Ultrastructural and immunohistochemical studies of intralobular fibroblasts in human submandibular gland: the recognition of a 'CD34 positive reticular network' connected by gap junctions

In order to clarify the ultrastructural and immunophenotypic characteristics of intralobular stromal cells in the human submandibular gland, normal tissues were examined from 25 patients by conventional histology of wax sections, light microscope immunohistochemistry, conventional thin-sectioning transmission electron microscopy, immuno-electronmicroscopy (for the CD34 antigen) and scanning electron microscopy. Spindled stromal cells had abundant rough endoplasmic reticulum cisternae typical of fibroblasts. Some actin filaments and microtubules were seen, but the filaments lacked the focal densities of smooth-muscle, nor was a lamina present. Fibroblasts exhibited long slender processes which associated with other fibroblasts by means of gap junctions and with mononuclear cells (lymphocytes, plasma cells, macrophages) by means of close appositions of membrane. Immunoelectron microscopy for CD34 revealed positive staining over the fibroblast surfaces but not over the mononuclear cells. The novel observation of gap junctions suggests a tissue closely organized as a network in physical and perhaps physiological terms. This network also incorporates mononuclear cells. The possible role in immunosurveillance of this CD34-positive reticular network is discussed.     

Gap junctions and nerve terminals among stromal cells in human fallopian tube ampullary mucosa

The purpose of the present study is to investigate the ultrastructure and immunohistochemistry of the stromal cells and terminal nerve fibers in human fallopian tube ampullar mucosa to achieve a detailed characterization of this tissue to permit a better assessment of possible functions. Tissues were obtained during surgery or at autopsy from 26 patients. Specimens were studied by the conventional histologic technique, immunohistochemistry (Cx43, synaptophysin, neurofilament proteins, and S-100 protein), and electron microscopy. Gap junction and nerve terminal frequency between stromal cells were studied by direct assessment on ultrathin sections in the transmission electron microscope. Gap junctions were observed between the cytoplasmic processes of subepithelial stromal cells. There were approximately 23 gap junctions per 73 nucleated stromal spindle cells. Immunohistochemistry using Cx43 antibody confirmed the dot-like distribution of gap junctions. The frequent and intimate association of stromal cell processes with nerve terminals was also demonstrated. Nerve terminals were immunostained by antibodies to nerve-specific molecules and ultrastructurally as axonal profiles containing dense-cored granules or empty vesicles. Analysis of nerve terminal frequency revealed 18 nerve profiles containing 51 axonal profiles per 73 nucleated stromal spindle cells. The present paper documents the participation of autonomic nerve endings and gap junctions in the stromal cell network in human fallopian tube stroma. Similarities to the unique anatomical unit referred to as the 'neuro-reticular complex' in bone marrow tissue (Yamazaki and Allen, 1990) are discussed.     

Sympathetic innervation and noradrenaline content of normal human thyroid tissue from fetal, young, and elderly subjects

In man, as well as in the mouse, there is morphologic and functional evidence for a direct, stimulatory influence of the sympathetic nervous system on the secretion of thyroid hormone by noradrenaline (NA), released from interfollicular adrenergic nerve terminals. In mice and rats, an age-related reduction of the sympathetic innervation of the thyoid has recently been observed. In the present study, possible age-related variations of the sympathetic innervation and the concentration of NA in human thyroid tissue were examined. Interfollicular adrenergic nerve terminals were studied by fluorescence histochemistry, and the tissue concentration of NA was measured by fluorometry. In apparently normal thyroid tissue, obtained from fetuses, young (20-45), and elderly (greater than 60) euthyroid people with thyroid cancer or hyperparathyroidism, the number of interfollicular adrenergic nerve terminals appeared to be reduced with increasing age, and the thyroid tissue concentration of NA was significantly lower in elderly than in young people. These findings may have functional importance.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Cell-cell interaction in prostate gene regulation and cytodifferentiation

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57+ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57+ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57+ cells are reconstituted with stromal cells. The CD44+ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44+ cells are cocultured with stromal cells. Our studies show that cell-cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.     

