The effect of fibroblast growth factor on distinct differentiation potential of cord blood-derived unrestricted somatic stem cells and Wharton's jelly-derived mesenchymal stem/stromal cells.

BACKGROUND AIMS: Perinatal tissues are considered an attractive source of mesenchymal stem/stromal cells (MSCs) and have unique characteristics depending on their origin. In this study, we compared the basic characteristics of unrestricted somatic stem cells isolated from cord blood (CB-USSCs) and MSCs isolated from Wharton's jelly of umbilical cords (WJ-MSCs). We also evaluated the effect of basic fibroblast growth factor (bFGF) supplementation on the growth and differentiation of these cells. METHODS: CB-USSCs and WJ-MSCs were isolated from the same individual (n = 6), and their morphology, cell surface antigens, proliferation, expression of stemness markers and adipogenic, osteogenic and chondrogenic differentiation potentials were evaluated. Their morphology, proliferation and differentiation potentials were then also compared in the presence of bFGF supplementation (10 ng/mL). RESULTS: Overall, CB-USSCs expressed DLK-1 and negative for all the HOX gene markers. The expression of cell surface antigen CD90, growth capacity and adipogenic differential potential of CB-USSCs were lower than those of WJ-MSCs. WJ-MSCs showed higher growth capacity, but the expression of CD73 and CD105 and their osteogenic differentiation potential were lower than those of CB-USSCs. The spindle morphology of both CB-USSCs and WJ-MSCs and the growth and adipogenic differentiation of CB-USSCs were improved by bFGF supplementation. However, the bFGF supplement did not have any positive effect on the tri-lineage differentiation potentials of WJ-MSCs. CONCLUSIONS: CB-USSCs and WJ-MSCs each had distinct characteristics including different growth capacity, distinguishable cell surface markers and distinct adipogenic and osteogenic potentials. bFGF supplementation improved the growth capacity and adipogenic differentiation of CB-USSCs.

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221.

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.

Oldenlandia diffusa Promotes Antiproliferative and Apoptotic Effects in a Rat Hepatocellular Carcinoma with Liver Cirrhosis.

Oldenlandia diffusa (OD) is commonly used with various diseases such as cancer, arthritis, and autoimmune disease. Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). Here, we show that the therapeutic effect of OD, which was investigated both in vitro and chemically, induced HCC model. OD significantly enhanced apoptosis and antiproliferative activity and reduced migration ability of HCC cells. In vivo, OD was treated twice a day for 28 days after confirmed HCC model through 2-[(18)F]-fluoro-2-deoxy-D-glucose ((18)F-FDG) imaging. The survival in OD treated groups was shown to have a greater therapeutic effect than the control group. 28 days after OD treatment, OD treated groups resulted in a significant reduction in tumor number, size, (18)F-FDG uptake, and serum levels such as alanine transaminase, aspartate transaminase, and alkaline phosphate compared to the control group. Also, proliferated cells in tumor sites by OD were reduced compared to the control group. Furthermore, several rats in OD treated group survived over 60 days and liver morphology of these rats showed the difference between tumor mass and normal tissue. These results suggest that OD may have antiproliferative activity, inhibition of metastasis, and apoptotic effects in chemically induced HCC model and can have the potential use for clinical application as anticancer drug of the herbal extract.

Focused low-intensity pulsed ultrasound enhances bone regeneration in rat calvarial bone defect through enhancement of cell proliferation.

A number of studies have reported the therapeutic potential of low-intensity pulsed ultrasound (LIPUS) for induction of bone repair. This study investigated whether bone regeneration might be enhanced by application of focused LIPUS to selectively stimulate fractured calvarial bone. To accomplish this, bone defects were surgically created in the middle of the skull of rats that were subsequently exposed to focused LIPUS. Bone regeneration was assessed by repeated computed tomography imaging after the operation, as well as histologic analysis with calcein, hematoxylin and eosin and proliferating cell nuclear antigen assay. At 6 wk after surgery, bone formation in the focused LIPUS-treated group improved significantly relative to the control. Interestingly, new bone tissue sprouted from focused LIPUS target points. Histologic analysis after exposure to focused LIPUS revealed that proliferating cells were significantly increased relative to the control. Taken together, these results suggest that focused LIPUS can improve re-ossification through enhancement of cell proliferation in calvarial defect sites.

