Changes in protein synthesis and breakdown rates and responsiveness to growth factors with ageing in human lung fibroblasts

The effects of insulin, epidermal growth factor, insulin-like growth factor-2 and fetal bovine serum on protein synthesis and protein breakdown have been measured in mid-passage and senescent cultures of human diploid lung fibroblasts. Each of the individual growth factors was a potent inhibitor of protein breakdown with no difference in either the maximum response or sensitivity evident between senescent cells and mid-passage cultures. Binding of 125I-labelled epidermal growth factor per mg of cell protein similarly showed no difference between senescent and confluent mid-passage fibroblasts, although sparse mid-passage cells bound more of the ligand. These results indicate normal binding and normal responsiveness to growth factors in senescent cultures. However, rates of protein synthesis are higher in confluent mid-passage cells and especially so in sparse mid-passage cells than in senescent cells of intermediate density. Furthermore, senescent cells differ from either growth state of mid-passage cells because protein synthesis in aged cells is much less responsive to any of the growth factors or to fetal bovine serum. It is suggested from these results that the reduced ability of serum or growth factors to initiate DNA synthesis or growth in ageing cells may be a consequence of an unresponsive protein synthesis pathway rather than a generalised defect in growth factor action.     

Age-dependent changes in DNA polymerase fidelity and proofreading activity during cellular aging.

DNA polymerase alpha and the 3'-->5' exonuclease involved in the proofreading of DNA synthesis were isolated from human diploid fetal lung fibroblast (TIG-1) cells at various population doubling levels (PDL). The final PDL of the TIG-1 cells used in these experiments was 70. The fidelity of DNA polymerase alpha remained high until late passage and fell suddenly just before the end of the life span between 65 and 69 PDL. The activities of the 3'-->5' exonuclease related to proofreading remained unchanged from 21 to 61 PDL, but the activity decreased rapidly in more aged cells. The 3'-->5' exonuclease activity at 69 PDL was about 50% of that in TIG cells at 21 PDL. In vitro DNA synthesis by DNA polymerase alpha from TIG-1 cells harvested at 69 PDL showed the amount of non-complementary nucleotides incorporated to be decreased by the addition of the 3'-->5' exonuclease from the same cells. However, not all errors were edited out since the ratio of DNA polymerase activity to 3'-->5' exonuclease activity was adjusted to reflect that in vivo and the infidelity of DNA synthesis by error-prone DNA polymerase alpha from aged cells was improved by the addition of the highly active 3'-->5' exonuclease from cells at 41 PDL. These results suggested that the mutation frequency rises just before the end of the life span of TIG-1 cells.     

Failure in S6 protein phosphorylation by serum stimulation of senescent human diploid fibroblasts, TIG-1

When quiescent young or senescent human diploid cells, TIG-1, were metabolically labeled with 32Pi and stimulated with 10% fetal bovine serum, the phosphorylation of ribosomal S6 protein was enhanced in young cells but not in senescent cells while that of some other proteins were increased in both cells. Inability to stimulate the phosphorylation of S6 protein in senescent cells after serum addition may be the primary cause of the failure of enhancement in protein synthesis followed by the block of prereplicative events dependent on protein synthesis and thus of the failure of cells to enter S phase. However, when the cell-free preparation from serum-stimulated senescent cells was incubated with [gamma-32P]ATP, S6-kinase activity was stimulated and S6 in ribosomal fraction was susceptible to phosphorylation as observed in young cells. Differences in S6 phosphorylation of senescent cells between in vivo and in vitro was discussed.     

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.     

Events blocked in prereplicative phase in senescent human diploid cells, TIG-1, following serum stimulation

When senescent human diploid cells, TIG-1, were stimulated with serum at the end of their proliferative life span, such biochemical events as uptakes of 2-deoxyglucose and uridine, and expression of c-myc, were enhanced. However, RNA synthesis, polyamine accumulation, thymidine uptake and DNA synthesis were not enhanced at all. Protein synthesis increased only moderately as compared with that observed in younger cells. These results indicated that the events in prereplicative phase known to be independent on protein synthesis are induced in senescent cells after the stimulation with serum, whereas those required protein synthesis failed to increase to the same extent as seen in young cells.     

