Beneficial effect of autologous cell transplantation on infarcted heart function: comparison between bone marrow stromal cells and heart cells

BACKGROUND: Cell transplantation may restore function after myocardial infarction, but the optimal cell type remains controversial. We compared autologous bone marrow stromal cells (BMCs) with autologous heart cells (HCs) in a porcine myocardial infarction model. METHODS: Yorkshire pigs underwent coil occlusion of the left anterior descending artery. Bone marrow stromal cells were obtained from sternal marrow and HCs were obtained by left ventricular biopsy, then cultured for 4 weeks. Four weeks after infarction, a 99mTc-sestamibi single-photon emission tomography (99mTc-MIBI SPECT) scan was performed and the pigs were then transplanted with BMCs (n = 7), HCs (n = 7), or culture medium (n = 14). Four weeks after transplantation, 99mTc-MIBI SPECT scanning was repeated to evaluate regional perfusion. Pressure-volume loops were constructed from micromanometer and conductance catheter data to evaluate left ventricular function. Hearts were evaluated histologically. RESULTS: Bone marrow stromal cells and HCs engrafted within the infarct and assumed a myocyte morphology. SPECT MIBI scans showed increased perfusion in the infarct in cell-transplanted pigs, while perfusion decreased in the control pigs. Heart cell transplantation improved preload-recruitable stroke work and HC and BMC transplantation both shifted the end-systolic pressure-volume relation to the left. Both BMCs and HCs prevented thinning and expansion of the infarct region, and some BMCs differentiated into endothelial cells in newly formed blood vessels perfusing the infarct. CONCLUSIONS: Both BMCs and HCs engrafted in the infarct region and improved let ventricular function by preventing infarct thinning. Bone marrow stromal cells demonstrated greater plasticity in vivo, and may offer a practical alternative to HC transplantation to restore function and perfusion after a myocardial infarction.     

骨髓单个核细胞移植治疗急性心肌梗死

目的探讨自体骨髓单个核细胞(BMMNCs)移植对急性心肌梗死后心脏功能及结构的影响。方法中国小型猪16头,随机分为对照组(n=6)及移植组(n=10)。建立急性心肌梗死模型后,分别注入BMMNCs/生理盐水。两组动物于术中监测血流动力学指标,术后进行影像学检查。移植组分别于2、4周后获取心脏标本,检测移植细胞标记物Hoechst33342。结果心梗4周后,移植组心梗范围较对照组显著缩小(P<0.01);血流动力学指标较对照组明显改善;局部室壁运动评分优于对照组,组间差异有统计学意义(P<0.05)。在术后2周获得的心肌标本内,可见多量Hoechst33342和MHC双阳性细胞。结论成体BMMNCs移植后,有可能抑止心室重构的发展,提高心室运动功能。     

Intravenous transplantation of mesenchymal stem cells improves cardiac performance after acute myocardial ischemia in female rats

Mesenchymal stem cells (MSCs) are potential sources of cells for tissue repairing. However, little information is available regarding the therapeutic potency of intravenously transplanted MSCs for myocardial ischemia (MI). In the present study, MSCs were isolated from bone marrow of male rats and expanded in vitro. Three hours after ligation of left anterior descending artery, the transplanted group received an infusion of MSCs through the tail vein. At the same time, a coronary-ligated control group was injected with culture medium. Homing of MSCs to the heart was assessed by expression of the Y chromosome sry gene using fluorescent in situ hybridization (FISH). At 1 week or 8 weeks after transplantation, sry positive cells were present in cardiac tissue in the transplanted group, but not in the hearts of control group. Cardiomyocytes, smooth muscle cells, and endothelial cells that bore sry gene were identified in transplanted group at 8 weeks after transplantation. Ultrastructural observation revealed that a large number of capillary and some immature myocytes were found to survive in the ischemia region. MSCs transplantation also decreased LVEDP pressure and -dP/dt, but increased LVSP and +dP/dt. The cardiac infarct size was significantly smaller in transplanted group than in control group. Our data suggest that intravenously transplanted MSCs improve cardiac performance and promote the regeneration of blood vessels and cardiomyocytes.     

