Silver birch telomeres shorten in tissue culture

Shortening of telomeres has been connected with ageing and loss of cell replication or regeneration capacity. The aim of the present study was to examine potential variation in the length of telomeric repeats in silver birch (Betula pendula Roth) using clonal materials consisting of different-aged outdoor trees and tissue cultures of seven genotypes. The overall average length of telomeres was 13.6 kb (±0.3), the minimum length of repeats in the different genotypes varied from 5.9 kb (±0.5) to 9.6 kb (±0.6), and the maximum length varied from 15.3 kb (±1.1) to 22.8 kb (±0.4). When germinated seeds and leaf and cambium samples from 15- and 80-year-old trees were compared, no correlation of ageing and the length of telomeric repeats was found. Positional variation in the telomere length was, however, observed in the cambium of mature trees, the stem base having longer repeats than the upper parts of the tree. Tissue cultures were found to have shorter telomeres than outdoor trees: prolonged culture, callus culture and stressful conditions were all observed to shorten telomeric repeats and should thus be avoided in birch micropropagation. There were significant differences among the studied silver birch genotypes in their telomere length, and these differences were consistent over the sample types. This is the first report on variation of telomeric repeats in a long-living organism studied with clonal materials.     

Mutation and Mutagenesis of thiol peroxidase of Escherichia coli and a new type of thiol peroxidase family.

A novel thioredoxin-linked thiol peroxidase (Px) from Escherichia coli has been reported previously (M. K. Cha, H. K. Kim, and I. H. Kim, J. Biol. Chem. 270:28635-28641, 1995). In an attempt to perform physiological and biochemical characterizations of the thiol Px, a thiol Px null (tpx) mutant and a functional-residue mutant of thiol Px were produced. The tpx mutant was viable in aerobic culture but grew more slowly than the wild-type cells. The difference in growth rate became more pronounced when oxidative-stress-inducing reagents, such as peroxides and paraquat, were added to the cultures. The viability of the individual tpx mutant under oxidative stress was much lower than that of wild-type cells. tpx mutants growing aerobically respond to paraquat with a sixfold greater induction of Mn-superoxide dismutase than that of the wild-type cells. The deduced amino acid sequence of the thiol Px was found to be from 42 to 72% identical to the sequences of proteins from Haemophilus influenzae (ToxR regulon), Vibrio cholerae (ToxR regulon), and three kinds of streptococci (coaggregation-mediating adhesins), suggesting that they all belong to a new thiol Px family. Alignment of the amino acid sequences of the thiol Px family members showed that one cysteine, which corresponds to Cys-94 in E. coli thiol Px, is perfectly conserved. The substitution of serine for this cysteine residue resulted in complete loss of Px activity. These results suggest that the members of the thiol Px family, including E. coli thiol Px, have a functional cysteine residue and function in vivo as peroxidases.     

Special symposium: In vitro plant recalcitrance do free radicals have a role in plant tissue culture recalcitrance?

Summary Free radicals have an important role in the metabolism and development of aerobic organisms; however, their uncontrolled production leads to oxidative stress. This paper explores the possibility that free radical mediated stress has a role in tissue culture recalcitrance. In the context of this paper, recalcitrance is considered to be the inabilit of plant tissue cultures to respond to culture manipulations; in its broadest terms, this study also concerns the time-related decline (i.e. in vitro aging) and loss of morphogenetic competence and totipotent capacity. Studies on a diverse range of in vitro plant systems have shown that tissue cultures produce free radicals, lipid peroxides and toxic, aldehydic lipid peroxidation products. Levels of these compounds vary in response to different tissue culture manipulations, but their production is enhanced during dedifferentation and antioxidant profiles also vary throughout different phases of culture. A hypothesis is presented which suggests that tissue culture manipulations cause major metabolic and developmental changes, some of which may predispose in vitro cultures to increased free radical formation. If antioxidant protection is compromised, oxidative stress ensues and free radicals and their reaction products react with macromolecules such as DNA, proteins and enzymes, causing cellular dysfuction and as a result, the cultures become recalcitrant.     

