Histochemical demonstration of collagen fibers in ascorbic-acid-fed cell cultures.

Nine cultures of fibroblast cell types and 13 epithelial-like cell types were maintained for 1 week in media supplemented with L-asborbic acid (50 microgram per ml). All fibroblast-like cultures produced extracellular fibers that stained positively by a silver-impregnation reticulin stain. Nine of the 13 epithelial-like cultures produced fibers that stained positively for reticulin. Nearly all cultures not supplemented with ascorbic acid showed no fiber staining. Those few lines that stained positively for reticulin in the absence of ascorbic-acid supplementation demonstrated only slight reticulin formation. Reticulin from one fibroblast culture and one epithelial culture was examined by electron microscopy, and the silver-impregnated fibrils were morphologically identical to collagen. The reticulin was digestible with collagenase, providing further evidence that the silver-impregnation reticulin stain identifies collagen in culture. The demonstartion of collagen can be performed easily in histology laboratories using Formalin-fixed cells, and provides a means of assaying a functional property of cells in culture which is characteristic of connective tissue fibroblasts in general as well as certain specialized epithelia.     

Detection of chick rRNA in the cytoplasm of heterokaryons containing reactivated chick red cell nuclei

Collagen fiber formation in vitro was morphologically investigated on 22 epithelial-like cell lines originating from normal rat livers mainly by using specific histochemical stainings. All the cell lines, eight of which were cloned formed typical “collagenous” and/or “reticulin” fibers in the histological sense when they had been left as a full sheet for more than 3 weeks without subculture. Only one of five fibroblastic cell lines serving as control formed fibers to a lesser degree. Different patterns were recognized in fibers appearing between the liver cell cultures and the fibroblast cultures.     

Promotion of collagen production by human fibroblasts with gastric cancer cells in vitro

To elucidate the histogenesis of gastric scirrhous cancer, the promotion of collagen production by normal human skin fibroblasts (HSF-1) with human gastric cancer cells (KATO-III, MKN-45 and MKN-28) was investigated by direct coculture and parabiotic culture. Argyrophilic collagenous fibers were demonstrated among fibroblasts on both direct cocultures and parabiotic cultures of the fibroblasts with gastric cancer cells. Microscopic examination showed that these fibers appeared earlier and were more abundant and thicker in direct cocultures and parabiotic cultures than in single cultures of fibroblasts. Gastric cancer cells in single or parabiotic culture did not form argyrophilic fibers. For quantitative proof of the promotion of collagen production by fibroblasts with gastric cancer cells, hydroxyproline produced by fibroblasts was measured. Much higher fibroblast hydroxyproline values were obtained in parabiotic cultures with gastric cancer cell lines than in single cultures of HSF-1. Moreover, the rate of collagen synthesis by HSF-1 was much higher than that of any gastric cancer cell line tested. These results demonstrate that gastric cancer cells enhance collagen production by fibroblasts in vitro. This finding suggests that they may produce a factor promoting fibroblast collagen synthesis and that this may contribute to the formation of stromal collagen in human gastric scirrhous cancer.     

New tetrachromic VOF stain (Type III-G.S) for normal and pathological fish tissues

A new VOF Type III-G.S stain was applied to histological sections of different organs and tissues of healthy and pathological larvae, juvenile and adult fish species (Solea senegalensis; Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the original Gutiérrez VOF stain, more acid dyes of contrasting colours and polychromatic/metachromatic properties were incorporated as essential constituents of the tetrachromic VOF stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histochemical approaches of the cell components. The VOF Type III -6.5 stain is composed of a mixture of several dyes of varying size and molecular weight (Orange G相似文献   

A combined elastic, fibrin and collagen stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified Verhoeff's elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by lissamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsin, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Stain Technology: A Combined Elastic, Fibrin and Collagen Stain

A method is described which demonstrates nuclei, elastic fibers, red blood cells, collagen and fibrin. Nuclei and elastic fibers are stained by a modified VerhoefPs elastic tissue stain which was previously developed and used in the elastic-Masson combination. Both early fibrin and red blood cells are shown by Hssamine fast yellow. Mature fibrin, some types of collagen and other cytoplasmic changes are stained by a combination of acid fuchsia, Biebrich scarlet and ponceau 2R, while old fibrin is demonstrated by the collagen stain. This method takes about 1 hr to perform and has the added advantage that several entities are clearly shown in a single slide.     

