Differences In Cellular Behavior Research Articles

P-155 MicroRNA-155 (miR-155) expression profile and developmental characteristics of bovine early embryos are altered when cultured in group versus individually

Abstract Study question Culturing bovine preimplantation embryos in group versus individually has an impact on the expression profile of miR-155 and developmental characteristics. Summary answer MiR-155 was significantly greater in its expression when embryos were cultured in groups versus singly. Developmental characteristics of blastocysts were similar regardless of culture condition. What is known already MiRNAs are short non-coding RNAs that play important roles in many biological pathways including embryogenesis and are potent effectors of post-transcriptional gene silencing. It has been estimated that approximately 60% of all proteins-coding genes are regulated by miRNAs. MiR-155 is involved in early events of embryogenesis; however, it has not been characterized in bovine embryos and this knowledge has not yet been clinically translated. Existing literature indicates that group culture yields superior embryo development as embryos can benefit from autocrine signaling and paracrine communication but embryos in groups may be exposed to negative effects of dying or delayed embryos. Study design, size, duration The study consisted of two groups: zygotes cultured to blastocysts in group and those cultured individually. Each sample consists of 3 blastocysts. Droplet digital PCR (dd-PCR) was used to examine the expression of miR-155. Gene Set Enrichment Analysis (GSEA) of miRNAs was carried out to identify functional Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that are enriched for the targets of one or more miRNAs. Participants/materials, setting, methods Oocytes were aspirated from antral follicles of bovine ovaries and matured in vitro. Twenty-four hours post retrieval, matured oocytes were inseminated with sperm from similar bull. Presumptive zygotes were cultured either in groups or individually until blastocyst stage, Blastocyst were collected and purified for RNA. Analysis for KEGG and GO Biological Process, Cellular Component and Molecular Functions were carried out on the identified target genes. Results were plotted according to level of enrichment for miR-155. Main results and the role of chance Five hundred and seventy-five oocytes were retrieved from 120 bovine ovaries. Cleavage rate and blastocyst formation after IVF were compared between those blastocysts cultured in groups versus individually. There was no significant difference among the two groups with regards to cleavage rate (69% vs. 54%) and blastocyst formation (42% vs.35%). ddPCR assessment showed significantly greater MiR-155 expression in those blastocysts cultured in groups (p = 0.002). The highest level of miR-151 expressed was 20 copies/sample. Every sample, regardless of the culture conditions, had detectable miR-155 level. The interaction of predicted miR-155 target clusters based on the GO Biological Processes shows that miR-155 plays a role in DNA binding, transcription factor binding, regulates transcription and affects cell adhesion. KEGG analysis revealed an association between miR-155 to steroidogenesis and controls apoptosis pathways. Limitations, reasons for caution The number of copies expressed for this miRNA was relatively low, which could suggest that miR-155 may not have a functional role at the blastocyst stage as the effectiveness of a miRNA depends on the relative number of binding site and target molecules. Blastocysts were pooled based on morphological assessment. Wider implications of the findings MiRNAs offer a novel mean to assess embryonic fitness. Further validation and more complex study designs are required to implement the profiling of embryonic miRNAs as a diagnostic test for embryo selection. Group culture increased miR-155 expression, possibly reflecting differences in cellular behavior between group cultured embryos and individually ones. Trial registration number Not applicable

3D Printing of Self-Assembling Nanofibrous Multidomain Peptide Hydrogels.

3D printing has become one of the primary fabrication strategies used in biomedical research. Recent efforts have focused on the 3D printing of hydrogels to create structures that better replicate the mechanical properties of biological tissues. These pose a unique challenge, as soft materials are difficult to pattern in three dimensions with high fidelity. Currently, a small number of biologically derived polymers that form hydrogels are frequently reused for 3D printing applications. Thus, there exists a need for novel hydrogels with desirable biological properties that can be used as 3D printable inks. In this work, the printability of multidomain peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations, is established. MDPs with different charge functionalities are optimized as distinct inks and are used to create complex 3D structures, including multi-MDP prints. Additionally, printed MDP constructs are used to demonstrate charge-dependent differences in cellular behavior in vitro. This work presents the first time that self-assembling peptides have been used to print layered structures with overhangs and internal porosity. Overall, MDPs are a promising new class of 3D printable inks that are uniquely peptide-based and rely solely on supramolecular mechanisms for assembly.

