SEPARATION OF TWO FACTORS AFFECTING THE AGGREGATION KINETICS FROM THE CONDITIONED MEDIUM

Two effective factors contained in medium "conditioned" with a large number of cultured cells were separated through Sephadex G-25 gel, by assaying the plating efficiency and growth rate of chicken embryonic cells in secondary culture. The first factor, which is contained in the heavy molecular weight fraction α, can promote plating efficiency of the inoculated cells at low density. The second is contained in the light molecular weight fraction β, and promotes the growth rate of sparsely cultured cells. The effects of these factors upon the aggregation kinetics of singly isolated cells were studied. The factor in the fraction α promotes aggregation to result in fast flocculation. The factor in the fraction β, while not effective by itself, affects the aggregation of the cells treated with the factor in α. In the co-presence of both α and β, a "lag" was found after the initial fast flocculation. The possible mechanisms of the interaction of these two factors on the cell surface are discussed.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Factors promoting the establishment of primary cultures of liver cells fromXenopus larvae

Summary The requirements for establishment and survival of primary cultures of larval amphibian liver cells were investigated.Plating efficiency was found to be enhanced by a collagen substrate, by diluted conditioned medium from an adultXenopus kidney cell line and by high initial cell densities. Plating efficiency was highest at a tonicity of 165–220 mOsm/kg. In cultures with undiluted conditioned medium the increase in cell number was 50–60% greater than in controls, where it was about 2-fold between day 3 and 6 of culture. Conditioned medium from theXenopus kidney cell line is assumed to contain at least two components, which are effective at different concentrations and stimulate either plating efficiency and cell aggregation or cell proliferation.In cultures without collagen sheets, cell flattening is greatly reduced, indicating that cell shape is also dependent upon the substrate.     

A factor produced by feeder cells which inhibits embryonal carcinoma cell differentiation. Characterization and partial purification

Medium conditioned by STO mouse fibroblast cells inhibited both the spontaneous differentiation of NG2 embryonal carcinoma cells and the differentiation of F9 embryonal carcinoma cells induced by retinoic acid. This effect was due to a differentiation retarding factor (DRF). Reduction in DRF activity in conditioned medium by boiling and by pronase treatment suggested the involvement of a polypeptide, which had an apparent molecular weight of 57000 on gel filtration. A 28-fold purification of DRF was achieved. DRF delayed but did not prevent the extensive differentiation observed after prolonged culture of NG2 colonies. Conditioned medium could be successfully used to replace feeder cells in NG2 stock cultures. Media conditioned by a variety of other cell types also contained differentiation retarding activity.     

Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture.

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.     

Cultures from adult rat liver cells. I. Establishment of monolayer cell-cultures from normal liver

Establishment of monolayer cell-cultures from adult normal rat liver is described using cells isolated by a collagenase-hyaluronidase perfusion technique. The cells plated originally were polygonal in shape and contained a large number of mitochondria. However, cells with less granules were also observed in the primary culture, after a few days of plating. In subsequent subcultures, the latter type of cells could be grown continuously. After an initial lag, the cells attained a doubling time of 55 hours and a plating efficiency of 20%. The cells are “normal” with respect to their karyology, morphology and growth pattern. Difficulties encountered during the culture and the possible uses of these cultured cells in studies on carcinogenesis in vitro are discussed.     

Development of a simple, repeatable, and cost-effective extracellular matrix for long-term xeno-free and feeder-free self-renewal of human pluripotent stem cells

Given the potential importance of human pluripotent stem cells (hPSCs) in translational research and regenerative medicine, the aim of the present study was to develop a simple, safe, and cost-effective substrate to expand hPSCs. We report the development of an extracellular matrix (ECM), designated “RoGel,” based on conditioned medium (CM) of human fibroblasts under serum- and xeno-free culture conditions. The long-term self-renewal of hPSCs on RoGel was also assessed. The results showed that self-renewal, pluripotency, plating efficiency, and cloning efficiency of hPSCs on this newly developed ECM were similar to those of Matrigel, the conventional mouse-cell line-derived ECM. The cells had the capability to passage mechanically on a cold surface, which resulted in their long-term maintenance with normal karyotype. We have demonstrated that CM-coated plates preserved for 1 year at room temperature maintained the capability of hPSC expansion. This ECM provides an attractive hPSC culture platform for both research and future therapeutic applications.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells

Background aimsThe purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo.MethodsCultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media.ResultsIsolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects.ConclusionsL-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism.     

