Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle

Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.     

受致死剂量照射模型小鼠输注骨骼肌卫星细胞后造血功能的变化

背景:骨骼肌卫星细胞是一种具有增殖、分化潜能的肌源性干细胞。近年,国外已有报道骨骼肌细胞具可被某种特定微环境因子激活分化为造血干细胞,从而具有造血重建的潜能。目的:初步验证肌源性的成体干细胞-骨骼肌卫星细胞分化为造血干细胞的能力。设计:验证性动物实验。单位:天津中医药大学基础医学院组胚教研室。材料:选用雌性昆明种成年小鼠65只,体质量25～28g。出生5d昆明种乳鼠5只,均由北京大学医学部实验动物中心提供。方法:实验于2001-08/2003-08在北京大学医学部人体解剖学与组织胚胎学系细胞培养室完成。胶原酶、胰蛋白酶消化分离5只乳鼠骨骼肌卫星细胞,分离5只成年昆明种小鼠骨髓单个核细胞。取成年雌性小鼠60只作为受体,用60Coγ8.0Gy照射,然后随机分为4组:对照组不做任何处理;培养液输注组小鼠尾静脉注射DMEM/F-12培养液;卫星细胞输注组小鼠尾静脉注射卫星细胞悬液0.3mL(细胞浓度1×109L-1);骨髓细胞输注组小鼠尾静脉注射骨髓细胞悬液0.3mL(细胞浓度1×109L-1)。主要观察指标:①观察各组小鼠14d存活率。②照射后14d处死存活下来的受体小鼠:肉眼计数脾脏表面结节数;对骨髓单个核细胞涂片进行Wright-Gimesa染色。结果:①脾集落形成单位的测定:照射后14d,卫星细胞注射组小鼠脾结节数与骨髓细胞注射组比较,差异无显著性意义(P>0.05)。②骨髓单个核细胞组织学鉴定结果:卫星细胞输注组与骨髓细胞输注组骨髓单个核细胞涂片中有相对较多的早期造血细胞出现,其形态符合早期造血细胞的生物学特性。③被照射鼠的存活情况:对照组和培养液输注组小鼠在被照射后9～13d全部死亡。照射后14d,卫星细胞输注组和骨髓细胞输注组分别有8和13只鼠存活。结论:骨骼肌卫星细胞具有诱导分化为造血干细胞的功能。     

Hematopoietic,vascular and cardiac fates of bone marrow-derived stem cells

Bone marrow contains many cell types, including stroma, vascular cells, adipocytes, osteoblasts and osteoclasts, as well as mesenchymal stem cells and hematopoietic stem cells. It was previously thought that cells within bone marrow solely functioned to regenerate cells within the marrow, as well as all circulating hematopoietic cells in peripheral blood. Recent reports, however, suggest that marrow-derived cells can also regenerate other cell types, including cardiac muscle, liver cell types, neuronal and non-neuronal cell types of the brain, as well as endothelial cells and osteoblasts. These multiple cell types could have originated from either of the stem cell populations within bone marrow or potentially other precursors. Therefore, it is not entirely clear whether each of these distinct cell lineages has a true progenitor within marrow or whether the marrow contains a multipotent population of cells that has been set aside during embryogenesis for postnatal repair and remodeling of a variety of tissues. It is clear, however, that directing the fate of bone marrow-derived progenitors (ie toward hematopoietic, vascular or cardiac cell fates) can only be accomplished if the phenotype of the stem cells is defined, and their homing and differentiation programs are elucidated. Much work is focused on these issues, wherein lie the key to harnessing the potential of adult stem cells for autologous cell and gene therapy.     

