Repair of Peripheral Nerve Sensory Impairments via the Transplantation of Bone Marrow Neural Tissue-Committed Stem Cell-Derived Sensory Neurons.

The present study aimed to investigate the efficacy of transplantation of bone marrow neural tissue-committed stem cell-derived sensory neuron-like cells for the repair of peripheral nerve sensory impairments in rats. Bone marrow was isolated and cultured to obtain the neural tissue-committed stem cells (NTCSCs), and the differentiation of these cells into sensory neuron-like cells was induced. Bone marrow mesenchymal stem cells (BMSCs), bone marrow NTCSCs, and bone marrow NTCSC-derived sensory neurons (NTCSC-SNs) were transplanted by microinjection into the L4 and L5 dorsal root ganglions (DRGs) in an animal model of sensory defect. On the 2nd, 4th, 8th, and 12th week after the transplantation, the effects of the three types of stem cells on the repair of the sensory functional defect were analyzed via behavioral observation, sensory function evaluation, electrophysiological examination of the sciatic nerve, and morphological observation of the DRGs. The results revealed that the transplanted BMSCs, NTCSCs, and NTCSC-SNs were all able to repair the sensory nerves. In addition, the effect of the NTCSC-SNs was significantly better than that of the other two types of stem cells. The general posture and gait of the animals in the sensory defect model exhibited evident improvement over time. Plantar temperature sensitivity and pain sensitivity gradually recovered, and the sensation latency was reduced, with faster sensory nerve conduction velocity. Transplantation of NTCSC-SNs can improve the repair of peripheral nerve sensory defects in rats.

Silencing of Proteasome 26S Subunit ATPase 2 Regulates Colorectal Cancer Cell Proliferation, Apoptosis, and Migration.

OBJECTIVE: Colorectal cancer (CRC) remains a major cause of cancer-related death worldwide. Proteasome 26S subunit ATPase 2 (PSMC2) plays vital roles in regulating cell cycle and transcription and has been confirmed to be a gene potentially associated with some human tumors. However, the expression correlation and molecular mechanism of PSMC2 in CRC are still unclear. This study aimed to investigate the role of PSMC2 in malignant behaviors in CRC. METHODS: The high protein levels of PSMC2 in CRC samples were identified by tissue microarray analysis. Lentivirus was used to silence PSMC2 in HCT116 and RKO cells; MTT and colony formation assay were performed to determine cell proliferation. Wound healing and Transwell assay were used to detect cell migration and invasion. Flow cytometry assay was applied to detect cell cycle and apoptosis. RESULT: The results showed that, among the 96 CRC patients, the expression of PSMC2 was a positive correlation with the clinicopathological features of the patients with CRC. Furthermore, the low PSMC2 expression group showed a higher survival rate than the high PSMC2 expression group. The expression levels of PSMC2 in cancer tissue were dramatically upregulated compared with adjacent normal tissues. In vitro, shPSMC2 was designed to inhibit the expression of PSMC2 in CRC cells. Compared with shCtrl, silencing of PSMC2 significantly suppressed cell proliferation, decreased single cell colony formation, enhanced apoptosis, and accelerated G2 phase and/or S phase arrest. CONCLUSION: Survival analysis indicated that high expression of PSMC2 in the CRC samples was associated with poorer survival rate than low expression of PSMC2, while the anti-tumor effect of PSMC2 silencing was also confirmed at the cellular level in vitro. Our results suggested that PSMC2 potentially worked as a regulator for CRC, and the silencing of PSMC2 may be a therapeutic strategy for CRC.

Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells.

