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Summary: In the bone marrow, CXCL12 and IL-7 are essential for B cell differentiation, whereas hematopoietic stem cell (HSC) maintenance requires SCF and CXCL12. Peri-sinusoidal stromal (PSS) cells are the main source of IL-7, but their characterization as a pro-B cell niche remains limited. Here, we characterize pro-B cell supporting stromal cells and decipher the interaction network allowing pro-B cell retention. Preferential contacts are found between pro-B cells and PSS cells, which homogeneously express HSC and B cell niche genes. Furthermore, pro-B cells are frequently located in the vicinity of HSCs in the same niche. Using an interactome bioinformatics pipeline, we identify Nidogen-1 as essential for pro-B cell retention in the peri-sinusoidal niche as confirmed in Nidogen-1−/− mice. Finally, human pro-B cells and hematopoietic progenitors are observed close to similar IL-7+ stromal cells. Thus, a multispecific niche exists in mouse and human supporting both early progenitors and committed hematopoietic lineages. : Balzano et al. show that bone marrow peri-sinusoidal stromal cells, which form the hematopoietic stem cell niche, also express B cell niche genes including IL-7 and Nidogen-1. Loss of Nidogen-1 expression specifically affects access of pro-B cells to IL-7, resulting in impaired expansion and differentiation of early B cells. Keywords: B cell development, hematopoietic stem cell, stromal cell niche, interaction network, bone marrow

Objective: Pharmacological activation of adenosine signaling has been shown to increase β-cell proliferation and thereby β-cell regeneration in zebrafish and rodent models of diabetes. However, whether adenosine has an endogenous role in regulating β-cell proliferation is unknown. The objective of this study was to determine whether endogenous adenosine regulates β-cell proliferation—either in the basal state or states of increased demand for insulin—and to delineate the mechanisms involved. Methods: We analyzed the effect of pharmacological adenosine agonists on β-cell proliferation in in vitro cultures of mouse islets and in zebrafish models with β- or δ-cell ablation. In addition, we performed physiological and histological characterization of wild-type mice and mutant mice with pancreas- or β-cell-specific deficiency in Adora2a (the gene encoding adenosine receptor A2a). The mutant mice were used for in vivo studies on the role of adenosine in the basal state and during pregnancy (a state of increased demand for insulin), as well as for in vitro studies of cultured islets. Results: Pharmacological adenosine signaling in zebrafish had a stronger effect on β-cell proliferation during β-cell regeneration than in the basal state, an effect that was independent of the apoptotic microenvironment of the regeneration model. In mice, deficiency in Adora2a impaired glucose control and diminished compensatory β-cell proliferation during pregnancy but did not have any overt phenotype in the basal state. Islets isolated from Adora2a-deficient mice had a reduced baseline level of β-cell proliferation in vitro, consistent with our finding that UK432097, an A2a-specific agonist, promotes the proliferation of mouse β-cells in vitro. Conclusions: This is the first study linking endogenously produced adenosine to β-cell proliferation. Moreover, we show that adenosine signaling via the A2a receptor has an important role in compensatory β-cell proliferation, a feature that could be harnessed pharmacologically for β-cell expansion and future therapeutic development for diabetes. Keywords: β-cell proliferation, Adenosine, Islet biology, Gestational diabetes

Summary: The cardinal property of bone marrow (BM) stromal cells is their capacity to contribute to hematopoietic stem cell (HSC) niches by providing mediators assisting HSC functions. In this study we first contrasted transcriptomes of stromal cells at different developmental stages and then included large number of HSC-supportive and non-supportive samples. Application of a combination of algorithms, comprising one identifying reliable paths and potential causative relationships in complex systems, revealed gene networks characteristic of the BM stromal HSC-supportive capacity and of defined niche populations of perivascular cells, osteoblasts, and mesenchymal stromal cells. Inclusion of single-cell transcriptomes enabled establishing for the perivascular cell subset a partially oriented graph of direct gene-to-gene interactions. As proof of concept we showed that R-spondin-2, expressed by the perivascular subset, synergized with Kit ligand to amplify ex vivo hematopoietic precursors. This study by identifying classifiers and hubs constitutes a resource to unravel candidate BM stromal mediators.