Survival of hypoxic human mesenchymal stem cells is enhanced by a positive feedback loop involving miR-210 and hypoxia-inducible factor 1.

The use of mesenchymal stem cells (MSCs) has emerged as a potential new treatment for myocardial infarction. However, the poor viability of MSCs after transplantation critically limits the efficacy of this new strategy. The expression of microRNA-210 (miR-210) is induced by hypoxia and is important for cell survival under hypoxic conditions. Hypoxia increases the levels of hypoxia inducible factor-1 (HIF-1) protein and miR-210 in human MSCs (hMSCs). miR-210 positively regulates HIF-1α activity. Furthermore, miR-210 expression is also induced by hypoxia through the regulation of HIF-1α. To investigate the effect of miR-210 on hMSC survival under hypoxic conditions, survival rates along with signaling related to cell survival were evaluated in hMSCs over-expressing miR-210 or ones that lacked HIF-1α expression. Elevated miR-210 expression increased survival rates along with Akt and ERK activity in hMSCs with hypoxia. These data demonstrated that a positive feedback loop involving miR-210 and HIF-1α was important for MSC survival under hypoxic conditions.

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells.

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1µM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the ß-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.

A Pilot Study for the Neuroprotective Effect of Gongjin-dan on Transient Middle Cerebral Artery Occlusion-Induced Ischemic Rat Brain.

In this study, we investigated whether gongjin-dan improves functional recovery and has neuroprotective effects on reducing the infarct volume after transient middle cerebral artery occlusion (MCAo). Infarct volume was measured using TTC staining and glucose utilization by F-18 FDG PET. Functional improvement was evaluated with the Rota-rod, treadmill, Garcia score test, and adhesive removal test. At 14 days after MCAo, neuronal cell survival, astrocytes expansion, and apoptosis were assessed by immunohistofluorescence staining in the peri-infarct region. Also, the expression of neurotrophic factors and inflammatory cytokines such as VEGF, BDNF, Cox-2, TNF-α, IL-1ß, and IL-1α was measured in ischemic hemisphere regions. The gongjin-dan-treated group showed both reduced infarct volume and increased glucose utilization. Behavior tests demonstrated a significant improvement compared to the control. Also in the gongjin-dan treated group, NeuN-positive cells were increased and number of astrocytes, microglia, and apoptotic cells was significantly decreased compared with the control group in the ischemic peri-infarct area. Furthermore, the expression of VEGF and BDNF was increased and level of Cox-2, TNF-α, IL-1ß, and IL-1α was decreased. These results suggest that gongjin-dan may improve functional outcome through the rapid restoration of metabolism and can be considered as a potential neuroprotective agent.

Electroacupuncture enhances motor recovery performance with brain-derived neurotrophic factor expression in rats with cerebral infarction.

OBJECTIVE: Electroacupuncture (EA) is a traditional medicine in patients with post-stroke rehabilitation. Brain-derived neurotrophic factor (BDNF) is a potent growth factor involved in recovery following cerebral injury. The aim of the present study was to investigate whether EA increases BDNF levels and facilitates functional recovery. METHODS: Occlusion of the middle cerebral artery was performed in rats (N=12) followed by reperfusion. EA was applied at the GV20 (Baihui) acupoint. Motor and sensory functions were monitored on the Garcia scale for 2 weeks. Expressions of BDNF and receptor tyrosine kinase B (trkB) were determined by immunoblotting and immunohistochemistry. RESULTS: Improvement of Garcia scores, particularly in motor performance, were noted in the group with EA stimulation (p<0.05). With EA application, BDNF was elevated in the ischaemic hemisphere with increased numbers of BDNF(+) cells. Increased expression of trkB was also detected. CONCLUSION: These results indicate that EA at GV20 improves motor recovery and stimulates BDNF/trkB expression in rats with cerebral ischaemia.

