Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.     

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.     

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.     

Distinct contribution of stem and progenitor cells to epidermal maintenance

The skin interfollicular epidermis (IFE) is the first barrier against the external environment and its maintenance is critical for survival. Two seemingly opposite theories have been proposed to explain IFE homeostasis. One posits that IFE is maintained by long-lived slow-cycling stem cells that give rise to transit-amplifying cell progeny, whereas the other suggests that homeostasis is achieved by a single committed progenitor population that balances stochastic fate. Here we probe the cellular heterogeneity within the IFE using two different inducible Cre recombinase–oestrogen receptor constructs targeting IFE progenitors in mice. Quantitative analysis of clonal fate data and proliferation dynamics demonstrate the existence of two distinct proliferative cell compartments arranged in a hierarchy involving slow-cycling stem cells and committed progenitor cells. After wounding, only stem cells contribute substantially to the repair and long-term regeneration of the tissue, whereas committed progenitor cells make a limited contribution.     

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.     

Myocyte renewal and ventricular remodelling.

Remaining young at heart is a desirable but elusive goal. Unbeknown to us, however, myocyte regeneration may accomplish just that. Continuous cell renewal in the adult myocardium was thought to be impossible, but multipotent cardiac stem cells may be able to renew the myocardium and, under certain circumstances, can be coaxed to repair the broken heart after infarction.     

Identification of adult nephron progenitors capable of kidney regeneration in zebrafish

Loss of kidney function underlies many renal diseases. Mammals can partly repair their nephrons (the functional units of the kidney), but cannot form new ones. By contrast, fish add nephrons throughout their lifespan and regenerate nephrons de novo after injury, providing a model for understanding how mammalian renal regeneration may be therapeutically activated. Here we trace the source of new nephrons in the adult zebrafish to small cellular aggregates containing nephron progenitors. Transplantation of single aggregates comprising 10-30 cells is sufficient to engraft adults and generate multiple nephrons. Serial transplantation experiments to test self-renewal revealed that nephron progenitors are long-lived and possess significant replicative potential, consistent with stem-cell activity. Transplantation of mixed nephron progenitors tagged with either green or red fluorescent proteins yielded some mosaic nephrons, indicating that multiple nephron progenitors contribute to a single nephron. Consistent with this, live imaging of nephron formation in transparent larvae showed that nephrogenic aggregates form by the coalescence of multiple cells and then differentiate into nephrons. Taken together, these data demonstrate that the zebrafish kidney probably contains self-renewing nephron stem/progenitor cells. The identification of these cells paves the way to isolating or engineering the equivalent cells in mammals and developing novel renal regenerative therapies.     

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts,which represent 50%of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes.     

Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors

Haematopoiesis is maintained by a hierarchical system where haematopoietic stem cells (HSCs) give rise to multipotent progenitors, which in turn differentiate into all types of mature blood cells. HSCs maintain themselves for the lifetime of the organism because of their ability to self-renew. However, multipotent progenitors lack the ability to self-renew, therefore their mitotic capacity and expansion potential are limited and they are destined to eventually stop proliferating after a finite number of cell divisions. The molecular mechanisms that limit the proliferation capacity of multipotent progenitors and other more mature progenitors are not fully understood. Here we show that bone marrow cells from mice deficient in three genes genetically downstream of Bmi1--p16Ink4a, p19Arf and Trp53 (triple mutant mice; p16Ink4a and p19Arf are alternative reading frames of the same gene (also called Cdkn2a) that encode different proteins)--have an approximately 10-fold increase in cells able to reconstitute the blood long term. This increase is associated with the acquisition of long-term reconstitution capacity by cells of the phenotype c-kit+Sca-1+Flt3+CD150-CD48-Lin-, which defines multipotent progenitors in wild-type mice. The pattern of triple mutant multipotent progenitor response to growth factors resembles that of wild-type multipotent progenitors but not wild-type HSCs. These results demonstrate that p16Ink4a/p19Arf and Trp53 have a central role in limiting the expansion potential of multipotent progenitors. These pathways are commonly repressed in cancer, suggesting a mechanism by which early progenitor cells could gain the ability to self-renew and become malignant with further oncogenic mutations.     

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.     

Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing

Mammalian ageing is associated with reduced regenerative capacity in tissues that contain stem cells. It has been proposed that this is at least partially caused by the senescence of progenitors with age; however, it has not yet been tested whether genes associated with senescence functionally contribute to physiological declines in progenitor activity. Here we show that progenitor proliferation in the subventricular zone and neurogenesis in the olfactory bulb, as well as multipotent progenitor frequency and self-renewal potential, all decline with age in the mouse forebrain. These declines in progenitor frequency and function correlate with increased expression of p16INK4a, which encodes a cyclin-dependent kinase inhibitor linked to senescence. Ageing p16INK4a-deficient mice showed a significantly smaller decline in subventricular zone proliferation, olfactory bulb neurogenesis, and the frequency and self-renewal potential of multipotent progenitors. p16INK4a deficiency did not detectably affect progenitor function in the dentate gyrus or enteric nervous system, indicating regional differences in the response of neural progenitors to increased p16INK4a expression during ageing. Declining subventricular zone progenitor function and olfactory bulb neurogenesis during ageing are thus caused partly by increasing p16INK4a expression.     

Platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell

The mitogens which modulate cell-cell interactions during development of the central nervous system are unknown. One of the few interactions sufficiently well understood to allow identification of such molecules involves the two glial lineages which make up the rat optic nerve. One population of glial cells in this tissue, the type-1 astrocytes, secrete a soluble factor(s) which promotes division of a second population of bipotential oligodendrocyte/type-2 astrocyte (O-2A) progenitor cells; these progenitors give rise to oligodendrocytes, which myelinate large axons in the CNS, and type-2 astrocytes, which enwrap bare axons at nodes of Ranvier. Type-1 astrocytes also promote progenitor motility, and inhibit the premature differentiation of progenitors into oligodendrocytes which occur when these cells are grown in the absence of type-1 astrocytes. We have now found that platelet-derived growth factor mimics the effects of type-1 astrocytes on O-2A progenitor cells, and antibodies to PDGF block the effects of type-1 astrocytes.     

Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9

Leukaemias and other cancers possess a rare population of cells capable of the limitless self-renewal necessary for cancer initiation and maintenance. Eradication of these cancer stem cells is probably a critical part of any successful anti-cancer therapy, and may explain why conventional cancer therapies are often effective in reducing tumour burden, but are only rarely curative. Given that both normal and cancer stem cells are capable of self-renewal, the extent to which cancer stem cells resemble normal tissue stem cells is a critical issue if targeted therapies are to be developed. However, it remains unclear whether cancer stem cells must be phenotypically similar to normal tissue stem cells or whether they can retain the identity of committed progenitors. Here we show that leukaemia stem cells (LSC) can maintain the global identity of the progenitor from which they arose while activating a limited stem-cell- or self-renewal-associated programme. We isolated LSC from leukaemias initiated in committed granulocyte macrophage progenitors through introduction of the MLL-AF9 fusion protein encoded by the t(9;11)(p22;q23). The LSC were capable of transferring leukaemia to secondary recipient mice when only four cells were transferred, and possessed an immunophenotype and global gene expression profile very similar to that of normal granulocyte macrophage progenitors. However, a subset of genes highly expressed in normal haematopoietic stem cells was re-activated in LSC. LSC can thus be generated from committed progenitors without widespread reprogramming of gene expression, and a leukaemia self-renewal-associated signature is activated in the process. Our findings define progression from normal progenitor to cancer stem cell, and suggest that targeting a self-renewal programme expressed in an abnormal context may be possible.     

Formation of a functional thymus initiated by a postnatal epithelial progenitor cell

The thymus is essential for the generation of self-tolerant effector and regulatory T cells. Intrathymic T-cell development requires an intact stromal microenvironment, of which thymic epithelial cells (TECs) constitute a major part. For instance, cell-autonomous genetic defects of forkhead box N1 (Foxn1) and autoimmune regulator (Aire) in thymic epithelial cells cause primary immunodeficiency and autoimmunity, respectively. During development, the thymic epithelial rudiment gives rise to two major compartments, the cortex and medulla. Cortical TECs positively select T cells, whereas medullary TECs are involved in negative selection of potentially autoreactive T cells. It has long been unclear whether these two morphologically and functionally distinct types of epithelial cells arise from a common bi-potent progenitor cell and whether such progenitors are still present in the postnatal period. Here, using in vivo cell lineage analysis in mice, we demonstrate the presence of a common progenitor of cortical and medullary TECs after birth. To probe the function of postnatal progenitors, a conditional mutant allele of Foxn1 was reverted to wild-type function in single epithelial cells in vivo. This led to the formation of small thymic lobules containing both cortical and medullary areas that supported normal thymopoiesis. Thus, single epithelial progenitor cells can give rise to a complete and functional thymic microenvironment, suggesting that cell-based therapies could be developed for thymus disorders.     

The earliest thymic progenitors for T cells possess myeloid lineage potential

There exists controversy over the nature of haematopoietic progenitors of T cells. Most T cells develop in the thymus, but the lineage potential of thymus-colonizing progenitors is unknown. One approach to resolving this question is to determine the lineage potentials of the earliest thymic progenitors (ETPs). Previous work has shown that ETPs possess T and natural killer lymphoid potentials, and rare subsets of ETPs also possess B lymphoid potential, suggesting an origin from lymphoid-restricted progenitor cells. However, whether ETPs also possess myeloid potential is unknown. Here we show that nearly all ETPs in adult mice possess both T and myeloid potential in clonal assays. The existence of progenitors possessing T and myeloid potential within the thymus is incompatible with the current dominant model of haematopoiesis, in which T cells are proposed to arise from lymphoid-. Our results indicate that alternative models for lineage commitment during haematopoiesis must be considered.     

CCL25/CCR9与骨髓祖细胞胸腺归巢

在骨髓中多种祖细胞有T细胞系潜能,但骨髓祖细胞胸腺归巢具有选择性.骨髓祖细胞胸腺归巢是多级粘附分子和趋化因子的级联过程,趋化因子CCL25及其受体CCR9在这一过程中发挥了重要作用.综述了CCL25/CCR9的结构特征及其对骨髓祖细胞胸腺归巢的作用和调节机制.