Background Aims: Advances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources., Methods: We obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit-fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels., Results: MSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential., Conclusions: Trabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.

We have studied the plasma membrane protein phenotype of human culture-amplified and native bone marrow mesenchymal stem cells (BM MSCs). We have found, using microarrays and flow cytometry, that cultured cells express specifically 113 transcripts and 17 proteins that were not detected in hematopoietic cells. These antigens define a lineage-homogenous cell population of mesenchymal cells, clearly distinct from the hematopoietic lineages, and distinguishable from other cultured skeletal mesenchymal cells (periosteal cells and synovial fibroblasts). Among the specific membrane proteins present on cultured MSCs, 9 allowed the isolation from BM mononuclear cells of a minute population of native MSCs. The enrichment in colony-forming units-fibroblasts was low for CD49b, CD90, and CD105, but high for CD73, CD130, CD146, CD200, and integrin alphaV/beta5. In addition, the expression of CD73, CD146, and CD200 was down-regulated in differentiated cells. The new marker CD200, because of its specificity and immunomodulatory properties, deserves further in-depth studies.

Background: Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases., Methods: BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed., Results: MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone., Discussion: MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

The Stro-1 antigen potentially defines a mesenchymal stem cell (MSC) progenitor subset. We here report on the role of human ex vivo-expanded selected Stro-1(+) or Stro-1(-) MSC subsets on the engraftment of human CD34(+) cord blood cells in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. The data show that cotransplantation of expanded Stro-1(-) cells with CD34(+) cells resulted in a significant increase of human CD45, CD34, CD19, and CD11b cells detected in blood or in bone marrow (BM) and spleen as compared with the infusion of CD34(+) cells alone. Infusion into mice of expanded Stro-1(+) and Stro-1(-) cells (without CD34(+) cells) showed that the numbers of Stro-1(+)-derived (as assessed by DNA analysis of human beta-globin with quantitative polymerase chain reaction [PCR]) were higher than Stro-1(-)-derived cells in spleen, muscles, BM, and kidneys, while more Stro-1(-)-derived than Stro-1(+)-derived cells were found in lungs. The transduction of expanded Stro-1(+) cells with an enhanced green fluorescent protein (eGFP) gene did not modify their cytokine release and their homing in NOD/SCID mouse tissues. The difference between the hematopoietic support and the homing capabilities of expanded Stro-1(+) and Stro-1(-) cells may be of importance for clinical therapeutic applications: Stro-1(+) cells may rather be used for gene delivery in tissues while Stro-1(-) cells may rather be used to support hematopoietic engraftment.

Endothelial progenitor cells (EPCs) were shown to be present in systemic circulation and cord blood. We investigated whether EPCs display specific properties compared with mature endothelial cells. Human cord blood CD34+ cells were isolated and adherent cells were amplified under endothelial conditions. Expression of specific markers identified them as endothelial cells, also called endothelial progenitor-derived cells (EPDCs). When compared to mature endothelial cells, human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (HBMECs), endothelial markers, were expressed to the same extent except for KDR, which is expressed more in EPDCs. They display a higher proliferation potential. Functional studies demonstrated that EPDCs were more sensitive to angiogenic factors, which afford these cells greater protection against cell death compared with HUVECs. Moreover, EPDCs exhibit more hematopoietic supportive activity than HUVECs. Finally, studies in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice demonstrated that human circulating EPCs are able to colonize a Matrigel plug. EPDCs display the morphology and phenotype of endothelial cells. Their functional features indicate, however, that although these cells have undergone some differentiation steps, they still have the properties of immature cells, suggesting greater tissue repair capabilities. Future use of in vitro amplified peripheral blood EPDCs may constitute a challenging strategy for cell therapy.

