Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so-called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti-PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6-P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6-P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis-supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6-P cells express a high level of the PA6 molecule, which is reactive with anti-PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.     

Monoclonal antibodies against a preadipose cell line (MC3T3-G2/PA6) which can support hemopoiesis

Monoclonal antibodies against a mouse preadipose cell line (MC3T3-G2/PA6:PA6), which can support hemopoiesis by direct cell-to-cell interaction, were produced and characterized. The antibodies react with PA6 but not PA6-M (a mutant cell line) which has the capacity neither to contact with hemopoietic stem cells (HSCs) nor to support hemopoiesis. Endosteal cells in the bone marrow show positive staining to these antibodies. They inhibit pseudoemperipolesis of PA6 to HSCs, resulting in a significant decrease in hemopoietic cell number. These findings suggest that the monoclonal antibodies bind to the stromal cell receptors for HSCs and block the binding of HSCs to stromal cells leading to suppression of hemopoiesis.     

Establishment of a murine pre-B cell clone dependent on interleukin-7 and stem cell factor.

To identify cytokines required for proliferation of murine pre-B cells, we established a pre-B cell clone MH11 (B220+ MB-1+ sIgM-) on a stromal cell line ST2 from day 13 fetal liver. The growth of MH11 is dependent on ST2. Another stromal cell line PA6, non-secretor of IL-7, could not support MH11 unless IL-7 was added. We investigated the effect of cytokines on proliferation of MH11 with or without stromal cells. IL-7 had a stimulatory effect on proliferation of MH11, but IL-7 alone could not support MH11 growth without ST2. Recombinant stem cell factor (rSCF) also had a positive effect on MH11. rSCF and rIL-7, when added together, could maintain the growth of MH11 in the absence of stromal cells. Moreover, the growth of MH11 on ST2 was inhibited almost completely by anti-c-kit monoclonal antibody (mAb). These results demonstrate that direct SCF/c-kit interaction is involved in the stimulation of pre-B cells.     

Analysis of the human fetal liver hematopoietic microenvironment

In the adult, hematopoietic stem cells (HSCs) are resident in the bone marrow (BM) compartment and are in direct association with the BM stromal microenvironment. However, human adult HSCs are largely quiescent and undergo limited self-renewal. This is in contrast to the higher frequency of cycling HSCs undergoing self-renewal during fetal development when hematopoiesis is transiently localized to the fetal liver (FL), suggesting that FL provides a more conducive microenvironment to support HSCs. Here, we provide phenotypic and molecular characterization of primary human FL stromal cells capable of supporting human repopulating progenitors. Qualitative and quantitative analysis revealed several properties unique to FL stromal cells compared to adult BM-derived stroma that included a greater than 10-fold enhanced proliferative capacity of FL stromal vs adult BM, and a 2-fold increase in the number of N-cadherin- and osteopontin-expressing cells. Supportive of extrinsic influences likely to modulate HSC expansion, global gene expression microarray analysis revealed that FL stroma has higher expression of regulators of the Wnt signaling pathway compared to adult BM stroma, which demonstrated an increased expression of the Notch signaling pathway. Our results suggest that human FL stromal cells provide a unique microenvironment to HSCs compared to adult BM stroma by controlling Wnt signaling of HSCs during human fetal hematopoietic development, while Notch signaling is tightly regulated by the HSC microenvironment in the adult. We propose that the human HSC niche is ontogenically controlled during human development to provide appropriate expansion of fetal HSCs and subsequent maintenance of adult HSCs.     

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.     

Significance of VLA-4–VCAM-1 interaction and CD44 for transendothelial invasion in a bone marrow metastatic myeloma model

In previous work, we established the B9/BM1 syngeneic murine bone marrow metastasis model. Interleukin (IL)-6-dependent, IL-1-producing B9/BM1 cells, which colonize the vertebral and femoral marrow after i.v. injection, show great similarity in cell surface phenotype to human myeloma cells, especially the expression of 3 adhesion molecules, CD44, VLA-4 and ICAM-1. Here we investigated the function of these adhesion molecules by binding and transendothelial invasion assays using a newly established bone marrow-derived endothelial cell line (BMEC). A combination of monoclonal antibodies against CD44 and VLA-4 significantly inhibited the adherence of B9/BM1 cells to BMEC and anti-CD44 mAb especially blocked B9/BM1 transendothelial invasion of unstimulated BMEC cells. Results of additional experiments, in which the cells were treated with anti-CD44 and hyaluronidase, demonstrated that the interaction of CD44 molecules on B9/BM1 cells with hyaluronan on BMEC cells was a critical factor in both adhesion and transendothelial invasion in this model. However, stimulation of BMEC with TNFα resulted in increased invasion by B9/BM1 cells, which was completely suppressed by anti-VCAM-1 mAb, implicating a significant role of this adhesion molecule in this process during inflammation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and functional characterization of the hepatic stellate cell CD38 cell surface molecule