The proarrhythmic risk of cell therapy for cardiovascular diseases

Stem cell therapy is an emerging therapeutic approach for the treatment of cardiovascular diseases. Experimental studies have demonstrated that different types of stem cells, including bone marrow-derived cells, mesenchymal stem cells, skeletal myoblasts, and cardiac progenitor cells and embryonic stem cells, can improve cardiac function after myocardial injuries. Nevertheless, the potential proarrhythmic risk after stem cell transplantation remains a major concern. Several mechanisms, including the immaturity of electrical phenotypes of the transplanted cardiomyocytes, poor cell–cell coupling and cardiac nerve sprouting, may contribute to arrhythmogenic risk after stem cell transplantation. This review summarizes the potential theoretical arrhythmogenic mechanisms associated with different types of stem cells for the treatment of cardiovascular diseases. Nevertheless, current experimental and clinical data on the proarrhythmic risk for different types of stem cell transplantation are limited, and await further experimental and clinical investigation.     

Lost and found: cardiac stem cell therapy revisited

Several clinical trials of bone marrow stem cell therapy for myocardial infarction are ongoing, but the mechanistic basis for any potential therapeutic effect is currently unclear. A growing body of evidence suggests that the potential improvement in cardiac function is largely independent of cardiac muscle regeneration. A study by Fazel et al. in this issue of the JCI provides evidence that bone marrow-derived c-kit+ cells can lead to an improvement in cardiac function in mutant hypomorphic c-kit mice that is independent of transdifferentiation into either cardiac muscle or endothelial cells, but rather is associated with the release of angiogenic cytokines and associated neovascularization in the infarct border zone (see the related article beginning on page 1865). These findings suggest the potential therapeutic effect of specific paracrine pathways for angiogenesis in improving cardiac function in the injured heart.     

The proarrhythmic risk of cell therapy for cardiovascular diseases

Stem cell therapy is an emerging therapeutic approach for the treatment of cardiovascular diseases. Experimental studies have demonstrated that different types of stem cells, including bone marrow-derived cells, mesenchymal stem cells, skeletal myoblasts, and cardiac progenitor cells and embryonic stem cells, can improve cardiac function after myocardial injuries. Nevertheless, the potential proarrhythmic risk after stem cell transplantation remains a major concern. Several mechanisms, including the immaturity of electrical phenotypes of the transplanted cardiomyocytes, poor cell-cell coupling and cardiac nerve sprouting, may contribute to arrhythmogenic risk after stem cell transplantation. This review summarizes the potential theoretical arrhythmogenic mechanisms associated with different types of stem cells for the treatment of cardiovascular diseases. Nevertheless, current experimental and clinical data on the proarrhythmic risk for different types of stem cell transplantation are limited, and await further experimental and clinical investigation.     

Therapeutic potential of adult progenitor cells in cardiovascular disease

Cardiovascular diseases are responsible for high morbidity/mortality rates worldwide. Advances in patient care have significantly reduced deaths from acute myocardial infarction. However, the cardiac remodeling processes induced after ischaemia are responsible for a worsening in the heart condition, which in many cases ends up in failure. In the last decade, a novel therapy based on stem cell transplantation is being intensively studied in animal models and some stem cell types (i.e., skeletal myoblasts and bone marrow-derived cells) are already being tested in clinical trials. A novel stem cell population isolated from the bone marrow, termed multipotent adult progenitor cells was characterised a few years ago by its ability to differentiate, at the single cell level, towards cells derived from the three embryonic germ layers. Later on, other pluripotent cell populations have been also derived from the bone marrow. In this overview, the authors outline different stem cell sources that have been tested for their cardiovascular potential and put the regenerative potential of multipotent adult progenitor cells in animal models of acute and chronic myocardial infarction into perspective.     

Therapeutic potential of adult progenitor cells in cardiovascular disease

Cardiovascular diseases are responsible for high morbidity/mortality rates worldwide. Advances in patient care have significantly reduced deaths from acute myocardial infarction. However, the cardiac remodeling processes induced after ischaemia are responsible for a worsening in the heart condition, which in many cases ends up in failure. In the last decade, a novel therapy based on stem cell transplantation is being intensively studied in animal models and some stem cell types (i.e., skeletal myoblasts and bone marrow-derived cells) are already being tested in clinical trials. A novel stem cell population isolated from the bone marrow, termed multipotent adult progenitor cells was characterised a few years ago by its ability to differentiate, at the single cell level, towards cells derived from the three embryonic germ layers. Later on, other pluripotent cell populations have been also derived from the bone marrow. In this overview, the authors outline different stem cell sources that have been tested for their cardiovascular potential and put the regenerative potential of multipotent adult progenitor cells in animal models of acute and chronic myocardial infarction into perspective.     