A cell-sourced biological pacemaker is a promising therapeutic approach for sick sinus syndrome (SSS) or severe atrial ventricular block (AVB). Adipose tissue-derived stem cells (ATSCs), which are optimal candidate cells for possible use in regenerative therapy for acute or chronic myocardial injury, have the potential to differentiate into spontaneous beating cardiomyocytes. However, the pacemaker characteristics of the beating cells need to be confirmed, and little is known about the underlying differential mechanism. In this study, we found that brown adipose tissue-derived stem cells (BATSCs) in mice could differentiate into spontaneous beating cells in 15% FBS Dulbecco's modified Eagle's medium (DMEM) without additional treatment. Subsequently, we provide additional evidence, including data regarding ultrastructure, protein expression, electrophysiology, and pharmacology, to support the differentiation of BATSCs into a cardiac pacemaker phenotype during the course of early cultivation. Furthermore, we found that silencing Tbx18, a key transcription factor in the development of pacemaker cells, terminated the differentiation of BATSCs into a pacemaker phenotype, suggesting that Tbx18 is required to direct BATSCs toward a cardiac pacemaker fate. The expression of Tbx3 and shox2, the other two important transcription factors in the development of pacemaker cells, was decreased by silencing Tbx18, which suggests that Tbx18 mediates the differentiation of BATSCs into a pacemaker phenotype via these two downstream transcription factors.

Sonic hedgehog and retinoic Acid induce bone marrow-derived stem cells to differentiate into glutamatergic neural cells.

Studies have showed that transplanted stem cells in the inner ear won't regenerate to replace the damaged sensory hair cells. They can spontaneously differentiate into mesenchymal cells and fibrocytes in the damaged inner ear. Only mature sensory cells of MSCs-derived possess the great potency for cell transplantation in the treatment of sensorineural hearing loss. So, we try to establish an efficient generation of the glutamatergic sensory neural phenotype for the cell transplantation of the hearing loss. We isolated MSCs from femoral and tibial bones according to their adherence to culture dishes. After purification, proliferation, and passaged, cells became homogeneous in appearance, showing more uniformity and grew in a monolayer with a typical spindle-shape morphology. The cell surface markers were assessed using FACS to characterize the isolated cells. For neural induction to harvest the glutamatergic sensory neurons, passage 3 MSCs were incubated with preinduced medium for 24 hr, and neural-induced medium for an additional 14 days. The cells exhibit a typical neural shape. RT-PCR analysis indicated that the mRNA levels of the neural cell marker nestin, Tau, MAP-2, ß-tubulin III, GluR-3, and GluR-4 were higher compared with primary MSCs. Immunohistochemistry and western-blotting proofed that nestin, MAP-2, ß-tubulin III, and GluR-4 proteins indeed exhibit their expression difference in the induced cells compared to the MSCs. We show an efficient protocol by the combined applications of Sonic Hedgehog (Shh) and Retinoic Acid (RA) to induce MSCs to differentiate into the glutamatergic sensory neuron which were identified from the morphological, biochemical, and molecular characteristics.

Applied anatomy of small branches of the portal vein in transverse groove of hepatic hilum.

PURPOSE: The objective of this study was to provide the morphological details on small branches of the portal vein in transverse groove of hepatic hilum. METHODS: According to the surgery significance, the small branches of portal vein in transverse groove of hepatic hilum were named as "Short hepatic portal veins (SHPVs)". SHPVs were minutely dissected in 30 adult cadaveric livers. The number, diameter, length, origin points, and entering liver sites of SHPVs were explored and measured. RESULTS: There were 181 SHPVs in 30 liver specimens, including 46% (83/181) from the left portal vein, 31% (56/181) from the bifurcation, and 23% (42/181) from the right portal vein. At the entering liver sites of SHPVs, 22% (40/181) supplied for segment IV, 9% (17/181) for segment V, 4% (7/181) for segment VI, 23% (41/181) for segment VII, and 42% (76/181) for segment I (caudate lobe). There were 6.0 ± 2.4 branches per liver specimen with range 3-12. The mean diameter of SHPVs was 2.25 ± 0.89 mm. The average length of SHPVs was 4.86 ± 2.12 mm. CONCLUSIONS: SHPVs widely existed in each liver specimen. The detailed anatomical study of SHPVs could be useful to avoid damaging the short portal branches during hepatic operations, such as isolated or combined caudate lobectomy.

Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

BACKGROUND: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. METHODS: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H2O2, high glucose/U0126 or normal glucose/H2O2/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. RESULTS: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H2O2 stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H2O2 conditions. CONCLUSIONS: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

Protective effects of hydrogen-rich saline on ulcerative colitis rat model.

