De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.     

Haemangioblast commitment is initiated in the primitive streak of the mouse embryo

Haematopoietic and vascular cells are thought to arise from a common progenitor called the haemangioblast. Support for this concept has been provided by embryonic stem (ES) cell differentiation studies that identified the blast colony-forming cell (BL-CFC), a progenitor with both haematopoietic and vascular potential. Using conditions that support the growth of BL-CFCs, we identify comparable progenitors that can form blast cell colonies (displaying haematopoietic and vascular potential) in gastrulating mouse embryos. Cell mixing and limiting dilution analyses provide evidence that these colonies are clonal, indicating that they develop from a progenitor with haemangioblast potential. Embryo-derived haemangioblasts are first detected at the mid-streak stage of gastrulation and peak in number during the neural plate stage. Analysis of embryos carrying complementary DNA of the green fluorescent protein targeted to the brachyury locus demonstrates that the haemangioblast is a subpopulation of mesoderm that co-expresses brachyury (also known as T) and Flk-1 (also known as Kdr). Detailed mapping studies reveal that haemangioblasts are found at highest frequency in the posterior region of the primitive streak, indicating that initial stages of haematopoietic and vascular commitment occur before blood island development in the yolk sac.     

Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors

Interaction between endothelial cells and mural cells (pericytes and vascular smooth muscle) is essential for vascular development and maintenance. Endothelial cells arise from Flk1-expressing (Flk1+) mesoderm cells, whereas mural cells are believed to derive from mesoderm, neural crest or epicardial cells and migrate to form the vessel wall. Difficulty in preparing pure populations of these lineages has hampered dissection of the mechanisms underlying vascular formation. Here we show that Flk1+ cells derived from embryonic stem cells can differentiate into both endothelial and mural cells and can reproduce the vascular organization process. Vascular endothelial growth factor promotes endothelial cell differentiation, whereas mural cells are induced by platelet-derived growth factor-BB. Vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they can incorporate as endothelial and mural cells and contribute to the developing vasculature in vivo. Our findings indicate that Flk1+ cells can act as 'vascular progenitor cells' to form mature vessels and thus offer potential for tissue engineering of the vascular system.     

Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair

Heart disease is a leading cause of death in newborn children and in adults. Efforts to promote cardiac repair through the use of stem cells hold promise but typically involve isolation and introduction of progenitor cells. Here, we show that the G-actin sequestering peptide thymosin beta4 promotes myocardial and endothelial cell migration in the embryonic heart and retains this property in postnatal cardiomyocytes. Survival of embryonic and postnatal cardiomyocytes in culture was also enhanced by thymosin beta4. We found that thymosin beta4 formed a functional complex with PINCH and integrin-linked kinase (ILK), resulting in activation of the survival kinase Akt (also known as protein kinase B). After coronary artery ligation in mice, thymosin beta4 treatment resulted in upregulation of ILK and Akt activity in the heart, enhanced early myocyte survival and improved cardiac function. These findings suggest that thymosin beta4 promotes cardiomyocyte migration, survival and repair and the pathway it regulates may be a new therapeutic target in the setting of acute myocardial damage.     

A peptide fusion protein inhibits angiogenesis and tumor growth by blocking VEGF binding to KDR

Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt-1) induces angiogenesis. In search of the peptides blocking VEGF binding to its receptor KDR/FLK1 to inhibit tumorangiogenesis and growth, we screened a phage display peptide library with KDR as target protein, and some candidate peptides were isolated. In this study, we cloned the DNA fragment coding the peptide K237 from the library, into a vector pQE42 to express fusion protein DHFR-K237 in E. coli M15. The affection of fusion protein DHFR-K237 on endothelial cell proliferation and angiogenesis was investigated. In vitro, DHFR-K237 could completely block VEGF binding to KDR and significantly inhibit the VEGF-mediated proliferation of the human vascular endothelial cells. In vivo, DHFR-K237 inhibited angiogenesis in chick embryo chorioallantoric membrane and tumor growth in nude mice. These results suggest that K237 is an effective antagonist of VEGF binding to KDR, and could be a potential agent for cancer biotherapy.     

Long-term culture of murine erythropoietic progenitor cells in the absence of an adherent layer

Replication of multipotential stem cells in long-term murine bone marrow cell culture is known to depend on the development of an adherent stromal cell layer. In these conditions, restricted haematopoietic progenitor cells have also been generated for up to several months1-3. However, maturation is observed only in the granulocyte/macrophage and megakaryocyte lineages; erythropoiesis appears to be blocked at the earliest burst-forming unit (BFU-E) stage. Addition of exogenous erythropoietin (Epo) or anaemic mouse serum results in full erythropoietic maturation, but it is transient. We describe here a culture system in which production of erythropoietic progenitor cells can be maintained for over 6 months in the absence of an adherent stromal layer and in the absence of added Epo, but in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWSCM). The data indicate that restricted erythroid progenitor cells exist which are capable of extensive self-renewal.     

