Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment

Haematopoietic progenitor cells proliferate and mature in semisolid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF). They also proliferate in association with marrow-derived stromal cells although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells. They are prime candidates for selectively retaining HCGFs in the stromal layer; they influence embryonic morphogenesis and cyto-differentiation and they may regulate haematopoiesis. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.     

Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia

Normal haematopoietic cell regulation involves interaction between marrow stromal cells and haematopoietic progenitor cells which may be facilitated by specific recognition and adhesion. Some leukaemogenic events might produce a selective growth advantage by altering this regulatory network, possibly by diminishing the capacities of cells to adhere to stromal elements. Using an in vitro culture system which allows investigation of adhesion to stromal layers and subsequent colony formation by blast colony-forming cells (B1-CFC) in normal marrow and Ph+ chronic myeloid leukaemic (CML) blood, we compared the adhesive properties of normal and malignant progenitor cells. We present evidence that altered adhesive interactions between primitive progenitor cells and marrow stromal cells occur in CML.     

Stimulation of multipotential, erythroid and other murine haematopoietic progenitor cells by adherent cell lines in the absence of detectable multi-CSF (IL-3)

It is well established that murine multipotential and committed erythroid progenitor cells require the presence of a glycoprotein, termed multi-CSF (multi-colony-stimulating factor, IL-3) for clonal proliferation and differentiation in vitro. The initial proliferation of these cells can also be stimulated by two other glycoproteins, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), although continued proliferation and differentiation requires the subsequent presence of multi-CSF. Here we report the stimulation of multipotential, erythroid and other haematopoietic progenitor cells by a number of adherent cell lines including a cloned bone marrow cell line (B.Ad). The positive cell lines, as feeder layers, exhibit colony-stimulating, erythropoietin-like and burst-promoting (BPA) activities. Optimal erythropoietic stimulation by the B.Ad line requires close cell-cell contact. The cell lines also support the in vitro clonal growth of multipotential colony-forming cells and progenitors of six other haematopoietic lineages. The biological activities observed seem not to be mediated by known multipotential or erythroid colony-stimulating factors (multi-CSF, IL-3, MCGF, HCGF, PSF, BPA).     

小鼠骨髓造血基质细胞体外培养及其功能的研究

采用细胞培养从正常LACA雄性小鼠骨髓中分离得到1株造血基质细胞（HSC），该细胞胞体较大，呈多角形，不吞噬酵母多糖，HSC粘壁层有其培养上清液均具有明显刺激CFU－GM体外增殖分化的功能，将HSC输注到经射线照射的雌性小鼠体内，PCR技术检测结果显示，HSC具有重建受体小鼠造血微环境的作用。     

Host origin of marrow stromal cells following allogeneic bone marrow transplantation

Although it is generally agreed that stromal cells are important in the regulation of haematopoietic cell development, the origin of these phenotypically diverse cells has been a subject for debate for more than 50 years. Data which support the concept of a separate origin for the haematopoietic stem cell and the marrow stroma are derived from cytogenetic or enzyme marker studies of explanted and expanded stromal cells grown under conditions that do not allow haematopoiesis in vitro. Recent evidence in man and in mouse suggesting that the stromal cells capable of transferring the haematopoietic microenvironment in vitro are transplantable seemingly questions this dichotomy, one interpretation being the existence of a common haematopoietic/stromal 'stem cell'. We used in situ hybridization to discriminate donor cells from host in blood and bone marrow samples obtained from patients with functioning sex-mismatched but HLA-identical allografts. Without exception, marrow-derived stromal cells that proliferate in long-term cultures were found to be of host genotype, whereas the macrophage component of the adherent layer in these cultures originated from the donor.     

Defective ability to self-renew in vitro of highly purified primitive haematopoietic cells

Stromal cells play a critical role in haematopoiesis, both in a permissive and, probably, in a directive manner. Study of the interactions between stromal cells and haematopoietic stem cells, however, is difficult to perform using whole bone marrow, in which stem cells are indistinguishable from precursor cells and maturing haematopoietic cells, and where stromal and haematopoietic cells co-exist in a heterogeneous mixture. We have purified primitive haematopoietic spleen colony-forming cells (CFU-S) using fluorescence-activated cell sorting (FACS) and produced CFU-S populations which approach 100% purity (ref. 6 and B.I.L. and E.S., in preparation). This cell population is devoid of significant stromal cells and mature haematopoietic cells. Here, we report that when purified CFU-S are seeded onto a stromal adherent layer in vitro, foci of haematopoietic cells develop within the stroma followed by production of a wave of maturing and mature progeny. However, self-renewal of CFU-S does not occur and haematopoietic activity rapidly declines, indicating that caution should be applied in the use of highly purified stem cells for human bone marrow transplantation.     