Transcoronary implantation of bone marrow stromal cells ameliorates cardiac function after myocardial infarction

OBJECTIVES: Bone marrow stromal cells are capable of differentiating into cardiomyogenic cells. We tested the hypothesis that transcoronary implantation of bone marrow stromal cells may regenerate infarcted myocardium and reduce cardiac dysfunction. METHODS: Isolated bone marrow stromal cells from the isogenic donor rats were transfected with LacZ reporter gene for cell labeling. To induce cardiomyogenic differentiation, the bone marrow stromal cells were treated with 5-azacytidine before implantation. Two weeks after left coronary ligation, these cells (1 x 10(6) in 150 microL) were infused into the briefly distally occluded ascending aorta of the recipient rats (n = 15) to simulate direct coronary infusion clinically. Control animals were infused with cell-free medium (n = 14). Cardiac function was evaluated by echocardiography at preimplantation and 4 and 8 weeks postimplantation. The hearts were then immunohistochemically studied to identify phenotypic changes of implanted bone marrow stromal cells. RESULTS: Immediately after cell infusion, the bone marrow stromal cells were trapped within coronary vessels in both infarcted and noninfarcted areas. However, after 8 weeks, most of the cells were identified in the scar and periscar tissue, expressing sarcomeric myosin heavy chain and cardiomyocyte-specific protein troponin I-C. Some bone marrow stromal cells were found to be connected to the adjacent host cardiomyocytes with gap junction. Two-way repeated-measures analysis of variance revealed significant improvement in fractional shortening and end-diastolic and end-systolic diameter of the left ventricle (P =.0465,.002,.0004, respectively) in the bone marrow stromal cell group. CONCLUSIONS: Although bone marrow stromal cells had been reported to improve cardiac function when injected directly into the myocardial scar, this study demonstrated for the first time that bone marrow stromal cells can be delivered via the coronary artery, as they are capable of targeted migration and differentiation into cardiomyocytes in the scar tissue to improve cardiac function.     

The coronary delivery of marrow stromal cells for myocardial regeneration: pathophysiologic and therapeutic implications

OBJECTIVES: Bone marrow stromal cells contain "adult stem cells." We tested the hypothesis that coronary-infused bone marrow stromal cells may populate the infarcted heart and undergo milieu-dependent differentiation to regenerate functional tissues with different phenotypic features. METHODS: Isogenic adult rats were used as donors and recipients to simulate autologous transplantation clinically. Myocardial infarction was created by proximal occlusion of left coronary artery in 12 recipient rats. Isolated bone marrow stromal cells were purified, expanded, and retrovirally transduced with LacZ reporter gene for cell labeling. Stromal cells were then infused into the briefly distally clamped ascending aorta of recipient rats 2 weeks after left coronary artery ligation. The hearts were harvested immediately (n = 2) or 4 weeks (n = 10) later to trace the implanted cells and identify their phenotypes. RESULTS: Viable cells labeled with LacZ reporter gene were identified in 8 recipient hearts. Immediately after cell infusion, the labeled cells were trapped within the coronary capillaries. After 4 weeks, they could be detected individually or in clusters within myocardial scar expressing fibroblastic phenotype or outside the infarction area with morphologic features of normal cardiomyocytes. Some were incorporated into endocardium and capillary endothelium. CONCLUSIONS: Our findings suggest that bone marrow stromal cells can traffic through the coronary system to the injured heart and form cardiomyocytes or fibroblasts, depending on the specific microenvironment. Endothelial progenitor cells in the stromal cell population may be involved in the postinfarction neovascularization process. Whether therapeutic use of bone marrow stromal cells can improve the myocardial healing and remodeling process after infarction is worthy of further investigation.     

Stromal cell-derived factor and granulocyte-monocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy

