A heparin sulfate-regulated human keratinocyte autocrine factor is similar or identical to amphiregulin.

A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.     

BALB and kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent BALB/c mouse epidermal keratinocyte lines

Clonal BALB/c mouse epidermal keratinocyte (BALB/MK) cell lines were established in tissue culture. Despite their aneuploid nature, the lines were nontumorigenic, and retained in vitro properties similar to those of primary diploid keratinocytes. These included the constitutive expression of keratin and terminal differentiation in response to a calcium concentration greater than 1.0 mM in the medium. The cells also demonstrated an absolute requirement for nanomolar concentrations of epidermal growth factor (EGF) for their proliferation. BALB or Kirsten murine sarcoma viruses are acute transforming retroviruses, which have been shown to transform fibroblastic and hematopoietic cells. Infection of BALB/MK or its clonal sublines with either virus leads to the rapid acquisition of EGF-independent growth. The cells concomitantly lose their sensitivity to calcium-induced terminal differentiation. Thus these retroviruses can rapidly confer upon epithelial keratinocytes in culture growth properties that resemble those of malignant epidermoid carcinoma cells.     

Stimulation by insulin-like growth factors is required for cellular transformation by type beta transforming growth factor

Medium conditioned by BRL-3A cells, a known source of insulin-like growth factor II (IGF-II), induced phenotypic transformation (anchorage-independent proliferation) of mouse BALB/c 3T3 fibroblasts but not rat NRK-49F fibroblasts, in the presence of 10% calf serum. A specific radioreceptor assay and a bioassay indicated that BRL-3A conditioned medium contained 0.5-1 ng/ml of type beta transforming growth factor (beta TGF). Purified IGF-II and beta TGF acting together reconstituted the transforming activity of BRL-3A conditioned medium on BALB/c 3T3 cells. Insulin was 5-10% as potent as IGF-II in supporting the transforming action of beta TGF on BALB/c 3T3 cells. NRK-49F cells were phenotypically transformed by beta TGF in the presence of EGF and 10% calf serum as the sole source of IGFs. However, transformation of NRK-49F cells under these conditions was inhibited by addition of purified IGF-binding protein. Addition of an excess of IGF-II prevented the inhibitory action of IGF-binding protein. The different sensitivity of the two cell lines to IGFs was correlated with lower levels of type I IGF receptor and higher levels of type II IGF receptor in NRK-49F cells as compared with BALB/c 3T3 cells. The results suggest that cellular stimulation by IGFs is a prerequisite for transformation of rodent fibroblasts by beta TGF. We propose that transformation of fibroblasts by beta TGF requires concomitant stimulation by the set of growth factors that support normal cell proliferation.     

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.     

Roles of various growth factors in growth of human osteosarcoma cells which can grow in protein-free medium.

The human osteosarcoma cell line (OST-1-PF) can grow in protein-free Coon's modified Ham's F12 medium. Growth of the cells in protein-free medium was partially density-dependent and partially depressed by medium change. An extract and conditioned medium of OST-1-PF cells contained high mitogenic activity for BALB/c3T3 cells. The growth factor in the cells was purified and identified as a basic fibroblast growth factor (bFGF)--like factor on the basis of its elution profile on heparin-affinity chromatography and the result of immunoblotting. An unidentified factor in a conditioned medium eliciting most of the DNA synthesis-stimulating activity showed a weak affinity for heparin. Various additions, including serum and growth factors, stimulated the growth of OST-1-PF cells in protein-free medium. Of these factors, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), acidic fibroblast growth factor (aFGF) and bFGF were the most potent mitogens. High-affinity receptors of EGF and FGF were found on the surface of these cells. These results indicate that autonomous growth of OST-1-PF cells in protein-free medium is mainly controlled by an intracellular mechanism.     