Effects of in vitro aging and cell growth on the viability and recovery of human diploid fibroblasts,TIG-1, after freezing and thawing

The viability and the recovery (cell attachment to the dish) after thawing of human diploid fibroblasts (TIG-1) frozen by four different methods were studied at different passages. Improved results were observed in a medium of 30% fetal bovine serum plus 15% glycerol, compared with the conventonal medium which contained 10% fetal bovine serum plus 10% glycerol. Centrifugation to remove glycerol immediately after thawing had a negative effect on the viability and recovery of cells. The recovery of cells after freezing and thawing showed a maximal value in the middle of phase II (PD 35) during the finite lifespan of the cell (average PD 67). This result indicates that the cells at early and late passages are sensitive to injury by freezing and thawing. The modified method yielded improved recovery, especially in the cells at early and late passages, except for the extremely senile stage. The recovery was also affected by the state of cell growth after inoculation.     

Effects of serum from rabbits of various ages on cell proliferation

The report of Carrel and Ebeling (J. Exp. Med., 34 (1921) 599-623) generally gives the impression that both serum and blood plasma from old animals inhibit cell proliferation. For confirmation of this, we examined the effects of serum from rabbits of various ages on rabbit fetal skin fibroblasts (RSF cell) and human fetal lung fibroblasts (TIG-1 cell). Serum from young rabbits 8 months of age stimulated proliferation of RSF cells just as did fetal bovine serum, but that from old rabbits 5-7.8 years of age was found to significantly increase proliferation more than serum from the young. This was also the case when using TIG-1 cells. The lesser effect on cell proliferation by young serum apparently does not arise from growth-inhibitory factor(s) in the blood components. An examination showed young serum to possibly contain fewer growth-stimulatory factor(s) than old serum. On the basis of our data, we concluded that old rabbit serum stimulates, not inhibits, the proliferation of RSF and TIG-1 cells.     

Hydrocortisone stimulation of DNA synthesis in bromodeoxyuridine-selected non-dividing WI-38 cells

Ostensibly noncycling WI-38 cells were selected by incubating growing cultures for 7 days with 10⁻⁵ M bromodeoxyuridine. These cells were then exposed to Hoechst dye 33258, and then visible light which kills cells that incorporated bromodeoxyuridine. Following selection, cultures were refed with medium containing 10% fetal bovine serum. By autoradiography, we determined that less than 10% of these cells could incorporate [³H] thymidine during the next 7 days. This value was increased to 25% or more by adding hydrocortisone to the medium. The proliferative response was also increased by refeeding cultures with hydrocortisone-containing medium conditioned 24 h by freshly seeded mid-, but not late, population doubling level (PDL) cultures. Medium conditioned without hydrocortisone did not stimulate incorporation of [³H] thymidine beyond the control value. These results show that: (1) cells that might otherwise not initiate DNA synthesis are stimulated to do so by hydrocortisone; (2)hydrocortisone-conditioned medium from mid-PDL culture increases this stimulation; and (3) hydrocortisone-conditioned medium from late PDL cultures is not stimulatory.     

Effects of serum collected from rats of different ages on in vitro cell proliferation

It has been reported by Carrel and his co-workers that serum from old hens inhibits cell growth in culture. However, as we had previously demonstrated contradictory results using serum from old rabbits, we examined whether serum from old rats would also show strong induction of cell proliferation. Sera from young and adult rats of either sex strongly stimulated the growth of rat fetal skin fibroblasts and human fetal lung fibroblasts (TIG-1). Sera of old female and male rats (24-29 months old) produced much greater fluctuations in growth-stimulatory activity than sera from young animals. Most samples of serum from old rats stimulated the growth of TIG-1 cells, as did fetal bovine serum and samples from younger rats, even when a higher concentration of serum (up to 50%) was used. On the other hand, a small proportion of samples repressed the growth of the cells. A study on the effects of serial mixtures of both different types of serum samples from old rats on cell growth suggested that this minor proportion of serum samples contain a large amount of inhibitory factor(s). The cell growth-stimulatory activity of serum did not correlate with the total protein and albumin concentrations, albumin/globulin ratio, and the levels of lipid peroxide in the sample. These results therefore seemed to imply that serum induced a striking increase in the heterogeneity of cell growth stimulatory activity with age, although most samples of serum from old rats of either sex stimulated cell proliferation as effectively as samples from younger rats. The biological significance of the small proportion of serum samples from old rats which do inhibit cell proliferation was discussed.     

