A novel three-dimensional adipose-derived stem cell cluster for vascular regeneration in ischemic tissue

Background aimsStem cells are one of the most powerful tools in regeneration medicine. However, many limitations remain regarding the use of adult stem cells in clinical applications, including poor cell survival and low treatment efficiency. We describe an innovative three-dimensional cell mass (3DCM) culture that is based on cell adhesion (basic fibroblast growth factor–immobilized substrate) and assess the therapeutic potential of 3DCMs composed of human adipose tissue–derived stromal cells (hASCs).MethodsFor formation of a 3DCM, hASCs were cultured on a substrate with immobilized fibroblast growth factor-2. The angiogenic potential of 3DCMs was determined by immunostaining, fluorescence-activated cell sorting and protein analysis. To evaluate the vasculature ability and improved treatment efficacy of 3DCMs, the 3DCMs were intramuscularly injected into the ischemic limbs of mice.ResultsThe 3DCMs released various angiogenic factors (eg, vascular endothelial growth factor and interleukin-8) and differentiated into vascular cells within 3 days in normal medium. Blood vessel and tissue regeneration was clearly observed through visual inspection in the 3DCM-injected group. hASC injection slowed limb necrosis after treatment, but 50% of the mice ultimately had limb loss within 28 days. Most mice receiving 3DCMs had limb salvage (89%) or mild limb necrosis (11%).Conclusions3DCM culture promotes the efficient vascular differentiation of stem cells, and 3DCM transplantation results in the direct vascular regeneration of the injected cells and an improved therapeutic efficacy.     

Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model

Multipotent germline stem (mGS) cells have been established from neonatal mouse testes. We previously reported that undifferentiated mGS cells are phenotypically similar to embryonic stem cells and that fetal liver kinase 1 (Flk1)⁺ mGS cells have a similar potential to differentiate into cardiomyocytes and endothelial cells compared with Flk1⁺ embryonic stem cells. Here, we transplanted these Flk1⁺ mGS cells into an ischemic heart failure mouse model to evaluate the improvement in cardiac function. Significant increase in left ventricular wall thickness of the infarct area, left ventricular ejection fraction and left ventricular maximum systolic velocity was observed 4 weeks after when sorted Flk1⁺ mGS cells were transplanted directly into the hearts of the acute ischemic model mice. Although the number of cardiomyocytes derived from Flk1⁺ mGS cells were too small to account for the improvement in cardiac function but angiogenesis around ischemic area was enhanced in the Flk1⁺ mGS cells transplanted group than the control group and senescence was also remarkably diminished in the early phase of ischemia according to β-galactosidase staining assay. In conclusion, Flk1⁺ mGS cell transplantation can improve the cardiac function of ischemic hearts by promoting angiogenesis and by delaying host cell death via senescence.     

Enhancement of cell-based therapeutic angiogenesis using a novel type of injectable scaffolds of hydroxyapatite-polymer nanocomposite microspheres

Background

Clinical trials demonstrate the effectiveness of cell-based therapeutic angiogenesis in patients with severe ischemic diseases; however, their success remains limited. Maintaining transplanted cells in place are expected to augment the cell-based therapeutic angiogenesis. We have reported that nano-hydroxyapatite (HAp) coating on medical devices shows marked cell adhesiveness. Using this nanotechnology, HAp-coated poly(l-lactic acid) (PLLA) microspheres, named nano-scaffold (NS), were generated as a non-biological, biodegradable and injectable cell scaffold. We investigate the effectiveness of NS on cell-based therapeutic angiogenesis.

Methods and Results

Bone marrow mononuclear cells (BMNC) and NS or control PLLA microspheres (LA) were intramuscularly co-implanted into mice ischemic hindlimbs. When BMNC derived from enhanced green fluorescent protein (EGFP)-transgenic mice were injected into ischemic muscle, the muscle GFP level in NS+BMNC group was approximate fivefold higher than that in BMNC or LA+BMNC groups seven days after operation. Kaplan-Meier analysis demonstrated that NS+BMNC markedly prevented hindlimb necrosis (P<0.05 vs. BMNC or LA+BMNC). NS+BMNC revealed much higher induction of angiogenesis in ischemic tissues and collateral blood flow confirmed by three-dimensional computed tomography angiography than those of BMNC or LA+BMNC groups. NS-enhanced therapeutic angiogenesis and arteriogenesis showed good correlations with increased intramuscular levels of vascular endothelial growth factor and fibroblast growth factor-2. NS co-implantation also prevented apoptotic cell death of transplanted cells, resulting in prolonged cell retention.

