Combined transplantation of bone marrow mesenchymal stem cells and pedicled greater omentum promotes locomotor function and regeneration of axons after spinal cord injury in rats

BACKGROUND： According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells （BMSCs） into neural cells. OBJECTIVE： Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETFING： A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007. MATERIALS： Fifteen male inbred Wistar rats, weighing （200±20） g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS： Cell culture： We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention： The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury （SCI） only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the Tl0 level for 30 seconds to produce the SCI model. 5 μ L PBS containing 10^5 BMSCs was injected into the injured site of the spinal cord in 60 seconds via a microsyringe in the cell group after SCI. In the combined group, after SCI and BMSC transplantation, an autograft pedicled greater omentum was transplanted onto the injured site of the spinal cord and fixed with a suture. SCI model and transplantation： Control group, SCI model without treatment; cell group, transplantation of BMSCs after SCI; combined group, combined transplantation of BMSCs and pedicled greater omentum after SCI. MAIN OUTCOME MEASURES： At days 1, 7, 14, 21 and 28 PO （post operation）, the Basso, Beattie and Bresnahan （BBB） scale was used to observe and evaluate the recovery of locomotor function. At day 29 PO, after transcardial perfusion using 4% paraformaldehyde, a spinal cord segment of 1 cm around the injury was harvested. A cryostat section was performed longitudinally in the horizontal plane and sections were chosen by systematic random sampling for staining. Anti-BrdU staining and counting was performed to measure survival rate of transplanted BMSCs; anti-BrdU-nestin and BrdU-glial fibrillary acidic protein （GFAP） double staining and counting measured neural differentiation of BMSCs; and anti-NF 200 staining was used to evaluate axonal regeneration. RESULTS： All 15 rats were included in the outcome analysis, without any loss. Changes in BBB scores： Combined transplantation of BMSCs and the pedicled greater omentum produced significantly higher BBB scores at 7-28 days post-injury than in the control group （P 〈 0.05）. BBB scores in the cell group were higher than in the control group at 28 days post-injury （P 〈 0.05）. Survival rate and neural differentiation of transplanted BMSCs： Immunostaining of BrdU demonstrated that transplanted BMSCs survived in the spinal cord and migrated cranially and caudally as far as 0.5 mm from the injection site in the cell group and combined group. Some of the transplanted BMSCs expressed nestin or GFAP which revealed neural differentiation of BMSCs in the combined group and cell group. Axonal regeneration： The areas of axonal NF200 staining in the cell group and control group were lower than that of the combined group （P 〈 0.01 ）. CONCLUSION： It is effective and feasible to transplant BMSCs with the pedicled greater omentum for regeneration of spinal cord after SCI compared with transplanting BMSCs alone. This method results in better locomotor outcomes and axonal regeneration.

Umbilical cord-derived mesenchymal stem cell transplantation combined with hyperbaric oxygen treatment for repair of traumatic brain injury

Transplantation of umbilical cord-derived mesenchymal stem cells（UC-MSCs） for repair of traumatic brain injury has been used in the clinic. Hyperbaric oxygen（HBO） treatment has long been widely used as an adjunctive therapy for treating traumatic brain injury. UC-MSC transplantation combined with HBO treatment is expected to yield better therapeutic effects on traumatic brain injury. In this study, we established rat models of severe traumatic brain injury by pressurized fluid（2.5–3.0 atm impact force）. The injured rats were then administered UC-MSC transplantation via the tail vein in combination with HBO treatment. Compared with monotherapy, aquaporin 4 expression decreased in the injured rat brain, but growth-associated protein-43 expression, calaxon-like structures, and CM-Dil-positive cell number increased. Following combination therapy, however, rat cognitive and neurological function significantly improved. UC-MSC transplantation combined with HBO therapyfor repair of traumatic brain injury shows better therapeutic effects than monotherapy and significantly promotes recovery of neurological functions.