OBJECTIVE: Ischemic heart failure is an increasingly prevalent global health concern with major morbidity and mortality. Currently, therapies are limited, and novel revascularization methods might have a role. This study examined enhancing endogenous myocardial revascularization by expanding bone marrow-derived endothelial progenitor cells with the marrow stimulant granulocyte-monocyte colony-stimulating factor and recruiting the endothelial progenitor cells with intramyocardial administration of the potent endothelial progenitor cell chemokine stromal cell-derived factor. METHODS: Ischemic cardiomyopathy was induced in Lewis rats (n = 40) through left anterior descending coronary artery ligation. After 3 weeks, animals were randomized into 4 groups: saline control, granulocyte-monocyte colony-stimulating factor only (GM-CSF only), stromal cell-derived factor only (SDF only), and combined stromal cell-derived factor/granulocyte-monocyte colony-stimulating factor (SDF/GM-CSF) (n = 10 each). After another 3 weeks, hearts were analyzed for endothelial progenitor cell density by endothelial progenitor cell marker colocalization immunohistochemistry, vasculogenesis by von Willebrand immunohistochemistry, ventricular geometry by hematoxylin-and-eosin microscopy, and in vivo myocardial function with an intracavitary pressure-volume conductance microcatheter. RESULTS: The saline control, GM-CSF only, and SDF only groups were equivalent. Compared with the saline control group, animals in the SDF/GM-CSF group exhibited increased endothelial progenitor cell density (21.7 +/- 3.2 vs 9.6 +/- 3.1 CD34 + /vascular endothelial growth factor receptor 2-positive cells per high-power field, P = .01). There was enhanced vascularity (44.1 +/- 5.5 versus 23.8 +/- 2.2 von Willebrand factor-positive vessels per high-power field, P = .007). SDF/GM-CSF group animals experienced less adverse ventricular remodeling, as manifested by less cavitary dilatation (9.8 +/- 0.1 mm vs 10.1 +/- 0.1 mm [control], P = .04) and increased border-zone wall thickness (1.78 +/- 0.19 vs 1.41 +/- 0.16 mm [control], P = .03). (SDF/GM-CSF group animals had improved cardiac function compared with animals in the saline control group (maximum pressure: 93.9 +/- 3.2 vs 71.7 +/- 3.1 mm Hg, P < .001; maximum dP/dt: 3513 +/- 303 vs 2602 +/- 201 mm Hg/s, P < .05; cardiac output: 21.3 +/- 2.7 vs 13.3 +/- 1.3 mL/min, P < .01; end-systolic pressure-volume relationship slope: 1.7 +/- 0.4 vs 0.5 +/- 0.2 mm Hg/microL, P < .01.) CONCLUSION: This novel revascularization strategy of bone marrow stimulation and intramyocardial delivery of the endothelial progenitor cell chemokine stromal cell-derived factor yielded significantly enhanced myocardial endothelial progenitor cell density, vasculogenesis, geometric preservation, and contractility in a model of ischemic cardiomyopathy.     

Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells

OBJECTIVE: To study the effect of bone marrow stromal cells administered intravenously to female rats subjected to traumatic brain injury. METHODS: We injected marrow stromal cells harvested from male rat bone marrow (n = 24) into the tail vein of the female rat (n = 8) 24 hours after traumatic brain injury; the rats were killed at Day 7 or 14 after treatment. The neurological function of the rats was evaluated using the rotarod test and the neurological severity score. The distribution of the male donor cells in brain, heart, lung, kidney, liver, muscle, spleen, and bone marrow of the female recipient rats was measured by identifying Y chromosome-positive cells using fluorescent in situ hybridization. RESULTS: We found that marrow stromal cells injected intravenously significantly reduced motor and neurological deficits compared with control groups by Day 15 after traumatic brain injury (P < 0.05, analysis of covariance for repeated measures). The transplanted cells preferentially engrafted into the parenchyma of the injured brain and expressed the neuronal marker NeuN and the astrocytic marker glial fibrillary acidic protein. Marrow stromal cells were also found in other organs in female rats subjected to traumatic brain injury without any obvious adverse effects. CONCLUSION: These data suggest that the intravenous administration of marrow stromal cells may be a promising therapeutic strategy that warrants further investigation for patients with traumatic brain injury.     

急性心梗后骨髓细胞心肌内移植促进心肌细胞再生的实验研究

目的 研究急性梗死心肌内移植骨髓细胞的促进心肌再生作用。方法 局部注射将骨髓细胞移植入大鼠急性心肌梗死模型的急性心肌梗死区域，1、2、4、8周后处死动物，取心肌标本行组织形态学检查和梗死面积测量。结果 梗死区心肌标本观察到带荧光的骨髓细胞，部分已分化成肌源性细胞，电镜观察到不同分化阶段的新生心肌细胞。与成熟心肌细胞以闰盘相联。骨髓细胞移植组梗死心肌面积明显小于对照组。结论 在梗死心肌内移植的骨髓细胞具有心肌再生能力，并能缩小梗死心肌面积。     

Infarct Size With Incremental Global Myocardial Ischemia Times: Cyclosporine A in Donation After Circulatory Death Rat Hearts

