Histological changes induced by Polyglycolic-Acid (PGA) scaffolds seeded with autologous adipose or muscle-derived stem cells when implanted on rabbit bladder

Purpose: To evaluate the morphological and histological changes induced by PGA scaffold seeded with autologous adipose or muscle derived stem cells implanted on rabbit bladder wall. Material and Methods: Adipose derived stem cells (ADSCs) were obtained from the inguinal fat of eight rabbits and muscle derived stem cells (MDSCs) from the anterior tibial muscle of other eight rabbits. After culture and isolation, the cells were stained with Vybrant Red CM DiI and then implanted at third passage. Two PGA scaffolds were implanted on the bladder submucosa of each animal. On the right bladder side was implanted unseeded PGA scaffold while on the left side was implanted ADSCs or skeletal MDSCs seeded PGA scaffold. ADSCs were implanted in eight animals and MDSC in other eight animals. The animals were sacrificed at four and eight weeks. Histological evaluation was performed with Hematoxylin and Eosin, Masson's Trichrome and smooth muscle α-actin. Results: We observed a mild inflammatory response in all the three groups. Seeded scaffolds induced higher lymphocytes and lower polimorphonuclear migration than controls. Fibrosis was more pronounced in the control groups. Smooth muscle α-actin was positive only in ADSC and MDSC seeded scaffolds. At four and eight weeks ADCSs and skeletal MDSCs labeled cells were found at the implant sites. Conclusions: The implantation of PGA scaffolds seeded with ADSC and MDSC induced less fibrosis than control and smooth muscle regeneration.     

Histological changes induced by Polyglycolic-Acid (PGA) scaffolds seeded with autologous adipose or muscle-derived stem cells when implanted on rabbit bladder

Purpose: To evaluate the morphological and histological changes induced by PGA scaffold seeded with autologous adipose or muscle derived stem cells implanted on rabbit bladder wall. Material and Methods: Adipose derived stem cells (ADSCs) were obtained from the inguinal fat of eight rabbits and muscle derived stem cells (MDSCs) from the anterior tibial muscle of other eight rabbits. After culture and isolation, the cells were stained with Vybrant Red CM DiI and then implanted at third passage. Two PGA scaffolds were implanted on the bladder submucosa of each animal. On the right bladder side was implanted unseeded PGA scaffold while on the left side was implanted ADSCs or skeletal MDSCs seeded PGA scaffold. ADSCs were implanted in eight animals and MDSC in other eight animals. The animals were sacrificed at four and eight weeks. Histological evaluation was performed with Hematoxylin and Eosin, Masson's Trichrome and smooth muscle α-actin. Results: We observed a mild inflammatory response in all the three groups. Seeded scaffolds induced higher lymphocytes and lower polimorphonuclear migration than controls. Fibrosis was more pronounced in the control groups. Smooth muscle α-actin was positive only in ADSC and MDSC seeded scaffolds. At four and eight weeks ADCSs and skeletal MDSCs labeled cells were found at the implant sites. Conclusions: The implantation of PGA scaffolds seeded with ADSC and MDSC induced less fibrosis than control and smooth muscle regeneration.     

Contractions but not AICAR increase FABPpm content in rat muscle sarcolemma

In the present study, it was investigated whether acute muscle contractions in rat skeletal muscle increased the protein content of FABPpm in the plasma membrane. Furthermore, the effect of AICAR stimulation on FAT/CD36 and FABPpm protein content in sarcolemma of rat skeletal muscle was evaluated. Methods Male wistar rats (150 g) were anesthetized and either subjected to in situ electrically induced contractions (hindlimb muscles: 20 min, 10–20 V, 200 ms trains, 100 Hz) or stimulated with the pharmacological activator of AMPK, AICAR. To investigate changes in the content of FABPpm and FAT/CD36 in the plasma membrane by these stimuli, the giant sarcolemma vesicle (GSV) technique was applied. The hindlimb muscles were removed and used for the production of GSV and lysates. All samples were analyzed using the western blotting technique. Results Electrical stimulation of rat hindlimb muscle resulted in an increase in FABPpm protein content in the GSV of 61% (P < 0.05) and in FAT/CD36 protein content in the GSV of 33% (P < 0.05). AICAR stimulation increased FAT/CD36 protein content in GSV by 22% (P < 0.05), whereas FABPpm protein content in GSV was unaffected by AICAR treatment. There was no change in total FAT/CD36 and FABPpm protein expression, measured in lysates with western blotting, by either stimulus. AMPK thr172 and ERK1/2 thr202/204 phosphorylation were significantly increased with muscle contractions (P < 0.05), whereas only AMPK thr172 phosphorylation was increased with AICAR stimulation (P < 0.05). Conclusion These data show that contractions increase both FAT/CD36 and FABPpm protein content in skeletal muscle plasma membrane, whereas only FAT/CD36 protein content is increased when muscle are stimulated with AICAR. This suggests that AMPK is involved in regulation of FAT/CD36, but not FABPpm in skeletal muscle. However, since both ERK1/2 thr202/204 and AMPK thr172 phosphorylation are increased during muscle contractions, the present study cannot rule out that both could play a significant role in regulation of FAT/CD36 and FABPpm during muscle contractions.     