The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis, because these cells are the main producers of extracellular matrix proteins in the liver and contribute to the modulation of inflammatory responses via the secretion of several cytokines and the expression of adhesion molecules. The goal of the present study was to characterize cell surface proteins that regulate HSC activation. To this end, a panel of monoclonal antibodies (mAbs) was generated. mAb 14.27 recognized a protein of 45 kd that was highly expressed on HSCs. Affinity purification of this protein followed by sequencing revealed that protein to be CD38. We subsequently demonstrated that CD38 was constitutively expressed by HSCs and that its expression increased after in vitro and in vivo activation. mAb 14.27 induced an increase in cytosolic Ca2+ levels in HSCs, showing that it functions as an agonistic antibody. Moreover, the effects mediated by the CD38 mAb included induction of the proinflammatory cytokine interleukin-6 and up-regulation of the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and neural cell adhesion molecule. Collectively, our data suggest that CD38 can act as a regulator of HSC activation and effector functions.     

Expression of CD34 in endothelial cells,hematopoietic progenitors and nervous cells in fetal and adult mouse tissues

The human cell surface molecule CD34 is selectively expressed on uncommitted and committed hematopoietic progenitor cells and on vascular endothelial cells. It has been suggested that CD34 regulates early events in blood cell migration and differentiation, possibly as a cell adhesion molecule. To characterize the patterns of expression of CD34 in the mouse embryo and in the adult, as well as to dissect the function of different portions of the extracellular domain of this molecule, we have generated the first monoclonal antibodies (mAb) specific for mouse CD34. The epitope(s) recognized by these mAb are not carbohydrate moieties, and are comprised either within the immunoglobulin-like domain or within a portion of the mucin domain, containing approximately half of the predicted O- and N-linked carbohydrate attachment sites. The specificity of the antibodies was established by ELISA and Western blotting. Western analysis revealed that these mAb recognize a protein of approximately 110 kDa in PA6 stromal cell lysates, which can be specifically blocked by the recombinant CD34 protein. To establish the reactivity of these mAb on different cell lineages, a panel of cell lines was stained. This analysis showed strong reactivities with 3T3 fibroblasts, stromal cell lines from fetal liver and with the endothelial cell line D10. Bone marrow hematopoietic progenitors were also stained by these mAb. Immunostaining of frozen sections from embryonic and adult tissues revealed a strong reactivity against vascular endothelial cells at different stages of development, including sinusoidal cells in the fetal liver, yolk sac, and in the fetal bone marrow, endothelial cells from adult lung and kidney, and neural cells, including those of the neural tube of midgestation embryos and neuronal bodies in adult brain.     

AGM区来源的基质细胞促进造血干细胞扩增的体外实验研究

目的： 探讨主动脉-性腺-中肾（aorta-gonad-mesonephros，AGM）来源的基质细胞对造血干细胞（HSC）增殖的促进作用，为探寻HSC的体外扩增方法奠定实验基础。 方法： 分别从孕11 d BALB/c小鼠胚胎AGM区及6周龄小鼠骨髓分离、培养基质细胞，流式细胞仪等对基质细胞进行鉴定；利用小鼠胚胎干细胞（ESC）向造血细胞定向分化的模型，结合高增殖潜能集落（HPP-CFC）、原始细胞集落（BL-CFC）形成实验及流式细胞仪分析CD34+、CD34+Sca-1+细胞比例，对比研究AGM及骨髓基质细胞对ESC来源的HSC的扩增作用。 结果： 小鼠AGM和骨髓基质细胞在形态及表型上基本相似，均符合基质细胞的特征。AGM和骨髓基质细胞均可促进ESC来源的HPP-CFC的形成，但AGM基质细胞还可促进ESC来源的 BL-CFC的形成；流式细胞仪检测发现：在骨髓基质细胞支持下，CD34+细胞增加了3-4倍，但CD34+/Sca-1+却无明显增加；而在AGM基质细胞支持下CD34+、CD34+Sca-1+细胞均明显增加了4-5倍。 结论： AGM基质细胞在有效扩增小鼠HSC同时，能很好地维持HSC自我更新及多向分化的潜能。     