BackgroundWe quantified the myocardial infarct size with varying global ischemia durations and studied the benefits of Cyclosporine A (CyA) in reducing cardiac injury in ex vivo and transplanted rat hearts.MethodsInfarct size was measured after 15, 20, 25, 30, and 35 minutes of in vivo global ischemia (n = 34) and compared with control beating-heart donor (CBD) hearts (n = 10). For heart function assessment, donation after circulatory death (DCD) rat hearts (n = 20) were procured after 25 minutes of in vivo ischemia and reanimated ex vivo for 90 minutes. Half of the DCD hearts received CyA (0.5 mM) at reanimation. The CBD hearts (n = 10) served as controls. A separate group of CBD and DCD (with or without CyA treatment) hearts underwent heterotopic heart transplantation; heart function was measured at 48 hours.ResultsInfarct size was 25% with 25 minutes of ischemia and increased significantly with 30 and 35 minutes to 32% and 41%, respectively. CyA treatment decreased infarct size in DCD hearts (15% vs 25%). Heart function in the transplanted DCD hearts was significantly better with CyA treatment and was comparable to CBD hearts.ConclusionsCyA administered at reperfusion limited infarct size in DCD hearts and improved their function in transplanted hearts.     

The effect of platelet-rich plasma on the cellular response of rat bone marrow cells in vitro.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on the proliferation and the differentiation of rat bone marrow cells (RBMCs). PRP, platelet-poor plasma (PPP), and bone marrow cells were derived from the rats (hearts and tibia) and the cells were cultured with or without PRP or PPP (0 [control]), 0.2 approximately 10 microL/mL). The proliferation of RBMCs was measured on days 2 and 4, and alkaline phosphatase (ALP) staining and activity measurement were evaluated to determine the effect of PRP on the differentiation on days 4 and 8. PRP enhanced the proliferation significantly compared to the control group (P < .05). These enhancements were greater than ones induced by the addition of PPP. ALP staining appeared to show that PRP decreased the number of ALP positive cells and ALP activity significantly (P < .05). Our results demonstrate that PRP stimulates the proliferation but suppresses the differentiation of RBMCs.     

Mobilization and homing of bone marrow stromal cells in myocardial infarction.

OBJECTIVE: Marrow stromal cells (MSCs) contain multipotent cells, which may participate in the repair of damaged organs. We tested the hypothesis that MSCs are recruited to the heart upon myocardial infarction (MI), and play pathophysiological roles in the healing and adaptation process. METHODS: Donor MSCs from isogenic Lewis rats were harvested, multiplied and labeled with Lac Z reporter gene. Ten million labeled cells were injected intravenously into the recipient rats (n=30). One week later, 10 rats were killed to examine the distribution of the labeled MSCs. Other rats underwent either coronary artery ligations (n=14) or sham operations (n=6). The hearts were removed at various time points (1-8 weeks) and stained for beta-galactosidase activity. Phenotypes of labeled cells were identified with immunohistochemical stains. RESULTS: In rats killed at 1 week, labeled cells had homed into the bone marrow of the recipients, and none found in their hearts. In the coronary ligated hearts, labeled cells were seen in and near the infarct at all time points studied (14/14), but none in the sham operated hearts (6/6). There was evidence for myogenic differentiation. Some of these labeled cells showed positive staining for cardiomyocyte specific troponin I-c at 4 weeks, while others appeared in the vascular walls expressing smooth muscle alpha-actin. CONCLUSIONS: Following myocardial infarction, MSC's are signaled and recruited to the injured heart, where they undergo differentiation, and may participate in the pathophysiology of post-infarct remodeling, angiogenesis, and maturation of the scar. Therapeutic implantation of MSCs thus may further enhance such effects.     

Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy

OBJECTIVES: Cellular cardiomyoplasty with isolated skeletal myoblasts and bone marrow mononuclear cells is an encouraging therapeutic strategy for heart failure. We investigated the achievements accomplished with combined cell therapy of skeletal myoblast and bone marrow mononuclear cell transplantation to the ischemic canine myocardium. METHODS: Autologous skeletal myoblasts (1 x 10(8)) and autologous bone marrow mononuclear cells (3 x 10(6)) were injected directly into the damaged myocardium of canine hearts that had undergone 2 weeks of left anterior descending coronary artery ligation. Treatment groups were as follows: skeletal myoblasts plus bone marrow mononuclear cells (combined cell therapy, n = 4), myoblasts (n = 4), bone marrow mononuclear cells (n = 4), and medium only (n = 4). In similarly designed supporting experiments, angiogenic factor expression was evaluated by enzyme-linked immunosorbent assay after cell transplantation in rat hearts that had undergone left anterior descending coronary artery ligation. RESULTS: Four weeks after cell implantation, echocardiography demonstrated better cardiac performance with reduced left ventricular dilation and significantly improved ejection fraction in the combined cell therapy group compared with that seen in the other groups (pretreatment, 37.7% +/- 1.1%, vs combined cell therapy, 55.4% +/- 8.6%; myoblasts, 47.4% +/- 7.4%; bone marrow mononuclear cells, 44.4% +/- 6.7%; medium only [control], 34.4% +/- 5.4%; P < .05). A significantly high number of neovessels were observed in the group receiving combined cell therapy only (combined cell therapy, 45.5 +/- 12 x 10(2)/mm2; myoblasts, 26.5 +/- 8 x 10(2)/mm2; bone marrow mononuclear cells, 30.7 +/- 15 x 10(2)/mm2; medium only [control], 7.1 +/- 1 x 10(2)/mm2; P < .05). Immunostained sections expressed the skeletal specific marker myosin heavy chain, although they did not express the cardiac specific marker troponin T. Results of enzyme-linked immunosorbent assay showed the highest expression of vascular endothelial growth factor (combined cell therapy, 2.9 +/- 0.7 ng/g tissue; myoblasts, 0.24 +/- 0.7 ng/g tissue; bone marrow mononuclear cells, 1.9 +/- 0.2 ng/g tissue; medium only [control], 0.19 +/- 0.004 ng/g tissue; P < .05) and hepatocyte growth factor in the combined cell therapy hearts. CONCLUSIONS: Combined autologous cellular therapy induced both myogenesis and angiogenesis with enhancement of cardiac performance and reduction of cardiac remodeling, suggesting a capable strategy for treating severe ischemic cardiomyopathy clinically.     

Optimal temporal delivery of bone marrow mesenchymal stem cells in rats with myocardial infarction.

OBJECTIVE: This study was designed to determine the optimal time point for bone marrow mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI). METHODS: MSCs from donor rats were labeled with DAPI before transplantation. The animals underwent MI by ligation of left anterior descending coronary artery, and received intramyocardial injection of MSCs at 1h, 1 week and 2 weeks after MI, respectively. Sham-operated and MI control groups received equal volume phosphate buffered saline. Cardiac function, histological analysis and immunoblot for troponin T were performed 4 weeks after cell transplantation. RESULTS: MSC transplantation attenuated left ventricular chamber dilation, reduced infarct size, and improved cardiac function in rats after MI. The greatest benefit was achieved in rats that received cells 1 week after MI, engrafted MSC survival, angiogenesis and functional cardiomyocytes in the injured hearts were more abundant in these rats than that in other transplantation groups. CONCLUSIONS: The optimal functional benefit of MSC transplantation was observed in 1-week transplantation group. At this time point scar formation has not occurred and the inflammation is reduced, which should facilitate integration of transplanted cells and functional recovery.     

Matrix metalloproteinase inhibition modifies left ventricular remodeling after myocardial infarction in pigs

BACKGROUND: Global and regional shape changes that occur within the left ventricular wall after myocardial infarction have been termed infarct expansion. A potential mechanism for this postinfarction remodeling is activation of the matrix metalloproteinases. Accordingly, the present study examined the effects of matrix metalloproteinase inhibition on left ventricular global geometry after myocardial infarction in pigs. METHODS: Myocardial infarction was created in pigs by means of occlusion of the first and second obtuse marginal branches of the circumflex coronary artery, resulting in a uniform left ventricular free wall infarct size of 21% +/- 2%. At 5 days after infarction, the pigs were randomized to undergo broad-spectrum matrix metalloproteinase inhibition (n = 9; PD166793, 20 mg. kg(-1). d(-1) by mouth) or myocardial infarction alone (n = 8). Ten pigs served as noninfarction control animals. Left ventricular end-diastolic area, determined by means of echocardiography, was measured 8 weeks after infarction. RESULTS: Left ventricular end-diastolic area increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and myocardial infarction only groups compared to reference control animals (3.7 +/- 0.2 cm(2)), but was reduced with broad-spectrum matrix metalloproteinase inhibition compared to myocardial infarction alone (4.5 +/- 0.2 vs 4.9 +/- 0.2 cm(2), respectively; P <.05). Regional radial stress within the infarct region increased in both infarction groups when compared to values obtained from reference control animals (599 +/- 152 g/cm(2)), but was attenuated in the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition group compared to the myocardial infarction alone group (663 +/- 108 vs 1242 +/- 251 g/cm(2), respectively; P <.05). Similarly, regional myocardial stiffness increased in both the myocardial infarction plus broad-spectrum matrix metalloproteinase inhibition and the myocardial infarction only groups compared with that observed in reference control animals (14 +/- 1 rkm, P <.05) but was lower with broad-spectrum matrix metalloproteinase inhibition than with myocardial infarction alone (42 +/- 6 vs 68 +/- 10 rkm, respectively; P <.05). CONCLUSIONS: Matrix metalloproteinase inhibition reduced postinfarction left ventricular dilation, reduced regional myocardial wall stress, and modified myocardial material properties. These unique findings suggest that increased myocardial matrix metalloproteinase activation after infarction contributes directly to the left ventricular remodeling process.     

