Fibroblastic Stromal Cells Research Articles

Vaginal and uterine stroma maintain their inductive properties following primary culture.

Vaginal and uterine stromal (VS and UtS) cells have been cultured in a collagen gel matrix, and the ability of the cells to retain their identity and interact normally with epithelia after culture was examined. Stromal explants from 2-d-old mice were plated onto an extracellular matrix covered with collagen, and maintained in Ham's F12:DMEM (1:1) containing 15% fetal bovine serum. The fibroblastic stromal cells invaded and eventually filled the overlying collagen during the 4-wk growth period, and the total DNA of the UtS and VS cultures increased 3.5- and 4-fold, respectively. To assess the ability of the cultured stroma to perform its normal functions after the in vitro period, recombinations of cultured stroma and fresh epithelia were prepared and transplanted under the renal capsule of female hosts and grown for 4 wk. The epithelium in recombinants of cultured VS + vaginal epithelium (VE) and cultured UtS + uterine epithelium (UtE) was histologically normal and proliferated in response to estrogen. Cultured stroma also instructively induced heterologous epithelium; VS induced UtE to undergo vaginal differentiation, and UtS induced VE to undergo uterine differentiation. These results indicate that UtS and VS retain their identity and do not irreversibly dedifferentiate in culture. Stromal cells grown in a collagen gel matrix form a functional stroma; they interact normally with epithelium after culture and express normal permissive and instructive inductive functions when reassociated with epithelium and grown in vivo.

Class II induction of human corneal fibroblasts by cell-free supernatants from HSV stimulated lymphocytes.

Lymphocytes obtained from patients with herpes simplex virus, type 1 (HSV) neutralizing antibodies were stimulated in vitro for 48 hr with heat inactivated HSV. Cell-free supernatant from these cultures was added to autologous or allogeneic HLA-DR-negative stromal (fibroblast) cells for 3 days. All stromal cultures were stained with monoclonal reagents by indirect immunofluorescence for Class I (beta 2) or Class II (HLA-DR) antigen expression. Supernatant from HSV-stimulated lymphocytes induced HLA-DR antigen on cultured stromal cells while supernatant from lymphocytes cultured in the absence of HSV antigen or from lymphocytes which did not proliferate in response to incubation with HSV antigen did not induce HLA-DR. The expression of Class II antigens induced by supernatant from HSV-stimulated lymphocytes was inhibited by the addition of purified antibodies to IFN.

Kinetics and differentiation of marrow stromal cells in diffusion chambers in vivo.

Rabbit marrow cells inoculated into diffusion chambers (10(7) cells/chamber) were implanted intraperitoneally into athymic mouse hosts and cultured in vivo for 20 days. A connective tissue consisting of bone, cartilage and fibrous tissues is formed by the stromal fibroblastic cells of marrow within the chambers. Cell kinetics and tissue differentiation have been studied using histomorphometric and biochemical analyses. Haemopoietic cell numbers decrease to less than 0.05% of the initial inoculum during the 20-day period. At 3 days an average of 15 stromal fibroblastic cells only are identifiable within the chambers. After 3 days there is a high rate of stromal cell proliferation with a doubling time of 14.5 h during the period from 3 to 8 days and an increase in the total stromal cell population by more than six orders of magnitude from 3 to 20 days. Thirteen to fourteen population doublings occur before expression of the first observable differentiation parameter, alkaline phosphatase activity. The data demonstrate that the mixture of connective tissues formed within the chamber is generated by a small number of cells with high capacity for proliferation and differentiation. This is consistent with the current hypothesis that stromal stem cells are present in bone marrow.

Bone lining cells and hematopoiesis: an electron microscopic study of canine bone marrow.

Hematopoietic bone marrow in the dog is enclosed by a nearly complete and rather complex layer of endosteum, consisting of a diverse group of cells collectively called bone lining cells (BLC). Cell types comprising BLC include osteoblasts and osteoclasts, and other cell types, among which are elongated, flat cells with a spindle-shaped nucleus, and small cytoplasmic vesicles. The composition and thickness of the layer of BLC varies along the perimeter of the marrow. The layer may be simple or stratified. Occasionally a zone of tightly packed regularly arranged collagenous fibers lies between the bone lining cells and bone. Hematopoiesis, particularly neutrophilic, often occurs in the bone marrow next to the BLC. Cytoplasmic processes of BLC occasionally extend into the hematopoietic spaces and stromal cells in the hematopoietic compartment may extend processes to the layer of BLC. Occasionally cells of the BLC are similar in appearance to stromal cells within the marrow. Our observations together with the experimental findings of others (that fibroblastic stromal cells contribute to the hematopoietic inductive microenvironment, that hematopoietic stem cells are concentrated subosteally, that cells responsible for regeneration of the marrow stroma are derived from the endosteal layer, and that high concentrations of hematopoietic colony-stimulating factors are produced there) indicate that the hematopoietic capacities of bone marrow may be regulated by BLC.

Establishment and characterization of multipotential state of chick embryo pigment epithelial cells [formula omitted]: [formula omitted], [formula omitted], [formula omitted]∗ and [formula omitted]∗. Natl. Inst. Basic Biol., Okazaki 444 and ∗Aichi Med. Univ., Aichi, Japan

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.

Ultrastructural evidence of vanadium-accumulating cell type in ascidian metamorphosis: [formula omitted] and [formula omitted]. II Chair of Histology and Embryology, University of Naples, via Mezzocannone 8, 80134 Naples, Italy

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.

Stimulation of Corneal Wound Healing With Mesodermal Growth Factor

Mesodermal growth factor (MGF) from mouse submaxillary glands was tested in vivo for stimulating effects on corneal wounds in rabbits. Intrastromal injection of 5 microgram of MGF induced widespread fibroblast activity and stromal cell division, and markedly stimulated stromal healing. At high doses (greater than 25 microgram), corneal destruction was indicated by extensive necrosis and perforation. When low doses (1 to 5 microgram) of MGF were applied to the lip of nonperforating knife wounds of the cornea, three major differences were noted between control and experimental wounds. In wounds treated with MGF, the depth of stromal healing was greater, as was the intensity of the fibroblast activity, and the width and depth of the epithelial plug were significantly decreased. These results establish that MGF is an effective growth-stimulating agent in vivo and that the initial stages of corneal wound healing may be accelerated in vivo.