Human alveolar type II cells: stimulation of DNA synthesis by insulin and endothelial cell growth supplement

Proliferation of type II cells is important for the recovery of the alveolar epithelium after acute lung injury. However, the factors that regulate the proliferation of human type II cells are unknown. Human alveolar type II cells were isolated from resected lung by dissociation with porcine pancreatic elastase and crystalline trypsin and purified by density-gradient centrifugation and serial differential adherence. The purity of the type II cells in the final adherent preparation was 84.4 +/- 1.1% type II cells by alkaline phosphatase and 87.7 +/- 2.8% by cytokeratin (n = 7). The medium MCDB-151 with 0.4% fetal bovine serum (FBS) was used to demonstrate the stimulatory effect of individual growth factors. Under these conditions, thymidine incorporation was stimulated by insulin, epidermal growth factor, endothelial cell growth supplement (ECGS), and acidic and basic fibroblast growth factors. Cholera toxin did not stimulate thymidine incorporation. The most effective stimulation was by the combination of insulin and ECGS. The incorporation of bromodeoxyuridine was used to identify the proportion of cells that were active in DNA synthesis. Insulin and ECGS increased the percentage of cells that incorporated bromodeoxyuridine from 8.5 +/- 1.3% to 21.3 +/- 2.4% (n = 6). Mitotic figures were seen in smears prepared from cultures incubated with insulin and ECGS. This observation was confirmed by electron microscopy, which demonstrated type II cells in metaphase. Increasing the concentration of FBS or human serum in the culture medium to 10% decreased the stimulatory effect of insulin and ECGS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells

The proliferation of alveolar type II cells is important for repair of the alveolar epithelium after lung injury. We have previously reported that epidermal growth factor (EGF), insulin, cholera toxin, and endothelial cell growth supplement (ECGS) stimulate DNA synthesis of rat alveolar type II cells in culture. ECGS is a crude extract from bovine neural tissue that contains heparin-binding growth factors, and in this report we have compared the effect of ECGS to purified heparin-binding growth factors. ECGS stimulated [3H]thymidine incorporation into type II cells by 3-fold with half-maximal stimulation at 50 micrograms/ml. The purified acidic, class I heparin-binding growth factors, alpha-endothelial cell growth factor (-ECGF) and beta-ECGF stimulated type II cell DNA synthesis by 10-fold and 5-fold, respectively, with half-maximal stimulation at 40 ng/ml. Acidic fibroblast growth factor (FGFa) stimulated [3H]thymidine incorporation by 16-fold with half-maximal stimulation at 20 ng/ml, whereas basic FGF (FGFb) only stimulated type II cell DNA synthesis by 3-fold. Heparin potentiates the mitogenic effect of the acidic heparin-binding growth factors for both endothelial cells and fibroblasts but was found to inhibit FGFa- and FGFb-induced [3H]thymidine incorporation in type II cells by 80% with half-maximal inhibition occurring with 0.4 micrograms/ml and 1.3 micrograms/ml, respectively. When type II cells were cultured in the absence of serum, the heparin-binding growth factors had very little effect on [3H]thymidine incorporation. Only rat high density lipoprotein (HDL), but not insulin, EGF, or transferrin, was found to act synergistically with FGFa in stimulating [3H]thymidine incorporation in type II cells cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in the migratory ability of human lung and skin fibroblasts during in vitro aging and in vivo cellular senescence.