De novo lipogenesis and desaturation of fatty acids during adipogenesis in bovine adipose-derived mesenchymal stem cells

Adipose-derived mesenchymal stem cells (ADSCs) are useful cell model to study adipogenesis and energy metabolism. However, the biological characteristics of bovine ADSCs (bADSCs) remain unclear. This study aimed to isolate and identify bADSCs and further investigate fatty acid (FA)-related gene expression and composition of FAs during adipogenesis. The growth curve showed the bADSCs of P5 cells had rapid proliferation superior to P10–P50. The colony formation assay showed colony number of P5 cells was higher than that of P50 cells (51.67 ± 3.06 vs 35.67 ± 6.43, P < 0.05). The immunofluorescence showed that bADSCs were positive for CD13, CD44, CD49d, CD90, CD105, and Vimentin while negative for CD34. The multipotential towards adipocyte, osteocyte, and chondrocyte was confirmed by specific histological staining and lineage gene expression. During adipogenic induction, the genes related to lipogenesis and lipolysis were assessed by real-time PCR and the FA composition was detected by GC-MS. Expression of lipogenesis-related genes showed coordinated regulation as peaking on day 7 and declining until induction ended, including PPARγ, SREBP1, ACC1, FAS, ELOVL6, SCD1, and FABP4. FA deposition-related genes (DGAT1 and ACAT1) increased until day 14. Lipolysis genes (CPT-1A, VLCAD, and ACO) showed a variant expression pattern. The profile of FAs showed that proportion of the FAs (C4–C15, ≥ C22) increased, but proportion of long-chain fatty acids (C16–C20) reduced after induction. And saturated FAs (SFA) decreased while monounsaturated FAs (MUFA) and polyunsaturated FAs (PUFA) increased during adipogenesis. These data suggest that bADSCs possess the characteristics of mesenchymal stem cells and have active de novo lipogenesis (DNL) and desaturation of FAs during adipogenesis.     

Choosing the right type of serum for different applications of human adipose tissue–derived stem cells: influence on proliferation and differentiation abilities

Background aimsAdipose tissue–derived stem cells (ADSCs) are thought to have great potential in regenerative medicine. A xenoprotein-free culture and handling system is desirable. To date, there is only little and contradictory information about the influence of the different types of human serum on ADSC proliferation and differentiation.MethodsFirst, ADSCs were cultured in media containing regular human serum (HS plus) or fetal calf serum (FCS plus) with supplementation of growth factors for three passages. During passage 4, ADSC proliferative activity and adipogenic, osteogenic and chondrogenic differentiation ability was quantified. Second, ADSCs were cultured with three different human sera (regular human serum [HS], human serum from platelet-poor plasma [SPPP] or human serum from platelet-rich plasma [SPRP]) without supplementation of platelet-derived growth factor and assessed accordingly. The growth factor content of the different types of human sera was determined by means of multiplex protein assay and enzyme-linked immunosorbent assay.ResultsThe different sera did not affect ADSC doubling time significantly (P < 0.05). Specific glycerol-3-phosphat-dehydrogenase activity was significantly lower in cultures with SPRP (P < 0.01) compared with the other media compositions. Extracellular calcium deposition was significantly higher in cells differentiated in cultures with HS or SPPP compared with those with SPRP, HS plus or FCS (P < 0.01). Glycosaminoglycan content and collagen 2 were highest in cells cultured with SPRP (P < 0.001).ConclusionsCulturing ADSCs in human serum appears to be a reasonable and efficient alternative compared with FCS. With respect to the outcome of a sighted clinical application, it appears to be feasible to handle the cells in a serum suitable for the intended later use.     