Properties of Wilms' tumor line (TuWi) and pig kidney line (LLC-PK1) typical of normal kidney tubular epithelium

Summary Two stable epithelial-like cell lines, the pig kidney strain (LLC-PK₁) and a Wilms' tumor line (TuWi), previously established in other laboratories, were found to exhibit a number of properties characteristic of kidney proximal tubular epithelium. Electron micrographs of LLC-PK₁ monolayers revealed cells forming rosettes reminiscent of tubules. Numerous elongated microvilli and an amorphous basal laminar material surrounded the cell membranes. Cell junctions were located between cell membranes at regions adjacent to the patent lumens. Wilms' cells in culture were similar in appearance to the pig kidney cells; they exhibited numerous microvilli, a thin basal laminar coating on the membrane, and desmonsomes between cells. No rosette formation was evident. Neither cell line was found to produce extracellular reticulin fibers when grown in the presence ofl-ascorbic acid for 1 week. Absence of stainable reticulin in cell monolayer culture after ascorbicacid treatment has been noted only in cell lines of apparent epithelial origin. Histochemically, both lines reacted positively for activities of a number of enzymes found in high amounts in normal kidney tubular epithelium. Pig kidney cells were highly positive for γ-glutamyl transpeptidase activity and moderately active for acid phosphatase and leucine aminopeptidase activities. Wilms' tumor cells were markedly active for γ-glutamyl transpeptidase, 5′-nucleotidase, ATPase, glucose-6-phosphatase, and acid phosphatase activities. These findings in conjunction with the ultrastructural observations indicate that these two lines in culture maintain many of the properties typical of proximal kidney tubular epithelium.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.     

A dual enzyme method for the establishment of long‐ and medium‐term primary cultures of epithelial and fibroblastic cells from Atlantic salmon gills

A dual enzyme disaggregation method using collagenase and then trypsin was developed that allowed the reproducible initiation of primary cultures from Atlantic salmon Salmo salar gills. Cultures had both epithelial and fibroblast morphology and persisted for an average of 20 passages. Growth was dependent upon a minimum concentration of 5% foetal calf serum (FCS) for fibroblasts and 10% FCS for epithelial cells. Growth was mostly independent of substrate, although epithelial cells showed increased growth on type I collagen gels. Matrigel? cell culture substrate produced reduced growth of fibroblasts and did not benefit epithelial cell growth. Epithelial cells reacted with monoclonal antibodies (MAbs) against mammalian cytokeratins, and fibroblast cells reacted with MAbs against mammalian fibronectin and type I collagen. The method also produced two long‐term cultures: one epithelial and one fibroblast that have been designated RGE‐2 and RGF respectively.     

Characterization of human mammary cell types in primary culture: Immunofluorescent and immunocytochemical indicators of cellular heterogeneity