A combined experimental and computational framework to evaluate the behavior of therapeutic cells for peripheral nerve regeneration.

Recent studies have explored the potential of tissue‐mimetic scaffolds in encouraging nerve regeneration. One of the major determinants of the regenerative success of cellular nerve repair constructs (NRCs) is the local microenvironment, particularly native low oxygen conditions which can affect implanted cell survival and functional performance. In vivo, cells reside in a range of environmental conditions due to the spatial gradients of nutrient concentrations that are established. Here we evaluate in vitro the differences in cellular behavior that such conditions induce, including key biological features such as oxygen metabolism, glucose consumption, cell death, and vascular endothelial growth factor secretion. Experimental measurements are used to devise and parameterize a mathematical model that describes the behavior of the cells. The proposed model effectively describes the interactions between cells and their microenvironment and could in the future be extended, allowing researchers to compare the behavior of different therapeutic cells. Such a combinatorial approach could be used to accelerate the clinical translation of NRCs by identifying which critical design features should be optimized when fabricating engineered nerve repair conduits.

Biocompatibility of Lithium Disilicate and Zirconium Oxide Ceramics with Different Surface Topographies for Dental Implant Abutments.

Gingivafibroblasts were cultured on lithium disilicate, on zirconia dioxide, and on titanium with two different surface roughnesses (0.2 µm and 0.07 µm); Proliferation (MTT), Living/Dead staining, cytotoxicity (LDH), proliferation (FGF2), and inflammation (TNFα) were analyzed after 1 day and 21 days. Furthermore, alteration in cell morphology (SEM) was analyzed. The statistical analysis was performed by a Kruskal–Wallis test. The level of significance was set at p < 0.05. There were no distinct differences in cellular behavior between the tested roughness. There were slight differences between tested materials. Cells grown on zirconia dioxide showed higher cytotoxic effects. Cells grown on lithium disilicate showed less expression of TNFα compared to those grown on zirconia dioxide or titanium. These effects persisted only during the first time span. The results indicate that the two tested high-strength ceramics and surface properties are biologically suitable for transmucosal implant components. The findings may help clinicians to choose the most appropriate biomaterial as well as the most appropriate surface treatment to use in accordance with specific clinical dental applications.