Conclusion

A novel and feasible injectable cell scaffold potentiates cell-based therapeutic angiogenesis, which could be extremely useful for the treatment of severe ischemic disorders.     

基于三维超声心动图对比分析扩张型心肌病与二尖瓣关闭不全左室构型和收缩功能的研究

摘要 目的：基于三维超声心动图对比分析扩张型心肌病（DCM）与二尖瓣关闭不全（MI）左室构型和收缩功能的研究。方法：收集我院2018年1月至2021年7月就诊患有左心室（LV）扩张的患者100例,其中DCM患者57例,MI患者43例。LV大小大致相仿,DCM组（43±5）mm/m²,MI组（42±5）mm/m²。另选取同时期50例健康受试者作为对照组。所有患者均进行常规超声心动图及三维超声心动图检查,测量指标主要包括左室大小（LVID）、左室后壁厚度（PWT）、左室舒张末期内径（LVEDD）、左室舒张末期室间间隔厚度（IVS）、左室舒张末期容积（LVEDV）、收缩末期容积（LVESV）、相对室壁厚度（RWT）、LV质量指数（LVMI）、三维左室射血分数（3D-LVEF）、三维舒张末期血流速度（3D-EDV）、二维或三维超声心动图球形指数（2D-SI/3D-SI）。结果：DCM组和MI组LVEDD均大于对照组,差异有统计学意义（P＜0.05）。DCM组比MI组患者心功能分级III/IV和心力衰竭的发生率更高,差异有统计学意义（P＜0.05）。DCM组和MI组患者的LVEDD、LVEDD指数、LVEDV、LVEDV指数、3D-EDV、3D-EDV指数均高于对照组,差异有统计学意义（P＜0.05）;但DCM组和MI组对比差异无统计学意义（P＞0.05）。DCM组和MI组患者的LV长度、LV长度指数、LVMI均高于对照组,差异有统计学意义（P＜0.05）;且MI组高于DCM组,差异有统计学意义（P＜0.05）。DCM组和MI组患者的LVESV、LVESV指数、2D-SI、3D-SI均高于对照组,差异有统计学意义（P＜0.05）;且DCM组高于MI组,差异有统计学意义（P＜0.05）。DCM组3D-LVEF、RWT均低于对照组和MI组,差异有统计学意义（P＜0.05）。ROC分析显示,3D-SI在评估左室扩大患者的左室重构方面优于其他变量,3D-SI的ROC曲线下面积为0.875,95%CI为0.816-0.920,3D-SI>0.62对于DCM和MI区分左室构型的特异性（81.66%）和敏感性（92.09%）较高。DCM和MI患者的3D-LVEF和3D-SI均呈线性负相关（r=-0.719,P=0.000;r=-0.682,P=0.000）。DCM和MI患者3D-SI检测心力衰竭的ROC曲线下面积均大于3D-LVEF的ROC曲线下面积,差异有统计学意义（P=0.000）。结论：与MI患者相比,尽管LV大小大致相仿,但DCM患者的左室几何形状更接近球形,且收缩功能更差。收缩功能与3D-SI显著相关,3D-SI较好地描述了左室重构,可能是LV扩张患者心力衰竭的较强指标。     

The Relative Contribution of Paracine Effect versus Direct Differentiation on Adipose-Derived Stem Cell Transplantation Mediated Cardiac Repair

Background

Recent studies have demonstrated that transplantation of adipose-derived stem cell (ADSC) can improve cardiac function in animal models of myocardial infarction (MI). However, the mechanisms underlying the beneficial effect are not fully understood. In this study, we characterized the paracrine effect of transplanted ADSC and investigated its relative importance versus direct differentiation in ADSC transplantation mediated cardiac repair.