The migration of human lung and skin fibroblasts was determined during in vitro aging and in vivo cellular senescence by measuring their migration from the edge of a denuded area of a monolayer. The migration of human fetal lung fibroblasts (TIG-1 and TIG-3) decreased only very slightly with increasing passage, whereas the migration of human fetal skin fibroblasts (TIG-3S) declined gradually: the difference in cell migratory ability between early and late passages was significant (P less than 0.05). The migratory patterns of skin fibroblasts from adult and elderly donors were also similar to that of fetal skin fibroblasts. Next, the migratory abilities of fibroblast lines from adult and elderly donor groups were compared, using relatively early passaged cells. The migratory ability of the elderly-donor skin fibroblast lines was significantly lower (P less than 0.05) than that of the adult-donor skin fibroblast lines. Addition of suramin and monensin suppressed the migration of fibroblasts from fetal, adult and elderly donors, which implies that fibroblast migration is regulated by growth factors and matrix substances. The relationships between the age-dependent decline of migratory ability, growth factors and the extracellular matrix are discussed.     

Effects of transforming growth factor beta1 and dexamethasone on the growth and chondrogenic differentiation of adipose-derived stromal cells

The effects of soluble mediators and medium supplements commonly used to induce chondrogenic differentiation in different cell culture systems were investigated to define their dose-response profiles and potentially synergistic effects on the chondrogenic differentiation of adipose-derived adult stromal (ADAS) cells. Human ADAS cells were suspended within alginate beads and cultured in basal medium with insulin, transferrin, and selenious acid (ITS+) or fetal bovine serum (FBS) and treated with different doses and combinations of TGF-beta1 (0, 1, and 10 ng/mL) and dexamethasone (0, 10, and 100 nM). Cell growth and chondrogenic differentiation were assessed by measuring DNA content, protein and proteoglycan synthesis rates, and proteoglycan accumulation. The combination of ITS+ and TGF-beta1 significantly increased cell proliferation. Protein synthesis rates were increased by TGF-beta1 and dexamethasone in the presence of ITS+ or FBS. While TGF-beta1 significantly increased proteoglycan synthesis and accumulation by 1.5- to 2-fold in the presence of FBS, such effects were suppressed by dexamethasone. In summary, the combination of TGF-beta1 and ITS+ stimulated cell growth and synthesis of proteins and proteoglycans by human ADAS cells. The addition of dexamethasone appeared to amplify protein synthesis but had suppressive effects on proteoglycan synthesis and accumulation.     

Platelet-derived growth factor and growth-related genes in rat lung. I. Developmental expression

The autocrine, paracrine, or systemic growth factors responsible for fetal lung cell growth are not completely defined. The progression-type insulin-like growth factors and epidermal growth factor, or transforming growth factor-alpha acting through the epidermal growth factor receptor, appear to act on the developing lung epithelium. The competence factors that facilitate the actions of progression factors during lung growth are unknown. Fetal rat lung cells in vitro synthesize a platelet-derived growth factor (PDGF)-like polypeptide, which we have hypothesized may play a paracrine role in normal lung development. Slot blot and Northern blot analyses of fetal rat lung mRNA have been used to determine if there is a relationship between expression of message for PDGF-A or PDGF-B chains, or their cognate receptors, and periods of maximal growth during late fetal rat lung development. Whole lung mRNA was extracted on 18, 19, 20, 21, and 22 days of gestation (term = 22 days). The peak of DNA synthesis, as assessed by expression of message for DNA polymerase alpha, histone 3, and the proto-oncogenes c-fos and c-myc, which are stimulated by binding of growth factors including PDGF, occurred during the canalicular stage of lung development on days 19 and 20 of gestation. Expression of message for PDGF-A and PDGF-B chains was low during the pseudoglandular stage on day 18, peaked during the canalicular stage on days 19 and 20, then fell again during the saccular stage at days 21 and 22 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane Depolarization was Required to Induce DNA Synthesis in Murine Macrophage Cell Line PU5-1.8