中药诱导小鼠胚胎干细胞分化为心肌细胞的研究进展

指出了胚胎干细胞(ES细胞)是一类可进行自我复制和更新并向骨骼、软骨、脂肪、神经、肌肉等多个胚层的组织分化的细胞.研究表明:ES细胞移植入缺血心脏后可以分化形成新生心肌细胞,血管内皮细胞等,还可以通过分泌多种生长因子,促进微循环的建立,提高梗死后心脏功能.综述了中药如黄芩苷、葛根素、淫羊藿苷、黄芪、丹参、皂苷、双龙方等诱导小鼠干细胞分化为心肌细胞的研究进展.     

Glioblastoma stem-like cells give rise to tumour endothelium

Glioblastoma (GBM) is among the most aggressive of human cancers. A key feature of GBMs is the extensive network of abnormal vasculature characterized by glomeruloid structures and endothelial hyperplasia. Yet the mechanisms of angiogenesis and the origin of tumour endothelial cells remain poorly defined. Here we demonstrate that a subpopulation of endothelial cells within glioblastomas harbour the same somatic mutations identified within tumour cells, such as amplification of EGFR and chromosome 7. We additionally demonstrate that the stem-cell-like CD133(+) fraction includes a subset of vascular endothelial-cadherin (CD144)-expressing cells that show characteristics of endothelial progenitors capable of maturation into endothelial cells. Extensive in vitro and in vivo lineage analyses, including single cell clonal studies, further show that a subpopulation of the CD133(+) stem-like cell fraction is multipotent and capable of differentiation along tumour and endothelial lineages, possibly via an intermediate CD133(+)/CD144(+) progenitor cell. The findings are supported by genetic studies of specific exons selected from The Cancer Genome Atlas, quantitative FISH and comparative genomic hybridization data that demonstrate identical genomic profiles in the CD133(+) tumour cells, their endothelial progenitor derivatives and mature endothelium. Exposure to the clinical anti-angiogenesis agent bevacizumab or to a γ-secretase inhibitor as well as knockdown shRNA studies demonstrate that blocking VEGF or silencing VEGFR2 inhibits the maturation of tumour endothelial progenitors into endothelium but not the differentiation of CD133(+) cells into endothelial progenitors, whereas γ-secretase inhibition or NOTCH1 silencing blocks the transition into endothelial progenitors. These data may provide new perspectives on the mechanisms of failure of anti-angiogenesis inhibitors currently in use. The lineage plasticity and capacity to generate tumour vasculature of the putative cancer stem cells within glioblastoma are novel findings that provide new insight into the biology of gliomas and the definition of cancer stemness, as well as the mechanisms of tumour neo-angiogenesis.     

Germline transmission of genetically modified primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.     

A clonogenic common myeloid progenitor that gives rise to all myeloid lineages

Haematopoietic stem cells give rise to progeny that progressively lose self-renewal capacity and become restricted to one lineage. The points at which haematopoietic stem cell-derived progenitors commit to each of the various lineages remain mostly unknown. We have identified a clonogenic common lymphoid progenitor that can differentiate into T, B and natural killer cells but not myeloid cells. Here we report the prospective identification, purification and characterization, using cell-surface markers and flow cytometry, of a complementary clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Common myeloid progenitors give rise to either megakaryocyte/erythrocyte or granulocyte/macrophage progenitors. Purified progenitors were used to provide a first-pass expression profile of various haematopoiesis-related genes. We propose that the common lymphoid progenitor and common myeloid progenitor populations reflect the earliest branch points between the lymphoid and myeloid lineages, and that the commitment of common myeloid progenitors to either the megakaryocyte/erythrocyte or the granulocyte/macrophage lineages are mutually exclusive events.     

Proliferating bipotential glial progenitor cells in adult rat optic nerve

We have shown previously that the rat optic nerve contains three types of macroglial cells--oligodendrocytes and two types of astrocytes--which develop as two distinct lineages. Type-1 astrocytes develop from one type of precursor cell beginning at embryonic day 16 (E16), while oligodendrocytes and then type-2 astrocytes develop from a common, bipotential progenitor cell beginning at birth (E21) and postnatal days 7-10 (P7-10), respectively. Here we report that proliferating bipotential oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells are present in the adult rat optic nerve, raising the possibility that these cells are produced continually from self-renewing stem cells throughout life.     

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.     