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

Overexpression of CDK5 in Neural Stem Cells Facilitates Maturation of Embryonic Neurocytes Derived from Rats In Vitro

To further understand the effects of cyclin-dependent kinase 5 (CDK5) on the differentiation of neural stem cells, which were cultured and transfected with CDK5-EGFP recombinant overexpression vector (OV-CDK5 Group), successful transfection was confirmed by RT-PCR and Western blot. Our results showed that the CDK5 mRNA expression significantly increased in 6 h after transfection. Increase in the levels of the CDK5 protein expression was observed in 72 h, compared with Empty Vector Control Group (EV-CTL Group) (P < 0.01). Furthermore, in OV-CDK5 Group, the percentage of S-phase cells was significantly higher than in EV-CTL Group (P < 0.01). Differentiated cells were showed with short processes in 24 h and with obviously enlarged cell body, and extended cellular processes in 72 h, in comparison to those in EV-CTL Group (P < 0.01). In 72 h under treatment with 10 μmol/L all-trans retinoic acid (ATRA), in OV-CDK5 Group, processes of the GFP-positive cells were reduced slightly and little GFP-positive debris was found. However, in the EV-CTL Group, processes of the GFP-positive cells were obviously shortened and deformed and much GFP-positive debris were found. Moreover, the percentage of G0/G1-phase cells was lesser, while the percentage of S-phase cells was higher than that in EV-CTL Group (P < 0.01, P < 0.01). In conclusion, our experiment suggested that CDK5 might promote proliferation and differentiation of neural stem cells, lengthen the processes of differentiated neurocytes, and accelerate morphological maturation of such cells. Furthermore, CDK5 might antagonize ATRA-induced inhibition against proliferation and differentiation in differentiated neurocytes.     

Nitric oxide contributes to the regulation of iron metabolism in skeletal muscle in vivo and in vitro

Nitric Oxide (NO) plays an important role in iron redistribution during exercise, while its molecular regulatory mechanism is still not clear. Our present studies were to investigate the effects of NO on iron metabolism and to elucidate the regulatory mechanism of iron transport in skeletal muscle both in vivo and in vitro. One group of male Wistar rats (300 ± 10 g) were subjected to an exercise of 30 min on a treadmill for 5 weeks (exercise group, EG, 6 rats) and the other one was placed on the treadmill without running (control group, CG, 6 rats). The cultured L6 rat skeletal muscle cells were treated with either 0.5 mM SNAP (NO donor) or not for 24 h, and their iron release and intake amount were examined by measuring radiolabelled ⁵⁵Fe. The results showed: (1) The NO content (CG, 1.09 ± 0.18 μmol/g vs. EG, 1.49 ± 0.17 μmol/g) and non-heme iron in gastrocnemius (CG, 118.35 ± 11.41 μg/g vs. EG, 216.65 ± 11.10 μg/g) of EG were significantly increased compared with CG. (2) The expression of DMT1 (IRE) and TfR1 of EG was increased. (3) The iron intake was increased in L6 cells treated with SNAP (P < 0.01). (4) Western blot results showed the protein level of both TfR1 and DMT1 (IRE) in SNAP cells were up-regulated, while the expression of FPN1 was down-regulated (P < 0.05). The data suggested that the induced elevation of NO level by exercise lead to the up-regulation of both TfR1 and DMT1 (IRE), which in turn increasing the iron absorption in skeletal muscle.     

Effects of Chromium-Loaded Chitosan Nanoparticles on Glucose Transporter 4, Relevant mRNA,and Proteins of Phosphatidylinositol 3-Kinase,Akt2-Kinase,and AMP-Activated Protein Kinase of Skeletal Muscles in Finishing Pigs