BACKGROUND: Ulcerative colitis (UC) is associated with enhanced production of reactive oxygen species and altered angiogenesis. Molecular hydrogen has been documented as a novel antioxidant to treat various reactive oxygen species-related diseases. The present study aimed to investigate the effects of hydrogen on UC using a rat model. MATERIALS AND METHODS: UC in rats was induced with intracolonically administrated acetic acid. Hydrogen was supplied through intraperitoneal injection of 10 or 20 mL/kg hydrogen-rich saline. The hydrogen treatment was performed once every 2 d and lasted 2 wk. The stool consistency and weight loss were used to evaluate UC development. Colonic mucosal damage at the end of the experiment was scored using the macroscopic and microscopic observations. Vascular endothelial growth factor expression in the colonic mucosa was determined using immunohistochemistry. RESULTS: The administration of acetic acid induced acute rat UC, as indicated by diarrhea, weight loss, and colonic mucosal damage. Treatment with hydrogen-rich saline reduced the weight loss and diarrhea and alleviated the colonic mucosal damage in the UC rats. In addition, the expression of vascular endothelial growth factor in the UC rats increased and could be inhibited by hydrogen treatment. CONCLUSIONS: Antioxidative hydrogen-rich saline effectively protected the rats from UC, which might be, at least in part, because of inhibition of vascular endothelial growth factor.

CNN2 silencing inhibits colorectal cancer development through promoting ubiquitination of EGR1.

Colorectal cancer (CRC) is one of the most commonly diagnosed malignant tumors of the digestive tract. H2-calponin (CNN2), an actin cytoskeleton-binding protein, is an isoform of the calponin protein family whose role in CRC is still unknown. Research based on clinical samples showed the up-regulation of CNN2 in CRC and its association with tumor development, metastasis, and poor prognosis of patients. Both in vitro loss-of-function and gain-of-function experiments showed that CNN2 participates in CRC development through influencing malignant cell phenotypes. In vivo, xenografts formed by CNN2 knockdown cells also showed a slower growth rate and smaller final tumors. Furthermore, EGR1 was identified as a downstream of CNN2, forming a complex with CNN2 and YAP1 and playing an essential role in the CNN2-induced regulation of CRC development. Mechanistically, CNN2 knockdown down-regulated EGR1 expression through enhancing its ubiquitination, thus decreasing its protein stability in a YAP1-dependent manner. In summary, CNN2 plays an EGR1-dependent promotion role in the development and progression of CRC, which may be a promising therapeutic target for CRC treatment.

Endothelin-induced differentiation of Nkx2.5⁺ cardiac progenitor cells into pacemaking cells.

The mechanisms governing the development of cardiac pacemaking and conduction system are not well understood. In order to provide evidence for the derivation of pacemaking cells and the signal that induce and maintain the cells in the developing heart, Nkx2.5(+) cardiac progenitor cells (CPCs) were isolated from embryonic heart tubes of rats. Endothelin-1 was subsequently added to the CPCs to induce differentiation of them towards cardiac pacemaking cells. After the treatment, Nkx2.5(+) CPCs displayed spontaneous beating and spontaneously electrical activity as what we have previously described. Furthermore, RT-PCR and immunofluorescence staining demonstrated that Tbx3 expression was increased and Nkx2.5 expression was decreased in the induced cells 4 days after ET-1 treatment. And the significantly increased expression of Hcn4 and connexin-45 were detected in the induced cells 10 days after the treatment. In addition, Nkx2.5(+) CPCs were transfected with pGCsi-Tbx3 4 days after ET-1 treatment in an attempt to determine the transcription regulatory factor governing the differentiation of the cells into cardiac pacemaking cells. The results showed that silencing of Tbx3 decreased the pacemaking activity and led to down-regulation of pacemaker genes in the induced cells. These results confirmed that Nkx2.5(+) CPCs differentiated into cardiac pacemaking cells after being treated with ET-1 and suggested that an ET-1-Tbx3 molecular pathway govern/mediate this process. In conclusion, our study support the notion that pacemaking cells originate from Nkx2.5(+) CPCs present in embryonic heart tubes and endothelin-1 might be involved in diversification of cardiomyogenic progenitors toward the cells.