Summary Parenchymal organoidal structures that were obtained from collagenase digestion of reduction mammoplasty specimens of apparently normal human breasts have been grown in short-term primary cultures, either on plastic or on floating gels of polymerized rat-tail collagen. Three morphologically distinct major cell types are readily observed in both systems: cuboidal cells, which occupy apical positions on collagen gels; larger, epithelioid, or basal cells on gels; and elongated cells which penetrate into the gel. In addition, a fourth cell type, that of a large, flat cell, is observed less readily by phase contrast microscopy on the surface of cultures grown on plastic. Immunofluorescent and immunocytochemical staining of cultures on plastic or histologic sections of cultures on gels have been undertaken with antisera and other histochemical reagents that stain the different parenchymal cell types in vivo. Thus antisera to epithelial membrane antigen(s), monoclonal antibodies (MABs) to the defatted mammary milk fat globule membrane, peanut lectin, and keratin MAB LE61, which preferentially stain the epithelial cells of ducts in vivo, also stain the cuboidal/apical cells in vitro. The large, flat cells are stained intensely by the first three reagents but not by the last one. Antisera to collagen IV, laminin, fibronectin, actin, keratin MAB LP34, MABs to the common acute lymphoblastic leukemia antigen, and MAB LICR-LON-23.10, which showed enhanced staining for the ductal myoepithelial cells in vivo, also stain the epithelioid/elongated cells in vitro. However, the effect of the last four reagents is reduced considerably in most elongated cells, and MAB LP34 stains the large, flat cells intensely. Heterogeneous cells of intermediate morphologies and staining patterns between the cuboidal/flat cells and large epithelioid cells have also been identified. The results suggest that the cuboidal cells and large, flat cells are related to mammary epithelial cells, whereas the large epithelioid/elongated cells have some characteristics of myoepithelial cells, and that intermediate forms may exist in culture between the two parenchymal cell types. This work was supported in part by the Ludwig Institute for Cancer Research and the Cancer and Polio Research Fund. Dr. M. J. Warburton is supported by the Cancer Research Campaign.     

Isolation of simian virus 40-transformed human mammary epithelial stem cell lines that can differentiate to myoepithelial-like cells in culture and in vivo

Simian Virus 40 (SV40) transformation of primary cultures of human mammary epithelial cells has yielded a cloned epithelial-like cell line and a representative, single-cell subclone. Although apparently homogeneous, both cloned cell lines can also yield small numbers of three other cell types. The more-elongated cell type can be obtained directly by replating cells from the medium of the epithelial-like cell cultures or by picking and culturing single cells to form representative lines. Immunofluorescent and immunocytochemical analysis of these cell lines growing on plastic or as tumor-nodules in nude mice for epithelial membrane antigens, various cytokeratins, various actins, laminin, Type IV collagen, the common acute lymphoblastic leukemia antigen (CALLA), and a 135-kDa glycoprotein confirm the epithelial nature of the epithelial-like cells and suggest a myoepithelial origin for the more-elongated cell type. Ultrastructural analysis largely confirms the results, although the myofilamental bundles can be scanty in the growing myoepithelial-like cells. The other two cell types are possibly related to the keratinizing and casein-secreting cells seen in the epithelial tumor-nodules before and after mating the mice, respectively. The myoepithelial-like cells produce 5- to 17-fold more laminin, Type IV collagen, CALLA, and the 135-kDa glycoprotein than the epithelial cells, and all of these antigens are preferentially found on myoepithelial cells in vivo. It is suggested that the SV40-transformed epithelial cell is an immortalized form of human mammary stem cell which can differentiate in culture and in vivo to myoepithelial-like cells.     

Mesenchymal precursor cells in the blood of normal individuals

Mesenchymal precursor cells found in the blood (BMPCs) of normal persons adhere to plastic and glass and proliferate logarithmically in DMEM-20% fetal calf serum (FCS) without growth factors. They form cells with fibroblast-like and stromal morphology, which is not affected by eliminating CD34, CD3, or CD14 cells. Osteogenic supplements (dexamethasone, ascorbic acid, and β-glycerophosphate) added to the culture inhibited fibroblast formation, and BMPCs assumed the cuboidal shape of osteoblasts. After 5 days in supplemented medium, the elutriated cells displayed alkaline phosphatase (AP), and the addition of bone morphogenetic protein (BMP)2 (1 ng) doubled AP production (P < 0.04). Two weeks later, 30% of the cells were very large and reacted with anti-osteocalcin antibody. The same cultures also contained sudanophlic adipocytes and multinucleated giant cells that stained for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors. Cultured BMPCs immunostain with antibodies to vimentin, type I collagen, and BMP receptors, heterodimeric structures expressed on mesenchymal lineage cells. In addition, BMPCs stain with anti-CD105 (endoglin), a putative marker for bone-marrow mesenchymal stem cells (MSCs).     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Abstract. Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, Ml8, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody Ml8 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Expression of epithelial antigens in primary cultures of normal human breast analysed with monoclonal antibodies