A common progenitor for haematopoietic and endothelial lineages in the zebrafish gastrula

It has been proposed that haematopoietic and endothelial cells share a common progenitor, termed the haemangioblast. This idea was initially conceived as a result of the observation that these two cell types develop in close proximity to each other within the embryo. Support for this hypothesis was provided by studies on single-cell-derived colonies that can produce both haematopoietic and endothelial cells in vitro. Although these data point towards the existence of a common progenitor for these two lineages, the presence of a bipotential progenitor cell has yet to be demonstrated in vivo. Through the construction of single-cell-resolution fate maps of the zebrafish late blastula and gastrula, we demonstrate that individual cells can give rise to both haematopoietic and endothelial cells. These bipotential progenitors arise along the entire extent of the ventral mesoderm and contribute solely to haematopoietic and endothelial cells. We also find that only a subset of haematopoietic and endothelial cells arise from haemangioblasts. The endothelial descendants of the haemangioblasts all clustered in a specific region of the axial vessels regardless of the location of their progenitors. Our results provide in vivo evidence supporting the existence of the haemangioblast and reveal distinct features of this cell population.     

Reduced function and disassembled microtubules of cultured cardiomyocytes in spaceflight

Lack of gravity during spaceflight has profound effects on cardiovascular system, but little is known about how the cardiomyocytes respond to microgravity. In the present study, the effects of spaceflight on the structure and function of cultured cardiomyocytes were reported. The primary cultures of neonatal rat cardiomyocytes were carried on Shenzhou-6 spacecraft and activated at 4 h in orbit. 8 samples were fixed respectively at 4, 48 and 96 h after launching for immunofluorescence of cytoskeleton, and 2 samples remained unfixed to analyze contractile and secretory functions of the cultures. Ground samples were treated in our laboratory in parallel. After 115 h spaceflight, video recordings displayed that the number of spontaneous beating sites in flown samples decreased significantly, and the cells in the beating aggregate contracted in fast frequency without synchrony. Radioimmunoassay of the medium showed that the atrial natriuretic peptide secreted from flown cells reduced by 59.6%. Confocal images demonstrated the time-dependant disassembly of mirotubules versus unchanged distribution and organization of microfilaments. In conclusion, above results indicate reduced function and disorganized cytoskeleton of cardiomyocytes in spaceflight, which might provide some cellular basis for further investigations to probe into the mechanisms underlying space cardiovascular dysfunction.     

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.     

Derivation of pluripotent epiblast stem cells from mammalian embryos

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.     

Current status of research and application in vascular stents

Cardiovascular diseases have been the leading cause of death in modern society. Using vascular stents to treat these coronary and peripheral artery diseases has been one of the most effective and rapidly adopted medical interventions. During the twenty-five years＇ development of vascular stents, revolutionary cardiovascular stents like drug eluting stents and endothelial progenitor cells capture stents have emerged. In this review, the evolution of vascular stents is summarized, aiming to provide a glimpse into the future of vascular stents. Advanced designs, focusing on the investigations of new substrates, new platforms, new drugs and new biomolecules are currently under evaluation with promising clinical studies. The concept of ＂time sequence functional stent＂ has been raised in this paper. It presents anti-proliferative properties in the first phase after implantation and subsequently support endothelialization. It also shows long-term inertness without release of toxic ions or toxic degradation products. The success of this concept is briefly presented with a clinical study in this model stents.     

Cell fusion-independent differentiation of neural stem cells to the endothelial lineage

Somatic stem cells have been claimed to possess an unexpectedly broad differentiation potential (referred to here as plasticity) that could be induced by exposing stem cells to the extracellular developmental signals of other lineages in mixed-cell cultures. Recently, this and other experimental evidence supporting the existence of stem-cell plasticity have been refuted because stem cells have been shown to adopt the functional features of other lineages by means of cell-fusion-mediated acquisition of lineage-specific determinants (chromosomal DNA) rather than by signal-mediated differentiation. In this study we co-cultured mouse neural stem cells (NSCs), which are committed to become neurons and glial cells, with human endothelial cells, which form the lining of blood vessels. We show that in the presence of endothelial cells six per cent of the NSC population converted to cells that did not express neuronal or glial markers, but instead showed the stable expression of multiple endothelial markers and the capacity to form capillary networks. This was surprising because NSCs and endothelial cells are believed to develop from the ectoderm and mesoderm, respectively. Experiments in which endothelial cells were killed by fixation before co-culture with live NSCs (to prevent cell fusion) and karyotyping analyses, revealed that NSCs had differentiated into endothelial-like cells independently of cell fusion. We conclude that stem-cell plasticity is a true characteristic of NSCs and that the conversion of NSCs to unanticipated cell types can be accomplished without cell fusion.