Methodology/Principal Findings

MI was experimentally induced in mice by ligation of the left anterior descending coronary artery. Either human ADSC, conditioned medium (CM) collected from the same amount of ADSC or control medium was injected into the peri-infarct region immediately after MI. Compared with the control group, both ADSC and ADSC-CM significantly reduced myocardial infarct size and improved cardiac function. The therapeutic efficacy of ADSC was moderately superior to ADSC-CM. ADSC-CM significantly reduced cardiomyocyte apoptosis in the infarct border zone, to a similar degree with ADSC treatment. ADSC enhanced angiogenesis in the infarct border zone, but to a stronger degree than that seen in the ADSC-CM treatment. ADSC was able to differentiate to endothelial cell and smooth muscle cell in post-MI heart; these ADSC-derived vascular cells amount to about 9% of the enhanced angiogenesis. No cardiomyocyte differentiated from ADSC was found.

Conclusions

ADSC-CM is sufficient to improve cardiac function of infarcted hearts. The therapeutic function of ADSC transplantation is mainly induced by paracrine-mediated cardioprotection and angiogenesis, while ADSC differentiation contributes a minor benefit by being involved in angiogenesis. Highlights 1 ADSC-CM is sufficient to exert a therapeutic potential. 2. ADSC was able to differentiate to vascular cells but not cardiomyocyte. 3. ADSC derived vascular cells amount to about 9% of the enhanced angiogenesis. 4. Paracrine effect is the major mechanism of ADSC therapeutic function for MI.     

顺铂联合VEGF抗体对食管癌移植瘤小鼠免疫调节以及癌细胞增殖和肺转移的影响

摘要 目的：研究顺铂联合血管内皮生长因子（vascular endothelial growth factor,VEGF）治疗对食管癌移植瘤小鼠免疫功能、癌细胞增殖以及肺转移的影响。方法：30只BALB/c小鼠通过皮下注射食管癌移植瘤模型。一周后,30只食管癌移植瘤模型小鼠被随机均分为3组,即模型组、顺铂组和联合组。模型组不进行治疗,顺铂组腹腔注顺铂治疗,联合组腹腔注射顺铂联合尾静脉注射VEGF抗体进行治疗,共治疗7周。比较各组小鼠体重,食管癌移植瘤体积和重量,卵巢癌细胞肺组织转移结节数、癌细胞转移面积和转移病灶总数,以及食管癌移植瘤外周血CD4⁺、CD8⁺以及CD4⁺/CD8⁺ T淋巴细胞比例。结果：（1）顺博组和联合组小鼠体重均显著高于对照组,而联合组小鼠体重显著高于顺铂组（P<0.05）;（2）顺铂组和联合组小鼠CD4+和CD8+细胞比例均显著低于对照组（P<0.05）,而CD4⁺/CD8⁺却显著高于对照组（P<0.05）;（3）联合组小鼠CD4⁺和CD8⁺细胞比例均显著高于对照组（P<0.05）,而CD4⁺/CD8⁺却显著低于顺铂组（P<0.05）;（4）顺铂组和联合组小鼠食管癌肿瘤组织体积和重量,肺转移结节数、转移面积和转移病灶数均显著低于对照组（P<0.05）,而联合组小鼠显著低于顺铂组（P<0.05）。结论：VEGF抗体可以显著增强顺铂在体内对食管癌的抗癌特性,并有助于增强食管癌移植瘤小鼠免疫功能、抑制癌细胞体内增殖和肺部转移。     

Ultrastructural Features of Ischemic Tissue following Application of a Bio-Membrane Based Progenitor Cardiomyocyte Patch for Myocardial Infarction Repair

Background and Objective

Implantation of cell-sheets into damaged regions of the heart after myocardial infarction (MI) has been shown to improve heart function. However, the tissue morphology following application of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) has not been studied in detail at the level afforded by electron microscopy. We hypothesized that increasing the number of CM derived from iPSC would increase the effectiveness of cell-sheets used to treat ischemic cardiomyopathy. We report here on the ultrastructural features after application of a bio-membrane ‘cell patch’.

