骨髓细胞使梗死后心肌再生

心肌梗死后可导致心肌细胞的丧失和心脏功能的降低 ,而残存的心肌又不能对已坏死的组织进行修复 ,且梗死后心脏功能的损害随时间的推延而加重。有文献报道 :干细胞可感知远处靶器官的损伤并移行到坏死区域进行分化 ,从而可促进坏死区域结构和功能的修复。干细胞的这种高度可塑性引发我们观察在大鼠心肌梗死模型中移植骨髓细胞是否可使坏死心肌进行再生。实验以转基因后表达增强型绿色荧光蛋白 (ＥＧＦＰ)的雄性Ｃ5 7ＢＬ/6大鼠为供体 ,收集其股骨和胫骨的骨髓细胞 ,通过荧光激活的细胞分离法 ,分离出血谱系阳性的骨髓干细胞 (Ｌｉｎ ｃ ｋｉ…     

心肌干细胞的研究进展

传统观点认为,心肌细胞出生后即向终末分化,不能够复制,并且成年心肌组织中没有储存的心肌祖细胞,因此心肌受损后心肌细胞不能再生,被疤痕组织代替,最终导致心脏的收缩功能受损[1].1994年,Soonpaa首次发现将小鼠胚胎干细胞移植到成年小鼠发生梗死的心肌中,能够存活并且能够限制疤痕发展及预防梗塞后心衰的发生[2],提示心肌干细胞治疗可能会从根本上解决心肌损伤后的恢复问题.     

骨髓干细胞移植与心肌修复

骨髓干细胞具有分化与修复心肌组织的潜力，与其它类型干细胞比较具有很多优势。骨髓Lin^-c—kit^POS细胞及间叶干细胞移植动物模型已成功修复心肌并改善心脏功能，并已应用于临床病人。自体骨髓干细胞动员修复损伤心肌具有更多优点。骨髓干细胞移植修复损伤心肌有望成为心脏修复的新手段。     

间质干细胞移植治疗心肌梗死

心肌细胞在成熟个体中属永久性细胞，发生局部心肌梗死后，不能再生而影响心功能。间质干细胞移植于心肌梗死部位，在局部诱导分化为心肌细胞、血管内皮细胞，以修复坏死的心肌，重建梗死部位的血运，而且可以将治疗性基因转染骨髓间质干细胞后进行移植，以最大限度恢复心功能。     

心肌细胞再生与细胞移植

成年心肌细胞并非终末分化细胞，通过细胞增殖与更新维持心肌细胞动态平衡及修复损坏心肌。再生心肌细胞可能来源于心肌干细胞，而干细胞则来源于体循环而非心脏本身。移植于心肌内的骨髓干细胞可分化为具有功能的心肌细胞及血管，替代瘢痕组织及改善心脏功能。     

骨髓间质干细胞治疗心肌梗死研究进展

以往认为,心肌细胞不能再生,心肌受损后只能形成瘢痕组织来修复。近年研究显示,在急性心肌梗死（AMI）患者的心脏中,特别在梗死周边区域,也有少量心肌细胞发生有丝分裂。虽然这些细胞的分裂能力有限,但心肌细胞具有分裂能力的新观念将干细胞与心肌细胞移植治疗联系起来,使干细胞移植治疗心肌梗死成为可能。现将骨髓间质干细胞移植重建受损心肌的研究进展综述如下。     

骨髓干细胞移植治疗缺血性心脏病的研究进展

细胞心肌成形术是通过细胞移植的方法替代或再生心肌细胞,为缺血受损心脏的细胞重建和功能恢复提供了具有里程碑意义的治疗策略.多种类型的干细胞被用于移植研究,其中骨髓干细胞移植最具潜力,已成为该领域的研究热点.     

心肌干细胞的研究进展

传统观点认为，心肌细胞出生后即向终末分化，不能够复制，并且成年心肌组织中没有储存的心肌祖细胞，因此心肌受损后心肌细胞不能再生，被疤痕组织代替，最终导致心脏的收缩功能受损。1994年，Soonpaa首次发现将小鼠胚胎干细胞移植到成年小鼠发生梗死的心肌中，能够存活并且能够限制疤痕发展     

骨髓干细胞移植治疗缺血性心脏病的研究进展

细胞心肌成形术是通过细胞移植的方法替代或再生心肌细胞，为缺血受损心脏的细胞重建和功能恢复提供了具有里程碑意义的治疗策略。多种类型的干细胞被用于移植研究，其中骨髓干细胞移植最具潜力，已成为该领域的研究热点。     