Primary cultures of rat aortic endothelial and smooth muscle cells: I. An in vitro model to study xenobiotic-induced vascular cytotoxicity

Summary Primary cultures of rat vascular endothelial and smooth muscle cells were developed as models to study xenobiotic-induced cytotoxicity. Endothelial and smooth muscle cells were isolated by enzymatic digestion and mechanical dissociation of rat thoracic aortae. Optimal cell growth and minimal fibroblast contamination in cultures of both cell types were obtained in Medium 199 supplemented with 10% fetal bovine serum. Cultured cells were characterized by distinctive morphologic features and growth patterns. Intercellular endothelial cell junctions were selectively stained with silver nitrate. Endothelial cells also exhibited a nonthrombogenic surface, as reflected by platelet-binding studies. Confluent cultures of smooth muscle cells, but not endothelial cells, contracted in response to norepinephrine (10 μM). Cultures of both cell types were exposed to acrolein (2, 5 or 50 ppm), an environmental pollutant, for 4 24 h. Morphologic damage, lactate dehydrogenase release, and cellular thiol content were used as indices of cytotoxicity. Acrolein-induced enzyme leakage and morpholgic alterations were dose- and time-dependent and more pronounced in cultures of smooth muscle cells than in endothelial cells. The total thiol content of endothelial cells exposed to acrolein (50 ppm) for 24 h was not significantly different from that of respective controls. In contrast, the content of treated smooth muscle cells was higher than that of controls. These observations show that primary cultures of vascular cells provide a useful model to evaluate xenobiotic-induced cytotoxicity. The information obtained using a cell culture system may be complemented by the use of other in vivo and in vitro models to determine the mechanisms by which xenobiotics cause vascular cell injury.     

Thiol protease and cathepsin D activities in selected tissues and cultured cells from normal and dystrophic mice

Thiol protease and cathepsin D activities were studied in extracts from hindlimb muscle of 60-day-old normal and dystrophic mice, strain 129 ReJ, and from cultured normal and dystrophic cells. Total thiol protease activity in dystrophic muscle extracts was 3.5 times higher than in normal muscle extracts, while cathepsin D, activity was 2.2 times greater in dystrophic muscle compared with normal muscle. Activation (pH 4.5, 30 degrees C) of latent thiol protease activity in extracts of muscle occurred concomitant with the inactivation or dissociation of endogenous protease inhibitors. Thiol protease assays revealed a higher ratio of active to inactive protease activity in extracts from dystrophic muscle than from normal muscle. Cultured myoblasts (L69/1) were found to contain 30-fold more thiol protease(s) and 6-fold more cathepsin D activity than whole muscle. Cells established from dystrophic muscle and grown in culture for periods up to 6 months were more responsive to thiol protease activation conditions than similar cultures derived from normal muscle. From data on the rate and extent of thiol protease activation in extracts from dystrophic cells and hindlimb muscle compared with normal tissue, it appears that cells and tissues from dystrophic mice contain a lower level of protease inhibitors than cells and tissues from normal mice.     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

DNA methylation and cellular ageing.

Methylated cytosine (m5C) in DNA appears to be an important modulator of the expression of some genes. There are several lines of evidence that gradual loss of m5C is relevant to in vitro cellular ageing: m5C loss occurs during cell culture; m5C loss is detectable at an early stage of culture; m5C loss appears to be related to cell division not just duration in culture; the rate of m5C loss appears to be related to in vitro lifespan of the cell strain in question; and the total loss of m5C during an in vitro lifespan is significant by comparison with induced-changes in m5C levels which effect cell growth, or cause cell-death in culture. Progressive loss of m5C in dividing cells may thus produce the multi-step cell division "clock" which underlies the Hayflick phenomenon.     