wnt3a but not wnt11 supports self-renewal of embryonic stem cells

wnt proteins (wnts) promote both differentiation of midbrain dopaminergic cells and self-renewal of haematopoietic stem cells. Mouse embryonic stem (ES) cells can be maintained and self-renew on mouse feeder cell layers or in media containing leukemia inhibitory factor (LIF). However, the effects of wnts on ES cells self-renewal and differentiation are not clearly understood. In the present study, we found that conditioned medium prepared from L cells expressing wnt3a can replace feeder cell layers and medium containing LIF in maintaining ES cells in the proliferation without differentiation (self-renewal) state. By contrast, conditioned medium from NIH3T3 cells expressing wnt11 did not. Alkaline phosphatase staining and compact colony formation were used as criteria of cells being in the undifferentiated state. ES cells maintained in medium conditioned by Wnt3a expressing cells underwent freezing and thawing while maintaining properties seen with LIF maintained ES cells. Purified wnt3a did not maintain self-renewal of ES cells for prolonged intervals. Thus, other factors in the medium conditioned by wnt3a expressing cells may have contributed to maintenance of ES cells in a self-renewal state. Pluripotency of ES cells was determined with the use of embryoid bodies in vitro. PD98059, a MEK specific inhibitor, promoted the growth of undifferentiated ES cells maintained in conditioned medium from wnt3a expressing cells. By contrast, the P38 MAPK inhibitor SB230580 did not, suggesting a role for the MEK pathway in self-renewal and differentiation of ES cells maintained in the wnt3a cell conditioned medium. Thus, our results show that conditioned medium from wnt3a but not wnt11 expressing cells can maintain ES cells in self-renewal and in a pluripotent state.     

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Macrophage differentiation inducing factor from human monocytic cells is equivalent to murine leukemia inhibitory factor

Human macrophage differentiation inducing factor (DIF) can induce differentiation of human myeloid leukemic cells into macrophage-like cells in vitro. A procedure is described for purification of DIF from serum-free human monocytic leukemia THP-1 cell-conditioned medium. The procedure included concentration of a conditioned medium by ultrafiltration, lentil lectin-Sepharose affinity chromatography, and fast protein liquid chromatography using Mono S and Mono Q. DIF-A of pI 9.0 and DIF-B of pI 8.8 were obtained after a final purification with a Mono Q column, and both DIF gave a single peak with a molecular weight of approximately 51,000 determined by gel chromatography. NH2-terminal amino acid analysis of DIF-A showed a noticeable homology with murine leukemia inhibitory factor. Human DIF-A was found to induce maturation of human and murine leukemic cells into both macrophage-like cells with nitro blue tetrazolium reducing activity and phagocytic cells, but was found to suppress proliferation of these leukemic cells.     

The calcium signal for BALB/MK keratinocyte terminal differentiation counteracts epidermal growth factor (EGF) very early in the EGF-induced proliferative pathway.

BALB/MK mouse epidermal keratinocytes require epidermal growth factor (EGF) for proliferation and terminally differentiate in response to high calcium concentrations. We show that EGF is an extremely potent mitogen, causing BALB/MK cultures to enter the cell cycle in a synchronous manner associated with a greater than 100-fold increase in DNA synthesis. Analysis of the expression of proto-oncogenes which have been reported to be activated during the cascade of events following growth factor stimulation of fibroblasts or lymphoid cells revealed a very rapid but transient 100-fold increase in c-fos RNA but little or no effect on the other proto-oncogenes analyzed. Exposure of EGF-synchronized BALB/MK cells to high levels of calcium was associated with a striking decrease in the early burst of c-fos RNA as well as the subsequent peak of cell DNA synthesis. Since the inhibitory effect of high calcium on c-fos RNA expression was measurable within 30 min, our studies imply that the EGF proliferative and calcium differentiation signals must interact very early in the pathway of EGF-induced proliferation. Our results also establish that c-fos RNA modulation is an important early marker of cell proliferation in epithelial as well as mesenchymal cells.     

Control of cytolysis of BALB/c-3T3 cells by platelet-derived growth factor: a model system for analyzing cell death