Investigating the Role of NHE1 in the Regulation of Changes in Cellular Functions Related to the Development of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by the thickening, stiffening, and scaring of tissue within the lungs. Affected individuals develop shortness of breath and the progressive reduction of respiratory function. Fibrosis ultimately results in low blood flow and hypoxia in the tissue, a microenvironment which is closely associated with the upregulation of the sodium-hydrogen exchanger isoform 1 (NHE1). NHE1 is a ubiquitously expressed protein that exchanges one extracellular Na+ for one intracellular H+. This regulates intracellular pH and cell progression through the cell cycle. When NHE1 is upregulated it supports increased cell proliferation and migration, indicating that it may play a vital role in the behavior of fibroblasts in the development of idiopathic pulmonary fibrosis. We hypothesized that inhibition of NHE1 activation would decrease cell progression towards a fibrotic phenotype in lung fibroblasts. For this assessment, we compared differences in cellular behavior in three cell lines to evaluate the role of NHE1 in these processes. LL29 cells are human fibroblasts derived from a patient diagnosed with idiopathic pulmonary fibrosis; WI-38 cells which are derived from healthy lung tissue; and PSN cells, which are derived from Chinese hamster lung fibroblasts and express only human NHE1.Transforming growth factor beta 1 (TGF-b1), Lysophosphatidic Acid (LPA) and Serotonin (5-hydroxytryptamine) were evaluated for their ability to alter cell proliferation, stress fiber formation, and myofibroblast differentiation in all three cell lines. TGF-b1 is a cytokine that is a growth factor implicated in the pathogenesis of pulmonary fibrosis and is involved in differentiation, proliferation, and matrix production of fibroblasts. Serotonin (5-HT) is a signaling molecule that binds to G-protein coupled receptors supporting the increase in TGF-b1 and its effects in cells. This hormone is involved in wound healing and stimulating inflammation. LPA is a lipid signaling molecule that plays a role in mediating inflammation and activating TGF-b1 signaling. All experiments were performed in the presence and absence of Ethylisopropylamiloride (EIPA), a potent inhibitor of NHE1, to evaluate the role of NHE1 in fibrotic transformation. In PSN cells, stimulation with 10 µM LPA increased stress fiber formation over 2-fold and this stimulation was absent in cells treated with 10 µM EIPA indicating a role for NHE1 in the regulation of stress fiber formation and related cellular processes.

Poly-l-Lactic Acid Nanotubes as Soft Piezoelectric Interfaces for Biology: Controlling Cell Attachment via Polymer Crystallinity.

It has become increasingly evident that the mechanical and electrical environment of a cell is crucial in determining its function and the subsequent behavior of multicellular systems. Platforms through which cells can directly interface with mechanical and electrical stimuli are therefore of great interest. Piezoelectric materials are attractive in this context because of their ability to interconvert mechanical and electrical energy, and piezoelectric nanomaterials, in particular, are ideal candidates for tools within mechanobiology, given their ability to both detect and apply small forces on a length scale that is compatible with cellular dimensions. The choice of piezoelectric material is crucial to ensure compatibility with cells under investigation, both in terms of stiffness and biocompatibility. Here, we show that poly-l-lactic acid nanotubes, grown using a melt-press template wetting technique, can provide a “soft” piezoelectric interface onto which human dermal fibroblasts readily attach. Interestingly, by controlling the crystallinity of the nanotubes, the level of attachment can be regulated. In this work, we provide detailed nanoscale characterization of these nanotubes to show how differences in stiffness, surface potential, and piezoelectric activity of these nanotubes result in differences in cellular behavior.

Different in vitro cellular responses to tamoxifen treatment in polydimethylsiloxane-based devices compared to normal cell culture.

Cell culture systems based on polydimethylsiloxane (PDMS) microfluidic devices offer great flexibility because of their simple fabrication and adaptability. PDMS devices also make it straightforward to set up parallel experiments and can facilitate process automation, potentially speeding up the drug discovery process. However, cells grown in PDMS-based systems can develop in different ways to those grown with conventional culturing systems because of the differences in the containers' surfaces. Despite the growing number of studies on microfluidic cell culture devices, the differences in cellular behavior in PDMS-based devices and normal cell culture systems are poorly characterized. In this work, we investigated the proliferation and autophagy of MCF7 cells cultured in uncoated and Parylene-C coated PDMS wells. Using a quantitative method combining solid phase extraction and liquid chromatography mass spectrometry we developed, we showed that Tamoxifen uptake into the surfaces of uncoated PDMS wells can change the drug's effective concentration in the culture medium, affecting the results of Tamoxifen-induced autophagy and cytotoxicity assays. Such changes must be carefully analyzed before transferring in vitro experiments from a traditional culture environment to a PDMS-based microfluidic system. We also found that cells cultured in Parylene-C coated PDMS wells showed similar proliferation and drug response characteristics to cells cultured in standard polystyrene (PS) plates, indicating that Parylene-C deposition offers an easy way of limiting the uptake of small molecules into porous PDMS materials and significantly improves the performance of PDMS-based device for cell related research.