小鼠骨髓干细胞分化为小胶质细胞的在体研究

将增强型绿色荧光蛋白转基因小鼠的骨髓细胞通过股静脉移植入实验小鼠制作成嵌合鼠模型,正规饲养8周后,通过免疫组织化学和免疫荧光染色法鉴定移植细胞在嵌合鼠脑内的分布和分化情况。结果利用免疫组织化学方法在嵌合鼠脑内发现了移植细胞,但只存在于小脑。利用免疫荧光双染方法证明了移植细胞仅表现为小胶质细胞表型。认为小鼠骨髓干细胞能够进入血脑屏障完整的正常小鼠小脑,并分化为小胶质细胞。     

An improved mouse Sca-1+ cell-based bone marrow transplantation model for use in gene- and cell-based therapeutic studies

This study sought to develop a murine bone marrow transplantation strategy that would yield consistently high levels of long-term engraftment without significant morbidity and mortality. Hematopoietic stem cell (HSC)-enriched Sca-1+ cells were used for transplantation because of their propensity of homing to bone marrow. Green fluorescent protein (GFP)-expressing transgenic mice were used as donors. Murine Sca-1+ cells were enriched 13-fold from whole bone marrow with immunomagnetic column chromatography. Retroorbital injections yielded highly reproducible and higher levels of engraftment compared with tail vein injections. The combination of W41/W41 recipient mice and sublethal irradiation preconditioning produced long-term engraftment with minimal morbidity and mortality. A 24-hour delay between the sublethal irradiation and transplantation did not affect the efficiency and level of engraftment, but provided flexibility with respect to the timing of transplantation. Based on these findings, a mouse Sca-1+ cell-based strategy, involving the retroorbital injection of Sca-1+ cells into sublethally irradiated, myelosuppressed W41/W41 recipient mice within 24 h after irradiation, was developed. Transplantation of lentiviral vector-transduced wild-type Sca-1+ cells expressing GFP by this strategy led to consistently high levels of long-term engraftment. In summary, this murine Sca-1+ cell-based strategy could be used in studies of HSC-based gene or cell therapies.     

Host-derived circulating cells do not significantly contribute to cardiac regeneration in heterotopic rat heart transplants

OBJECTIVES: The aim of this study was to investigate the contribution of host-derived circulating cells to cardiac repair after tissue damage using the model of heterotopic heart transplantation between transgenic recipient rats expressing green fluorescent protein (GFP) and wild-type donors. METHODS: Unlabeled donor rat hearts, some of which underwent prolonged cold ischemia pretreatment, were transplanted into the abdominal cavity of GFP+ transgenic recipient rats and were analyzed 15 and 90 days after surgery. An additional experimental group underwent heart transplantation following administration of granulocyte-colony stimulatory factor (G-CSF) to mobilize bone marrow cells. RESULTS: Most transplants contained GFP+ mature cardiomyocytes. However, systematic counting in the transplants showed that the proportion of GFP+ cardiomyocytes was only 0.0005% to 0.008% of all cardiomyocytes. These relative proportions did not change after G-CSF treatment, despite evidence for sustained marrow cell mobilization. Confocal image analysis showed that the majority of GFP+ cardiomyocytes contained a high number of nuclei, suggesting that these cells may derive from fusion events. Very rarely, small GFP+ undifferentiated cells, expressing GATA-4, were also identified. Occasionally, GFP+ endothelial cells, but not smooth muscle cells, were detected in blood vessels of some transplants. CONCLUSIONS: Our results demonstrate that cardiomyocytes expressing a host transgenic marker are detectable in heterotopic heart transplants; however, they do not significantly contribute to repopulation of the damaged myocardium.     

Granulocyte-colony stimulating factor increases donor mesenchymal stem cells in bone marrow and their mobilization into peripheral circulation but does not repair dystrophic heart after bone marrow transplantation

BACKGROUND: Hereditary disordered cardiac muscle could be replaced with intact cardiomyocytes derived from genetically intact bone marrow (BM)-derived stem cells. METHODS AND RESULTS: Cardiomyopathic mice with targeted mutation of delta-sarcoglycan gene underwent intra-BM-BM transplantation (IBM-BMT) from transgenic mice expressing green fluorescence protein. The host BM and the peripheral blood were completely reconstituted by donor-derived hematopoietic cells by IBM-BMT. Treatment with granulocyte-colony stimulating factor (G-CSF) markedly increased donor-derived mesenchymal stem cells (MSC) in the BM and their mobilization into the peripheral blood after IBM-BMT. Treatment with isoproterenol (iso) for 7 days caused myocardial damage and left ventricular (LV) dysfunction in the cardiomyopathic mice. Co-treatment with iso and G-CSF increased donor BM cell recruitment to the heart and temporarily improved LV function in the cardiomyopathic mice with or without IBM-BMT. However, the cardiac muscle was not replaced with donor BM-derived cardiomyocytes in the cardiomyopathic mice with or without IBM-BMT, and this was associated with no improvement of LV function of mice aged 20 weeks. CONCLUSIONS: These results suggest that G-CSF enhances engraftment of donor MSC in the BM and their mobilization into the peripheral circulation after IBM-BMT but MSC recruited to the heart do not differentiate into cardiomyocytes and do not repair the dystrophic heart.     