Liver becomes the predominant site of hematopoiesis by 11.5 dpc (days after coitus) in the mouse and 15 gestational weeks in humans and stays so until the end of gestation. The reason the liver is the major hematopoietic site during fetal life is not clear. In this work, we tried to define which of the fetal liver microenvironmental cell populations would be associated with the development of hematopoiesis and found that a population of cells with mixed endodermal and mesodermal features corresponded to hematopoietic-supportive fetal liver stroma. Stromal cells generated from primary cultures or stromal lines from mouse or human fetal liver in the hematopoietic florid phase expressed both mesenchymal markers (vimentin, osteopontin, collagen I, alpha smooth muscle actin, thrombospondin-1, EDa fibronectin, calponin, Stro-1 antigens, myocyte-enhancer factor 2C) and epithelial (alpha-fetoprotein, cytokeratins 8 and 18, albumin, E-cadherin, hepatocyte nuclear factor 3 alpha) markers. Such a cell population fits with the description of cells in epithelial-to-mesenchymal transition (EMT), often observed during development, including that of the liver. The hematopoietic supportive capacity of EMT cells was lost after hepatocytic maturation, induced by oncostatin M in the cell line AFT024. EMT cells were observed in the fetal liver microenvironment during the hematopoietic phase but not in nonhematopoietic liver by the end of gestation and in the adult. EMT cells represent a novel stromal cell type that may be generated from hepatic endodermal or mesenchymal stem cells or even from circulating hematopoietic stem cells (HSCs) seeding the liver rudiment.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell-dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis. (Blood. 2000;95:3758-3764)

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

We examined long bones from 42 human embryos and fetuses whose gestational ages ranged from 6 to 28 weeks. Bone rudiment sections were stained using a panel of monoclonal antibodies directed at antigens expressed by hematopoietic cells, endothelial cells, smooth muscle cells, fibro-blasts, and stromal cells, to describe the events preceding and accompanying the onset of hematopoiesis in the diaphyseal region. Five distinct stages were identified. Stage I (6.6 to 8.5 gestational weeks [gw]) was that of entirely cartilaginous rudiments: chondrocytes were dilated and capillaries with CD34+ endothelial cells were observed in the perichondral limb mesenchyme. At stage II (8.5-9 gw) chondrolysis was actively proceeding; numerous CD68+ cells were observed, interspersed within the marrow cavity. Stage III (9 to 10.5 gw) was characterized by the development of the vascular bed in the absence of detectable hematopoiesis. At mid-diaphysis, specific structures that we named primary logettes were discernible; they consisted of small chambers of connective tissue, framed by a loose network of CD45-negative cells organized around an arteriole and limited from the surrounding sinus by a clearcut lining of CD34+ endothelial cells flanked on their abluminal side by alpha SM actin+ myoid cells. Stage IV (10.5-15 gw) was characterized by the onset of hematopoiesis. Hematopoietic cells were found exclusively in the primary logettes that had considerably increased in size. Logettes filled with hematopoietic cells were immersed within large and almost empty vascular sinuses. Logettes were attached by a short pedicle to connective tissue adjacent to bone/cartilage remaining formations; this tissue contained very rare hematopoietic cells. Logettes were few, usually less than 10 per long bone, and found solely in the diaphyseal area. Most hematopoietic cells found inside logettes were CD15+ myelocytes; rarely seen were glycophorin A+ immature erythroblasts and CD34+ nonendothelial cells. Hematopoietic cells within the logettes were in contact with alpha SM actin+ myoid cells and flattened endothelial-like (although consistently CD34-negative), aligned cells limiting small capillary lumina. Stage V (16 gw onward) was that of final organization of the long bones with areas of fully calcified bone and areas of dense hematopoiesis where logettes were no longer visible. This study shows three major features of incipient long bone hematopoiesis: 1) absence of CD34+ hematopoietic precursors before the onset of hematopoiesis and extreme rarity of those in the emerging blood-forming marrow, 2) predominance of granulopoiesis, and 3) exclusive development in specific structures organized by vascular cells. This study also suggests that CD68+ cells are instrumental in the chondrolysis process while vascular cells (endothelial and myoid cells) may be the critical microenvironment at the onset of hematopoiesis.