Establishment and characterization of murine bone marrow stromal macrophage line

The authors established a murine bone marrow stromal cell line QXMSC1 by discarding the suspensible hematopoietic cells and passaging the adherent stromal cells many times in long-term bone marrow culture. QXMSC1 cells have been passaged 65 times for 15 months and immortalized. The mean number of QXMSC1 chromosomes is 66 ± 4. The appearance of the cells is elliptical. There are many pseudopods and mononuclei under the optical microscope. Many lisosomes and phagosomes in cytoplasm exist under the transmission electron microscope. There are no desmosome junction between the cells, no lipid drops, no intermediate filament. Vimentin is positive and keratin is negative by stain of immunocytochemistry. Nonspecific lipase staining is positive. These results indicate that QXMSC1 cells are murine bone marrow macrophages.     

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.     

贴壁及悬浮培养的Trex-293细胞生长及其重组腺病毒制备产率的比较分析

为了提高病毒产率并避免制备的重组腺病毒产物中残留的动物血清,将贴壁生长的Trex-293细胞用无动物血清的CD293培养基在搅拌式培养瓶中悬浮培养。分析比较其生长特征及不同条件下的病毒产率。结果表明悬浮培养的细胞生长为稳定状态的时间比贴壁培养时稍长,但每次以(4—6)×105cells/mL密度传代。(3—4)d后基本达到2×106cells/mL,21 d达3×106cells/mL以上,比贴壁细胞密度约高3倍。培养初期,悬浮细胞存活率稍低,培养14 d开始维持90%以上,细胞倍增时间约32 h。而贴壁细胞存活率是基本维持在95%以上。重组腺病毒以200,500和1 000 VP/cell的比例感染悬浮细胞所获产率均值各为18 000,14 000和990 VP/cell,而1 000 VP/cell的比例感染贴壁细胞所获产率均值为10 500 VP/cell。结论为Trex-293细胞用无动物血清的培养基在搅拌式培养瓶中可悬浮培养,当重组腺病毒感染比例为200 VP/cell,感染后48 h,细胞存活率50%时收获有利于提高病毒产率,且高于感染贴壁细胞所获产率。     

A novel human lymphokine that inhibits haematopoietic progenitor cell proliferation

T lymphocytes in culture synthesize and secrete a variety of factors that activate and guide the differentiation, replication and maturation of haematopoietic cells in vitro. Malignant T-cell lines as well as T-cell hybridomas producing several of these factors have been established. We report here a factor produced by a human cell line that exerts a potent inhibitory effect on the growth of bone marrow progenitor cells. The properties of this factor, which we have termed colony-inhibiting lymphokine ( CIL ), differ from other inhibitors of haematopoietic progenitor cell proliferation, but resemble those of a T-cell-derived factor causally linked with some cases of severe aplastic anaemia in humans. Sensitivity of cells to this factor appears to correlate positively with expression of HLA-DR surface antigens.     

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.     

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.     

Erythropoietin-stimulated erythropoiesis in long-term bone marrow culture.

The proliferation of multipotential haematopoietic stem cells (CFU-S) is possible in some long-term bone marrow cultures. Granulocyte and macrophage progenitor (CFU-C) and megakaryocyte precursor cells (CFU-M) are present in these cultures and undergo full development into mature cells. In contrast, while immature erythroid progenitors ('early' BFU-E) are maintained in long-term culture, none of the more differentiated progeny (CFU-E) have been detected, and no morphologically recognisable erythroid cells have been observed. We now describe a modified culture system in which the 'early' BFU-E develop into 'late' BFU-E in response to added erythropoietin. Further maturation of these cells into CFU-E and non-nucleated erythrocytes can be achieved by mechanical agitation of the long-term cultures or by transferring the cells into dishes which do not allow cell attachment to occur.     