Connexin43 gap junctions in normal, regenerating, and cultured mouse bone marrow and in human leukemias: their possible involvement in blood formation

Communicating channels called gap junctions are thought to play a ubiquitous part in cell growth and development. Based on earlier work, we have recently found functional evidence of their presence in human and mouse bone marrow. In this study we studied the cell-type association of the gap junction channel-forming protein, connexin, in mouse and human bone marrow under different physiological and pathological conditions and tested the pathway of communication in bone marrow cultures. For high-resolution antigen demonstration we took advantage of semi-thin resin sections, antigen retrieval methods, immunofluorescence, and confocal laser scanning microscopy. Connexin43 (Cx43) and its mRNA were consistently expressed in human and rodent marrow. Cx37 was found only in the arteriolar endothelium, but neither Cx32 nor -26 were expressed. In tissue sections, the immunostained junctions appeared as dots, which were digitally measured and counted. Their average size was 0.40 mm in human and 0.49 mm in mice marrow. There were at least twice as many gap junctions in the femoral midshaft of 6-week-old mice (1.75 x 10(5)/mm3) as in those older than 12 weeks (0.89 x 10(5)/mm3). Most Cx43 was associated with collagen III+ endosteal and adventitial stromal cells and with megakaryocytes. Elsewhere, they were few and randomly distributed between all kinds of hematopoietic cells. In the femoral epiphysis of juvenile mice, stromal cell processes full of Cx43 enmeshed three to six layers of hematopoietic cells near the endosteum. The same pattern was seen in the midshaft of regenerating mouse marrow 3 to 5 days after cytotoxic treatment with 5-fluorouracil. Functional tests in cultures showed the transfer of small fluorescent dyes, Lucifer Yellow and 2',7'-bis-(2-carboxyethyl)-5, 6-carboxyfluorescein, between stromal cells and in rare cases between stromal and hematopoietic cells too. The stromal cells were densely packed with Cx43 and we found aggregates of connexon particles in their membrane replicas. In normocellular human bone marrow, gap junctions were as rare as in adult mouse and similarly distributed, except that they were also on adipocytic membranes. In a few leukemic samples, characterized by an increased stromal/hematopoietic cell ratio, there were two- to fourfold more Cx43 (2.8 x 10(5) to 3.9 x 10(5)/mm3) than in the normal (1.0 x 10(5) to 1.2 x 10(5)/mm3). The cases included a hypoplastic acute lymphoblastic leukemia, an acute myeloid leukemia (French-American-British classification M4-5), a case of myelodysplastic syndrome with elevated number of megakaryocytes, and a CD34+ acute hemoblastosis, probably acute myeloid leukemia (French-American-British classification M7). Taken together, our results indicate that direct cell-cell communication may be involved in hematopoiesis, ie, in developmentally active epiphyseal bone marrow and when there is a demand for progenitors in regeneration. However, gap junctions may not play as important a role in resting adult hematopoiesis and in leukemias.     

Comparison of angiomyofibroblastoma and aggressive angiomyxoma in both sexes: four cases composed of bimodal CD34 and factor XIIIa positive dendritic cell subsets