胎儿骨髓和肝脏间充质干细胞的表型和生物学性状研究

为研究胎儿骨髓和肝脏间充质干细胞的表型和生物学性状,取胎龄为4-5个月水囊引产胎儿,将骨髓和肝脏细胞在SF（含2?S）培养基中培养,进行电镜观察,测定生长曲线,用流式细胞术对培养细胞进行表型测定。细胞周期分析。用SA方法测定Ⅰ,Ⅲ型胶原和vWF因子表达。结果表明;从胎儿骨髓和肝脏培养出的间充质干细胞,两在形态学,生长特性,免疫表型上是相似的,肝脏间充质干细胞有更好的支持造血的功能。结论提示,从胎儿骨髓和肝脏可分离培养出间充质干细胞。在体外有效扩增且保持其低分化状态。     

Cardiomyocytes derived from stem cells

One way to restore failing heart function following myocardial infarction would be to replace lost or damaged cardiac cells by local or systemic injection. The sources of replacement cells presently discussed include embryonic stem cells, hematopoietic and non‐hematopoietic stem cells from bone marrow or cord blood and small stem cell populations thought to reside in the heart itself or in skeletal muscle. Here we review this area of stem cell research with focus particularly on recent laboratory advances towards producing cardiomyocytes from embryonic stem cells. We conclude that embryonic stem cells and cardiac progenitors in the heart itself are the only proven sources of cardiomyocytes and that reported clinical effects of bone marrow stem currently undergoing validation are likely mediated by other mechanisms.     

核转染对成体骨髓源性干细胞的影响

背景:成体骨髓源性干细胞是实施细胞治疗的重要细胞来源。对成体骨髓源性干细胞的示踪,是研究其移行、分化的规律与机制并进一步阐明再生潜能、疗效的关键。目的:探讨经转染后的成体骨髓呈克隆样干细胞,在细胞表型、增殖能力、心肌分化潜能是否存在的影响。方法:应用核转染技术利用U-23程序将编码maxGFP报告基因的载体对成体骨髓呈克隆样干细胞进行转染,应用MTT法对核转染前后的生长曲线进行测定,使用3μmol/L5-氮胞苷对核转染前后成体骨髓呈克隆样干细胞进行向心肌分化诱导,并利用RT-PCR对向心肌分化的特异性标记物GATA4与MLC-2v的表达进行测定。使用成年SD大鼠心肌梗死左前降支结扎模型,对使用经maxGFP转染的成体骨髓呈克隆样干细胞施心内注射并于注射后的第2天和第7天对经注射心脏行冰冻切片并观察maxGFP在体内的表达情况。结果与结论:经核转染24h后,第47代及第119代成体骨髓呈克隆样干细胞的转染率为49.4%和43.1%,转染后5h,开始出现呈绿色荧光阳性的细胞,经核转染后仍能保持小圆球形、可贴壁、呈克隆样生长。MTT检测结果显示:核转染前后第47代及第119代均具有相似的生长曲线。核转染前第47代及119代细胞平均群体倍增时间分别为8.57,10.28h;核转染后分别为9.42,10.42h,各代前后比较差异无显著性意义(P=0.551,P=0.774)。RT-PCR检测结果显示:核转染前5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强;核转染后5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强,上述2个向心肌分化指标于转染前后变化并不明显。体内实验结果:心内注射经核转染后的成体骨髓呈克隆样干细胞,第2天及第7天可在经注射心肌中发现有少量绿色荧光阳性的细胞。提示,核转染可快速地将外源基因导入细胞,经核转染后的成体骨髓呈克隆样干细胞仍能保持其细胞形态、增殖能力及向心肌分化潜能,然而,经maxGFP基因核转染的成体骨髓呈克隆样干细胞,移植入心肌后只有少数细胞能表达经转染基因。     