Methods

iPSC-derived progenitor cells were transduced using lentivirus vectors with or without NCX1 promoter. iPSC-CM sheets were transplanted over the transmural MI region in a mouse model of regional ischemic cardiomyopathy. Mice were divided into four groups, 1) Sham; 2) MI; 3) MI + iPSC without NCX1 treated cells (MI + iPSC^Null) and 4) MI + iPSC receiving NCX1 promoter treated cells (MI + iPSC^NCX1). Echocardiography was performed 4 weeks after cell patch application, followed by histological and transmission electron microscopy (TEM) analysis.

Results

Large numbers of transplanted CM were observed with significant improvements in left ventricular performance and remodeling in group 4 as compared with group 3. No teratoma formation was detected in any of the treatment groups.

Conclusion

Manipulation of iPSC yields large numbers of iPSC-CM and favorable morphological and ultrastructural tissue changes. These changes have the potential to enhance current methods used for restoration of cardiac function after MI.     

MicroRNA-377 Regulates Mesenchymal Stem Cell-Induced Angiogenesis in Ischemic Hearts by Targeting VEGF

MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSC^miR-377) or knockdown microRNA-377 (MSC^Anti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSC^Null). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSC^Anti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSC^miR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSC^Null-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.     

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31⁺, KDR⁺, and CD34⁺. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.     

Catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats by targeting HIF-1α/VEGF

BackgroundThe initial factor in the occurrence, development, and prognosis of cerebral ischemia is vascular dysfunction in the brain, and vascular remodeling of the brain is the key therapeutic target and strategy for ischemic tissue repair. Catalpol is the main active component of the radix of Rehmannia glutinosa Libosch, and it exhibits potential pleiotropic protective effects in many brain-related diseases, including stroke.PurposeThe present study was designed to investigate whether catalpol protects vascular structure and promotes angiogenesis in cerebral ischemic rats and to identify its possible mechanisms in vivo and in vitro.Study designCerebral ischemic rats and oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to study the therapeutic potential of catalpol in vivo and in vitro.MethodsFirst, neurological deficits, histopathological morphology, infarct volume, vascular morphology, vessel density, and angiogenesis in focal cerebral ischemic rats were observed to test the potential treatment effects of catalpol. Then, oxygen-glucose deprivation-exposed brain microvascular endothelial cells were used to mimic the pathological changes in vessels during ischemia to study the effects and possible mechanisms of catalpol in protecting vascular structure and promoting angiogenesis.ResultsThe in vivo results showed that catalpol reduced neurological deficit scores and infarct volume, protected vascular structure, and promoted angiogenesis in cerebral ischemic rats. The in vitro results showed that catalpol improved oxygen-glucose deprivation-induced damage and promoted proliferation, migration, and in vitro tube formation of brain microvascular endothelial cells. The HIF-1α (hypoxia-inducible factor 1α)/VEGF (vascular endothelial growth factor) pathway was activated by catalpol both in the brains of cerebral ischemic rats and in primary brain microvascular endothelial cells, and the activating effects of catalpol were inhibited by SU1498.ConclusionThe results of both the in vivo and in vitro studies proved that catalpol protects vascular structure and promotes angiogenesis in focal cerebral ischemic rats and that the mechanism is dependent on HIF-1α/VEGF.     

Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel‐mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 μl) or phosphate‐buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI‐PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure–volume loops after 4 weeks. There is no significant difference in infarct size between MI‐matrigel (MI‐M; 21.48 ± 1.49%, n= 10) and MI‐PBS hearts (20.98 ± 1.25%, n= 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI‐M (0.72 ± 0.02 mm, n= 10) compared with MI‐PBS (0.62 ± 0.02 mm, n= 10). MI‐M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high‐power field [HPF; 400×], n= 6) than MI‐PBS hearts. c‐Kit⁺ stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c‐Kit⁺ cells per HPF [630×], n= 5, P < 0.05) and CD34⁺ cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34⁺ cells per HPF [630×], n= 5, P < 0.01) were significantly more numerous in MI‐M than in MI‐PBS in the infarcted hearts (n= 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34⁺ and c‐Kit⁺ stem cells.     