In vitro and in vivo effects of bone marrow stem cells on cardiac structure and function

It is hypothesized that the protection of bone marrow stem cells (BMSCs) on ischemic myocardium might be related to the anti-apoptotic effect via paracrine mechanisms. In this study, a wide array of cytokines including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor-1 (SDF-1) and insulin growth factor-1 (IGF-1) were detected in the BMSCs cultured medium by ELISA. Myocyte apoptosis was assayed by DNA fragmentation and annexin-V staining. Myocardial infarction model was produced by ligation of mouse left anterior descending coronary artery (LAD). Before LAD ligation, mice were myoablated by irradiation and transplanted with bone marrow cells from transgenic mice expressing green fluorescent protein (GFP). After LAD ligation, animals were administered stem cell factor (SCF, 200 mug/day/kg, i.p.) or saline for 6 days. Animals were sacrificed at 4 weeks after SCF treatment. Apoptotic cardiomyocytes were analyzed by TUNEL. Myocardial function was analyzed by echocardiography and pressure-volume system. Bcl-2 protein was analyzed by Western blotting. Our results showed that cultured BMSCs released VEGF, bFGF, SDF-1 and IGF-1. Hypoxia-induced cell apoptosis was diminished in cardiomyocytes co-cultured with BMSCs. Smaller LV dimension and increased LV ejection fraction were seen in SCF-treated animals. SCF significantly reduced cardiomyocytes apoptosis within peri-infarct area and increased up-regulation expression of Bcl-2 in ischemic area. Moreover, conditioned medium from cultured BMSCs also induced up-regulation of Bcl-2 protein in cardiomyocytes. It is concluded that paracrine mediators secreted by BMSCs might be involved in early repair of ischemic heart by preventing cardiomyocytes apoptosis and improving cardiac function.     

Green Fluorescent Protein-Transgenic Rat as a Tool for Study of Transplantation and Regeneration in Myocardium

Cell transplantation and regeneration therapy are potentially new therapeutic approaches for cardiovascular disease. Transgenic (tg) animals for reporter genes would be useful to follow the cell lineage and differentiation during development and regeneration processes. In the present study, we developed green fluorescent protein (GFP)-tg rats and evaluated them as a tool for the study of cardiomyocyte transplantation and regeneration. The myocardium and bone marrow cells derived from GFP-tg rats strongly expressed GFP. Because neonatal rat cultured cardiomyocytes also strongly expressed GFP, we transplanted GFP-tg rat-derived cardiomyocytes in a rat myocardial infarction (MI) model. Survival of GFP-tg rat-derived cardiomyocytes was confirmed. We further investigated whether bone marrow cells could differentiate into cardiomyocytes using this GFP-tg rat-derived bone marrow cells in vitro and in vivo. GFP-tg rat-derived bone marrow cells differentiated into cardiomyocyte- like cells (cardiac troponin I-expressed cells) by co-culture with wild rat cultured cardiomyocytes in vitro. Furthermore, differentiation of bone marrow cells into cardiomyocyte-like cells was observed by injection of GFP-tg rat-derived bone marrow cells in a rat MI model in vivo. These findings suggest that GFP-tg rats are a useful and valuable tool for the study of transplantation and regeneration in myocardium.     