Cardiomyocytes derived from stem cells

One way to restore failing heart function following myocardial infarction would be to replace lost or damaged cardiac cells by local or systemic injection. The sources of replacement cells presently discussed include embryonic stem cells, hematopoietic and non-hematopoietic stem cells from bone marrow or cord blood and small stem cell populations thought to reside in the heart itself or in skeletal muscle. Here we review this area of stem cell research with focus particularly on recent laboratory advances towards producing cardiomyocytes from embryonic stem cells. We conclude that embryonic stem cells and cardiac progenitors in the heart itself are the only proven sources of cardiomyocytes and that reported clinical effects of bone marrow stem currently undergoing validation are likely mediated by other mechanisms.     

A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1⁺ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1^– cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1⁺ cells did not. We therefore believe that the SSEA-1⁺ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.     

Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration

Vascular progenitors were previously isolated from blood and bone marrow; herein, we define the presence, phenotype, potential, and origin of vascular progenitors resident within adult skeletal muscle. Two distinct populations of cells were simultaneously isolated from hindlimb muscle: the side population (SP) of highly purified hematopoietic stem cells and non-SP cells, which do not reconstitute blood. Muscle SP cells were found to be derived from, and replenished by, bone marrow SP cells; however, within the muscle environment, they were phenotypically distinct from marrow SP cells. Non-SP cells were also derived from marrow stem cells and contained progenitors with a mesenchymal phenotype. Muscle SP and non-SP cells were isolated from Rosa26 mice and directly injected into injured muscle of genetically matched recipients. SP cells engrafted into endothelium during vascular regeneration, and non-SP cells engrafted into smooth muscle. Thus, distinct populations of vascular progenitors are resident within skeletal muscle, are derived from bone marrow, and exhibit different cell fates during injury-induced vascular regeneration.     

Differential myocardial infarct repair with muscle stem cells compared to myoblasts.

Myoblast transplantation for cardiac repair has generated beneficial results in both animals and humans; however, poor viability and poor engraftment of myoblasts after implantation in vivo limit their regeneration capacity. We and others have identified and isolated a subpopulation of skeletal muscle-derived stem cells (MDSCs) that regenerate skeletal muscle more effectively than myoblasts. Here we report that in comparison with a myoblast population, MDSCs implanted into infarcted hearts displayed greater and more persistent engraftment, induced more neoangiogenesis through graft expression of vascular endothelial growth factor, prevented cardiac remodeling, and elicited significant improvements in cardiac function. MDSCs also exhibited a greater ability to resist oxidative stress-induced apoptosis compared to myoblasts, which may partially explain the improved engraftment of MDSCs. These findings indicate that MDSCs constitute an alternative to other myogenic cells for use in cardiac repair applications.     

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.     

Correction of the dystrophic phenotype by in vivo targeting of muscle progenitor cells

Successful gene therapy for most inherited diseases will require stable expression of the therapeutic gene. This can be addressed with integrating or self-replicating viruses by targeting postmitotic cells that have a long lifetime or stem cells that can replenish defective tissue with corrected cells. In this study, we explore the possibility of targeting a muscle stem cell population in situ through in vivo administration of vector. To develop this concept, we selected a mouse model of muscular dystrophy (mdx mice) that undergoes rapid turnover of muscle fibers. In vivo targeting of muscle progenitor cells, notably satellite cells, with a pseudotyped lentiviral vector encoding the minidystrophin restores dystrophin expression and provides functional correction in skeletal muscle of mdx mice. This study shows that progenitor cells can be genetically engineered in vivo and subsequently proliferate into terminally differentiated tissue carrying the genetic graft in a way that stably corrects function.     

