young mice. In conclusion, ageing causes several structural and functional changes/impairments in lung epithelial cells. CR induces several potentially beneficial changes in lung epithelial cells, even when it is initiated at an older age, including reversal of some ageing-induced changes.

High fat diet activates adult mouse lung stem cells and accelerates several aging-induced effects.

High fat diet (HFD) decreases the lifespan of mice, and is a risk factor for several human diseases. Here, we investigated the effects of a HFD on lung epithelial and stem cells and its interaction with aging. Young and old mice were fed with either a standard diet (SD) or a HFD then their trachea and lung were examined for histological changes, inflammation, and mitochondrial function. Their stem cell function was examined using the in vitro organoid/colony forming efficiency (CFE) assay. Aging reduced the number of tracheal basal and alveolar type-2 (AT2) cells. HFD significantly increased the number of AT2 cells. Aging also caused a significant increase in lung inflammation, and HFD caused a similar increase, in young mice. Aging reduced mitochondrial mass and function, and increased reactive oxygen species. In young mice, HFD caused mitochondrial changes similar to the aging-induced changes. Organoid culture of tracheal and lung epithelial cells collected from both young and old HFD-fed mice showed higher CFE compared to SD-fed mice. Switching the HFD to low calorie/fat diet (LCD) efficiently reversed several of the HFD-induced effects. Thus, HFD induces several histological, inflammatory, and functional changes in the lung, and exacerbates the aging-induced lung inflammation and mitochondrial deterioration. LCD can reverse many of the HFD-induced effects.

Sonic hedgehog and androgen signaling in tumor and stromal compartments drives epithelial-mesenchymal transition in prostate cancer.

OBJECTIVE: Sonic hedgehog (Shh) signaling, androgens and epithelial-mesenchymal transition (EMT) are related to prostate cancer (PCa) progression. The aim of this study was to investigate how Shh and androgen [dihydrotestosterone (DHT)] signaling act in prostate epithelial and stromal compartments and whether this signaling pathway drives EMT and promotes PCa progression. MATERIAL AND METHODS: LNCaP, normal prostate fibroblast (NPF) and cancer-associated prostate fibroblast (CPF) cells were studied with DHT and/or the Shh signaling inhibitor cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate the expressions of a potential Shh target gene, osteonectin (ON) and EMT-associated markers (E-cadherin, N-cadherin and vimentin). Immunohistochemical studies using PCa prostatectomy samples were performed to assess the expression levels of ON, Gli-1, androgen receptor, Shh, E-cadherin, N-cadherin and vimentin. RESULTS: While DHT enhanced cell proliferation in CPF more than LNCaP or NPF, cyclopamine inhibited cell proliferation enhanced by DHT in CPF. Real-time RT-PCR showed whereas both Shh and DHT induced N-cadherin and vimentin, DHT also induced the expression of osteonectin in LNCaP and cyclopamine blocked these expressions in osteonectin, N-cadherin and vimentin (p = 0.0084, 0.0002 and 0.0373, respectively). Immunohistochemistry showed that high expression of stromal, but, not epithelial, ON was significantly correlated with serum prostate-specific antigen (PSA) (p = 0.031), and high expression of Gli-1 and low expression of stromal ON with PSA recurrence (p = 0.0114 and p = 0.0005, respectively). CONCLUSIONS: Shh and androgen signaling in prostate tumor and stromal compartments drives EMT, and thus may play some role in PCa progression. Cyclopamine may be one therapeutic strategy for PCa.

Possible role of sonic hedgehog and epithelial-mesenchymal transition in renal cell cancer progression.

PURPOSE: Sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) are both known to relate to cancer progression. The purpose of this study was to investigate the role of Shh signaling and EMT in renal cell carcinoma (RCC). MATERIALS AND METHODS: Cell proliferation was assayed in RCC cell lines in the presence or absence of a Shh signaling stimulator, recombinant Shh (r-Shh) protein, or a Shh signaling inhibitor, cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the expression of EMT markers (E-cadherin, N-cadherin, and vimentin) and osteonectin. The expression of Ki-67, Gli-1, osteonectin, and EMT markers in nephrectomy specimens from RCC patients was also measured by immunohistochemical (IHC) staining. RESULTS: RCC cells showed enhanced cell proliferation by r-Shh protein, whereas cell proliferation was suppressed by the addition of cyclopamine in RenCa cells. Real-time RT-PCR showed that r-Shh suppressed the expression of E-cadherin and that this suppression was partly blocked by cyclopamine alone in RenCa cells. In the IHC results, osteonectin significantly correlated with vein sinus invasion (p=0.0218), and the expression of vimentin significantly correlated with lymphatic invasion (p=0.0392). CONCLUSIONS: Shh signaling and EMT play roles in RCC progression, and the Shh signaling inhibitor cyclopamine might be a possible molecular targeted therapeutic strategy for RCC.

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue, bone marrow, and skeletal muscle tissue.

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.