Aggressive angiomyxoma (AA) is a distinctive, locally aggressive, fibromyxoid tumor of the pelvic and genital soft tissues. AA is of unknown histogenesis but the cytologically bland spindled tumor cells, which surround characteristic variegated blood vessels, show fibroblastic or myofibroblastic features. AA may be related to angiomyofibroblastoma (AMF), another cytologically bland fibromyxoid genital spindle cell tumor with variable myoid differentiation that does not, as a rule, recur. Recently, CD34+ primitive fibroblasts and factor XIIIa+ dendritic histiocytes have been found in varying combination in many fibrovascular, fibrohistiocytic, and myxoid soft tissue tumors. Both cells belong to the microvascular unit, a tissue responsible for stromal repair and remodeling and angiogenesis. To determine if these ubiquitous stromal cells participate in the histogenesis of AA and AMF, we examined two scrotal tumors, one AA with multiple recurrences and one AMF, for the presence of CD34+ and FXIIIa+ dendritic cell subsets. For comparison, a vaginal AMF and a pararectal AA in a woman were included. We also studied actins and desmin to detect myofibroblastic differentiation, and, through double labeling studies, assessed hormone receptors and the cell cycle marker Ki 67 in the different cell subsets. The AA showed unusual cytologic atypia and was initially diagnosed as liposarcoma. It massively recurred four times over 12 years, the first time after seven years. The histologic appearance was fairly constant over the years. The scrotal AMF was a circumscribed 6 cm mass in a 37 year old man. In both cases, most tumor cells were wavy and fibrillar, spindled, stellate, or polygonal fibroblast-like CD34+ dendritic cells. Depending on the area examined, a 20-50% subset of dendritic cells showed both nuclear and cytoplasmic staining for FXIIIa. Actin+ cells were rare but vessels had actin+ myopericytes, although a small focus of the initial male AA was desmin positive. The recurring AA expressed androgen receptors and had Ki 67 index of 10-20% in "hot spots" of the primary and up to 30% in recurrent tumors. The scrotal AMF widely expressed androgen and progesterone receptors with focal estrogen receptor positivity and the Ki 67 index was 10%. Both CD34+ fibroblasts and FXIIIa+ histiocytes were present in the Ki 67+ cycling fraction in both the male AA and AMF and both cell types expressed androgen receptors. The female pararectal AA had more focal CD34 reactivity, particularly in perivascular fibroblasts and these cells were admixed with small FXIIIa+ cells. The vaginal AMF was strongly desmin+ and variably to weakly CD34+ with 20% FXIIIa+ dendritic cells and Ki 67 index of 2%. The vaginal AMF strongly expressed estrogen, progesterone, and androgen receptors. In conclusion our data suggest that at least some AA and AMF are myxoid fibrohistiocytic tumors composed of CD34+ fibroblasts and FXIIIa+ dendritic histiocytes. In our tumors, neoplastic CD34+ dendritic fibroblasts showed predominantly myxo-collagenous differentiation with prominent myofibroblastic differentiation in only one desmin+ vaginal AMF. Our results support the notion that AMF and AA are part of a morphologic and histogenetic continuum of myxofibrous and myoid tumors that may arise due to interactions between microvascular CD34+ fibroblasts and FXIIIa+ histiocytes. CD34 and FXIIIa reactivity may be underappreciated in these tumors and is more important when considered histogenetically and biologically rather than in classifying individual neoplasms. Hormonal stimulation of proliferating pelvico-gential microvascular dendritic cells appears to play a role in the morphogenesis of both tumors.     

Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules

We have investigated the role of specific components of the thymic stroma during development of CD4-8-T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+ 44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44- precursors, develop to the CD4+ 8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.     

Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.     

In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential

Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38- cells. CD34+CD38- cells cocultivated on the murine stromal line S17 generated predominantly CD19(+) B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the immunophenotypically uncommitted CD19- cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38- cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38- cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38- cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.     

Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha

In situ estrogen synthesis by hormone-dependent breast cancers could potentially regulate cellular proliferation through autocrine or paracrine mechanisms. Several biochemical studies have demonstrated activity of the enzyme aromatase, the rate-limiting step for estrogen synthesis, in breast tumor homogenates. Prior immunohistochemical studies in breast neoplasms demonstrated aromatase antibody binding to both stroma and parenchyma, but biochemically measured enzyme activity significantly correlated only with the level of staining in the stromal component. The present study sought to provide more direct evidence of the predominant role for stromal cell aromatase in breast tumor tissue. Accordingly, breast tumor stromal and epithelial cells were examined for aromatase enzyme activity and messenger ribonucleic acid (mRNA) expression. Stromal and epithelial cells from benign tissue served as a means of comparing activity and regulation in benign and tumor tissue. Enzyme activity in stromal cells from breast tumor tissue was low basally, but increased by 30- to 1200-fold when induced by dexamethasone. Combining dexamethasone with phorbol esters and cAMP produced an additional 1.2- to 4.1-fold stimulation. Analyses of exons III/V and exons IX/X demonstrated that aromatase mRNA expression was also substantially increased by these treatments. Increases in enzyme activity and mRNA expression in cells from benign breast stroma paralleled those observed in tumor stroma, although the increases in enzyme activity were generally lower. In contrast to the responses observed in stromal cells, epithelial cells from breast tumor or nonmalignant breast tissue were unresponsive to dexamethasone, either added alone or in combination with phorbol esters and cAMP. This study provides direct biochemical evidence that aromatase is present in stroma within breast tumors, as in surrounding tissues, and suggests that estrogen synthesis within the tumor may modulate tumor growth via a paracrine mechanism.     

Gastrointestinal autonomic nerve tumor: further observations regarding an ultrastructural and immunohistochemical analysis of six cases

Gastrointestinal autonomic nerve tumor (GANT) is a specialized form of stromal neoplasm whose ultrastructural features support a myenteric plexus derivation and provide the basis for its diagnosis. GANT actual frequency, relationship to skeinoid fibers, and CD34 expression status are some of the controversial aspects of this entity. Out of 14 gastrointestinal stromal tumors gathered during a 1-year period, six (42%) instances were diagnosed as GANT by electron microscopic study of at least five ultrathin sections per case. Additionally, GANTs were immunohistochemically investigated with a panel of nine antibodies including CD34. Ultrastructurally, every GANT case showed diagnostic findings and evidence of skeinoid fibers, whereas immunohistochemically all except one were CD34 positive. Immunoreactivity for neuron-specific enolase, synaptophysin, and vimentin was a common occurrence as well. In conclusion, GANT seems to be more frequent than hitherto recognized, skenoid fibers are a regular feature of GANT, and a positive CD34 immunoreaction does not discriminate between GANT and other non-smooth muscle, non-schwannian neoplasms.     

Direct demonstration of cytokeratin filaments by electron microscopy in K562 cell lines in liquid culture

Our previous study suggested the presence of cytokeratins in the supernatant of human K562 erythroleukemic cell line. In this study we confirm, by using an electron microscopy technique, that K562 cells contain typical intermediate tonofilaments with the characteristics of cytokeratins. After variable intervals of liquid culture, K562 cells have been examined for their clonogenic ability by immunostaining and ultrastructural study. K562 cells showed variable amounts of medium-sized filaments (intermediate filaments) of 10 nm mean size, having the submicroscopic pattern consistent with tonofilaments, arranged as electron-dense curve-shaped bundles, in perinuclear position independently from the different growth phases. A pool of monoclonal antibodies against cytokeratins confirmed the presence of cytokeratins in immunostaining, while the cytofluorimetric analysis showed an unexpected positivity of the CD34 antigen, associated with the typical adhesion molecule VLA5. In conclusion, the immunostaining with monoclonal antibodies and the ultrastructural findings suggest the expression of epithelial features in human K562 leukemic cells.     

Cardiac risk in surgery. A review and guidelines for risk evaluation and reduction of cardiac risk in general surgery

We report herein the phenotypic and functional analysis of human bone marrow and thymus derived early T cells. Commitment to T cell lineage is acquired during CD7 antigen expression by CD34+ precursors in human bone marrow and before thymus colonization. Early thymocytes show similar phenotypic characteristics as bone marrow T cells. They rapidly acquire CD4 before the dual expression of CD4 and CD8. Their expansion and differentiation is regulated by two major factors: thymic stroma and cytokines produced by these stroma cells or by thymocytes themselves. Among cytokines, IL1 and sCD23 produced by thymic epithelial cells support in vitro early T cell development.     