Expression of sphingosine kinase 1 in amoeboid microglial cells in the corpus callosum of postnatal rats.

Sphingosine kinase 1 (SphK1), a key enzyme responsible for phosphorylating sphingosine into sphingosine-1-phosphate (S1P) has been shown to be expressed in monocytes and monocyte-derived peripheral macrophages. This study demonstrates SphK1 immunoexpression in amoeboid microglial cells (AMC), a nascent monocyte-derived brain macrophage in the corpus callosum of developing rat brain. SphK1 immunofluorescence expression, which appeared to be weak in AMC in normal brain, was markedly induced by lipopolysaccharide (LPS) or hypoxia treatment. Western blot analysis also showed increased expression level of SphK1 in the corpus callosum rich in AMC after LPS treatment. Detection of SphK1 mRNA and its upregulation after LPS treatment was confirmed in primary culture AMC by RT-PCR. Administration of N, N-dimethylsphingosine (DMS), a specific inhibitor of SphK1, effectively reduced upregulated SphK1 immunoexpression in AMC both in vivo and in vitro. This was corroborated by western blot which showed a decrease in SphK1 protein level of callosal tissue with DMS pretreatment. Remarkably, LPS-induced upregulation of the transcription factor NFκB was suppressed by DMS. We conclude that SphK1 expression in AMC may be linked to regulation of proinflammatory cytokines via an NFκB signaling pathway.

The role of bFGF in preventing the shrinkage of cardiac progenitor cell-engineered conduction tissue by downregulating α-SMA expression.

AIMS: Engineered conduction tissues (ECTs) fabricated from cardiac progenitor cells (CPCs) and collagen sponges were precisely targeted for the treatment of atrioventricular conduction block in our previous studies. However, obvious shrinkage and deformation of ECTs was observed during in vitro culture. According to the literature, it can be speculated that basic fibroblast growth factor (bFGF) may downregulate alpha-smooth muscle actin (α-SMA) produced by CPCs to prevent the shrinkage of CPC-engineered conduction tissues. MAIN METHODS: In this study, culture media with or without bFGF were used for both cell culture and 3D tissue construction. The expression of α-SMA and the size change of engineered tissue were analyzed to evaluate the feasibility of adding bFGF to regulate α-SMA expression and shrinkage of constructs. In addition, cardiac-specific examinations were performed to evaluate the effect of bFGF on cardiac tissue formation. KEY FINDINGS: Supplementation with bFGF efficiently relieved shrinkage of engineered tissue by downregulating the expression of α-SMA at both the cellular and 3D tissue levels. Moreover, bFGF had a positive influence on cardiac tissue formation in terms of cell viability, tissue organization and electrical conduction velocity. SIGNIFICANCE: This study provides a guide for both shape control and quality improvement of CPC-engineered cardiac tissues.

Intramuscular innervations of muscle flaps that are commonly used in clinical settings.

PURPOSE: Abductor hallucis, latissimus dorsi, gracilis, rectus abdominis, sartorius and pectoralis minor are muscle flaps that are commonly used in clinic, but their intramuscular innervation has seldom been systematically investigated. METHODS: Five Chinese fresh human cadavers were included in the study and abductor hallucis, latissimus dorsi, gracilis, rectus abdominis, sartorius and pectoralis muscles were dissected. After gross anatomy measurement, the specimens were then stained by Sihler's staining technique. Intramuscular innervation was observed and the number as well as distribution was recorded. RESULTS: Intramuscular nerves were clearly visualized by Sihler's staining technique. Based on the shape and muscle-tendon morphology, Lim et al. in Muscle Nerve 29:523-530, 2004 grouped the muscles into trapezoidal-shaped (type I), spindle-shaped (type II), and combination-shaped (type III). According to Lim's study the abductor hallucis was a type IIb muscle and was divided into two compartments by the distal tendon. Latissimus dorsi was a type I muscle, divided into 3-4 compartments by intramuscular nerve branches. Gracilis was a type IIa muscle and the distal part was divided into two compartments by intramuscular nerve branches. Rectus abdominis was a type III muscle and the four bellies comprised four compartments, each of which could be designated as a subunit. Sartorius was a type II muscle and it could be divided into 2-3 compartments along the long axis. Pectoralis minor was a type I muscle that was divided into two compartments by extramuscular terminal nerves. CONCLUSIONS: The six muscles are divided into several compartments by the tendon or nerve branches, and all of them make good donor tissue for muscle compartment transfer in reconstructive surgery.