Glucocorticoid regulation of chloroquine nonsensitive insulin degradation in cultured fetal rat hepatocytes

The influence of cortisol and other culture conditions on insulin degradation by the chloroquine-sensitive pathway and the chloroquine-nonsensitive pathway (CNP) was investigated in fetal rat hepatocytes during 3 days of culture. The proportions of the chloroquine nonsensitive release of 125I-insulin degradation products into the conditioned medium/h increased from the 1st to the 3rd day of culture, i.e. from 19 to 50% by cells grown in the presence of cortisol and from 17 to 82% by those grown in the absence of cortisol. Replacement of the conditioned medium with the respective fresh medium dramatically enhanced cellular insulin degradation by CNP, i.e. from 22 to 58%, and 19 to 85% in cells grown for 2 days in the presence and absence of cortisol, respectively. Thus, the conditioned medium contained some factor(s) that inhibited CNP. Therefore, we used the inhibited insulin and alpha-casein degradation by papain in vitro as an assay to investigate the nature of the putative anti-(insulin) protease. Cycloheximide completely prevented the appearance of anti-papain activity in the medium. Conditioned medium obtained from cells grown in the presence of cortisol contained about 2-fold more anti-papain activity than the medium that was obtained in the absence of the steroid. The release of anti-papain activity also declined with time from 1 to 3 days of culture and showed an inverse relationship with the magnitude of cellular insulin degradation by CNP. The inhibition of papain-mediated insulin degradation by the anti-(insulin) protease was noncompetitive. The anti-(insulin) protease was nondialyzable (up to the 10-kDa exclusion limit) and inactivated by heat treatment at 50 degrees C for 30 min. These results suggest that fetal hepatocytes synthesize and secrete a glucocorticoid-regulated heat-labile low molecular mass (less than 25 kDa) anti-(insulin) protease, which may contribute to the suppression of insulin degradation caused by the enzymes involved in CNP.     

Isolation and culture of the terminally differentiated adult mammalian ventricular cardiac muscle cell

Summary We have carried out systematic studies to optimize and standardize methodology to isolate and culture the adult rat ventricular cardiac muscle cell. Four hearts were perfused simultaneously with a calcium-free medium containing collagenase. The ventricular tissue was then minced and further digested to liberate individual cells. Approximately 16 million rod-shaped muscle cells were obtained. The plating efficiency has been greatly improved by culturing the cells in a conditioned medium prepared from a rabbit corneal cell line. This medium also contained added fetal bovine serum, essential and nonessential amino acids, vitamins, insulin, transferrin, and 25 trace minerals. The culture flasks were precoated with rat-tail collagen. Fibroblast contamination was virtually eliminated by including cytosine arabinoside in the medium during the first 7 d of culture. After this time the cells could be cultured in the absence of serum in a chemically defined medium composed of MEM, vitamins, nonessential amino acids, and trace minerals. They continued to contract spontaneously and do well in this medium for at least 3 d thereafter. This improved methodology resulted in a reproducible culture system with improved plating efficiency. It provided a new and unique system to study the structure and function of the adult mammalian ventricular cardiac muscle cell. This investigation was supported by Grant HL 25873 from the National Institutes of Health, Bethesda, MD.     