A novel sub-population of bone marrow-derived myocardial stem cells: potential autologous cell therapy in myocardial infarction.

BACKGROUND: Several studies have identified beta2-microglobulin-negative (beta2M(-)) cells as a potential stem cell fraction in the bone marrow of rats and humans. We studied the ability of bone marrow-derived beta2M(-) cells to differentiate into cardiomyocytes and reconstitute the myocardium in a model of myocardial infarction. METHODS: beta2M(-) cells were purified from bone marrow of Lewis rats using a magnetic activated cell-sorting technique. beta2M(-) cells, 2.5 x 10(6) cells in 100 microl of phosphate-buffered saline (PBS), were transplanted 7 days after infarction into a transmural myocardial scar induced by cryoinjury in Lewis rats (n = 9). Control Group 1(n = 10) received a 100-microl injection of PBS, and Control Group 2 (n = 15) received no injection. The beta2M(-) cells were labeled before transplantation, using the membrane fluorescent intercalated dye, PKH26. Repopulation was examined at 6 and 8 weeks after transplantation. Differentiation of beta2M(-) cells into cardiac myocytes was determined by the colocalization of troponin and PKH26 to the same cell, utilizing immunohistochemistry, ultraviolet photomicroscopy and fluorescence microscopy on 6-microm serial sections. Area of engraftment within the scar was calculated by planimetry. RESULTS: The treatment group had multiple islands of de novo-formed myocardium within the fibrous matrix of the transmural scar (mean area 35 +/- 4.2% of scar area at 6 and 8 weeks). These cells colocalized cardiac-specific troponin and PKH26. Using these techniques, no myocardial islands were seen in the control groups. Before transplantation, beta2M(-) cells were troponin-negative. CONCLUSIONS: This study demonstrates that beta2M(-) cells represent a novel sub-population of bone marrow-derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de novo formation of cardiac myocytes. The functional significance of this observation is being studied.     

探讨脑缺血大鼠骨髓基质细胞移植后减少神经细胞凋亡的作用

目的探讨大鼠局灶性脑缺血后骨髓基质细胞移植对神经细胞凋亡及相关蛋白表达的影响。方法取大鼠股骨髓基质细胞,分化培养为骨髓基质细胞源神经干细胞。将32只健康SD大鼠随机平均分为4组。A组不建立局灶性脑缺血模型,不做任何移植,其余步骤同其他组。B、C、D组均建立局灶性脑缺血再灌注模型。B组将1mlPBS经尾静脉注入大鼠体内;C组将1ml3×106个骨髓基质细胞经尾静脉注入大鼠体内;D组将1ml3×106个骨髓基质细胞源神经干细胞经尾静脉注入大鼠体内。分别在移植后7d和14d行脑灌注固定取材,应用免疫组织化学染色检测脑组织中Bcl2、Bax蛋白表达阳性的细胞,原位末端脱氧核糖核酸转移酶标记法(TUNEL)检测神经细胞凋亡数。结果C组和D组各时点的神经细胞凋亡数均少于B组(P<0.01),C组和D组移植14d时,神经细胞凋亡数显著少于移植7d时(P<0.01),移植14d时D组神经细胞凋亡数显著少于C组(P<0.05)。C组和D组Bcl2表达阳性的细胞数显著高于B组(P<0.01)。C组和D组Bax蛋白表达阳性的细胞数明显低于B组(P<0.01)。结论骨髓基质细胞源神经干细胞可能通过上调Bcl2蛋白,下调Bax蛋白的表达,减少神经细胞凋亡,从而对脑缺血再灌注损伤后的神经细胞起保护作用。     