The role of membrane potential (Em) on the initiation of DNA synthesis in murine macrophage cell line PU5-1.8 was investigated with fluorescent probes bis-oxonol and diS-C_3-(5). Incubation of PU5-1.8 cells in high K⁺-HEPES buffer or with gramicidin at 37°C for lh that depolarized the membrane induced L3H]-thymidine incorporation and expression of early response gene such as c-myc and c-fos. When PU5-1.8 cells were treated with a number of agents including fetal calf serum (FCS), lipopolysaccharide (LPS), epidermal growth factor (EGF), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and bradykinin (BK), only FCS caused DNA synthesis and membrane depolarization. Other agents had no effect on these events. the FCS-mediated DNA synthesis in PU5-1.8cells was inhibited by clamping the membrane potential with valinomycin. Moreover, intracellular alkalinization induced by nigericin at pH 7.9, which is believed to be a permissive signal for mitogenesis, caused membrane depolarization. On the other hand, challenge of cells with phorbol 12-myristate 13 acetate (PMA) suppressed the K⁺-mediated DNA synthesis. However, the treatment of cells with PMA did not change the membrane potential but suppressed the gramicidin-mediated membrane depolarization. These observations suggest that there is a correlation between membrane depolarization and initiation of DNA synthesis in PUS-1.8 cells. PKC may be acting as a modulator in this transducing pathway.     

Inhibition of angiotensin II and platelet-derived growth factor-induced vascular smooth muscle cell proliferation by calcium entry blockers

Summary Structural changes within the blood vessel wall such as hyperplasia and hypertrophy of vascular smooth muscle cells are important factors in the pathogenesis of hypertension. Humoral growth factors such as angiotensin II (AII) and platelet-derived growth factor BB (PDGF-BB) may participate in the remodelling of the blood vessel wall. Whether and by which mechanisms antihypertensive treatment is capable of influencing the structural blood vessel alterations to date remains unclear. In the present study, the effect of nifedipine and diltiazem on AII- and PDGF-BB-induced vascular smooth muscle cell proliferation was examined. Nifedipine and diltiazem at a concentration of 10 M did not affect baseline DNA synthesis in isolated vascular smooth muscle cells in culture. AII (final concentration 100 nM) and PDGF-BB (50 ng/ml) stimulated DNA synthesis by approximately 9.0- and 4.6-fold, respectively. Both AII- and PDGF-BB-induced DNA synthesis was significantly blunted by diltiazem and nifedipine in a concentration of 10 M, while no significant influence was seen with concentrations from 10 nM up to 1 M. In contrast, no significant influence of these drugs could be observed on fetal calf serum 5%-induced DNA synthesis. The findings indicate that calcium antagonists possess antimitogenic potential and that they may thus contribute to the regression of structural changes of the blood vessels associated with hypertension.Abbreviations PDGF-BB platelet-derived growth factor BB - AII angiotensin II - FCS fetal calf serum - VSMC vascular smooth muscle cells - ACE angiotensin I converting enzyme This work was supported by HEXAL-PHARMA, Holzkirchen, Federal Republic of Germany     

Receptor for epidermal growth factor retains normal structure and function in aging cells

Cellular responsiveness to epidermal growth factor (EGF) and the structure of the receptor for epidermal growth factor (EGF-R) were compared in young and senescent human fibroblast (HF) cells. Biosynthetic labeling of HF cells with [35S] methionine followed by immunoprecipitation with EGF-R antibody revealed the presence of Mr 170 000 EGF-R in cells from both stages. Autophosphorylation of EGF-R in response to EGF was identical in young and senescent cells. Phosphoamino acid analysis of the autophosphorylated EGF-R indicated that tyrosine residues were phosphorylated in each preparation. Two-dimensional peptide mapping of [125I]EGF-R from young and senescent cells showed essentially the same pattern, indicating that EGF-R does not apparently undergo detectable changes in senescent human fibroblasts. The responsiveness of aging HF cells to EGF for the induction of ornithine decarboxylase activity and for the production of secretory proteins was measured. Young and senescent HF cells showed about a three-fold induction of collagenase activity upon addition of EGF. Ornithine decarboxylase activity was also stimulated by EGF to a comparable level in young and senescent cells. Our results indicate that the responsiveness of HF cells to EGF for these two biochemical parameters does not decline with the loss of proliferative activity.     

Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells

Three methods can induce reversible arrest of the growth of cultured human keratinocytes in the G1 phase of the cell cycle. These include the incubation of cells in medium containing transforming growth factor (TGF)-beta or ethionine, or in isoleucine-deficient medium. The current studies were performed to determine if the growth arrest induced by these methods occurs at a common or at a distinct G1 state(s). We first evaluated the relative time interval required for arrested cells to initiate DNA synthesis after growth restimulation with mitogenic medium. The results show that ethionine arrested cells require 22 to 28 hours to initiate DNA synthesis and that a maximum rate of DNA synthesis occurs at 46 hours. Cells arrested by isoleucine deficiency required 10 to 12 hours to initiate DNA synthesis with peak DNA synthesis occurring at 24 hours. Finally, TGF-beta arrested cells require only 6 to 8 hours to initiate DNA synthesis and show a maximum rate of DNA synthesis at 18 hours. We next evaluated if cells that were growth arrested at these states were differentially capable of initiating DNA synthesis in different types of potentially mitogenic medium. The results show that if TGF-beta arrested cells were refed TGB-beta free serum containing medium with ethionine or similar medium with isoleucine deficiency, no DNA synthesis occurred. In contrast, if cells whose growth was arrested in ethionine-containing medium or in isoleucine-deficient medium were refed mitogenic medium containing TGF-beta, significant DNA synthesis was detected. These results suggest that least two different types of reversible growth arrest states exist in cultured human keratinocytes. One appears to be mediated by receptor-dependent processes, such as that induced by TGF-beta, and the other appears to be mediated by other types of metabolic events, such as those induced by ethionine treatment or by isoleucine deficiency.     

Macromolecular synthesis in the isolated rat myointima in vitro.

The myointima, which forms within the lumen of the carotid artery of the rat in response to the removal of the endothelium by a balloon catheter, could be extruded from the vessel as early as 6 days after denudation. The mean concentration of DNA in the isolated myointima increased as the myointima grew in vivo until 12 to 14 days after denudation, when the arterial lumen was filled with the myointima; thereafter the mean concentration of DNA declined. DNA and protein synthesis, incorporation of 3H-glucosamine and 3H-fucose into macromolecules, occurred in the myointima during incubation in vitro in the absence of fetal bovine serum, and with the exception of 3H-glucosamine, failed to be stimulated by it. These data indicate that the cells of the intact myointima do not require exogenous macromolecular growth factors for the synthesis of macromolecules in vitro.     

Development of defined media for the serum-free expansion of primary keratinocytes and human embryonic stem cells

Primary keratinocyte (Kc) cells and human embryonic stem (hES) cells are routinely propagated on a mouse fibroblast feeder layer in media containing fetal bovine serum or other nondefined factors. One disadvantage of using these nondefined factors is that they may inadvertently contaminate the culture system with infectious agents; thus, there remains a need to develop safe culture conditions free from poorly defined and/or animal products. Our laboratory has discovered that growth factors (GFs) and vitronectin (VN) can bind to each other resulting in synergistic short-term functional effects in several cell types. The aim of the current study was to determine whether primary Kc and hES cells can be established and serially propagated serum-free using medium containing VN, insulin-like growth factor-I, and insulin-like growth factor binding protein-3 (VN:GF). Here we demonstrate that primary Kc cells can be isolated, established, serially propagated, and re-form an epidermal layer using the VN:GF combination. Additionally, cell proliferation studies indicate that the Kcs proliferate using the VN:GF combination at a rate comparable to cells grown using serum. Similarly, we verified that this VN:GF combination could be employed for the serial propagation of hES cells. Importantly, both the Kc and hES cells retain their undifferentiated phenotype when cultured using the VN:GF combinations as a serum-free medium for up to 4 passages for Kc and at least 10 passages for hES cells as indicated by the expression of a range of cell surface markers. This study demonstrates that the novel, fully defined VN:GF medium is a viable alternative to media containing serum and highlights the potential of this technology for generating therapeutically viable cells and tissues.