Effect of leukemia inhibitory factor (LIF) on in vitro produced bovine embryos and their outgrowth colonies

In vitro produced (IVP) bovine embryos were subjected to in vitro culture with or without 1000 U/ml human recombinant leukemia inhibitory factor (LIF) added to the culture medium from Days 5 to 8 post insemination (p.i.). Resulting blastocysts were subsequently plated intact on mouse feeder cells in a medium with or without LIF. Significantly more embryos reached the hatched blastocyst stage, and the number of blastocysts with excellent morphology was significantly higher, when LIF was omitted. At Day 8 p.i., total cell count (TCC) and inner cell mass (ICM) cell count was significantly higher in embryos cultured without LIF. In embryos cultured with LIF, cytoplasmic vesicles and lipid droplets were abundant and a decreased expression of both Oct4 and laminin could be observed. Initial hypoblast formation was revealed in almost 1/3 of the LIF-cultured blastocysts whereas this feature was evident in 2/3 of the blastocysts cultured in the absence of LIF. Overall, almost 60% of the blastocysts cultured without LIF formed outgrowth colonies (OCs) when plated on feeders, whereas this phenomenon was only observed in 30% of the blastocysts cultured in the presence of LIF. A tendency for retaining a tightly packed central growth of putative ICM-derived cells was observed, when attachment to the feeder layer was initiated close to the embryonic pole of the blastocyst. At Day 8 of outgrowth culture, approximately 20% of the colonies contained a central core of putative ICM-derived cells appearing large enough for mechanical isolation and further subculture. Immunohistochemical labeling for Oct4 revealed staining of both trophectodermal and ICM-derived cells. The presence of LIF in the outgrowth culture medium did not have any apparent effect on the plating efficiency or colony type. In conclusion, LIF had an adverse effect on in vitro embryonic development when added to the culture medium in the period from Days 5 to 8 p.i., whereas it had no apparent effect on the OCs subsequently formed from such embryos.     

Effect of Conditioned Media from Several Cell Types on Invasion by Eimeria adenoeides Sporozoites1

ABSTRACT. The effect of conditioned media (media aspirated from a variety of cell cultures after 4 d of growth) on cellular invasion by sporozoites of the turkey coccidium, Eimeria adenoeides, was examined. Conditioned medium from turkey kidney cells and baby hamster kidney cells failed to alter invasion. However, conditioned medium from turkey cecal cell cultures produced a significant (P ≤ 0.05), two-fold increase in invasion over control medium in a variety of cell types. Retentates of conditioned medium from the turkey cecal cells that were passed through microconcentrators having molecular mass cutoffs of 50, 100, and 300 kDa similarly enhanced invasion over retentates from control medium. However, retentates from microconcentrators with a cutoff of 1,000 kDa failed to enhance invasion. Pretreatment in conditioned medium, followed by washing of sporozoites prior to inoculation into cultures, did not result in enhanced invasion. Moreover, when the interval between inoculation of sporozoites into cells and fixation of cultures was reduced to less than 3 h, no enhancement of invasion occurred. Conditioned medium from turkey cecal cells that were grown in the presence of ³⁵S-translabel had at least two labeled bands at 150 kDa and > 200 kDa that were absent in conditioned media from turkey kidney and baby hamster kidney cells.     

Studies on in vitro colony formation by mouse bone-marrow cells using different sources of colony-stimulating factor

Summary Conditioned media of a primary mouse embryo and a mouse cell line were compared as sources of colony-stimulating factor. The incorporation of embryo cell conditioned medium into semisolid cultures of mouse bone-marrow cells induced the formation of a larger number of in vitro colonies than did the addition of equal volumes of LM cell conditioned medium. This finding did not appear to be the result of quantitative differences in the levels of C.S.F. between the sources since concentration of the LM cell conditioned preparation did not enhance effectively the number of colonies produced. Both the colonial morphology and the cellular components of the developing colonies were found to differ in accordance with the C.S.F. source employed for stimulation. Colonies that developed under the influence of embryo cell conditioned medium were typically larger and more disperse than were those produced in cultures stimulated with the LM cell conditioned source. The latter colonies were smaller, more compact and contained fewer cells. Differences also were noted in the relative proportion of granulocytes to mononuclear cells comprising the colonies. Those colonies stimulated with LM cell conditioned medium rapidly underwent a transition from primarily a granulocytic composition to one comprised principally of mononuclear cells. Cultures stimulated with embryo cell conditioned medium contained a greater number of granulocytic colonies which persisted for a protracted period during cultivation. The addition of 2-mercaptoethanol to cultures stimulated with embryo cell conditioned medium increased the number of colonies produced. Such synergy did not occur in cultures stimulated with the LM cell conditioned medium. Supported by United States Public Health Service Research Grant CA 13752.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Schwann cell proliferation in vitro is under negative autocrine control