Improvement of biomaterials used in tissue engineering by an ageing treatment

Biomaterials based on crosslinked sponges of biopolymers have been extensively used as scaffolds to culture mammal cells. It is well known that single biopolymers show significant change over time due to a phenomenon called physical ageing. In this research, it was verified that scaffolds used for skin tissue engineering (based on gelatin, chitosan and hyaluronic acid) express an ageing-like phenomenon. Treatments based on ageing of scaffolds improve the behavior of skin-cells for tissue engineering purposes. Physical ageing of dry scaffolds was studied by differential scanning calorimetry and was modeled with ageing kinetic equations. In addition, the physical properties of wet scaffolds also changed with the ageing treatments. Scaffolds were aged up to 3 weeks, and then skin-cells (fibroblasts) were seeded on them. Results indicated that adhesion, migration, viability, proliferation and spreading of the skin-cells were affected by the scaffold ageing. The best performance was obtained with a 2-week aged scaffold (under cell culture conditions). The cell viability inside the scaffold was increased from 60 % (scaffold without ageing treatment) to 80 %. It is concluded that biopolymeric scaffolds can be modified by means of an ageing treatment, which changes the behavior of the cells seeded on them. The ageing treatment under cell culture conditions might become a bioprocess to improve the scaffolds used for tissue engineering and regenerative medicine.     

A study of mitotic division fidelity and numerical chromosome changes in ageing Syrian hamster dermal cells

Events associated with culture ageing in Syrian hamster dermal cells have been studied from the time of culture isolation during continuous passage until they senesced and died. Microscopic examination of mitotic cells using differential staining of chromosome and spindle apparatus assessed the faithfulness of cell division. Other indicators of the quality of cell division were obtained from chromosome counts, micronucleus frequencies and incidences of binucleate cells. A loss of spindle fidelity and an increase in aneuploidy corresponded to the period of culture senescence. The data presented indicate that the loss of division fidelity and chromosome number instability is an important indicator of the progression of a mammalian culture to senescence under in vitro conditions. Such information may provide the basis of a model for the study of factors which modify mitotic fidelity and senescence and provide a methodology for monitoring the suitability of mammalian cultures for commercial usage.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Trace metal levels in commercially prepared tissue culture media

Four trace elements, lead, copper, tin and zinc, in addition to certain electrolytes, were measured in 11 commercially prepared tissue culture media. Glass media bottles and plastic tissue culture dishes and flasks were treated with a HCl acid solution to determine the amounts of trace metals leached from their surfaces. Zinc, lead and copper were detected in all media. Tin was detected only in RPMI Medium 1640, fetal bovine serum, minimum essential medium and penicillin-streptomycin. It is possible that a major cause of variability in tissue culture experimental results may be due to effects on growth caused by fluctuation in trace element contamination from batch to batch. Variability in establishing primary cultures of corneal endothelial cells was traced to high lead levels in commercially prepared tissue culture media. A strong case is made for continued diligent efforts to expand analytical horizons and our definition of substances in culture media.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (≥ 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ≤ 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing.     

Morphogenesis from cultured leaf tissue ofSorghum bicolor-culture initiation

Summary In this paper we present further studies on the generation of tissue cultures from leaves of the cerealSorghum bicolor (L.) Moench. It could be shown that during differentiation the leaf tissue rapidly loses the ability to respond to conventional tissue culture techniques. This was probably related to a loss of sensitivity towards 2,4-D, an otherwise most potent growth regulator in tissue culture. The immature tissue which proved to be sensitive proliferated over a wide range of concentration with a broad optimum of about 0.6–6 mg 1^–1 2,4-D. This concentration range appears to be only slightly higher than that described for many dicotyledonous tissue cultures. The relevance of these findings is discussed with reference to the well known dual function of 2,4-D, namely as a selective herbicide and a potent artificial auxin. The implications of these attributes to the practical application of cereal tissue culture is stressed.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - 4-CPA 4-Chlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA 3-Indoleacetic acid - Kinetin 6-Furfurylaminopurine - 6-BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - ABA Abscisic acid - MS Murashige and Skoog     