骨髓间充质细胞和单个核细胞移植对糖尿病小鼠胰岛功能的影响

目的 比较骨髓间充质细胞移植和单个核细胞移植对糖尿病小鼠胰岛功能影响的差异.方法 建立糖尿病小鼠模型并分成3组:对照组(n=14)通过尾静脉注射磷酸盐缓冲液(PBS);单个核细胞组(n=14)通过尾静脉移植骨髓单个核细胞;间充质细胞组(n=14)通过尾静脉移植骨髓间充质细胞.观察移植后1周(n=6)和移植后6周(n=8),各组小鼠血糖的变化、胰岛数量、胰腺组织形态学特征及相关标记物的表达.结果 移植后1周,间充质细胞组小鼠血糖出现显著下降(16.6±1.6)mmol/L,与对照组(26.3±0.5)mmol/L和单个核细胞移植组(24.4±1.3)mmol/L比较差异有统计学意义(P＜0.05),并一直维持到移植后第6周,血糖下降到(16.5±1.5)mmol/L,与对照组(27.7±0.1)mmol/L比较差异有统计学意义(P＜0.05);移植后1周,间充质细胞组小鼠胰岛数目(21.2±1. 1)和胰岛β细胞数目(415.9±25.4)显著增加,与对照组(11.2±1.3)/(65.9±7.1)和单个核细胞组(12.2±1.3)/(64.1±6.5)比较差异均有统计学意义(P均＜0.05).单个核细胞组和间充质细胞组小鼠胰岛中均发现BrdU(+)Insulin(+)细胞和BrdU(+)Insulin(-)细胞.结论 骨髓间充质细胞移植改善糖尿病小鼠胰岛功能的效果优于骨髓单个核细胞移植.移植后胰岛的再生既来源于胰岛β细胞的增殖,也可能来源于胰岛干细胞的分化.

Abstract：

Objective To compare the different effects of bone marrow mononuclear cells vs mesenchymal cells transplantation on islets function of diabetic mice. Methods Mouse diabetic models were created by multiply peritoneal injection of low-dose streptozotocin (STZ) and divided into three groups:control group ( n = 14) , bone marrow mononuclear cells group ( n = 14) , and bone marrow mesenchymal cells group (n = 14). Blood glucose was measured weekly after transplantation by glucometer. Histochem istry and immunofluorescence were performed to characterize pancreatic histology, morphology and markers expressed in receipt pancreas. Results Compared with control group and bone marrow mesenchymal cells group, blood glucose levels in bone marrow mesenchymal cells group were significantly reduced at first week after transplantation[( 16. 6 ± 1.6 ) vs ( 26. 3 ± 0. 5 ) / ( 24. 4 ± 1.3 ) mmol/L, P ＜ 0. 05]and sustained to reduce at 6th week after transplantation[( 16. 5 ± 1.5 ) vs ( 27.7 ± 0. 1 ) mmol/L in control group,P＜0. 05]. One week after transplantation, the islets number in bone marrow mesenchymal cells group was larger than in control group ( 21.2 ± 1. 1vs 11.2 ± 1.3, P ＜ 0. 05 ) and bone marrow mononuclear cells group ( 21.2 ± 1. 1vs 12. 2 ± 1.3 ,P ＜0. 05 ). One weeks after transplantation, the beta cell number in bone marrow mesenchymal cells group was larger than in control group (415.9 ± 25.4 vs 65.9 ±7. 1,P＜0.05) and bone marrow mononuclear cells group (415.9 ±25.4 vs 64. 1 ±6.5,P＜0.05). In bone marrow mononuclear cells and bone marrow mesenchymal cells groups, there were several BrdU ( + )Insulin( - ) cells and BrdU( + )Insulin( - ) cells in the islets. Conclusion The effect of bone marrow mesenchymal cells transplantation to improve diabetic islet function is more satisfactory than bone marrow mononuclear cells transplantation. Bone marrow mesenchymal cells transplantation can initiate pancreatic islets β cells regeneration by both proliferation of β cells and differentiation of pancreatic stem cells.     

Effect of simvastatin on bone morphogenetic protein-2 expression and alkaline phosphatase activity of bone marrow stromal cell]

OBJECTIVE: To study the effect of simvastatin on the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphates (ALP) activity in the primary cultured bone marrow stromal cells, and to elucidate the mechanism of the anabolic osteogenetic effect of simvastatin. METHODS: Bone marrow stromal cells in femur and tibia of adult mouse were cultured in vitro. after treated with different concentrations of simvastatin (0, 0.1, 0.2, 0.5 and 1.0 mumol/L) or recombinant human BMP-2 for 72 hours, ALP activity of bone marrow stromal cells was determined. BMP-2 expression of bone marrow stromal cells was analyzed by using immunocytochemistry and Western blotting. RESULTS: After treated with simvastatin for 72 hours, BMP-2 expression increased, while little BMP-2 expression could be observed in the control group. ALP activity also increased in a dose-dependent manner; t-test showed that ALP activity in the group which concentrations of simvastatin were 0.5 mumol/L (t = 2.35, P = 0.041), 1.0 mumol/L (t = 2.348, P = 0.041) had significant difference when compared with control group. CONCLUSION: Simvastatin lead to high expression of BMP-2 in bone marrow stromal cells, via the increased auto- or para-crine of BMP-2, and ALP activity increased. These may be parts of the mechanism on the anabolic osteogenetic effect of simvastatin.     