Dynamic behaviors of the non-neural ectoderm during mammalian cranial neural tube closure

The embryonic brain and spinal cord initially form through the process of neural tube closure (NTC). NTC is thought to be highly similar between rodents and humans, and studies of mouse genetic mutants have greatly increased our understanding of the molecular basis of NTC with relevance for human neural tube defects. In addition, studies using amphibian and chick embryos have shed light into the cellular and tissue dynamics underlying NTC. However, the dynamics of mammalian NTC has been difficult to study due to in utero development until recently when advances in mouse embryo ex vivo culture techniques along with confocal microscopy have allowed for imaging of mouse NTC in real time. Here, we have performed live imaging of mouse embryos with a particular focus on the non-neural ectoderm (NNE). Previous studies in multiple model systems have found that the NNE is important for proper NTC, but little is known about the behavior of these cells during mammalian NTC. Here we utilized a NNE-specific genetic labeling system to assess NNE dynamics during murine NTC and identified different NNE cell behaviors as the cranial region undergoes NTC. These results bring valuable new insight into regional differences in cellular behavior during NTC that may be driven by different molecular regulators and which may underlie the various positional disruptions of NTC observed in humans with neural tube defects.

Controlling the kinetics of thiol-maleimide Michael-type addition gelation kinetics for the generation of homogenous poly(ethylene glycol) hydrogels.

The development of synthetic hydrogels analogs for the extracellular matrix has proven a useful and important tool to study the role of specific signals on biological outcomes invitro and to serve as scaffolds for tissue repair. Although the importance of physical properties (e.g. microstructure and stiffness) in the micro and nano scale on cell fate has been widely reported, bulk modulus measurements are typically used to characterize hydrogels. Thus, the physical properties of hydrogels have not been widely tested for their controlled physical properties in the nano and micron scales. In this report, we show that although fast Michael-type addition crosslinked hydrogels appear uniform by bulk modulus readings and visual inspection, they are non-uniform in the micron scale, with high and low crosslinking regions. Further, we show that these regions of high and low crosslinking result in differences in cellular behavior. Since these regions are random in density and shape, this leads to misleading cellular responses. These inconsistences are most widely observed when the gel forms faster than the material can be mixed. This study slows the gelation rate of thiol-maleimide cross-linked hydrogels in order to overcome the cellular response variability between batches.

Deep Dermal Fibroblasts Refractory to Migration and Decorin-Induced Apoptosis Contribute to Hypertrophic Scarring

Hypertrophic scar (HTS) represents the dermal equivalent of fibroproliferative disorders. Fibroblasts from the deep dermis are implicated in the development of HTS after injuries that involve deeper areas of the skin. However, fibroblasts that reside in the superficial layer of the skin show antifibrotic properties, and injuries limited to this area heal with little or no scarring. Previously, cellular and molecular characteristics of superficial fibroblasts and deep dermal fibroblasts that may influence HTS formation were analyzed. In this study, differences in cellular behavior between superficial fibroblasts and deep dermal fibroblasts that may also affect the development of HTS or tissue fibrosis were further characterized. Immunostaining and migration, adhesion, apoptosis, and cell viability assays were performed in fibroblasts from the superficial and deep dermis. Reverse-transcription polymerase chain reaction was used to examine the gene expression of molecules involved in cell death after treatment of fibroblasts with decorin. When compared with superficial fibroblasts, deep dermal fibroblasts showed lower migration rates. Although all the fibroblasts tested showed no difference in adhesion to fibronectin, superficial fibroblasts demonstrated increased apoptotic and dead cells when treated with decorin. Decorin resulted in a significant increase in the expression of apoptosis markers, histone-1, caspase-1, caspase-8, and p53 in superficial fibroblasts when compared with deep dermal fibroblasts. Taken together, the findings suggest that reduced migration, lack of decorin, and resistance of deep dermal fibroblasts to decorin-induced apoptosis may result in hypercellularity in injuries involving the deep dermis, leading to deposition of excess extracellular matrix and HTS formation.