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2^−/− than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2^−/− mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2^−/− mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2^−/− EPC intramyocardially into mice with induced MI. Per2^−/− reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2^−/− EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.     

Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Loss of Sirt3 Limits Bone Marrow Cell-Mediated Angiogenesis and Cardiac Repair in Post-Myocardial Infarction

Sirtuin-3 (Sirt3) has a critical role in the regulation of human aging and reactive oxygen species (ROS) formation. A recent study has identified Sirt3 as an essential regulator of stem cell aging. This study investigated whether Sirt3 is necessary for bone marrow cell (BMC)-mediated cardiac repair in post-myocardial infarction (MI). In vitro, BMC-derived endothelial progenitor cells (EPCs) from wild type (WT) and Sirt3KO mice were cultured. EPC angiogenesis, ROS formation and apoptosis were assessed. In vivo, WT and Sirt3 KO mice were subjected to MI and BMCs from WT and Sirt3 KO mice were injected into ischemic area immediately. The expression of VEGF and VEGFR2 was reduced in Sirt3KO-EPCs. Angiogenic capacities and colony formation were significantly impaired in Sirt3KO-EPCs compared to WT-EPCs. Loss of Sirt3 further enhanced ROS formation and apoptosis in EPCs. Overexpression of Sirt3 or treatment with NADPH oxidase inhibitor apocynin (Apo, 200 and 400 microM) rescued these abnormalities. In post-MI mice, BMC treatment increased number of Sca1⁺/c-kit⁺ cells; enhanced VEGF expression and angiogenesis whereas Sirt3KO-BMC treatment had little effects. BMC treatment also attenuated NADPH oxidase subunits p47^phox and gp91^phox expression, and significantly reduced ROS formation, apoptosis, fibrosis and hypertrophy in post-MI mice. Sirt3KO-BMC treatment did not display these beneficial effects. In contrast, Sirt3KO mice treated with BMCs from WT mice attenuated myocardial apoptosis, fibrosis and improved cardiac function. Our data demonstrate that Sirt3 is essential for BMC therapy; and loss of Sirt3 limits BMC-mediated angiogenesis and cardiac repair in post-MI.     

Adipose Stromal Cells Amplify Angiogenic Signaling via the VEGF/mTOR/Akt Pathway in a Murine Hindlimb Ischemia Model: A 3D Multimodality Imaging Study

Although adipose-derived stromal cell (ADSC) transplantation has been demonstrated as a promising therapeutic strategy for peripheral arterial disease (PAD), the mechanism of action behind the observed therapeutic efficacy of ADSCs remains unclear. This study was designed to investigate the long-term outcome and therapeutic behavior of engrafted ADSCs in a murine hindlimb ischemia model using multimodality molecular imaging approaches. ADSCs (1.0×10⁷) were isolated from Tg(Fluc-egfp) mice which constitutively express dual-reporter firefly luciferase and enhanced green fluorescent protein (Fluc⁺-eGFP⁺, mADSCs^Fluc+GFP+), then intramuscularly injected into the hindlimb of BALB/c-nu mice after unilateral femoral artery ligation and excision. Abbreviated survival (∼5 weeks) of post-transplant mADSCs within the ischemic hindlimb was longitudinally monitored using noninvasive bioluminescence imaging (BLI), fluorescence imaging (FRI), and bioluminescence tomography with micro-computed tomography (BLT/micro-CT). Use of the BLT/micro-CT system enabled quantitative 3-dimensional (3D) imaging of the cells’ distribution and kinetics in vivo. Engrafted mADSCs improved blood perfusion recovery, ambulatory performance and prognosis of the ischemic hindlimb, probably by inducing angiogenesis and formation of collateral vessels, which could be visualized using laser Doppler perfusion imaging (LDPI), micro-CT angiography, vascular-cast imaging, and immunofluorescence. mADSCs augmented activation of the pro-angiogenic VEGF/mTOR/Akt pathway in vivo, even though the cells failed to incorporate into the host microvasculature as functional components. Downregulation of VEGF/mTOR/Akt signaling using small molecule inhibitors counteracted mADSC-induced angiogenesis and perfusion restoration. This study demonstrates for the first time the spatiotemporal kinetics and functional survival of transplanted mADSCs in a PAD model using in vivo 3D multimodality imaging. Our study indicates that mADSCs potentiate pro-angiogenic signal amplification via a VEGF/mTOR/Akt-dependent pathway, and thereby promote recovery from hindlimb ischemia.     