c-kit is required for cardiomyocyte terminal differentiation

c-kit, the transmembrane tyrosine kinase receptor for stem cell factor, is required for melanocyte and mast cell development, hematopoiesis, and differentiation of spermatogonial stem cells. We show here that in the heart, c-kit is expressed not only by cardiac stem cells but also by cardiomyocytes, commencing immediately after birth and terminating a few days later, coincident with the onset of cardiomyocyte terminal differentiation. To examine the function of c-kit in cardiomyocyte terminal differentiation, we used compound heterozygous mice carrying the W (null) and W(v) (dominant negative) mutations of c-kit. In vivo, adult W/W(v) cardiomyocytes are phenotypically indistinguishable from their wild-type counterparts. After acute pressure overload adult W/W(v) cardiomyocytes reenter the cell cycle and proliferate, leading to left ventricular growth; furthermore in transgenic mice with cardiomyocyte-restricted overexpression of the dominant negative W(v) mutant, pressure overload causes cardiomyocytes to reenter the cell cycle. In contrast, in wild-type mice left ventricular growth after pressure overload results mainly from cardiomyocyte hypertrophy. Importantly, W/W(v) mice with pressure overload-induced cardiomyocyte hyperplasia had improved left ventricular function and survival. In W/W(v) mice, c-kit dysfunction also resulted in an approximately 14-fold decrease (P<0.01) in the number of c-kit(+)/GATA4(+) cardiac progenitors. These findings identify novel functions for c-kit: promotion of cardiac stem cell differentiation and regulation of cardiomyocyte terminal differentiation.     

Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells

Aims/hypothesis Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas.Methods Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation.Results In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas.Conclusions/interpretation Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.Abbreviations STZ streptozotocin - EGFP enhanced green fluorescent protein - GP guinea-pig - vWF von Willebrand Factor - BrdU bromodeoxyuridine - GFP green fluorescent protein - IPGTT Intraperitoneal glucose tolerance test     

Haematopoietic stem cells participate in muscle regeneration

It has previously been shown that bone marrow cells contribute to skeletal muscle regeneration, but the nature of marrow cell(s) involved in this process is unknown. We used an immunocompetent and an immunocompromised model of bone marrow transplantation to characterize the type of marrow cells participating regenerating skeletal muscle fibres. Animals were transplanted with different populations of marrow cells from Green Fluorescent Protein (GFP) transgenic mice and the presence of GFP(+) muscle fibres were evaluated in the cardiotoxin-injured tibialis anterior muscles. GFP(+) muscle fibres were found mostly in animals that received either CD45(-), lineage(-), c-Kit(+), Sca-1(+) or Flk-2(+) populations of marrow cells, suggesting that haematopoietic stem cells (HSC) rather than mesenchymal cells or more differentiated haematopoietic cells are responsible for the formation of GFP(+) muscle fibres. Mac-1 positive population of marrow cells was also associated with the emergence of GFP(+) skeletal muscle fibres. However, most of this activity was limited to either Mac-1(+) Sca(+) or Mac-1(+)c-Kit(+) cells with long-term haematopoietic repopulation capabilities, indicating a stem cell phenotype for these cells. Experiments in the immunocompromised transplant model showed that participation of HSC in the skeletal muscle fibre formation could occur without haematopoietic chimerism.     

A Novel Approach to Transplanting Bone Marrow Stem Cells to Repair Human Myocardial Infarction: Delivery via a Noninfarct‐relative Artery

Bone marrow stem cells are able to repair infarcted human myocardium following intracoronary transplantation via the infarct‐relative artery. However, traditional reperfusion strategies fail to open the artery in some patients, making effective delivery impossible. Our previous study demonstrated a safe and efficient approach to delivering bone marrow stem cells via a noninfarcted artery in an animal myocardial infarction model. The objective of the present study was to evaluate the safety and feasibility of autologous bone marrow mesenchymal stem cell transplantation via such an approach in patients with acute myocardial infarction (AMI). Sixteen patients with anterior AMI who had successfully undergone percutaneous coronary intervention (PCI) were enrolled in this pilot, randomized study. Three weeks after PCI, cultured bone marrow mesenchymal stem cells were injected into the myocardium via either the infarct‐relative artery (left anterior descending branch artery, LAD) or a noninfarct‐relative artery (right coronary artery, RCA). The safety and feasibility of the cell infusion were evaluated during the procedure and during 6 months of follow‐up. In addition, 2D echocardiography, technetium‐99m methoxyisobutylisonitrile (99mTc‐MIBI) and 18F‐deoxyglucose single photon emission computed tomography were employed to examine cardiac function, myocardial perfusion, and viable cardiomyocytes, respectively, at day 4 after PCI and 6 months after the cell infusion. There were no arrhythmia and any other side‐effects, including infections, allergic reactions or adverse clinical events, during, immediately after, or 6 months after cell transplantation. Cardiac function and myocardial perfusion had improved 6 months after PCI/bone marrow stem cells transplantation. Viable cardiomyocytes metabolism was detected in the infarcted areas in both groups after the cell infusion, as demonstrated by 18F‐deoxyglucose. Intracoronary infusion of autologous bone marrow mesenchymal stem cells via a noninfarct‐relative artery appears safe and feasible in the treatment of patients with AMI.