Propofol promotes spinal cord injury repair by bone marrow mesenchymal stem cell transplantation

Propofol is a neuroprotective anesthetic. Whether propofol can promote spinal cord injury repair by bone marrow mesenchymal stem cells remains poorly understood. We used rats to investigate spinal cord injury repair using bone marrow mesenchymal stem cell transplantation combined with propofol administration via the tail vein. Rat spinal cord injury was clearly alleviated; a large number of newborn non-myelinated and myelinated nerve fibers appeared in the spinal cord, the numbers of CM-Dil-labeled bone marrow mesenchymal stem cells and fluorogold-labeled nerve fibers were increased and hindlimb motor function of spinal cord-injured rats was markedly improved. These improvements were more prominent in rats subjected to bone marrow mesenchymal cell transplantation combined with propofol administration than in rats receiving monotherapy. These results indicate that propofol can enhance the therapeutic effects of bone marrow mesenchymal stem cell transplantation on spinal cord injury in rats.

Science Letters:Brain natriuretic peptide: A potential indicator of cardiomyogenesis after autologous mesenchymal stem cell transplantation?

We observed in a pilot study that there was a transient elevation of brain natriuretic peptide (BNP) level shortly after the transplantation in the patient with ischemic heart failure, which is unexplainable by the simultaneous increase of the cardiac output and six-minute walk distance. Similar findings were observed in the phase I trial. We postulated on the basis of the finding of Fukuda in vitro that this transient elevation of BNP level against the improvement of cardiac function and exercise capacity might indicate cardiomyogenesis in patients after mesenchymal stem cell transplantation. Further study is warranted to verify the hypothesis.

Editor's Choice--Umbilical cord mesenchymal stem cell transplantation for the treatment of peripheral nerve injury

Schwann cells are the predominant seed cells for cell transplantation in the treatment of peripheral nerve injury. However, the source of Schwann cells is limited and amplification remains difficult. Studies have shown that mesenchymal stem cells, an alternative cell type, can be used for transplantation treatment of peripheral nerve defects. Umbilical cord mesenchymal stem cells are pluripotent stem cells derived from newborn umbilical cord tissues.

Editor's Choice——Umbilical cord blood mesenchymal stem cell differentiation and transplantation in neural regeneration

Previous in vivo experiments have shown that human umbilical cord blood mesenchymal stem cells can promote the proliferation and differentiation of damaged celts, and help to repair damaged sites, Recent studies have reported that umbilical cord blood-derived mesenchymal stem cells can differentiate into neurons and glial cells. Recent studies have reported that the repair mechanisms underlying cord blood stern cells involve the replacement of damaged cells and mediation of the local micro-environment.

Improvement of neurological function in rats with spinal cord injury after the transplantation of neural stem cells directly differentiated from bone marrow mesenchymal stem cells

Objective To study the effect and mechanism of neurological function recovery in rats with spinal cord injury ( SCI) rats after transplantation of neural stem cells which are directly differentiated from bone marrow mesenchymal stem cells ( BMSC ) ,and to investigate the suitable engraftment time. Methods BMSC at 3rd passage were differentiated into neural stem cells ( NSC) , and immunofluorescence staining was used to

Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury

Medialization thyroplasty or injection laryngoplasty for unilateral vocal fold paralysis cannot restore mobility of the vocal fold. Recent studies have shown that transplantation of mesenchymal stem cells is effective in the repair of nerve injuries. This study investigated wheth- er adipose-derived stem celt transplantation could repair recurrent laryngeal nerve injury. Rat models of recurrent laryngeal nerve injury were established by crushing with micro forceps. Adipose-derived mesenchymal stem cells （ADSCs; 8 ×105） or differentiated Schwann-like adipose-derived mesenchymal stem cells （dADSCs; 8×105） or extracellular matrix were injected at the site of injury. At 2, 4 and 6 weeks post-surgery, a higher density of myelinated nerve fiber, thicker myelin sheath, improved vocal fold movement, better recovery of nerve conduction capacity and reduced thyroarytenoid muscle atrophy were found in ADSCs and dADSCs groups compared with the extracellu- lar matrix group. The effects were more pronounced in the ADSCs group than in the dADSCs group. These experimental results indicated that ADSCs transplantation could be an early interventional strategy to promote regeneration after recurrent laryngeal nerve injury.