干细胞与运动性骨骼肌细胞凋亡

背景:运动性骨骼肌细胞凋亡业已成为当前运动医学领域的研究重点,干细胞应用于运动性伤病的恢复和防治也有报道,但将干细胞用于细胞凋亡干预作用的相关研究还很少.目的:总结干细胞防治运动性骨骼肌细胞凋亡的作用及其机制,为科学的运动训练和体育锻炼提供依据.方法:通过计算机检索PubMed数据库1991-01/2009-10的相关文献,检索词为"Exercise Training,Sports,Skeletal Muscle,Apoptosis",并限定文章语言种类为English.同时计算机检索中国期刊全文数据库1994-01/2009-10的相关文献,检索词"干细胞、运动、骨骼肌、细胞凋亡",并限定文章语言种类为中文.纳入标准:①文章所述内容应与干细胞及其骨骼肌细胞凋亡的研究密切相关.②同一领域选择近期发表或在权威杂志上发表的文章.排除标准:①重复性研究.②Meta分析.结果与结论:共检索到360篇文献,对资料进行初审,文献的来源主要是通过对干细胞及其在运动医学领域的研究进展,以及骨骼肌细胞凋亡的变化与发展趋势等的应用情况进行汇总分析,共选取31篇文献,其中21篇为综述,其余均为临床或基础实验研究.大强度的运动可引起骨骼肌细胞出现凋亡,而运用干细胞技术可在一定程度上起到预防细胞凋亡的作用,干细胞可通过调节Bcl-2和Bax蛋白表达来防治运动过程中出现的骨骼肌细胞凋亡,从而促进运动机体骨骼肌的早期恢复.     

肝细胞生长因子对骨髓间充质干细胞生物学特性的影响

骨髓间充质干细胞的增殖、分化及迁移等生物学特性受骨髓微环境的调节.肝细胞生长因子（HGF）是骨髓微环境分泌的主要因子之一,但它对骨髓间充质干细胞生物学特性的影响并不清楚.本研究目的是观察重组人HGF对骨髓MSC生物学特性的影响.用免疫组织化学方法检测c-Met的表达,用MTT法测定细胞增殖,定量测定ALP的活性,进行细胞迁移分析和失巢凋亡（anoikis）诱导MSC凋亡分析.结果表明,重组人HGF不影响骨髓间充质干细胞的增殖和向成骨细胞的分化,但可明显促进骨髓间充质干细胞的迁移并抑制anoikis诱导的细胞凋亡;PI-3K和MAPK途径参与了HGF促进细胞迁移的作用.结论：肝细胞生长因子通过受体c-Met影响骨髓间充质干细胞的生物学特性.     

SDF-1α as a therapeutic stem cell homing factor in myocardial infarction

Myocardial infarction is associated with persistent muscle damage, scar formation and depressed cardiac performance. Recent studies have demonstrated the clinical significance of stem cell-based therapies after myocardial infarction with the aim to improve cardiac remodeling and function by inducing the reconstitution of functional myocardium and formation of new blood vessels. Stem cell homing signals play an important role in stem cell mobilization from the bone marrow to the ischemic cardiac environment and are therefore crucial for myocardial repair. To date, the most prominent stem cell homing factor is the chemokine SDF-1α/CXCL12. This protein was shown to be significantly upregulated in many experimental models of myocardial infarction and in patients suffering from ischemic cardiac diseases, suggesting the involvement in the pathophysiology of these disorders. A number of studies focused on manipulating SDF-1α and its receptor CXCR4 as central regulators of the stem cell mobilization process. Targeted expression of SDF-1α after myocardial infarction was shown to result in increased engraftment of bone marrow-derived stem cells into infarcted myocardium. This was accompanied by beneficial effects on cardiomyocyte survival, neovascularization and cardiac function. Thus, the SDF-1/CXCR4 axis seems to be a promising novel therapeutic approach to improve post-infarction therapy by attracting circulating stem cells to remain, survive and possibly differentiate in the infarct area. This review will summarize clinical trials of stem cell therapy in patients with myocardial infarction. We further discuss the basic findings about SDF-1α in stem cell recruitment and its therapeutic implications in experimental myocardial infarction.