南方鲇卵巢滤泡细胞和卵膜生成的超微结构研究

滤泡细胞来源于卵巢基质细胞,其发育过程分为:零散卵泡膜细胞期、单层扁平卵泡膜细胞期、多层扁平 卵泡膜细胞期、立方形颗粒细胞期、柱状颗粒细胞期、颗粒细胞分泌期6个时期.多层细胞的滤泡可以分为外膜 层、鞘膜细胞层、粗纤维层、细纤维层、滤泡细胞层5个亚层.颗粒细胞具丰富的微丝、线粒体、内质网、核糖 体,高尔基体也丰富而发达.滤泡细胞具有生成次级卵膜、合成卵黄蛋白并加工成卵黄前体颗粒和中间颗粒、产 生类固醇激素和协助排卵等作用. 卵膜包括初级卵膜和次级卵膜.初级卵膜分为3个亚层,由卵母细胞依次产生的3个亚层最终融合并致密化 形成放射带.而次级卵膜在卵黄合成后期由滤泡细胞产生.     

Fibronectin inhibits the terminal differentiation of human keratinocytes

In the epidermis proliferation of keratinocytes is restricted to the basal layer, which is in contact with the basement membrane, and cells undergo terminal differentiation as they move upwards through the suprabasal layers. In stratified cultures of human keratinocytes, upward migration is a consequence, not a cause, of terminal differentiation and occurs because keratinocytes become less adhesive to their substratum and to one another. Most keratinocytes can be induced to differentiate to completion by placing them in suspension in methylcellulose: within 12 h DNA synthesis is irreversibly inhibited and by 24 h most cells express involucrin (ref 4; P. A. Hall, J.C.A. and F.M.W., unpublished observations). Here we report that when fibronectin is added to the methylcellulose, keratinocytes still withdraw from the cell cycle, but induction of involucrin expression is largely inhibited. The effect of fibronectin is concentration- and time-dependent and is mediated by a receptor of the integrin family. These results provide an explanation for why overt terminal differentiation is normally restricted to suprabasal cells, whereas cell-cycle withdrawal occurs within the basal layer; they also have important implications for the mechanism of epidermal wound healing. Furthermore, our data show that the binding of an extracellular matrix protein to its receptor can regulate differentiated gene expression in the absence of changes in cell shape.     

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.     

Evidence for migration of oligodendrocyte--type-2 astrocyte progenitor cells into the developing rat optic nerve

Formation of myelinated tracts in central nervous system (CNS) regions such as the optic nerve seems to depend on two glial cell types, both of which derive from a common progenitor cell. This oligodendrocyte--type-2 astrocyte (O-2A) progenitor cell gives rise to oligodendrocytes, which produce internodal myelin sheaths, and to type-2 astrocytes, which extend fine processes in the region of the nodal axolemma. The optic nerve also contains a third glial cell, the type-1 astrocyte, which derives from a separate precursor. These three glial cells develop in a fixed sequence over a two-week period: type-1 astrocytes appear at embryonic day 16 (E16), oligodendrocytes at the day of birth (E21 or postnatal day P0), and type-2 astrocytes between P8 and P10. Type-1 astrocytes secrete a potent mitogen which causes expansion of the O-2A progenitor cell population in vitro. Here, we report that dividing O-2A progenitor cells are highly motile and seem to migrate from the brain into the optic nerve, beginning at its chiasmal end. Our results indicate that long-distance migration along the neural axis is characteristic only of progenitors of the O-2A lineage and may serve to distribute these cells to regions of the CNS that will become myelinated. These results also suggest that the intrinsic neuroepithelial cells of the optic stalk may be even more restricted than previously thought, giving rise only to type-1 astrocytes.     

A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium

We have identified a cell type in 7-day-old rat optic nerve that differentiates into a fibrous astrocyte if cultured in the presence of fetal calf serum and into an oligodendrocyte if cultured in the absence of serum. In certain culture conditions some of these cells acquire a mixed phenotype, displaying properties of both astrocytes and oligodendrocytes. These observations suggest that fibrous astrocytes and oligodendrocytes develop from a common progenitor cell and provide a striking example of developmental plasticity and environmental influence in the differentiation of CNS glial cells.