Magnetic resonance evaluation of transplanted mesenchymal stem cells after myocardial infarction in swine

Objectives To trace and evaluate intracoronary transplanted mesenchymal stem cells(MSCs) labeled with superparamagnetic iron oxide(SPIO) by using magnetic resonance imaging(MRI) in a swine model of myocardial infarction (MI).Methods MSCs were transfected with a lentiviral vector carrying the gene encoding green fluorescent protein (GFP) and labeled in vitro with SPIO.Two weeks after MI, swine were randomized to intracoronary transplantation of dual -labeled MSCs(n = 10),MSCs-GFP(n = 10) and saline(n = 5).MRI examination was performed with a 1.5T clinical scanner at 24 hours,3 weeks and 8 weeks after cells transplantation. Signal intensity(SI) changes,cardiac function and MI size were measured using MRI.Correlation between MR findings and histomorphologic findings was also investigated. Results The labeling efficiency at a combination of 25μg Fe/ml SPIO and 0.8 pi/ml Lipofectamine 2000 reached 100%.SPIO labeling did not affect GFP fluorescence and dual-labeling did not affect cell proliferation(P】0.05). Multipotentiality was not affected especially for cardiomyocyte-like cells differentiation.Cardiac cell marker of a-MHC and actinin were positively expressed by immunofluorescence staining after induction.SI on T2 * WI decreased substantial- ly in the interventricular septum 24 hours after injection of MSCs.The intensity of hypo-intense signals appeared to increase throughout the later time points.Changes in SI at 24 hours,3 weeks and 8 weeks were 52.98%±10.74%,21.53%±5.40%and 6.23%±2.01%,respectively(P【0.01).DE-MRI demonstrated both dual-labeled MSCSs and MSCs-GFP could dramatically reduce the size of MI and improve cardiac function. Histological data revealed that prussian blue stain-positive cells were found mainly in the border zone which also showed green fluorescence but negative for macrophage marker(CD68).Gross pathologic examination revealed that engrafted MSCs dramatically reduce the extent of necrotic myocardium and promote the regeneration of new,contractile myocardium along the subendocardial surface of the MI. Conclusions MSCs could be efficiently and safely labeled with SPIO and GFP,and could be detected reproducibly and noninvasively in vivo using cardiac MRI.Intracoronary transplantation of dual-labeled MSCs could increase cardiac function and reduce the size of MI.

Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice

BACKGROUND：Cell therapy has been promising for various diseases.We investigated whether transplantation of human umbilical cord mesenchymal stem cells（h UCMSCs）has any therapeutic effects on D-galactosamine/lipopolysaccharide（Gal N/LPS）-induced fulminant hepatic failure in mice.METHODS：h UCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with Gal N/LPS-induced fulminant hepatic failure.After transplantation,the localization and differentiation of h UCMSCs in the injured livers were investigated by immunohistochemical and genetic analy- ses. The recovery of the injured livers was evaluated histologi- cally. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test. RESULTS： hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adip- ogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the for- mation of hUCMSCs-derived hepatocyte-like cells in vivo.CONCLUSIONS： hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUC- MSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

Effects of allogeneic mouse adipose-derived mesenchymal stem cell-microporous sheep acellular dermal matrix on healing of wound with full-thickness skin defect in mouse and the related mechanism