The effect of bleeding on hematopoietic stem cell cycling and self-renewal

Hematopoietic stem cells (HSCs) divide and give rise to more committed progenitors, which ultimately produce all lineages of blood cells. HSCs can be induced to enter the cell cycle in vitro and in vivo by stimulatory cytokines and in vivo by ablation of bone marrow (BM) cells with irradiation or chemotherapeutic agents. Although it has been postulated that rates of HSC proliferation increase with normal hematopoietic stresses, such as infection or hemorrhage, this hypothesis has never been directly tested. The ability to analyze HSCs prospectively by cell-surface phenotype c-kit(+), Thy1.1(lo), Sca-1(+), Linage(neg/lo) has allowed us to perform a detailed examination of the effects of bleeding on the cell cycle kinetics of HSCs. Our results demonstrate for the first time that HSCs in both the BM and the spleen proliferate and self-renew in response to tail-vein bleeding in mice. This response was suppressed when red blood cells, but not when white blood cells, were transferred after bleeding. Thus, regulators of HSC proliferation can sense and respond to red blood cell levels.     

c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation

The activity of adult stem cells is essential to replenish mature cells constantly lost due to normal tissue turnover. By a poorly understood mechanism, stem cells are maintained through self-renewal while concomitantly producing differentiated progeny. Here, we provide genetic evidence for an unexpected function of the c-Myc protein in the homeostasis of hematopoietic stem cells (HSCs). Conditional elimination of c-Myc activity in the bone marrow (BM) results in severe cytopenia and accumulation of HSCs in situ. Mutant HSCs self-renew and accumulate due to their failure to initiate normal stem cell differentiation. Impaired differentiation of c-Myc-deficient HSCs is linked to their localization in the differentiation preventative BM niche environment, and correlates with up-regulation of N-cadherin and a number of adhesion receptors, suggesting that release of HSCs from the stem cell niche requires c-Myc activity. Accordingly, enforced c-Myc expression in HSCs represses N-cadherin and integrins leading to loss of self-renewal activity at the expense of differentiation. Endogenous c-Myc is differentially expressed and induced upon differentiation of long-term HSCs. Collectively, our data indicate that c-Myc controls the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSCs and their niche.     

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.     

Stromal cell lines which support lymphocyte growth: characterization, sensitivity to radiation and responsiveness to growth factors

Stromal cells which grow as an adherent layer of Whitlock-Witte cultures are thought to be an essential component of the lymphohemopoietic microenvironment. Stromal cell lines from bone marrow (BM) and spleen have been obtained by treatment of cultures with 5-fluorouracil and selected for their lymphocyte support capacity by measuring the clonal growth of stromal cell-dependent lymphocyte lines in methyl cellulose. Established stromal cell lines differed significantly from stromal cells in primary Whitlock-Witte cultures with respect to expression of certain hemopoietic cell surface markers. For example, the Thy-1 and Mac-3 antigens were expressed by stromal cell lines obtained from BM and spleen, but not by stromal cells in primary cultures. Features common to all stromal cells include synthesis of actins, the neural adhesion molecule N-CAM, and a variety of collagens. Two types of common leukocyte antigens were not significantly expressed. The proliferation and total protein synthetic capacity of lymphocyte-supportive stromal cell lines was sensitive to ionizing radiation. After exposure of the cells to 200 rads, the incorporation of either [3H]thymidine or [3H]Leucine was reduced to less than 50% of control values, but the growth of lymphocytes was augmented in the presence of an irradiated stromal cell layer. The proliferation of stromal cell lines was also affected by exposure to a variety of growth factors. Addition of epidermal growth factor or endothelial cell growth factor augmented BM or spleen-derived stromal cell proliferation, while interferon-gamma had the opposite effect. In general, but not exclusively, lymphocyte growth was inhibited by factors which augmented the proliferation of stromal cells. Novel methods are described for isolating stromal cells and determining their capacity to support lymphocyte growth in vitro. Evidence is presented that this ability is not restricted to BM-derived stromal cells. The function of stromal cells was not dependent on their ability to proliferate, and this may be modulated by immunoregulatory and other growth factors.     

Microenvironmental organization and stromal cell associations of B lymphocyte precursor cells in mouse bone marrow