In culture medium supplemented with 10% clotted blood serum, the saturation density of BALB/c-3T3 cells is determined jointly by cell replication and cell loss. By prelabelling cellular DNA with ³H-thymidine and also by time lapse photography, we studied cell loss independently of replication. Cell loss was accelerated when BALB/c-3T3 cells were transferred from serum-supplemented medium, which contains the platelet derived growth factor (PDGF), to medium supplemented with platelet-poor plasma which lacks it. Loss occurred via the disintegration of cell attached to the surface of the tissue culture dish. Cytolysis of individual cells occurred rapidly; less than 15 minutes transpired between the first indication of a perturbance (by phase contrast microscopy) and fragmentation of the cell cytoplasm. Kinetic analysis was consistent with random cell death rather than a fixed lifetime. The percentage of cells undergoing cytolysis was governed by the cell density; at high densities, such as are present in confluent cultures, a higher percentage of cell loss was noted than at low density. Cell death was antagonized by partially purified or electrophoretically homogenous preparations of-PDGF. Pure PDGF stimulated cell survivial at ng/ml in a concentration dependent fashion. The process of cell replication was not necessary for survival because PDGF prevented cytolysis in the presence of methotrexate, an inhibitor of DNA synthesis. A brief (4 hour) treatment with PDGF prevented cell death; such PDGF treated cells displayed increased survival after being taken up with trypsin and planted onto a fresh surface in plasma supplemented medium. Pituitary fibroblast growth factor, a functional analogue of PDGF for induc of DNA synthesis in BALB/c-3T3 cells, also functioned as an anticytolytic agent. By contrast, epidermal growth factor and insulin did not. Cytolysis of SV40-transformed cells occurred at a constitutively low rate and was insensitive to PDGF.     

Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium

A 6-kDa alpha-transforming growth factor (TGF) was purified 100,000-fold to homogeneity from the culture fluid conditioned by normal bovine anterior pituitary-derived cells. Initial purification of the acid-soluble TGF from concentrated conditioned medium was achieved by Bio-Gel P-60 gel filtration (apparent molecular mass of 9 kDa). After the Bio-Gel step, three different steps of reverse-phase fast-protein liquid chromatography on the same Pharmacia C18 column, using linear acetonitrile gradients, gave complete purification. The ion-pairing agents used in the three consecutive steps were: 0.1% trifluoroacetic acid, 0.13% heptafluorobutyric acid, and again, 0.1% trifluoroacetic acid at a shallower gradient. Homogeneity was confirmed by reverse-phase high performance liquid chromatography, and by polyacrylamide gel electrophoresis, where TGF visualization was facilitated by autoradiography of 125I-TGF. The 125I-TGF bound to epidermal growth factor (EGF) receptors and after elution ran identically to the starting material. The molecular mass of TGF is 6 kDa by polyacrylamide gel electrophoresis and 6.6 kDa by amino acid analysis. The amino acid composition of bovine TGF is similar to that of rat or human alpha TGF and distinct from epidermal growth factor. Colony-stimulating activity was lost after purification, but the TGF retained its ability to stimulate thymidine uptake by quiescent cells. This mitogenic activity could be blocked completely by anti-EGF-receptor monoclonal antibodies, indicating that the activity was mediated through the EGF-receptor.     

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.     

A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.     

Loss of viability in hybridoma cell culture—A kinetic study

In the cultivation of hybridoma cells HB8178 in suspension, exponential growth was followed by cell death after reaching a maximum cell concentration. We examined the cause of such transition from growth to death. Exogenously added lactate and ammonium did not cause cell death at the concentrations observed at the end of exponential growth. The rate of cell death decreases by diluting the conditioned medium with phosphate buffer saline, suggesting the presence of inhibitory factor(s) in the conditioned medium. This inhibitory factor(s) is dialyzable. Furthermore, the conditioned medium obtained from HB8178 culture also causes transition to death phase for another hybridoma cell line AFP.     

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

Secretion of DNA synthesis factor (DSF) by A431 cells that can grow in protein-free medium

A431 cells grew in protein-free Coon's modified Ham's F12 medium at a similar rate to that in medium supplemented with calf serum and secreted a growth factor capable of stimulating DNA synthesis in BALB/c3T3 cells. This factor had strong affinity for heparin and was partially purified from the conditioned medium by heparin-Sepharose affinity chromatography and molecular sieving on Bio-Gel P-60. The apparent molecular weight of the factor was 20-30K. Its activity was inhibited by heparin at concentrations of above 0.03 microgram/ml.     

Age dependent production of a competence factor by human fibroblasts

Several cell types such as Balb/c 3T3 have been shown to require platelet-derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin-C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12-wk embryo, newborn, and 3-yr-old were exposed to serum-free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3-yr-old cells with the addition of plasma alone, whereas conditioned medium from the 3-yr-old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3-yr-old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3-yr-old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age-dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and supplemented with plasma alone.