Wnt signaling regulates hemopoiesis through stromal cells.

Hemopoietic cells develop in a complex milieu that is made up of diverse components, including stromal cells. Wnt genes, which are known to regulate the fate of the cells in a variety of tissues, are expressed in hemopoietic organs. However, their roles in hemopoiesis are not well characterized. In this study, we examined the roles of Wnt proteins in hemopoiesis using conditioned medium containing Wnt-3a. This conditioned medium dramatically reduced the production of B lineage cells and myeloid lineage cells, except for macrophages in the long-term bone marrow cultures grown on stromal cells, although the sensitivity to the conditioned medium differed, depending on the hemopoietic lineage. In contrast, the same conditioned medium did not affect the generation of B lineage or myeloid lineage cells in stromal cell-free conditions. These results suggested that Wnt proteins exert their effects through stromal cells. Indeed, these effects were mimicked by the expression of a stabilized form of beta-catenin in stromal cells. In this study, we demonstrated that Wnt signaling regulates hemopoiesis through stromal cells with selectivity and different degrees of the effect, depending on the hemopoietic lineage in the hemopoietic microenvironment.     

Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene

Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by “unmarked” progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.     

Flare-up of delayed-type hypersensitivity initially induced by murine cloned helper T cells

Delayed-type hypersensitivity (DTH) reactions were induced in mice by cloned helper T cells directed against methylated bovine serum albumin (mBSA). The DTH reactions were induced either by local injection of the helper T cells together with the antigen in the hind feet or by intravenous (iv) administration of the cloned T cells and local injection of the antigen. Local or systemic (oral or iv) administration of mBSA after waning of the DTH induced by the cloned helper T cells caused a flare-up reaction. This indicates that functional helper T cells persist at the inflammation site. The inflammations were quantified in a foot swelling assay and were examined histologically. The inflammation measured in the flare-up reaction was generally lower than in the acute reaction. Histologically the acute inflammation showed edema and a large proportion of granulocytes, whereas the flare-up reaction appeared more histiocytic and showed less edema.     

Multilineage potential of adult human mesenchymal stromal cells derived from bone marrow of patients with polycytaemia vera

Human mesenchymal stromal cells were isolated from the bone marrow of patients with polycyteamia vera (the myeloproliferative disorder) with the aim to characterize the properties of the mesenchymal stromal cells originating from the pathologically affected bone marrow. Their in vitro growth and potential to differentiate were determined. Isolated mesenchymal stromal cells were able to differentiate into three mesenchymal lineages under appropriate cultivation conditions.     

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.     

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.     

Transgenic twin lambs cloned by granulosa cells

The development potential of transgenic adult cells after nuclear transfer (NT) was evaluated. Primary ovine granulosa cells (GC(S)) from a slaughter ovary were transfected with pEGFP-N1 plasmid DNA. Three G418-resistance cell lines (A2, B2 and B4) were used as donor cells in NT. A total of 162 NT blastocysts were then frozen with ethylene glycol solution and stored for five months before transplanted into recipients. Twenty-nine frozen thawed NT blastocysts were transferred into 15 synchronized recipients. Twin lambs (6.9%) derived from B2 line were delivered by cesarean section on day 143 but died after birth. A tumor consisting of lung tissues was found on the surface of left lung of the 4-kg lamb and histological analysis indicated that it resembles a hamartoma. DNA analysis confirmed that two lambs were genetically identical to B2 donor cells. Gene insertion and expression have been detected in fibroblasts cells derived from muscle tissues of the lambs. This study indicates that granulosa cell is a suitable cell type for producing transgenic animals by nuclear transfer. Offspring were produced after long-term storage of NT blastocysts.     

Inhibition of hemopoiesis in murine marrow cell cultures by recombinant murine tumor necrosis factor alpha: evidence for long-term effects on stromal cells

The addition of recombinant murine tumor necrosis factor alpha (rmTNF-alpha) to serum-free methylcellulose cultures inhibited macrophage colony formation stimulated by purified colony stimulating factor-1 (CSF-1), recombinant granulocyte-macrophage-CSF (rmGM-CSF), and recombinant interleukin 3 (rmIl-3). The concentration of rmTNF-alpha inhibiting colony formation by 50% (IC50) was between 2 and 20 ng/ml. Erythroid colony formation in cultures with erythropoietin (EPO) alone or EPO, rmIl-3, and rmGM-CSF in combination were reduced to a much lesser extent. In established long-term marrow cultures (LTMC), addition of 20 and 200 ng/ml of rmTNF-alpha resulted in release of cells from the adherent layer during the first week. Treatment of cultures with rmTNF-alpha for 4 consecutive weeks led to prolonged inhibition of cell production lasting up to 8 weeks after cessation of treatment. One day after addition of a low dose of TNF (2 ng/ml), "fat" cells were no longer observed in the adherent layer. Our results indicate that TNF inhibition of hemopoiesis occurs both at the progenitor cell and stromal cell levels.     