Objective:To explore the effects of allogeneic mouse adipose-derived mesenchymal stem cell(ADSC)-microporous sheep acellular dermal matrix(ADM)on wound healing of full-thickness skin defect in mice and the related mechanism.Methods:One Kunming mouse was sacrificed by cervical dislocation to collect adipose tissue from the inguinal region.Mouse ADSCs were isolated from the adipose tissue and cultured in vitro.Cells in the third passage were identified by cell adipogenic and osteogenic differentiation.The expressions of CD34,CD73,CD90,and CD105 were analyzed by flow cytometer.After one sheep was sacrificed with the skin of its back cut off,microporous sheep ADM was prepared by using acellular processing and freeze-thaw method.A round and full-thickness skin defect wound,with a diameter of 12 mm,was made on the back of each of 36 Kunming mice.The wounds were covered by microporous sheep ADM.The mice were divided into ADSC group and control group with 18 mice in each group according to the random number table method after surgery.A volume of 0.2 ml of DMEM/F12 culture medium containing 1×10^(6)ADSCs was injected between microporous sheep ADM and the wound of each mouse in ADSC group,while 0.2 ml of DMEM/F12 culture medium was injected between microporous sheep ADM and the wound of each mouse in control group.At post-surgery day(PSD)12 and 17,the wound healing rate in each group was calculated respectively;wound vascularization in 2 groups of mice was observed under the reverse irradiation of back light;and the granulation tissue in the wound in ADSC group was observed by means of hematoxylin-eosin staining.At PSD 7,the thickness of the granulation tissue in the wound was measured in each group of mice.At PSD 12 and 17,the immunohistochemical method was used to detect the expression of VEGF in each group of mice.The number of samples was 6 in each group at each time point in the above experiments.The data obtained were processed with t-test and factorial design ANOVA.Results:(1)After 7 days of adipogenic induction,red lipid droplets were observed in the cytoplasm with oil red O staining.After 21 days of osteogenic induction,black calcium deposition was observed in the medium stained with silver nitrate.The expression levels of CD73,CD90,CD 105 and CD34 in cells were 97.82%,99.32%,97.35%and 5.88%respectively.The cells were identified as ADSCs.(2)The wound healing rates of ADSC group at PSD 12 and 17[(78±6)%,(98±3)%]were significantly higher than those of control group at PSD 12 and 17[(60±9)%,(90±4)%,t=4.26,4.46,p<.01].(3)At PSD 7,no vessels obviously grew into the center of the wound in both groups of mice,while the granulation tissue already covered the wound in ADSC group.At PSD 12,the wound in ADSC group was more well-perfused than control group.At PSD 17,it was observed that large vessels were crossing through the whole wound in ADSC group,while large vessels were observed without crossing through the whole wound in control group.(4)In ADSC group,at PSD 7,the wound was covered with thin granulation tissue,and the granulation tissue was obviously thickened at PSD 12.At PSD 17,the granulation tissue was covered by epidermis.At PSD 7,the thickness of the granulation tissue in the wound in ADSC group[(0.62±0.05)mm]was significantly greater than that in control group[(0.31±0.04)mm,t=12.27,p<.01].(5)At PSD 12 and 17,the expression levels of VEGF in the wound in ADSC group[(80.7±2.2),(102.8±2.6)/mm^(2)]were significantly than those in control group[(59.5±2.4),(81.5±2.6)/mm^(2),t=15.95,14.14,p<.01].Conclusions:Allogeneic mouse ADSC-microporous sheep ADM can promote angiogenesis and the growth of granulation tissue in the wound with full-thickness skin defect in mice,thus accelerating wound healing.The mechanism is probably related with the increase in the expression of VEGF.

Potential advantages of a combination of Chinese Medicine and bone marrow mesenchymal stem cell transplantation for removing blood stasis and stimulating neogenesis during ischemic stroke treatment

Combined treatment of ischemic stroke with Chinese medicine and exogenous bone marrow mesenchymal stem cell(BMSC) transplantation may improve the removal of blood stasis and stimulation of neogenesis.Chinese medicines that remove blood stasis not only promote blood circulation but also calm the endopathic wind,remove heat,resolve phlegm,remove toxic substances and strengthen body resistance.The medicinal targeting effect of Chinese medicine can promote the homing of BMSCs,and the synergistic therapeutic effects of drugs can contribute to BMSC differentiation.As such,exogenous BMSC transplantation has potential advantages for neogenesis.Chinese medicines and exogenous BMSCs provide complementary functions for the removal of blood stasis and stimulation of neogenesis.Therefore,a combination of Chinese medicine and transplantation of exogenous BMSCs may be particularly suited to ischemic stroke treatment.

Vascular endothelial growth factor expressing mesen-chymal stem cells improves cardiac function in chronic myocardial infarction in pigs

Transplantation of mesenchymal stem cells （MSCs） for myocardial reconstruction has shown promise in both animal models and human phase 1 clinical studies. Vascular endothelial growth factor （VEGF） is a strong therapeutic agent for treating ischaemia by inducing angiogenesis. The feasibility of ex vivo MSCs mediated gene transfer is documented. Matsumoto and colleagues have recently reported genetically engineered MSCs carrying VEGF165 delivery for revascularization in a model of acute myocardial infarction （MI）. The promising data from our laboratory in both angiogenesis and MSCs transplantation in cunicular heart model of acute MI have prompted us to attempt the combined and simultaneous application of the two strategies.