Normal human bone marrow stromal cells (BMSC) were isolated from Dexter-type long-term cultures according to their capacity to adhere to plastic and to their lack of hematopoietic antigens. The BMSC displayed a homogeneous appearance and a myofibroblastic phenotype in culture. The stromal cells (SC) were shown to support the proliferation of purified CD34+ hematopoietic progenitors and permitted us to maintain myeloid cells for several weeks in culture. In addition, the BMSC induced the proliferation of purified CD10+ s mu- fetal BM B-cell precursors (BCP). The capacity of the BMSC to induce the proliferation of early myeloid cells was shared by several other human fibroblastic-like cell types. In contrast, the BMSC were far superior to other adherent cells for induction of BCP proliferation. This capacity was largely mediated by endogenously produced interleukin-7 (IL-7), because it could be inhibited by anti-IL-7 antibody. In line with this finding, addition of IL-7 considerably enhanced BCP proliferation in cocultures with skin fibroblasts or synoviocytes. Thus, production of IL-7 appears to be a critical parameter that determines the ability of fibroblastic-like cells to induce BCP proliferation. Taken together, our data show that normal human myofibroblastic BMSC induce the proliferation of both early myeloid and B-lymphoid cells in the absence of accessory hematopoietic cells. The present system should constitute a model to study interactions between native human BM myofibroblastic stroma and various hematopoietic cell subsets.

Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.

In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle-specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha-SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.

This study was designed to assess the presence of endogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) within adherent layers of human Dexter-type cultures and to investigate the effect on granulomonopoiesis of adding exogenous GM-CSF to the culture medium. The presence of GM-CSF was demonstrated using a bioassay, in which adherent layers from normal bone marrows gave rise to endogenous granulocyte-macrophage colony-forming units (CFU-GM) that were specifically inhibited by increasing amounts of an anti-GM-CSF neutralizing antibody. Using an immunoassay, the estimated amounts of GM-CSF were less than or equal to 40 pg per flask in adherent layers, while remaining undetectable in supernatants. The addition of 10 ng or purified recombinant GM-CSF per milliliter of culture medium increased slightly the CFU-GM output over a 5-week culture period. The addition of 50 ng/mL decreased significantly the CFU-GM output after 5 weeks of culture. This decrease was associated with major modifications of the adherent layer cell composition. Large round or ovoid macrophages were generated at the expense of the interdigitated and elongated stromal cells and the extracellular fibronectin network was no longer observed. These studies suggest that GM-CSF production by accessory cells (stromal cells and/or monocytes) is almost equal to its consumption by hematopoietic cells, a situation similar to that found in long-term cultures of murine marrows. They also show that the maintenance of granulomonopoiesis is decreased by adding more than 10 ng/mL of exogenous GM-CSF to the culture medium, which is related to the induction of adherent macrophages, the disappearance of the major smooth-muscle-like stromal cell component of the adherent layer, and that of the fibronectin extracellular matrix.

The CGA-7, a monoclonal antibody that reacts with smooth muscle cell actin but not with endothelial cell or fibroblast actin, and HHF, a monoclonal antibody that reacts with smooth muscle, skeletal muscle, and cardiac muscle actin, both recognize microfilaments present within adherent cells from actively hematopoietic human long-term marrow cultures. Macrophages, monocytes, and cultured marrow fibroblasts do not react with either antibody. These data suggest that the anti-actin antibodies may serve as useful markers for in vitro microenvironmental cells and lend support to the hypothesis that stromal cells from long-term marrow cultures are different from marrow fibroblasts and may constitute a unique cell lineage.

Adherent cells from long-term marrow cultures from 23 individuals were transformed with wild-type simian virus 40 (SV40). After transformation, cloned cell lines were developed that even after rigorous subcloning invariably produced both stromal cells and round cells. The stromal cells expressed cytoskeletal filaments similar to those of long-term marrow culture adherent cells and produced interstitial and basal lamina collagen types. The round cells had the electron microscopic appearance of primitive hematopoietic cells and when examined with cytochemical stains and monoclonal antibodies to hematopoietic differentiation antigens had reaction patterns suggestive of cells from several lineages. Most round cells expressed the pan-hematopoietic T-200 determinant, and lesser percentages expressed the early T cell antigens CD-1 and CD-3, HLA-DR determinants, the monocytic antigen recognized by Leu M3, and the myeloid antigens detected by monoclonal antibodies 1G10 and 12.8. In addition, when plated in semisolid medium in the presence of a source of colony-stimulating activity, up to 11% of the cells formed colonies consisting of blastlike cells that also expressed hematopoietic cell surface determinants. The data suggest that adherent cells in long-term marrow cultures contain a cell that after transformation by SV40 obligately produces cells with hematopoietic as well as stromalike features.