Primary cultures of normal human breast were stained with monoclonal antibodies to see if antigens characteristic of luminal epithelial cells are retained in culture. Three monoclonal antibodies were used, LICR-LON-M8, LICR-LON-M18, and LICR-LON-M24, all specific for the cell surface of luminal epithelial as opposed to myoepithelial or stromal cells in the breast, and each staining a different subset of the epithelial cells in the intact tissue. Cultures were prepared from reduction mammoplasty samples by digestion with collagenase. The surface layer of cells was stained by immunofluorescence without fixation. (Cells underneath the surface layer were not accessible to this mode of staining). The antibodies stained patches of cells resembling flattened epithelium. These patches of cells cannot be distinguished by phase contrast microscopy without reference to the staining, in fact the boundaries of the cells are not usually resolved by phase contrast microscopy. Electron microscopy of sections through these cells show they are very flattened. They lie on top of the polygonal and elongated cells that dominate the phase contrast image. Two of the antibodies, M8 and M24, stain subsets of these epithelial-like cells at all stages of culture. The third antibody, M18, stains such cells initially, but after the first few days staining is predominantly found on the polygonal and elongated cells, then this also gradually disappears. It is possible that the cells stained by antibody M18 are converting from the epithelial-like morphology to the cuboidal and elongated morphology. Many cells are not stained by any of the antibodies, so appear either to by myoepithelial in origin or to have lost their luminal epithelial surface antigens at an early stage. This analysis draws attention to the variety of cell types in these cultures and the limitations of phase contrast microscopy as a means of analysing them.     

Collagen processing, crosslinking, and fibril bundle assembly in matrix produced by fibroblasts in long-term cultures supplemented with ascorbic acid

Human foreskin fibroblasts were cultured for up to 6 weeks in medium supplemented with ascorbic acid. During this time, the cells produced an extensive new connective tissue matrix in which the accumulated collagen (mostly type I) amounted to about 0.25 mg/10(6) cells. The matrix was highly differentiated as shown by complete processing of procollagen to collagen alpha-chains and covalent crosslinking of the collagen. Alignment of collagen fibrils occurred as the fibrils were deposited between cells, and binding of adjacent fibrils to the cell surface appeared to hold the fibrils in register. Groups of aligned fibrils were subdivided into bundles by cell-surface folds. If beta-aminopropionitrile was added to the medium, collagen crosslinking was inhibited, but not collagen synthesis or fibril bundle organization. If ascorbic acid was omitted from the culture medium, the extensive new connective tissue matrix was not produced. Our results indicate that fibroblasts in long-term cultures supplemented with ascorbic acid produce a connective tissue matrix with many in vivo-like properties including supermolecular organization of collagen.     

Long-term culture of fetal rat hepatocytes in media supplemented with fetal calf-serum Ultroser SF or Ultroser G

Fetal hepatocytes cultured in medium supplemented with fetal calf serum (FCS) or Ultroser SF do not maintain production of albumin or transferrin beyond one week of culture. When dexamethasone (10(-7) M) is present, secretion of albumin and transferrin can be extended to two weeks, however, levels are extremely low. By three weeks, neither plasma protein can be detected in the culture medium in either conditions of culture. In contrast, hepatocytes maintained in medium supplemented with Ultroser G continue to produce albumin and transferrin at high levels for the entire three week period of this study. The morphology of the cultures are different. In FCS and Ultroser SF supplemented medium there are many more fibroblast and epithelial-like cells and relatively fewer cells which are distinctly hepatocytes when compared with Ultroser G supplemented medium. The level of tyrosine aminotransferase, which is a dexamethasone inducible enzyme, is found to be much higher in Ultroser G cultures, with no further increase demonstrable by addition of dexamethasone. In contrast, dexamethasone induces the enzyme by about eight-fold in cultures maintained in FCS supplemented medium. Therefore it appears that Ultroser G already contains sufficient steroid activity to maximize the level of tyrosine aminotransferase. A comparison between Ultroser C and SF (steroid-free) suggests that the mixture of steroid and steroid derivatives in the G formulation must be important in the maintenance of differentiated functions of hepatocytes in culture. However, supplementation of FCS cultures with dexamethasone, which is known to be present in Ultroser G, does not allow hepatocytes to retain their differentiated functions over an extended period. Therefore it is concluded that other components besides dexamethasone must be important.     