Therapeutic Effects of Acupuncture through Enhancement of Functional Angiogenesis and Granulogenesis in Rat Wound Healing.

Acupuncture regulates inflammation process and growth factors by increasing blood circulation in affected areas. In this study, we examined whether acupuncture has an effect on wound healing in injured rat. Rats were assigned randomly into two groups: control group and acupuncture group. Acupuncture treatment was carried out at 8 sites around the wounded area. We analyzed the wound area, inflammatory cytokines, proliferation of resident cells, and angiogenesis and induction of extracelluar matrix remodeling. At 7 days after-wounding the wound size in acupuncture-treat group was decreased more significantly compared to control group. In addition, the protein levels of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were significantly decreased compared to the control at 2 and 7 days post-wounding. Also, we analyzed newly generated cells by performing immunostaining for PCNA and using several phenotype markers such as CD-31, α-SMA, and collagen type I. In acupuncture-treated group, PCNA-positive cell was increased and PCNA labeled CD-31-positive vessels, α-SMA- and collagen type I-positive fibroblastic cells, were increased compared to the control group at 7 days post-wounding. These results suggest that acupuncture may improve wound healing through decreasing pro-inflammatory response, increasing cell proliferation and angiogenesis, and inducing extracellular matrix remodeling.

Motor-evoked potential confirmation of functional improvement by transplanted bone marrow mesenchymal stem cell in the ischemic rat brain.

This study investigated the effect of bone marrow mesenchymal stem cells (BMSCs) on the motor pathway in the transient ischemic rat brain that were transplanted through the carotid artery, measuring motor-evoked potential (MEP) in the four limbs muscle and the atlantooccipital membrane, which was elicited after monopolar and bipolar transcortical stimulation. After monopolar stimulation, the latency of MEP was significantly prolonged, and the amplitude was less reduced in the BMSC group in comparison with the control group (P < .05). MEPs induced by bipolar stimulation in the left forelimb could be measured in 40% of the BMSC group and the I wave that was not detected in the control group was also detected in 40% of the BMSC group. Our preliminary results imply that BMSCs transplanted to the ischemic rat brain mediate effects on the functional recovery of the cerebral motor cortex and the motor pathway.

Assessment of di (2-ethylhexyl) phthalate exposure by urinary metabolites as a function of sampling time.

OBJECTIVES: In most DEHP exposure assessment studies, single spot urine sample was used. It could not compare the exposure level among studies. Therefore, we are going to represent the necessity of selection of proper sampling time of spot urine for assessing the environmental DEHP exposure, and the association urinary DEHP metabolites with steroid hormones. METHODS: We collected urine and plasma from 25 men. The urine sampling times were at the end of the shift (post-shift) and the next morning before the beginning of the shift (pre-shift). Three metabolites of DEHP {mono(2-ethylhexyl) phthalate [MEHP], mono-(2-ethyl-5-hydroxyhexyl)phthalate [MEHHP], and mono(2-ethyl-5-oxohexyl)phthalate [MEOHP]} in urine were analyzed by HPLC/MS/MS. Plasma luteinzing hormone, follicle stimulating hormone, testosterone, and 17ß-estradiol were measured at pre-shift using a ELISA kit. A log-transformed creatinine-adjusted urinary MEHP, MEHHP, and MEOHP concentration were compared between the post- and pre-shift. The Pearson's correlation was calculated to assess the relationships between log-transformed urinary MEHP concentrations in pre-shift urine and hormone levels. RESULTS: The three urinary metabolite concentrations at post-shift were significantly higher than the concentrations in the pre-shift (p<0.0001). The plasma hormones were not significantly correlated with log-transformed creatinine - adjusted DEHP metabolites. CONCLUSIONS: To assess the environmental DEHP exposure, it is necessary to select the urine sampling time according to the study object. There were no correlation between the concentration of urinary DEHP metabolites and serum hormone levels.