The murine stromal cell line AFT024 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro

A stromal cell line derived from murine fetal liver (AFT024) has been demonstrated to maintain long-term repopulating murine stem cells for up to 7 weeks in vitro. We evaluated the ability of AFT024 to maintain the immunophenotype and function of primitive human progenitors in vitro by comparing the cocultivation of CD34+CD38 cells on AFT024 with that on primary human stroma (HS). We have previously reported that within the CD34+CD38- population of bone marrow and cord blood, a highly primitive progenitor subpopulation can be identified functionally by its ability to generate colony forming unit-cells (CFU-Cs) in extended long-term culture (ELTC), that is, beyond 60 days of stromal cocultivation. Cocultivation of bone marrow and cord blood CD34+CD38-cells on AFT024 produced significantly greater cell expansion (p=0.0002) and CFU-C output (p=0.0007) during the ELTC period compared with culturing on HS. CFU-C production continued up to 9 weeks longer on AFT024 stroma. After 3 to 4 weeks of bulk culture on either AFT024 or HS, cells were replated in a limiting dilution to measure the number of cobblestone area-forming cells (CAFCs) maintained on each stroma. AFT024 maintained significantly more CAFCs than did HS (n=3, p=0.002). Fluorescence-activated cell sorter analysis of AFT024 and HS cocultures showed that both the frequency (p=0.018) and absolute number (p=0.027) of CD34+CD38- cells were significantly higher in cultures on AFT024 than in those on HS (n=9). The effects of AFT024 on preservation of primitive progenitors were not seen in transwell (noncontact) cultures. Thus, AFT024 acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors currently identifiable by in vitro assays.     

Human CD34(+) bone marrow cells regulate stromal production of interleukin-6 and granulocyte colony-stimulating factor and increase the colony-stimulating activity of stroma

Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34(+) cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34(+) cells for 3 to 5 weeks. Coculture of more mature CD15(+)/CD14(-) myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34(+) nor CD15(+)/CD14(-) cells produced IL-6, G-CSF, IL-1beta, or tumor necrosis factor alpha. When CD34(+) cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34(+) cells or when CD34(+) cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit-granulocyte-macrophage-stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34(+) progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.     

Reemergence of gentamicin-susceptible strains of methicillin-resistant Staphylococcus aureus: roles of an infection control program and changes in aminoglycoside use

We have tested the effect of stromal cells on the proliferation in long- and short-term cultures of primitive (Thy-1+, CD34+, CD33-, CD38- , HLA-DR , adherent in vitro and quiescent in vivo) progenitors in normal human bone marrow. These primitive cells produce granulocyte-macrophage colony-forming cells (CFU-GM) that are measured in secondary clonogenic assays. Addition of stromal cells to normal adherent haemopoietic progenitor cells reduced CFU-GM production by 80% (P =0.0002) after 1 week of incubation. In long-term culture (LTC), in the presence of stroma. the normal adherent cells did not produce significant numbers of CFU-GM until 3-4 weeks later which suggests that stromal cells reduce the probability of quiescent cell activation. This effect could not be attributed to soluble inhibitory factors and was specific to stroma grown with, rather than without, methylprednisolone. It was blocked by heparanase (H'ase) II treatment of stromal cells, by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment of progenitor cells, by antibody blocking of beta1 integrin molecules or by exposure to glucose/N-acetyl-D-glucosamine/alpha-methyl-D-mannoside, but not by exposure to galactose or fructose. Moreover, these interventions enabled the progenitor cells to respond to stimulatory factors in the culture supernatant. We interpret these results as support for a model involving primitive progenitor cell binding to stroma by PI-CAM/HS, beta1 integrin activation via lectin-like interactions and the transduction of signals which reduce the ability of primitive cells to respond to ambient stimulators. This model provides a mechanism for the maintenance of the quiescent state of stem cells by adhesion to stromal cells.     

Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.