Comparison of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes with Human Mesenchymal Stem Cells following Acute Myocardial Infarction

Introduction

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have recently been shown to express key cardiac proteins and improve in vivo cardiac function when administered following myocardial infarction. However, the efficacy of hiPSC-derived cell therapies, in direct comparison to current, well-established stem cell-based therapies, is yet to be elucidated. The goal of the current study was to compare the therapeutic efficacy of human mesenchymal stem cells (hMSCs) with hiPSC-CMs in mitigating myocardial infarction (MI).

Methods

Male athymic nude hyrats were subjected to permanent ligation of the left-anterior-descending (LAD) coronary artery to induce acute MI. Four experimental groups were studied: 1) control (non-MI), 2) MI, 3) hMSCs (MI+MSC), and 4) hiPSC-CMs (MI+hiPSC-derived cardiomyocytes). The hiPSC-CMs and hMSCs were labeled with superparamagnetic iron oxide (SPIO) in vitro to track the transplanted cells in the ischemic heart by high-field cardiac MRI. These cells were injected into the ischemic heart 30-min after LAD ligation. Four-weeks after MI, cardiac MRI was performed to track the transplanted cells in the infarct heart. Additionally, echocardiography (M-mode) was performed to evaluate the cardiac function. Immunohistological and western blot studies were performed to assess the cell tracking, engraftment and cardiac fibrosis in the infarct heart tissues.

Results

Echocardiography data showed a significantly improved cardiac function in the hiPSC-CMs and hMSCs groups, when compared to MI. Immunohistological studies showed expression of connexin-43, α-actinin and myosin heavy chain in engrafted hiPSC-CMs. Cardiac fibrosis was significantly decreased in hiPSC-CMs group when compared to hMSCs or MI groups. Overall, this study demonstrated improved cardiac function with decreased fibrosis with both hiPSC-CMs and hMSCs groups when compared with MI group.     

Context- and Cell-Dependent Effects of Delta-Like 4 Targeting in the Bone Marrow Microenvironment

Delta-like 4 (Dll4) is a ligand of the Notch pathway family which has been widely studied in the context of tumor angiogenesis, its blockade shown to result in non-productive angiogenesis and halted tumor growth. As Dll4 inhibitors enter the clinic, there is an emerging need to understand their side effects, namely the systemic consequences of Dll4:Notch blockade in tissues other than tumors. The present study focused on the effects of systemic anti-Dll4 targeting in the bone marrow (BM) microenvironment. Here we show that Dll4 blockade with monoclonal antibodies perturbs the BM vascular niche of sub-lethally irradiated mice, resulting in increased CD31⁺, VE-Cadherin⁺ and c-kit⁺ vessel density, and also increased megakaryocytes, whereas CD105⁺, VEGFR3⁺, SMA⁺ and lectin⁺ vessel density remained unaltered. We investigated also the expression of angiocrine genes upon Dll4 treatment in vivo, and demonstrate that IGFbp2, IGFbp3, Angpt2, Dll4, DHH and VEGF-A are upregulated, while FGF1 and CSF2 are reduced. In vitro treatment of endothelial cells with anti-Dll4 reduced Akt phosphorylation while maintaining similar levels of Erk 1/2 phosphorylation. Besides its effects in the BM vascular niche, anti-Dll4 treatment perturbed hematopoiesis, as evidenced by increased myeloid (CD11b⁺), decreased B (B220⁺) and T (CD3⁺) lymphoid BM content of treated mice, with a corresponding increase in myeloid circulating cells. Moreover, anti-Dll4 treatment also increased the number of CFU-M and -G colonies in methylcellulose assays, independently of Notch1. Finally, anti-Dll4 treatment of donor BM improved the hematopoietic recovery of lethally irradiated recipients in a transplant setting. Together, our data reveals the hematopoietic (BM) effects of systemic anti-Dll4 treatment result from qualitative vascular changes and also direct hematopoietic cell modulation, which may be favorable in a transplant setting.     