B lymphocyte precursor cells expressing B220 glycoprotein have been examined in mouse bone marrow (BM) by the in vivo binding of monoclonal antibody (mAb) 14.8 visualized by light and electron microscope radio autography. Young mice were injected intravenously with 125I-labeled mAb 14.8 and then perfused to remove unbound antibody. Quantitative analysis of radioauto graphic sections of femoral BM revealed many labeled mAb 14.8-binding cells which were situated both singly and in groups throughout the extravascular BM parenchyma. Groups of large 14.8+ cells were located in patchy areas in the peripheral regions of the BM near the endosteum. These cells were shown to include proliferating precursor B cells by using mice given vincristine sulfate to stop cells in metaphase and mice treated from birth with anti-IgM antibodies to delete mature B lymphocytes. Electron microscopy revealed clusters of 14.8+ cells intimately associated with the processes of stromal reticular cells. Other 14.8+ cells were in close contact with macrophages; in some instances the intervening cell membranes were indistinct and the macrophages contained 14.8+ material in their cytoplasm. In addition, 14.8+ small lymphocytes were highly concentrated within the lumen of some sinusoids. The present method of detecting B lineage precursor cells in situ has led to a working model of the microenvironmental organization of primary B cell genesis in vivo. The model proposes (a) a centrally directed sequence of differentiation initiated by early precursor cells situated peripherally near the surrounding bone; (b) close associations between precursor B cells and stromal reticular cells; (c) deletion of ineffective B cells by macrophages, and (d) an intravascular maturation phase before B lymphocytes are finally delivered into the blood stream.     

Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors

Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively, and this may influence their biologic properties. In this study, we have compared human MSC isolated from bone marrow (BM) using two culture conditions, from cord blood (CB), and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays, enzyme-linked immunosorbent assay, and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations, but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11, N-cadherin, vascular cell adhesion molecule 1, neural cell adhesion molecule 1, and integrins were highly expressed in MSC preparations derived from BM and CB. Thus, MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article.     

B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation

B lymphopoiesis supporting activities of two stromal cell clones, MC3T3-G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock-Witte-type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre-B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell-dependent pre-B cell lines. Among four ST2-dependent pre-B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra-marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.     

Transinteractome analysis reveals distinct niche requirements for isotype-based plasma cell subsets in the bone marrow

Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR⁺) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype.     

New type of matrix support for bone marrow cell cultures: in vitro culture and in vivo transplantation experiments

A new type of bone marrow cell culture system was developed by using a highly porous substrate matrix, i.e., porous polyvinyl formal (PVF) resin. Murine bone marrow (BM) cells were cultured without the use of exogenous growth factors in a three-dimensional matrix support made of collagen coated porous PVF resin. To examine the optimal conditions for highest stromal cell density, short-term and long-term in vitro culture experiments using PVF were performed. In the short-term culture experiments, it was found that cubes of PVF (10 x 10 x 2 mm and 130 microm in pore size) coated with type I collagen with a seeding density of 2x10(7) BM cells offered the most appropriate culture conditions. In the long-term cultures, BM cells in PVF maintained their viability for up to 6 weeks. In another series of re-inoculation experiments, freshly isolated BM cells were inoculated onto the already developed stromal layer. In this study, a higher cell density of the stromal layer was obtained in the PVF culture compared with those in the control dish culture. Based upon the results of in vitro experiments, in vivo transplantation studies were also performed. Histologic examinations of the subcutaneously transplanted PVF with stroma revealed host derived hematopoiesis inside the PVF matrix. Moreover, survival of approximately 15% of the transplanted BM cells that were cultured in PVF were confirmed in X-ray irradiated recipients. From these results, it is suggested that PVF resin is a promising three-dimensional substrate for BM cell culture and that it can maintain hematopoietic stem cells or progenitor cells after transplantation.     

NTera2: a model system to study dopaminergic differentiation of human embryonic stem cells

NTera2, a human embryonal carcinoma (EC) stem cell line, shares many characteristics with human embryonic stem cells (hESCs). To determine whether NTera2 can serve as a useful surrogate for hESCs, we compared global gene expression between undifferentiated NTera2, multiple undifferentiated hESC cell lines, and their differentiated derivatives, and we showed that NTera2 cells share multiple markers with hESCs. Similar to hESCs, NTera2 cells differentiated into TH-positive cells that express dopaminergic markers including AADC, DAT, Nurr1, TrkB, TrkC, and GFRA1 when co-cultured with PA6 cells. Flow cytometry analysis showed that tyrosine hydroxylase (TH) and neural cell adhesion molecule (NCAM) expression increased, whereas SSEA4 expression decreased as cells differentiated. Medium conditioned by PA6 cells stimulated differentiation of NTera2 cells to generate TH-positive cells that expressed dopaminergic markers. Flow cytometry selected polysialylated (PSA-NCAM) cells responded to medium conditioned by PA6 cells by differentiating into TH-positive cells and expressed dopaminergic markers. Sorted cells differentiated for 4 weeks in PA6 cell conditioned media included functional neurons that responded to neurotransmitters and exhibited electronic excitability. Therefore, NTera2 cell dopaminergic neuronal differentiation and PSA-NCAM enrichment provides a useful system for the future study of hESCs.     

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-β1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-β1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-β1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-β1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-β1 with anti-TGF-β1 antibodies or with Ly-364947 (a specific inhibitor TGF-β1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-β1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.