The transforming growth factor-βs (TGF-βs) comprise a family of pleiotropic members that signal through two types of serine/threonine kinase receptors, named TGFRI (TGF-β type I receptor) and TGFRII (TGF-β type II receptor). We previously demonstrated that cortical neurons increase the astrocyte maturation marker, glial fibrillary acidic protein (GFAP), and thus, astrocyte differentiation, by inducing TGF-β1 secretion by astrocytes in vitro. Although TGF-β receptor expression has been described in different brain regions and cell types, their localization is still a subject of discussion. In the present work, we analyzed TGFRII expression in cultured cortical astrocytes from embryonic and newborn animals by immunocytochemistry, Western blot, and reverse transcriptase–polymerase chain reaction (RT-PCR). We report for the first time expression of TGFRII in embryonic glia. TGFRII immunostaining was punctual and spread throughout the cellular membrane of embryonic and newborn astrocytes. Western blot and RT-PCR assays revealed similar levels of the receptor in astrocytes from different ages. Identification of TGFRII in embryonic astrocytes is novel and might point to the multipotent precursor cell, radial glia, as a potential target for TGFβ1 during astrocyte development.

The significantly higher incidence of anterior cruciate ligament (ACL) injuries in collegiate women compared with men may result from relative ligament laxity. Differences in estrogen and relaxin activity, similar to that seen in pregnancy, may account for this. We quantified estrogen receptors by flow cytometry and relaxin receptors by radioligand binding assay in human ACL cells and compared the presence of these receptors in males and females. ACL stumps were harvested from seven males and eight females with acute ACL injuries. The tissue was placed in M199 cell culture medium. Outgrowth cultures were obtained, and passage 2 cells were used for all studies. Estrogen receptor determination was performed using flow cytometry. Relaxin binding was performed in ACL cells derived from five female and male patients using I¹²⁵-labeled relaxin. Estrogen receptors were identified by flow cytometry in 4 to 10% of ACL cells. Mean fluorescence of cells expressing estrogen receptors was approximately twice that of controls, with no significant differences between males and females. Relaxin studies showed low-level binding of I¹²⁵-relaxin–labeled ACL cells. Relaxin binding was present in four out of five female ACL cells versus one out of five male ACL cells.

Osteoclasts are bone-resorbing cells that differentiate from macrophage precursors in response to receptor activator of NF-κB ligand (RANKL). In vitro models of osteoclast differentiation are principally based on primary cell cultures, which are poorly suited to molecular and transgene studies because of the limitations associated with the use of primary macrophage. RAW264.7 is a transfectable macrophage cell line with the capacity to form osteoclast-like cells. In the present study, we have identified osteoclast precursors among clones of RAW264.7 cells. RAW264.7 cell were cloned by limiting dilution and induced to osteoclast differentiation by treatment with recombinant RANKL. Individual RAW264.7 cell clones formed tartrate resistant acid phosphatase (TRAP)-positive multinuclear cells to various degrees with RANKL treatment. All clones tested expressed the RANKL receptor RANK. Each of the clones expressed the osteoclast marker genes TRAP and cathepsin-K mRNA with RANKL treatment. However, we noted that only select clones were able to form large, well-spread, TRAP-positive multinuclear cells. Clones capable of forming large TRAP-positive multinuclear cells also expressed β₃ integrin and calcitonin receptor mRNAs and were capable of resorbing a mineralized matrix. All clones tested activated NF-κB with RANKL treatment. cDNA expression profiling of osteoclast precursor RAW264.7 cell clones demonstrates appropriate expression of a large number of genes before and after osteoclastic differentiation. These osteoclast precursor RAW264.7 cell clones provide a valuable model for dissecting the cellular and molecular regulation of osteoclast differentiation and activation.

In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT₁ and AT₂. Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor– deficient mice were bred with an Immortomouse®, which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F₂ mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor–deficient cell lines, (AT_1A [−/−], Tag [ /−]), (AT_1B [−/−], Tag [ /−]), and (AT_1A [−/−], AT_1B [−/−], Tag [ / ]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; −3 to 3 μA/cm²), basal monolayer conductance (G, 2 to 10 mS/cm²), Na₃ –phosphate cotransport (ΔIsc of 2 to 3 μA/cm² at 1 mM), and Na₃ –succinate cotransport (ΔIsc of 1 to 9 μA/cm² at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II–stimulated β-arrestin 2 translocation and showed an Ang II–mediated response in wild type but not (AT_1A [−/ −], AT_1B [−/−]) cells. Cell lines were amplified, yielding a virtually unlimited supply of highly differentiated, transport-competent, angiotensin receptor–deficient PCT cell lines.