Collagen synthesized and modified by aging fibroblasts in culture.

Collagen is produced by WI-38 diploid human fibroblast cultures throughout their life cycle. It is examined by a sensitive method based on the analysis of specific peptides obtained after digestion with bacterial collagenase. The production and hydroxylation of the collagen is strongly dependent upon the age (population doublings) of the culture and the presence of ascorbic acid. Young cultures (passage 26) produce large amounts of collagen in the absence of ascorbic acid, and this collagen is about 50% hydroxylated compared to that produced by young cultures in the presence of ascorbic acid. Ascorbic acid reduces to about one-half the amount of collagen produced by these young cultures. The young confluent cultures also depend strongly on ascorbic acid for hydroxylation of proline. The dependence declines rapidly with the age of the culture. The collagen produced by young cultures supplied with ascorbic acid is very similar to the type I collagen produced by normal individuals and has about the same degree of hydroxylation of its prolyl residues. The amount of collagen produced by "older" cultures is unaffected by ascorbic acid, but the degree of hydroxylation is normal only if ascorbic acid is present, and is decreased to about 60 to 70% in the absence of the vitamin. "Senescent" cultures showed little, if any, dependency on ascorbic acid, and the collagen produced, with and without the vitamine, is about 80% hydroxylated. The prolyl hydroxylation system of the WI-38 cells and the various controls on the system are age-dependent.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.     

Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells

Human liposuction aspirates contain pluripotent adipose-derived mesodermal stem cells that have previously been shown to differentiate into various mesodermal cell types, including osteoblasts and chondrocytes. To develop an autologous research model of bone and cartilage tissue engineering, the authors sought to determine whether rat inguinal fat pads contain a similar population of osteochondrogenic precursor cells. It was hypothesized that the rat inguinal fat pad contains adipose-derived multipotential cells that resemble human adipose-derived mesodermal stem cells in their osteochondrogenic capacity. To test this, the authors assessed the ability of cells isolated from the rat inguinal fat pad to differentiate into osteoblasts and chondrocytes by a variety of lineage-specific histologic stains.Rat inguinal fat pads were isolated and processed from Sprague-Dawley rats into a fibroblast-like cell population. Cell cultures were placed in pro-osteogenic media containing dexamethasone, ascorbic acid, and beta-glycerol phosphate. Osteogenic differentiation was assessed at 2, 4, and 6 weeks. Alkaline phosphatase activity and von Kossa staining were performed to assess osteoblastic differentiation and the production of a calcified extracellular matrix. Cell cultures were also placed in prochondrogenic conditions and media supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid. Chondrogenic differentiation was assessed at 2, 7, and 14 days by the presence of positive Alcian blue staining and type II collagen immunohistochemistry. Cells placed in osteogenic conditions changed in structure to a more cuboidal shape, formed bone nodules, stained positively for alkaline phosphatase activity, and secreted calcified extracellular matrix by 2 weeks. Cells placed in chondrogenic conditions formed cartilaginous nodules within 48 hours that stained positively for Alcian blue and type II collagen. The authors identified the rat inguinal fat pad as a source of osteochondrogenic precursors and developed a straightforward technique to isolate osteochondrogenic precursors from a small animal source. This relatively easily obtained source of osteochondrogenic cells from the rat may be useful for study of tissue engineering strategies and the basic science of stem cell biology.