Proliferation of human leukemic pre-B cells induced by human bone marrow stromal cells and murine fibroblasts.

Aim of the study was the establishment of a standardized in vitro culture system for studies on proliferation and differentiation of human leukemic pre - B cells. Coincubation with human stromal cells led to a significantly higher proliferation of the cytokine - sensitive leukemic pre - B cell line BLIN-1. Clones from the murine fibroblast cell line L929 provided identical results. Coincubation with the murine cells also resulted in a significantly higher numbers of viable cells in 5 of 8 patient samples with newly diagnosed B lineage ALL. The results show that in vitro bone marrow stromal cells can be substituted by murine fibroblasts and form the basis for a simpler and more reproducible assay system.     

The effect of Arbas Cashmere goat bone marrow stromal cells on production of transgenic cloned embryos

The aim of this study was to develop a method for the in vitro separation and culture of Arbas Cashmere goat bone marrow stromal cells (gBMSCs). Arbas Cashmere gBMSCs were isolated and cultured in vitro, and cell surface markers were identified immunohistochemically. The gBMSCs were differentiated into neurocytes and osteoblasts, and the expression of neuron-specific enolase and osteocalcin was identified by immunohistochemistry. The gBMSCs and goat fetal fibroblast cells (gFFCs) were compared for transient transfection efficiency and fluorescent colony-forming efficiency with Arbas Cashmere gFFCs as a control. pDsRed2-1 encodes DsRed2, a variant of the Discosoma sp. red fluorescent protein (DsRed). In addition, the coding sequence for DsRed2 contains a series of silent base-pair changes for higher expression in mammalian cells. Of the gBMSCs-pDsRed2-1, one fraction was tested for pluripotency, whereas the other fraction was manipulated using somatic cell nuclear transfer, and the in vitro growth status of transgenic embryos derived from gBMSCs-pDsRed2-1 and gFFCs-pDsRed2-1 was compared. The findings showed that gBMSCs were isolated and amplified to express CD29, CD44, and CD90 through adherent culture, with no marked signs of aging after multiple passages. Expression of neuron-specific enolase and osteocalcin by gBMSCs and gBMSCs-pDsRed2-1 was strongly induced by neuronal and osteogenic differentiation, whereas the integrated exogenous genes did not influence pluripotency (P > 0.05). The transient transfection efficiencies of gBMSCs and gFFCs after 48 hours were not significantly different; however, the fluorescent colony-forming efficiency of gBMSCs-pDsRed2-1 after G418 screening was approximately 13% higher than that of gFFCs-pDsRed2-1. The convergence and cleavage rates of cloned embryos derived from gBMSCs-pDsRed2-1 were higher than those derived from gFFCs-pDsRed2-1, whereas their eight-cell and blastocyst rates were similar. The red fluorescent protein expression levels were higher in transgenic embryos derived from gBMSCs-pDsRed2-1 compared with those derived from gFFCs-pDsRed2-1 (48.8% vs. 31.1%, respectively) (P < 0.01). Real-time quantitative Polymerase Chain Reaction analysis showed that DsRed2-1 messenger RNA expression of cloned embryos derived from gBMSCs was 2.24 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). Similarly, Western blot analysis showed that DsRed2 protein expression of cloned embryos derived from gBMSCs-pDsRed2-1 was 1.29 greater than that of embryos derived from gFFCs-pDsRed2-1 (P < 0.01). In this study, gBMSCs were also used for somatic cell nuclear transfer and shown to provide effective nuclear donor cells for breeding new genetically modified varieties of livestock.     

Erythroid lineage-specific activity in conditioned medium derived from cloned human marrow stromal cells (CFU-RF)

Using long-term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self-renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU-RF. In this paper, a method is described for subculturing cells from individual CFU-RF-derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage-specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose-response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony-stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.     

Collagen synthesis by cloned pulmonary artery endothelial cells

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.     

Characterization of stem cells and progenitors of hemopoiesis by cell sorting.

Cell sorting has been used as a method for characterizing hemopoietic stem cells and progenitors. Fluorescent antibody-surface labels and changes in fluorescence polarization induced by in vitro stimulation with potential hemopoietic regulators were used. As detected by significant enrichment of CFU-S (pluripotent stem cells) in fluorescence-activated cell sorting, some CFU-S bear 'unique antigens' recognized by rabbit anti-human brain sera, human anti-human sperm sera, and 129 anti-F9 serum, but not A . TH anti-A . TL (Ia) ascites. Significant changes in fluorescence polarization induced by in vitro stimulation of mouse bone marrow with potential hemopoietic regulators were also observed; further, progenitors of human T-lymphocyte colonies were observed to exhibit a significantly decreased mean polarization value after short-term stimulation with PHA-LCM (phytohemagglutinin-stimulated leukocyte conditioned medium).     