In healthy adult peripheral nerve, Schwann cells are believed to be generally quiescent. Similarly, cultures of isolated rat sciatic nerve Schwann cells hardly proliferate in serum-supplemented medium. The possibility that Schwann cells negatively regulate their own proliferation was supported by the demonstration that conditioned media from Schwann cell cultures inhibited the proliferation of mitogen- stimulated test cultures. The inhibition could be complete, was dose dependent, and was exhibited when the test Schwann cells were under the influence of different types of mitogens such as cholera toxin, laminin, and living neurons. The inhibition of proliferation was completely reversible and a rapid doubling of cell number resulted when treatment with conditioned medium was withdrawn from mitogen-stimulated Schwann cells. Conditioned medium from cholera toxin-stimulated and immortalized Schwann cell cultures contained less antiproliferative activity than that found in medium from quiescent Schwann cell cultures. However, media conditioned by two actively proliferating rat Schwannoma cell lines were rich sources of antiproliferative activity for Schwann cells. Unlike the mitogen-stimulated Schwann cells, whose proliferation could be inhibited completely, the immortalized and transformed Schwann cell types were nearly unresponsive to the antiproliferative activity. The antiproliferative activity in Schwann and Schwannoma cell conditioned media was submitted to gel filtration and SDS-PAGE. The activity exists in at least two distinct forms: (a) a high molecular weight complex with an apparent molecular mass greater than 1,000 kD, and (b) a lower molecular weight form having a molecular mass of 55 kD. The active 55-kD form could be derived from the high molecular weight form by gel filtration performed under dissociating conditions. The 55-kD form was further purified to electrophoretic homogeneity. These results suggest that Schwann cells produce an autocrine factor, which we designate as a "neural antiproliferative protein," which completely inhibits the in vitro proliferation of Schwann cells but not that of immortalized Schwann cells or Schwannoma lines.     

Evaluation of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 on bovine inner cell mass outgrowth in vitro

Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%). compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 microg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 microg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 microg/ml TIMP-2 was greater (P < 0.05) compared with 0 microg/ml TIMP-2 (16.2 +/- 4.8 versus 6.7 +/- 1.4 x 10(-3) IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.     

Control of Melanoblast Differentiation in Amphibia by α-Melanocyte Stimulating Hormone,a Serum Melanization Factor,and a Melanization Inhibiting Factor