Changes in brain FDG metabolism induced by acupuncture in healthy volunteers.

BACKGROUND: With the aid of newly developed functional brain imaging studies, studies are ongoing to see if acupuncture first acts on specific brain areas to induce effects on the human body. PURPOSE: To examine if stimulation at specific acupuncture points changes brain glucose metabolism patterns, including the limbic system and specific brain areas related to the acupuncture effect in healthy volunteers using fluorodeoxyglucose positron emission tomography combined computed tomography (FDG-PET/CT). MATERIAL AND METHODS: Twenty healthy volunteers (11 men and 9 women; mean age 49.1+/-7.3 years, age range 35-62 years) were included. Two sets of PET/CT scans were obtained from each volunteer, with and without stimulation by acupuncture. Two classic acupoints, LR3 (liver meridian) and ST44 (stomach meridian) were tested at the same time, using disposable sterile stainless steel needles. After initial acupuncture, the needle was kept in place without further stimulation. FDG-PET/CT scan of the brain began 45 min after FDG injection (185-222 MBq). RESULTS: After stimulation of LR3 and ST44 by acupuncture, glucose metabolism in the brain was increased in the left insula (BA 13), bilateral thalami, superior frontal region of the right frontal lobe, and the inferior frontal region of left frontal lobe compared with baseline. On the other hand, glucose metabolism was decreased after acupuncture in the cingulate and parahippocampal (BA 36) regions of the left limbic lobe. CONCLUSION: Changes of glucose metabolism in specific brain areas following stimulation by acupuncture on LR3 and ST44 were documented using FDG-PET/CT.

Effect of combination therapy with sodium ozagrel and panax ginseng on transient cerebral ischemia model in rats.

Sodium ozagrel (SO) prevents platelet aggregation and vasoconstriction in the cerebral ischemia. It plays an important role in the prevention of brain damage induced by cerebral ischemia/reperfusion. Recently, many animal studies have suggested that the Panax ginseng (PG) has neuroprotective effects in the ischemic brain. In this study, we assessed the neuroprotective effects that come from a combination therapy of SO and PG in rat models with middle cerebral artery occlusion (MCAO). Animals with MCAO were assigned randomly to one of the following four groups: (1) control (Con) group, (2) SO group (3 mg/kg, intravenously), (3) PG group (200 mg/kg, oral feeding), and (4) SO + PG group. The rats were subjected to a neurobehavior test including adhesive removal test and rotarod test at 1, 3, 7, 10, and 15 days after MCAO. The cerebral ischemic volume was quantified by Metamorph imaging software after 2-3-5-triphenyltetrazolium (TTC) staining. The neuronal cell survival and astrocytes expansion were assessed by immunohistofluorescence staining. In the adhesive removal test, the rats of PG or SO + PG group showed significantly better performance than those of the control group (Con: 88.1 ± 24.8, PG: 43.6 ± 11, SO + PG: 11.8 ± 7, P < .05). Notably, the combination therapy group (SO + PG) showed better performance than the SO group alone (SO: 56 ± 12, SO + PG: 11.8 ± 7, P < .05). In TTC staining for infarct volume, cerebral ischemic areas were also significantly reduced in the PG group and SO + PG group (Con: 219 ± 32, PG: 117 ± 8, SO + PG: 99 ± 11, P < .05). Immunohistofluorescence staining results showed that the group which received SO + PG group therapy had neuron cells in the normal range. They also had a low number of astrocytes and apoptotic cells compared with the control or SO group in the peri-infarction area. During astrocytes staining, compared to the SO + PG group, the PG group showed only minor differences in the number of NeuN-positive cells and quantitative analysis of infarct volume. In conclusion, these studies showed that in MCAO rat models, the combination therapy with SO and PG may provide better neuroprotective effects such as higher neuronal cell survival and inhibition of astrocytes expansion than monotherapy with SO alone.