Comparative research of the thin transverse sectional anatomy and the multislice spiral CT on Pterygopalatine Fossa.

AIM: To explore a method to obtain sub-millimeter data of the thin transverse section of the pterygopalatine fossa (PPF), and to study the thin transverse sectional anatomy of the adult pterygopalatine fossa and its communicating structure for providing anatomic gist for the imaging diagnosis and minimal invasive operation when PPF diseased. MATERIAL AND METHODS: Two heads of adult cadaver without macroscopic trauma (four sides of PPF) were selected to observe. Images of 0.6 mm-thick multi-planar construction (MPR) were obtained with multislice spiral CT (MSCT) based on the superior orbitomeatal line. Then, the specimens were sliced into 0.1 mm serial section on the transverse plane with the computerized milling machine, the figures were taken with digital camera and the sectional data were stored in the computer. Lastly, the thin transversal section of PPF was investigated and compared with multislice spiral CT images acquired by MPR technique to explore and discuss the anatomy of the thin transverse section of the internal structure of PPF. RESULTS: PPF was divided into four portions: infrapterygopalatine portion, pterygopalatine ganglionic one, suprapterygopalatine one and roof of PPF according to the structural characteristics of the transverse section of PPF. The infrapterygopalatine portion communicated laterally with the infratemporal fossa through the pterygomaxillary fissure and communicated downwards with the oral cavity via palatine greater and lesser canals. The pterygopalatine ganglion was shown clearly in the pterygopalatine ganglionic portion, and its dimensions were 3.91x1.92 mm at the best layer. In the suprapterygopalatine portion, the sphenopalatine foramen and artery were obviously shown on the medial wall, while the palatovaginal canal and artery, the pterygoid canal and artery, and the foramen rotundum and maxillary nerve were shown from the inferiomedial to laterosuperior on the posterior wall. The vomerovaginal canal and artery were located at the slightly superior portion of the medial side of the palatovaginal canal. CONCLUSION: Figures of thin transverse section and multislice spiral CT have highly consistency for the display of PPF. Both of them can correctly identify the micro-structure, the complex relationship of the connectivity and the spatial localization in the narrow space of PPF. It can provide reference gist for the imaging diagnosis and minimal invasive operation.

Olig2 positive cells derived from hair follicle neural crest stem cells in rats.

Motor neuron disease (MND) is a kind of common clinical nervous system disease with typical characteristic of progressive motor neurons degeneration or death. Motor neuron derived from stem cells or motor neuron progenitor cells will be a good choice to be used for treatment of the disease. In this study, we used the combination of 5 small molecular including CHIR99021 (CHIR), SB431542 (SB), DMH1 (DMH), retinoic acid (RA) and Purmorphamine (Pur) to induce hair follicles neural crest stem cells (hfNCSCs) to motor neurons progenitors (MNPs). Valproic acid (VPA) was used to make MNPs proliferation. RA and Pur were used to try to induce MNPs toward motor neurons (MNs) and CpdE was tried for MNs maturation. Nestin, ß-tubulin Ш (Tuj1), microtubule associated protein 2 (MAP2), Olig2, choline acetyltransferase (ChAT)and TUBB3 were examined at protein and mRNA levels by immunofluoresence cytochemistry, western blot and real time PCR at 6, 16 and 22 days. Our data showed cells changed into bipolar or multipolar shape forming the cell clusters like scattered rosettes. Nestin expression decreased significantly at 22 days. Compared to 6 days, percentage of Olig2 + MNPs was higher, (88.53 ± 6.67)%, and Olig2 expression at protein and gene level was lower at 22 days. Percentage of MAP2 positive cells increased to (90.62 ± 2.31) % and ChAT positive cells increased to (83.29 ± 6.62) % at 22 days. But no expression of ChAT was examined by western blot and real time PCR. It indicates that these 5 molecular can differentiate hfNCSCs into Olig2 positive cells with a unipotent differentiation toward motor neurons.