The study was conducted to evaluate the effects of chromium-loaded chitosan nanoparticles (Cr-CNP) on glucose transporter 4 (GLUT4), relevant messenger RNA (mRNA), and proteins involved in phosphatidylinositol 3-kinase (PI3K), Akt2-kinase, and AMP-activated protein kinase (AMPK) of skeletal muscles in finishing pigs. A total of 120 crossbred barrows (BW 65.00 ± 1.26 kg) were randomly allotted to four dietary treatments, with three pens per treatment and 10 pigs per pen. Pigs were fed the basal diet supplemented with 0, 100, 200, or 400 μg/kg of Cr from Cr-CNP for 35 days. After the feeding trials, 24 pigs were slaughtered to collect longissimus muscle samples for analysis. Cr-CNP supplementation increased GLUT4 messenger RNA (mRNA) (quadratically, P < 0.01) and total and plasma membrane GLUT4 protein contents (linearly and quadratically, P < 0.001) in skeletal muscles. Glycogen synthase kinase 3β (GSK-3β) mRNA was decreased linearly (P < 0.001) and quadratically (P < 0.001). Supplemental Cr-CNP increased insulin receptor (InsR) mRNA quadratically (P < 0.01), Akt2 total protein level linearly (P < 0.01) and quadratically (P < 0.001), and PI3K total protein was increased significantly (P < 0.05) in 200 μg/kg treatment group. The mRNA of AMPK subunit gamma-3 (PRKAG3) and protein of AMPKα₁ was significantly increased (P < 0.001) with the addition of Cr-CNP. The results indicate that dietary supplementation of Cr-CNP may promote glucose uptake by leading to recruitment of GLUT4 to the plasma membrane in skeletal muscles, and these actions may be associated with the insulin signal transduction and AMPK.     

Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Evaluation of CD49f as a novel surface marker to identify functional adipose‐derived mesenchymal stem cell subset

ObjectivesCD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue‐derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified.Materials and methodsThe effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f⁺ cells were selected from rat ADSCs (rADSCs) by magnetic‐activated cell sorting (MACS), and the cellular functions of CD49f⁺ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities, were compared.ResultsCD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF‐α and IFN‐γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti‐CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f⁺ ADSCs. CD49f⁺ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities than unsorted ADSCs.ConclusionIn the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f ⁺ ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)‐based therapies.     

Distribution of antibodies to the troponin complex,troponin-C and troponin-I in chicken skeletal muscle as determined by a simplified method for immuno-electron microscopy

Antisera against the troponin complex, troponin-C and troponin-I have been utilized to locate these proteins in normal, adult chicken skeletal muscle and in filaments prepared from chicken acetone dried powder. The antisera had been previously characterized by immunochemical methods and were employed to ascertain the distribution of the proteins by a simple method for immuno-electron microscopy. Glycerinated chicken breast muscle was treated with the antisera, unreacted antibody was washed from the muscle, and a goat anti-rabbit γ-globulin was added to enhance the electron density of the antigen-antibody complexes. A periodic distribution of anti-troponin-C at a mean interval of 389 Å was observed along the thin filaments in the sectioned tissue. Anti-troponin-I was deposited every 399 Å (P < 0.01). Thin filaments were prepared from acetone dried powder and reacted with the antisera. The anti-troponin-C was located every 386 Å; anti-troponin-I, every 399 Å (P < 0.01). Our technique for immuno-electron microscopy is compared with that used by others, and the significance of the findings is discussed.     

An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p?<?0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.     

Effects of Synbiotic2000™ Forte on the Intestinal Motility and Interstitial Cells of Cajal in TBI Mouse Model

The main objective of this study was to investigate the effects of Synbiotic2000? Forte on the intestinal motility and interstitial cells of Cajal (ICC) in traumatic brain injury (TBI) mouse model. Kunming mice were randomly divided into sham operation group (S group), enteral nutrition group with TBI (E group), and Synbiotic2000? Forte group with TBI (P group). The contractile activity of the intestinal smooth muscle, densities and ultrastructure of the ICC, kit protein concentration, weight, and defecation of mice were monitored and analyzed. TBI markedly suppressed contractile activity of the intestinal smooth muscle (P < 0.01), which led to a reduction of defecation (P < 0.01) and weight (P < 0.01). However, application of Synbiotic2000? Forte significantly improved contractile activity of the small intestine (P < 0.01), which may be related to protective effects to the interstitial cells of Cajal, smooth muscle cells, and enteric neurons. TBI impaired ICC networks and densities (P < 0.01), events that were protected by the application of Synbiotic2000? Forte. Synbiotic2000? Forte may attenuate TBI-mediated inhibition of the kit protein pathway. Synbiotic2000? Forte may improve intestinal motility and protect the ICC in the TBI mouse. These findings provide a novel support for the application of Synbiotic2000? Forte in intestinal motility disturbance after TBI.