Plant regeneration from embryo-derived tissue cultures of soybeans

Summary Routine regeneration of fertile plants from a tissue culture of soybean has been achieved. Serially propagated embryogenic cultures were initiated from immature embryos of many genotypes. Organized tissues developed only on the cotyledons of embryos. Genotypic variation in the frequency of initiation of embryogenic tissue was noted. However, embryogenic tissue cultures were generated from all genotypes tested. Embryogenic tissue was serially increased and underwent morphogenesis. Whole fertile plants were recovered. Cultures have been maintained for two years without loss of morphogenic competency. Editor's Statement This procedure for initiating embryogenic tissue cultures from commerical cultivars of soybean and the subsequent development of fertile plants establishes a framework for studying the processes of embryogenesis and embryogenyin vitro as well as providing a system for tissue culture propagation and in vitro modification of soybean. Robert B. Horsch     

Nitrogen Utilization in N-limited Barley During Vegetative and Generative Growth: III. POST-ANTHESIS KINETICS OF NET NITRATE UPTAKE AND THE ROLE OF THE RELATIVE ROOT SIZE IN DETERMINING THE CAPACITY FOR NITRATE ACQUISITION

Barley (Hordeum vulgare L., cvs Golf, Mette, and Laevigatum)was grown under nitrogen limitation in solution culture untilnear maturity. Three different nitrogen addition regimes wereused: in the ‘HN’ culture the relative rate of nitrate-Naddition (RA) was 0·08 d^–1 until day 48 and thendecreased stepwise to, finally, 0·005 d^–1 duringgrain-filling; the ‘LN’ culture received 45% ofthe nitrogen added in HN; the ‘CN’ culture was maintainedat RA 0·0375 d^–1 throughout. Kinetics of net nitrateuptake were measured during ontogeny at 30 to 150 mmol m^–3external nitrate. V_max (which is argued to reflect the maximuminflux rate in these plants) declined with age in both HN andLN cultures. A pronounced transient drop was observed just beforeanthesis, which correlated in time with a peak in root nitrateconcentration. Similar, but less pronounced, trends were observedin CN. The relative V_max (unit nitrogen taken up per unit nitrogenin plants and day) in all three cultures declined from 1·3–2·3d^–1 during vegetative growth to 0·1–0·7d^–1 during generative growth. These values are in HN andLN cultures 15- to more than 100-fold in excess of the demandset by growth rates throughout ontogeny. Predicted balancingnitrate concentrations (defined as the nitrate concentrationrequired to support the observed rate of growth) were below6·0 mmol m^–3 in HN and LN cultures before anthesisand then decreased during ontogeny. In CN cultures the balancingnitrate concentration increased during grain-filling. Apartfrom the transient decline during anthesis, most of the effectof ageing on relative V_max can be explained in terms of reducedcontribution of roots to total biomass (R:T). The loss in uptakeper unit root weight is largely compensated for by the declinewith time in average tissue nitrogen concentrations. The quantitativerelationships between relative V_max and R:T in ageing plantsare similar to those observed for vegetative plants culturedat different RAs. The data support the contention that the capacity for nitrateacquisition in N-limited plants is under general growth control,rather than controlled by specific regulation of the biochemicalpathway of nitrate assimilation. Key words: Barley, nitrogen concentration, root: total plant biomass ratio, V_max     

Some observations on contamination of animal cell cultures by the fungus Aspergillus fumigatus and suggested control measures

The fungus Aspergillus fumigatus Fres. was found to be a most troublesome contaminant of animal cell cultures (hybridomas). Conditions in a carbon dioxide incubator (37 degrees C; about 100% relative humidity) are very favourable for its growth. Unlike bacteria it spreads by means of passively-released aerial spores and of filaments (mycelium) growing within and between wells of tissue culture vessels. It has the potential to cause mycotic infections and allergic reactions in laboratory workers. Various measures for controlling it in the tissue culture laboratory are briefly discussed, such as use of antifungal antibiotics, fumigation with formaldehyde and heat sterilisation of the incubator.     