Isolation, culture and characterization of cardiac progenitor cells derived from human embryonic heart tubes.

A variety of studies have reported on the isolation and expansion of cardiac stem cells from adult hearts. However, there is little information concerning cardiac stem/progenitor cells derived from embryonic hearts/heart tubes. To provide more evidence for embryonic heart-derived stem/progenitor cells, Nkx2.5+ human cardiac progenitorcells (hCPCs) were isolated and cloned from human heart tubes. The cells stained positive for Nkx2.5 and Oct-4, and negative for alpha-smooth muscle actin (alpha-SMA), cytokeratin, factor-VIII, alpha-sarcomeric actin and c-Kit. GATA-4 expression of Nkx2.5+ hCPCs was higher than that of embryonic limb bud mesenchymal cells of the control group (p < 0.05). These cells were passaged continuously for >3 months (23 passages) and proliferated actively in vitro. After being treated with 5-azacytidine, Nkx2.5+ hCPCs underwent cardiomyogenic differentiation. Ultrastructural observation confirmed that the longitudinal section of these cardiomyogenic differentiation cells clearly revealed typical sarcomeres and intercalated discs. alpha-MHC, alpha-sarcomeric actin and GATA-4 levels were increased in Nkx2.5+ hCPCs treated with 5-azacytidine compared to untreated cells. Nkx2.5+ hCPCs exhibited positive staining and had a higher expression for alpha-SMA when cocultured with canine vascular endothelial cells. After Nkx2.5+ hCPCs were treated with endothelin-1, all cells displayed spontaneous electrical activity and spontaneous beating. Connexin-40 and -45 were stained positive in the treated cells. In conclusion, Nkx2.5+ hCPCs derived from heart tubes have been isolated and cloned in vitro. These cells are capable of long-term self-renewal and possess a potential to differentiate into cardiac muscle-like cells, cardiac pacemaking cells and smooth muscle-like cells. They could have a significant impact on cardiac regeneration medicine and developmental biology.

[Differentiation of bone marrow mesenchymal stem cells co-cultured with endothelial cells under shear stress].

Differentiation of bone marrow mesenchymal stem cells (BMSCs) co-cultured with endothelial cells (ECs) under shear stress was studied. BMSCs and ECs were co-cultured on the two sides of PET membrane, and 20 dyn/cm2 shear stress produced by parallel plate flow chamber was performed after 72 hours. Cell morphology was observed under phase-difference microscope, and the expressions of smooth muscle-alpha-actin (SM-alpha-actin), calponin and smooth muscle myosin heavy chain (SMMHC) of BMSCs were detected by fluorescence immunocytochemistry. The co-cultured BMSCs became smooth muscle-like cells gradually; after 24 hours, the BMSCs started to express SM-alpha-actin. After 48 hours, they expressed SM-alpha-actin and calponin obviously. After 72 hours, obvious expressions of SM-alpha-actin and calponin, but not of SMMHC, were detected. Further static co-culture had no effect on SM-alpha-actin, calponin and SMMH expression of BMSCs; after 24 hours, shear stress induced feeble expression of SM-alpha-actin and obvious expression of SMMHC in co-cultured BMSCs, but it had no effect on the expression of calponin. The results suggest that shear stress may potentiate the differentiation of BMSCs (co-cultured with ECs) into mature smooth muscle-like cells.

Protective Effects of Methane-Rich Saline on Rats with Lipopolysaccharide-Induced Acute Lung Injury.

