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.     

Continuous in vitro generation of multipotential stem cell clones from src-infected cultures

A molecular recombinant of Rous sarcoma virus and murine amphotropic leukaemia virus, src(MoMuLV), where the avian src oncogene has been placed under the influence of a murine virus promoter sequence, has been reported. Infection of long-term marrow cultures with this virus led to a dramatic change in the relative numbers of stem cells, granulocyte-macrophage progenitor cells and mature cells found in normal haematopoietic cell development. However, although the balance between self-renewal, differentiation and development was disturbed, injection of the cultured cells into irradiated syngeneic recipients did not lead to the development of leukaemia. Thus, although the control had been 'loosened', the host regulatory mechanisms were sufficient to impose a restraint on unlimited growth of the cells. We now show that the stem cells from the src-infected cultures show a remarkably increased capacity for self-renewal in vitro in situations which are inimical to the maintenance of self-renewal in normal uninfected stem cells and that self-renewal/differentiation can be modified by the culture conditions.     

Identification of the human analogue of a regulator that induces differentiation in murine leukaemic cells

We have recently purified murine granulocyte colony-stimulating factor (G-CSF), a regulatory glycoprotein which stimulates granulocyte colony formation from committed murine precursor cells in semi-solid agar cultures. G-CSF is one of a family of colony-stimulating factors that regulate the growth and differentiation of granulocytes and macrophages. While the other murine CSFs (granulocyte-macrophage (GM)-CSF, macrophage (M)-CSF and multi-CSF) show little or no differentiation-inducing activity on murine myelomonocytic leukaemia cell lines, G-CSF (or MGI-2(6)) is able to induce the production of terminally differentiated cells from WEHI-3B and other myeloid leukaemia cell lines. More importantly, G-CSF-containing materials suppress the self-renewal of myeloid leukaemia stem cells in vitro and the leukaemogenicity of treated myeloid leukaemic cells in vivo. It is important to identify the human analogue of murine G-CSF so that its effectiveness on human myeloid leukaemia cells can be assessed. Here we show that an analogue of G-CSF does exist among the CSFs produced by human cells and that the murine and human molecules show almost complete biological and receptor-binding cross-reactivities to normal and leukaemic murine or human cells. The human G-CSF analogue is identified as a species of CSF that we have previously described as CSF-beta.     

Stem cells, cancer, and cancer stem cells.

Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.     

Multi-genetic events collaboratively contribute to Pten-null leukaemia stem-cell formation

Cancer stem cells, which share many common properties and regulatory machineries with normal stem cells, have recently been proposed to be responsible for tumorigenesis and to contribute to cancer resistance. The main challenges in cancer biology are to identify cancer stem cells and to define the molecular events required for transforming normal cells to cancer stem cells. Here we show that Pten deletion in mouse haematopoietic stem cells leads to a myeloproliferative disorder, followed by acute T-lymphoblastic leukaemia (T-ALL). Self-renewable leukaemia stem cells (LSCs) are enriched in the c-Kit(mid)CD3(+)Lin(-) compartment, where unphosphorylated beta-catenin is significantly increased. Conditional ablation of one allele of the beta-catenin gene substantially decreases the incidence and delays the occurrence of T-ALL caused by Pten loss, indicating that activation of the beta-catenin pathway may contribute to the formation or expansion of the LSC population. Moreover, a recurring chromosomal translocation, T(14;15), results in aberrant overexpression of the c-myc oncogene in c-Kit(mid)CD3(+)Lin(-) LSCs and CD3(+) leukaemic blasts, recapitulating a subset of human T-ALL. No alterations in Notch1 signalling are detected in this model, suggesting that Pten inactivation and c-myc overexpression may substitute functionally for Notch1 abnormalities, leading to T-ALL development. Our study indicates that multiple genetic or molecular alterations contribute cooperatively to LSC transformation.     

Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells

An emerging concept in the field of cancer biology is that a rare population of 'tumour stem cells' exists among the heterogeneous group of cells that constitute a tumour. This concept, best described with human leukaemia, indicates that stem cell function (whether normal or neoplastic) might be defined by a common set of critical genes. Here we show that the Polycomb group gene Bmi-1 has a key role in regulating the proliferative activity of normal stem and progenitor cells. Most importantly, we provide evidence that the proliferative potential of leukaemic stem and progenitor cells lacking Bmi-1 is compromised because they eventually undergo proliferation arrest and show signs of differentiation and apoptosis, leading to transplant failure of the leukaemia. Complementation studies showed that Bmi-1 completely rescues these proliferative defects. These studies therefore indicate that Bmi-1 has an essential role in regulating the proliferative activity of both normal and leukaemic stem cells.     

Somatic support cells restrict germline stem cell self-renewal and promote differentiation

Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.     

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.     

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.     

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.     

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.     

Wnt signalling in stem cells and cancer

The canonical Wnt cascade has emerged as a critical regulator of stem cells. In many tissues, activation of Wnt signalling has also been associated with cancer. This has raised the possibility that the tightly regulated self-renewal mediated by Wnt signalling in stem and progenitor cells is subverted in cancer cells to allow malignant proliferation. Insights gained from understanding how the Wnt pathway is integrally involved in both stem cell and cancer cell maintenance and growth in the intestinal, epidermal and haematopoietic systems may serve as a paradigm for understanding the dual nature of self-renewal signals.     

Isolation of cell lines possessing functional and serological properties resembling those of thymocyte precursors

Thymocytes develop from a committed haematopoietic progenitor, referred to as a prothymocyte. They are uniquely capable of migrating to and restoring the thymus of a lethally irradiated host, a property which has been exploited as a specific assay for these cells. Like other committed haematopoietic progenitors, prothymocytes are found only in small numbers in even the richest sources (0.05-1.0% of the nucleated cells in bone marrow). Purification has proved difficult both in terms of finding a suitable starting material and in the degree of enrichment achieved. We now report the isolation of cloned lines of cells with some of the serological and functional properties of prothymocytes. One of these lines has been in continuous culture for almost 2 years. When injected into irradiated recipients, cells from this line migrate to the thymus and there develop into cells which resemble normal cortical thymocytes.     

Identification and characterization of an inhibitor of haemopoietic stem cell proliferation

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.     

Purification and unique properties of mammary epithelial stem cells

Elucidation of the cellular and molecular mechanisms that maintain mammary epithelial tissue integrity is of broad interest and paramount to the design of more effective treatments for breast cancer. Evidence from both in vitro and in vivo experiments suggests that mammary cell differentiation is a hierarchical process originating in an uncommitted stem cell with self-renewal potential. However, analysis of the properties and regulation of mammary stem cells has been limited by a lack of methods for their prospective isolation. Here we report the use of multi-parameter cell sorting and limiting dilution transplant analysis to demonstrate the purification of a rare subset of adult mouse mammary cells that are able individually to regenerate an entire mammary gland within 6 weeks in vivo while simultaneously executing up to ten symmetrical self-renewal divisions. These mammary stem cells are phenotypically distinct from and give rise to mammary epithelial progenitor cells that produce adherent colonies in vitro. The mammary stem cells are also a rapidly cycling population in the normal adult and have molecular features indicative of a basal position in the mammary epithelium.     

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.     

Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.

Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.