Chitinase production in pine callus (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.): a defense reaction against endophytes?

In shoot tip-derived tissue cultures of Scots pine (Pinus sylvestris L.), browning and subsequent degeneration of the culture is accompanied by lipid peroxidation and lignification of cells, which are characteristic features of a plant defense reaction. Since chitinases are enzymes acting primarily in plant defense, their expression was studied in pine callus in order to elucidate the defense reaction. Chitinases were present diversely in tissue cultures originating from shoot tips and embryos of P. sylvestris, in contrast to Pinus nigra embryogenic callus, where production of chitinases or browning was not detected. Because endophytic microbes had earlier been detected in buds of Scots pine, their subsequent presence in the tissue cultures was considered a potential cause of the defense reaction. Therefore, the presence of endophytes in the tissue cultures was examined by in situ hybridization. Endophytes were found to colonize heavily in 45% of the tissue cultures of P. sylvestris and to form biofilms, while the P. nigra callus was not found to contain any microbes. The endophytes seemed to propagate uncontrollably once a tissue culture of P. sylvestris was initiated. Regardless of the high level of chitinase production in the callus, the control of the endophytes presumably becomes inadequate during the tissue culture of P. sylvestris.     

Consistent and Heritable Alterations of DNA Methylation Are Induced by Tissue Culture in Maize

Plants regenerated from tissue culture and their progenies are expected to be identical clones, but often display heritable molecular and phenotypic variation. We characterized DNA methylation patterns in callus, primary regenerants, and regenerant-derived progenies of maize using immunoprecipitation of methylated DNA (meDIP) to assess the genome-wide frequency, pattern, and heritability of DNA methylation changes. Although genome-wide DNA methylation levels remained similar following tissue culture, numerous regions exhibited altered DNA methylation levels. Hypomethylation events were observed more frequently than hypermethylation following tissue culture. Many of the hypomethylation events occur at the same genomic sites across independent regenerants and cell lines. The DNA methylation changes were often heritable in progenies produced from self-pollination of primary regenerants. Methylation changes were enriched in regions upstream of genes and loss of DNA methylation at promoters was associated with altered expression at a subset of loci. Differentially methylated regions (DMRs) found in tissue culture regenerants overlap with the position of naturally occurring DMRs more often than expected by chance with 8% of tissue culture hypomethylated DMRs overlapping with DMRs identified by profiling natural variation, consistent with the hypotheses that genomic stresses similar to those causing somaclonal variation may also occur in nature, and that certain loci are particularly susceptible to epigenetic change in response to these stresses. The consistency of methylation changes across regenerants from independent cultures suggests a mechanistic response to the culture environment as opposed to an overall loss of fidelity in the maintenance of epigenetic states.     

Growth induction by serum or polyoma virus inhibits the aggregation of trypsinised suspensions of BHK21 tissue culture fibroblasts

BHK21 hamster tissue culture fibroblasts, when brought into suspension with trypsin, aggregate spontaneously in serum-free medium. The amount of aggregation appears to depend on the proportion of quiescent (growth inhibited) cells which were present in the culture. It is greatest in cells from cultures whose growth is inhibited, either by high cell density or by low serum concentration. When serum is added to low serum cultures, growth is induced and S phase and mitosis follow at 14 and 20 h respectively. It has now been found that aggregation ceases less than 2 h after the addition of serum. Growth can also be induced in such cultures by infection with high multiplicities of polyoma virus. Infected cells enter S phase about 28 h after infection. It has been found that aggregation ceases between 12 and 20 h after infection. Thus in both these cases loss of aggregation precedes S phase by about 12 h. It is concluded that the absence of aggregation in suspensions derived from polyoma virus-transformed lines of BHK21 cells is a consequence of their resistance to density-dependent inhibition of growth. Failure to undergo spontaneous aggregation appears to be an indicator of an early surface change associated with the induction of growth.