Objective. The aim of this research is to evaluate the protective effects of methane-rich saline (MS) on lipopolysaccharide- (LPS-) induced acute lung injury (ALI) and investigate its potential antioxidative, anti-inflammatory, and antiapoptotic activities. Methods. LPS-induced (20 mg/kg) ALI rats were injected with MS (2 ml/kg and 20 ml/kg) before the initiation of LPS induction. Survival rate was determined until 96 h after LPS was induced. Lung injury was assayed by oxygenation index, lung permeability index (LPI), wet-to-dry weight (W/D), and histology. The cells in the bronchoalveolar lavage fluid (BALF) were counted. Oxidative stress was examined by the level of malondialdehyde (MDA) and superoxide dismutase (SOD). Inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in BALF were determined by ELISA. Lung tissue apoptosis was detected by TUNEL staining and western blotting of caspase-3. Results. It was found that methane significantly prolonged the rat survival, decreased the lung W/D ratio and the content of the inflammatory factors, and reduced the amount of caspase-3 and apoptotic index. In addition, MS increased the level of SOD and decreased the level of MDA significantly. Conclusions. MS protects the LPS-challenged ALI via antioxidative, anti-inflammatory, and antiapoptotic effect, which may prove to be a novel therapy for the clinical management of ALI.

Construction of vascularized pacemaker tissues by seeding cardiac progenitor cells and endothelial progenitor cells into Matrigel.

AIMS: Transplantation of a tissue engineered cardiac pacemaker (TECP) may represent a novel therapy for cardiac sinus node dysfunction. We previously reported that cardiac progenitor cells (CPCs) derived from embryonic heart tubes could differentiate into cardiac pacemaking cells after endothelin-1 treatment. We aimed to examine the feasibility of TECP fabricated from CPCs-derived pacemaking cells and vascularization of TECP fabricated from CPCs-derived pacemaking cells and endothelial progenitor cells (EPCs) in vitro and in vivo implantation. MAIN METHODS: TECP created using CPCs-derived pacemaking cells and vTECP created by mixing CPCs and EPCs in vitro were implanted into rat hearts. Sinus node damaged was induced by formaldehyde insult. KEY RESULTS: Spontaneous beating tissues, namely TECP, were obtained after seeding CPCs-derived pacemaking cells into Matrigel. ECG and epicardial multielectrode array (MEA) measurements confirmed implanted TECP have electrical activity. TECP implantation promoted individual survival in sinus node damage models (15/22 animals lived versus 0/17 control). vTECP fabricated by mixing the both EPCs and CPCs-derived pacemaking cells with Matrigel in equal proportions optimally formed pre-vascularization in vitro. The implantation of vTECP enhanced electrical activity in vivo, which may correlate with increased vascularization. PI3K-Akt-VEGF/VEGFR signaling was involved with vascular ingrowth in vTECP. SIGNIFICANCE: Our data supports the therapeutic potential of TECP fabricated with the CPCs-derived pacemaking cells for sinus node dysfunction. Vascularization by the addition of EPCs is an important factor to sustain viability of the TECP in vivo.

Construction of Tissue-Engineered Nerve Conduits Seeded with Neurons Derived from Hair-Follicle Neural Crest Stem Cells.

Tissue-engineered nerve conduits are widely used for the study of peripheral nerve injury repair. With regard to repairing long nerve defects, stem-cell-derived neurons are recommended as seed cells. As hair-follicle neural crest stem cells (hfNCSCs) are easily to be harvested from patients and have the potential to differentiate into neuronal cells, hfNCSCs-derived neurons are an ideal candidate choice. Acellular nerve grafts, a type of biological material scaffold, with intact collagen structure, with biocompatibility and less toxicity are obtained through removing live cells with 1 % lysolecithin, are also an ideal choice. In the present report, we describe a tissue-engineered nerve conduit seeded with rat hfNCSCs-derived neurons into the beagle acellular sciatic nerve scaffold. Our goal is to provide a novel engineered therapeutic for repairing peripheral nerve injury with long distance defects.