Multilineage potential research of Beijing duck amniotic mesenchymal stem cells

Amnion, which is usually discarded as medical waste, is considered as abundant sources for mesenchymal stem cells. In human and veterinary medicine, the multipotency of mesenchymal stem cells derived from amnion (AMSCs) together with their plasticity, self-renewal, low immunogenicity and nontumorigenicity characteristics make AMSCs a promising candidate cell for cell-based therapies and tissue engineering. However, up till now, the multipotential characteristics and therapeutic potential of AMSCs on preclinical studies remain uncertain. In this work, we successfully obtained AMSCs from Beijing duck embryos in vitro, and also attempted to detect their biological characteristics. The isolated AMSCs were phenotypically identified, the growth kinetics and karyotype were tested. Also, the cells were positive for MSCs-related markers (CD29, CD71, CD105, CD166, Vimentin and Fibronection), while the expression of CD34 and CD45 were undetectable. Additionally, AMSCs also expressed the pluripotent marker gene OCT4. Particularly, when appropriately induced, AMSCs could be induced to trans-differentiate into adipocytes, osteoblasts, chondrocytes and neurocytes in vitro. Together, these results demonstrated that the isolated AMSCs maintained their stemness and proliferation in vitro, which may be useful for future cell therapy in regenerative medicine.     

The osteogenic differentiation of human bone marrow stromal cells induced by nanofiber scaffolds using bioinformatics

This article aims to investigate the mechanism of behaviors of human bone marrow stromal cells (hBMSCs) affected by scaffold structure combining Monte Carlo feature selection (MFCS), incremental feature selection (IFS) and support vector machine (SVM). The specific differentially expressed genes (DEGs) of hBMSCs cultured on nanofiber (NF) scaffolds and freeform fabrication (FFF) scaffolds were obtained. Key genes were screened from common genes between osteogenic DEGs and NF specific DEGs with MFCS, IFS and SVM. The results demonstrated that NF scaffolds induced hBMSCs to express more genes related to osteogenic differentiation. Finally, 16 key genes were identified among the common genes. The common genes were significantly enriched in Rap1 signaling pathway, extracellular matrix and ossification. The results in this study suggested that the gene expression of hBMSCs was sensitive to NF scaffolds and FFF scaffolds, and the osteogenic differentiation of hBMSCs could be enhanced by NF scaffolds.     

Multilineage differentiation of muscle-derived stem cells from GFP transgenic mice

It is unclear whether green fluorescent protein (GFP) expression is maintained during the course of multilineage differentiation of muscle-derived stem cells (MDSCs). We isolated MDSCs from GFP-transgenic mice and transferred them to chondrogenic, neurogenic or myogenic media. Multilineage differentiation was examined by morphological observation, histological staining, immunocytochemical staining, real-time RT-PCR and Western blot. Both differentiated cells and non-differentiated cells maintained stable GFP expression until the cells exhibited a senescent phenotype. Thus, MDSCs from GFP-transgenic mice have multilineage potential in vitro and that GFP expression does not influence the multilineage potential of MDSCs (or vice versa).     

Multilineage differentiation of Cbfa1-deficient calvarial cells in vitro

We characterized calvaria-derived cells of Cbfa1-deficient mice to determine their stages of differentiation. In long-term culture, Cbfa1-deficient calvarial cells did not acquire osteoblastic phenotypes, but numerous adipocyte foci appeared with an increase in the expression of marker genes for adipocyte differentiation. In culture with BMP-2, Cbfa1-deficient calvarial cells still failed to generate bone nodules but differentiated into chondrocytes and further to terminal hypertrophic chondrocytes, and adipocyte foci were decreased. Cbfa1-deficient calvarial cells transplanted into the peritoneal cavity of athymic mice using BMP-2-coated diffusion chambers generated cartilage but not bone. These data indicate that Cbfa1-deficient calvarial cells completely lack the ability to differentiate into mature osteoblasts and Cbfa1 has an inhibitory function in adipocyte differentiation. As Cbfa1-deficient calvarial cells were enriched with immature mesenchymal cells, which can differentiate into adipocytes and chondrocytes, it is suggested that Cbfa1 plays an essential role in determining the lineage of multipotential mesenchymal precursor cells.     

Role of stromal microenvironment in the regulation of bone marrow hemopoiesis after curantyl administration

Role of the stromal microenvironment in regulation of bone marrow hemopoiesis at the administration of the thrombocyte disaggregant curantyl was studied by the method of heterotopic transplantation of the mice bone marrow. It is shown that the action of curantyl on hemopoiesis is realised through the stem stromal cells of the bone marrow. It is noted that the inhibitory action of the preparation on proliferation of osteogenic precursor-cells is followed by activation of bone resorption processes in regenerating ectopic hemopoietic organ. Under the action of curantyl at low bone marrow cellularity in the focus of heterotopic hemopoiesis and femur an increase of mitotic activity in hemopoietic elements is noted. It is revealed that a phenomenon of ineffective megakaryocytopoiesis with intramedullary destruction of megakaryocytes leads to the local excretion of the thrombocyte released growth factor (TRGF) which has a compensatory character.