Bone marrow-independent adventitial macrophage progenitor cells contribute to angiogenesis

Pathological angiogenesis promotes tumor growth, metastasis, and atherosclerotic plaque rupture. Macrophages are key players in these processes. However, whether these macrophages differentiate from bone marrow-derived monocytes or from local vascular wall-resident stem and progenitor cells (VW-SCs) is an unresolved issue of angiogenesis. To answer this question, we analyzed vascular sprouting and alterations in aortic cell populations in mouse aortic ring assays (ARA). ARA culture leads to the generation of large numbers of macrophages, especially within the aortic adventitia. Using immunohistochemical fate-mapping and genetic in vivo-labeling approaches we show that 60% of these macrophages differentiate from bone marrow-independent Ly6c⁺/Sca-1⁺ adventitial progenitor cells. Analysis of the NCX^−/− mouse model that genetically lacks embryonic circulation and yolk sac perfusion indicates that at least some of those progenitor cells arise yolk sac-independent. Macrophages represent the main source of VEGF in ARA that vice versa promotes the generation of additional macrophages thereby creating a pro-angiogenetic feedforward loop. Additionally, macrophage-derived VEGF activates CD34⁺ progenitor cells within the adventitial vasculogenic zone to differentiate into CD31⁺ endothelial cells. Consequently, depletion of macrophages and VEGFR2 antagonism drastically reduce vascular sprouting activity in ARA. In summary, we show that angiogenic activation induces differentiation of macrophages from bone marrow-derived as well as from bone marrow-independent VW-SCs. The latter ones are at least partially yolk sac-independent, too. Those VW-SC-derived macrophages critically contribute to angiogenesis, making them an attractive target to interfere with pathological angiogenesis in cancer and atherosclerosis as well as with regenerative angiogenesis in ischemic cardiovascular disorders.Subject terms: Stem-cell differentiation, Stem-cell niche, Adult stem cells, Cell signalling     

Transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model

Background aimsThe purpose of this study was to investigate therapeutic potential of green fluorescent protein expressing porcine embryonic stem (pES/GFP⁺) cells in A rat model of spinal cord injury (SCI).MethodsUndifferentiated pES/GFP⁺ cells and their neuronal differentiation derivatives were transplanted into the contused spinal cord of the Long Evans rat, and in situ development of the cells was determined by using a live animal fluorescence optical imaging system every 15 days. After pES/GFP⁺ cell transplantation, the behavior functional recovery of the SCI rats was assessed with the Basso, Beattie, and Bresnahan Locomotor Rating Scale (BBB scale), and the growth and differentiation of the grafted pES/GFP⁺ cells in the SCI rats were analyzed by immunohistochemical staining.ResultsThe relative green fluorescent protein expression level was decreased for 3 months after transplantation. The pES/GFP⁺-derived cells positively stained with neural specific antibodies of anti-NFL, anti-MBP, anti-SYP and anti-Tuj 1 were detected at the transplanted position. The SCI rats grafted with the D18 neuronal progenitors showed a significant functional recovery of hindlimbs and exhibited the highest BBB scale score of 15.20 ± 1.43 at week 24. The SCI rats treated with pES/GFP⁺-derived neural progenitors demonstrated a better functional recovery.ConclusionsTransplantation of porcine embryonic stem (pES)-derived D18 neuronal progenitors has treatment potential for SCI, and functional behavior improvement of grafted pES-derived cells in SCI model rats suggests the potential for further application of pES cells in the study of replacement medicine and functionally degenerative pathologies.