A mutant strain of drosophila (D. subobscura) has two types of mitochondrial genomes: a small population (20%) identical to that of the wild strain (15.9 kb) and a predominant population (80%) which has undergone a 5-kb deletion affecting more than 30% of the coding zone. Two cell lines were established from homogenates of embryos from mutant and wild strains. The activities of the respiratory complexes measured in the different cell lines are much lower than in the flies, indicating a glycolytic metabolism. Various modifications of the medium composition did not change this metabolic pathway. The mutant cell line has two types of populations of mitochondrial genomes and the heteroplasmy is equivalent to that measured in the mutant strain. However, the biochemical characteristics differ from those observed in the flies (i.e., the decrease of complex I and III activities), and the various systems of compensation for the consequences of the deletion that are showed in the mutant strain are no longer observed. Furthermore, in contrast with observations made on mutant flies, the heteroplasmy appears unstable in the mutant cell lines: after 60 or so generations, it progressively decreases until it disappears completely. The limited importance of mitochondrial energy metabolism in cells may explain the low impact of the mutation on the established cell line, in contrast to what is seen in the mutant strain.

Long-term cultures (LTC) producing dendritic cells (DC) have been previously established from spleen. LTC support the development of nonadherent cells comprising small DC progenitors and immature DC. Similarly, the splenic stroma STX3, derived from a LTC which ceased DC production, can support DC development from precursors in overlaid bone marrow. The STX3 stroma is an immortalised mixed population of endothelial cells and elongated spindle-shaped cells, thought to be fibroblasts. The stromal cell components of STX3 have been studied here. A panel of 102 cell lines was established by single-cell sorting. A range of clone morphology, including cobblestone cells and elongated spindle-shaped cells, was reflective of heterogeneity in STX3. However, similar expression levels for the endothelial genes ACVRL1/ ALK1, COL18A1, and MCAM in 13 splenic stromal cell lines suggested that both cell types had endothelial origin. The hematopoietic support function of stromal clones was tested in coculture assays with a bone marrow cell overlay. Splenic stromal cell lines with different morphology were both supporters and nonsupporters of hematopoiesis, in terms of foci formation or release of suspension cells. Cloning of STX3 led to the isolation of a panel of splenic endothelial cell lines heterogeneous in terms of morphology and hematopoietic support function.

Previously, it was found that senescent cells can undergo a modified cell cycle with mitotic cells as the end results. The major cycling events started with polyploidization, followed by depolyploidization to multinucleated cells (MNCs). These latter cells produced mononuclear offspring cells that could express mitotic cell divisions. In this report the emphasis is on late senescent fibroblasts that exhibited the senescence-associated change in cell morphology to large flat cells. Prior to live cell photography, flat cell cultures were maintained for months in the same culture flasks and therefore judged to be in a late senescent phase. All of the cellular events outlined above were present in these old cell cultures. Time lapse pictures showed movements of mitotic daughter cells away from each other and alignment of the chromosomes on the metaphase plate was visible in other mitotic cells. These data challenge the common view that cell senescence is irreversible and, therefore, an antitumor mechanism. A new finding was that the spike in polyploid cells in the near senescent phase consisted of cells with pairs of sister chromosomes from endoreduplication of DNA (two rounds of DNA synthesis and no mitosis). The lack of cells with 92 single chromosomes (e.g., G2 tetraploid cells) suggested that these polyploid cells also went through a changed cell cycle. The question now is whether these atypical polyploid cells are a subpopulation in senescence that can undergo the cycling from polyploidy to genome-reduced mitotic cells.

Ulmus davidiana Planch (Ulmaceae) (UD) long has been known to have anti-inflammatory and protective effects on damaged tissue, inflammation, and bone among other functions. The herbal medicine also is being used in Oriental medicine to treat osteoporosis. In a preliminary study, treatment of osteoclasts containing long bone cells with the water extract of UD bark prevented the intracellular maturation of cathepsin K (cat K), and thus, it was considered that UD is a pro-drug of a potent bone-resorption inhibitor. To further clarify the role of UD in ossification, we investigated the effects of UD on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, we assessed the effects of UD on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. UD enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the UD was observed at relatively low doses (significant at 5–50 μg/ml and maximal at 50 μg/ml). Northern blot analysis showed that UD (100 μg/ml) increases in bone morphogenic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. UD slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that UD has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.