A ventrally localized melanization inhibiting factor (MIF) has been suggested to play an important role in the establishment of the dorsal-ventral pigment pattern in Xenopus laevis [Fukuzawa and Ide: Dev. Biol., 129:25–36, 1988]. To examine the possibility that melanoblast expression might be controlled by local putative MIF and melanogenic factors, the effects of α-melanocyte stimulating hormone (α-MSH), a serum melanization factor (SMF) from X. laevis or Rana pipiens, and MIF on the “outgrowth” and “melanization” of Xenopus neural crest cells were studied. Outgrowth represents the number of neural crest cells emigrating from cultured neural tubes, and melanization concerns the percentage of differentiated melanophores among the emigrated cells. MSH or SMF stimulate both outgrowth and melanization. The melanogenic effect of Xenopus serum in this system is more than twice that of Rana serum. The actions of MSH and Xenopus serum on melanization seem to be different: 1) Stronger melanization is induced by Xenopus serum than by MSH, and the onset of melanization occurs earlier with Xenopus serum; 2) MSH stimulates melanization only in the presence of added tyrosine; and 3) MSH causes young melanophores to assume a prominent state of melanophore dispersion during culture, while Xenopus serum (10%) had only a slight dispersing effect and not until day 3. A fraction of Xenopus serum presumably containing molecules of a smaller molecular weight (MW <30 kDa) than that of a pigment promoting factor reported in calf serum [Jerdan et al.: J. Cell Biol., 100:1493–1498, 1985] produces the same remarkable melanogenic effects as does intact serum. While this fraction stimulates outgrowth, another fraction presumably containing larger molecules (MW > 100 kDa) does not. MIF contained in Xenopus ventral skin conditioned medium (VCM) inhibits both outgrowth and melanization dose dependently. When VCM is used in combination with MSH, the stimulating effects of MSH on both outgrowth and melanization are completely inhibited. In contrast, the stimulatory effects of Xenopus serum are not completely inhibited when combined with VCM, although melanization is reduced to approximately 40% that of controls. MIF activity was also found to be present in ventral, but not in dorsal, skin conditioned media of R. pipiens when tested in the Xenopus neural crest system. We suggest that ventrally localized MIF plays an important role in amphibian pigment pattern formation and that the interacting effects of MIF and melanogenic factors influence melanoblast differentiation, migration, and/or proliferation of neural crest cells to effect the expression of pigmentary patterns.     

CHANGES IN THE CAPACITY FOR CLONAL GROWTH AND DIFFERENTIATION IN VITRO OF THE VERTEBRAL CARTILAGE CELLS WITH EMBRYONIC DEVELOPMENT II. VITALIZING EFFECT OF CONDITIONED MEDIUM ON THE CELLS OF YOUNGER EMBRYOS

Most of the younger cartilage cells taken from chick embryos at the stage 35 were known to regress after the initial cell multiplication in in vitro clonal culture. A partial supplementation of conditioned medium (CM) to the standard medium was effective to permit colony formation from many of these younger cells. Most colonies thus derived from the younger cells by the aid of CM expressed differentiative traits as cartilage, as the cells from older embryos did. CM exerted multiple effects on clonal development of the younger cells. It vitalized the cells destined to regress, to promote cell multiplication and to flatten the shape of the colony. The experimental results suggested that these effects were associated with an independent factor each.     

Human fibroblast-conditioned medium contains a 100K dalton glucose-regulated cell surface protein

This report describes the identification and partial characterization of a 100K dalton “glucose-regulated” cell surface protein of human diploid fibroblasts (HDF). This protein is released into and can be recovered virtually intact from the surrounding culture medium. At the present level of analysis, the protein recovered from the culture medium (“conditioned medium”) is indistinguishable from the protein extracted directly from the cell surface by 1 M urea treatment. Both proteins have molecular weights of 100K daltons when analyzed by gel electrophoresis. The protein is readily labeled at the cell surface via lactoperoxidase-catalyzed iodination, and the label can be chased into the released form of this protein in conditioned medium. Antiserum raised against the medium form of the protein reacts with the surface form of the protein but does not react with fibronectin, the major cell surface protein of HDF. Conditioned medium from SV40-transformed human fibroblasts does not contain the 100K protein, but instead contains a component that has a slightly lower molecular weight (97K daltons). The lower molecular weight band does not iodinate at the cell surface and is apparently an underglycosylated form of the 100K protein. Its molecular weight is shifted back to 100K by growing transformed cells in medium containing excess glucose. After the shift, the component becomes accessible to the radioiodine label. We suggest that the 100K protein is a glucose-regulated protein (Shiu, Pouyssegur and Pastan, 1977; Pouyssegur and Yamada, 1978) that is released into the culture medium. An underglycosylated form of the same glycoprotein is released from transformed cells.