Myocardial repair achieved by the intramyocardial implantation of adult cardiomyocytes in combination with bone marrow cells

Various cytokines produced by bone marrow cells can protect adult cardiomyocytes against apoptosis. Thus, we investigated the feasibility of implanting adult cardiomyocytes in combination with bone marrow cells for myocardial repair. Ventricular cardiomyocytes were isolated from adult rats and cocultured with bone marrow cells. Using a rat model of doxorubicin-induced cardiomyopathy, we injected 6 x 10(5) adult cardiomyocytes, 3 x 10(7) bone marrow cells, or both into damaged hearts, for myocardial repair. Coculture of the cardiomyocytes with the bone marrow cells enhanced the expression of integrin-beta1D and focal adhesion kinase in cardiomyocytes, resulting in increased survival and decreased apoptosis of the cardiomyocytes after 7 days of culture. Compared with the baseline levels, cardiac function was preserved by the implantation of bone marrow cells alone and by the implantation of cardiomyocytes in combination with bone marrow cells, but it was decreased significantly 28 days after the implantation of cardiomyocytes alone. Furthermore, apoptosis of the host cardiomyocytes was decreased significantly after the implantation of bone marrow cells alone, or in combination with cardiomyocytes, compared with that after the implantation of cardiomyocytes alone (p < 0.01). Interestingly, the implantation of adult cardiomyocytes in combination with bone marrow cells resulted in a dramatic increase in the survival of donor cardiomyocytes, and induced the myogenic differentiation of donor bone marrow stem cells. Our findings indicate that cardiomyocytes and bone marrow cells can assist and compliment each other; thus, the implantation of adult cardiomyocytes in combination with bone marrow cells shows promise as a feasible new strategy for myocardial repair.     

Human cord blood cells and myocardial infarction: effect of dose and route of administration on infarct size

There is no consensus regarding the optimal dose of stem cells or the optimal route of administration for the treatment of acute myocardial infarction. Bone marrow cells, containing hematopoietic and mesenchymal stem cells, in doses of 0.5 x 10(6) to >30 x 10(6) have been directly injected into the myocardium or into coronary arteries or infused intravenously in subjects with myocardial infarctions to reduce infarct size and improve heart function. Therefore, we determined the specific effects of different doses of human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic and mesenchymal stem cells, on infarct size. In order to determine the optimal technique for stem cell administration, HUCBC were injected directly into the myocardium (IM), or into the LV cavity with the ascending aorta transiently clamped to facilitate coronary artery perfusion (IA), or injected intravenously (IV) in rats 1-2 h after the left anterior coronary artery was permanently ligated. Immune suppressive therapy was not given to any rat. One month later, the infarct size in control rat hearts treated with only Isolyte averaged 23.7 +/- 1.7% of the LV muscle area. Intramyocardial injection of HUCBC reduced the infarct size by 71% with 0.5 x 10(6) HUCBC and by 93% with 4 x 10(6) HUCBC in comparison with the controls (p < 0.001). Intracoronary injection reduced the infarction size by 47% with 0.5 x 10(6) HUCBC and by 80% with 4 x 10(6) HUCBC (p < 0.001), and IV HUCBC reduced infarct size by 51% with 0.5 x 10(6) and by 75-77% with 16-32 million HUCBC (p < 0.001) in comparison with control hearts. With 4 x 10(6) HUCBC, infarction size was 65% smaller with IM HUCBC than with IA HUCBC and 78% smaller than with IV HUCBC (p < 0.05). Nevertheless, IM, IA, and IV HUCBC all produced significant reductions in infarct size in comparison with Isolyte-treated infarcted hearts without requirements for host immune suppression. The present experiments demonstrate that the optimal dose of HUCBC for reduction of infarct size in the rat is 4 x 10(6) IM, 4 x 10(6) IA, and 16 x 10(6) IV, and that the IM injection of HUCBC is the most effective technique for reduction in infarct size.