The Oriented Emergence of Axons from Retinal Ganglion Cells Is Directed by Laminin Contact In Vivo

SummaryHow the site of axon emergence is specified during neural development is not understood. Previous studies disagree on the relative importance of intrinsic and extrinsic mechanisms. The axons of retinal ganglion cells (RGCs) emerge basally in vivo, yet because RGCs develop from polarized neuroepithelial cells within a polarized environment, disentangling intrinsic and extrinsic influences is a challenge. We use time-lapse imaging to demonstrate that Laminin acting directly on RGCs is necessary and sufficient to orient axon emergence in vivo. Laminin contact with the basal processes of newborn RGCs prevents the cells from entering a stochastic Stage 2 phase, directs the rapid accumulation of the early axonal marker Kif5c560-YFP, and leads to the formation of axonal growth cones. These results suggest that contact-mediated cues may be critical for the site of axon emergence and account for the differences in cellular behavior observed in vitro and in vivo.Video

Evaluation of polymer scaffolds to be used in a composite injectable system for intervertebral disc tissue engineering

Adult porcine nucleus pulposus cells were seeded onto gelatin, demineralized bone matrix (DBM), and polylactide scaffolds and cultured in vitro. Cellular behavior in response to the scaffolds was analyzed using biochemical assays, histology, and real-time quantitative reverse transcriptase-polymerase chain reaction. Scanning electron microscopy showed pronounced differences in surface texture of the scaffolds. Nucleus pulposus cells attached and assumed an elongated fibroblast-like morphology on the gelatin and DBM scaffolds. The cells cultured on the gelatin and DBM were metabolically active and expressed types I and II collagen and aggrecan. Detached cellular aggregates with a rounded morphology were noted in the culture tubes containing the polylactide scaffolds. Both surface chemistry and texture likely had a role in causing differences in cellular behavior in response to scaffold material. Promising results were observed using the gelatin and demineralized bone scaffolds, but the behavior of cells cultured on these scaffolds will need to be characterized further. This initial research will be used to direct future work involved in developing this composite injectable tissue engineering system.

Differences between fibroblasts cultured from oral mucosa and normal skin: implication to wound healing

It is generally agreed that oral mucosa heals faster with less scar than skin does, and hypertrophic scar or keloid is very rare in the oral cavity. Fibroblasts are thought to play an important role in wound healing and scar formation, whose control is mediated by growth factors. We have studied whether there are any differences in the cellular behavior of fibroblasts between oral mucosa and skin, and in their response to growth factors. Oral mucosal fibroblasts proliferated slightly more than dermal fibroblasts on average. Dermal fibroblasts in collagen gel possessed greater contraction potency than oral mucosal fibroblasts, irrespective of the presence of growth factors; however, oral mucosal fibroblasts showed an earlier collagen gel contraction with or without TGF-β1. There were no differences in basal collagen synthetic rate between dermal and oral mucosal fibroblasts, while the latter synthesized more collagen than dermal fibroblasts when they were stimulated with TGF-β1. Our study showed that oral mucosal fibroblasts and dermal fibroblasts had selective differences in cellular behavior and in their responses to growth factors, which seems to contribute to the differences in wound healing.

Different behavior of human osteoblast-like cells isolated from normal and heterotopic bone In vitro.

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM beta-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3-6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells.

Microcirculatory homeostasis 1930-1990: insight into microcirculatory readjustments provided by studies on the peripheral circulatory insufficiency of the shock syndrome.

The present overview is intended to serve several purposes. It highlights efforts over the years to resolve the difficult key issues raised by August Krogh in the 1930s and by the urgent need created in the 1940s and 1950s by World War II to come to grips with the basic problems underlying the collapse of tissue perfusion in the syndrome of circulatory shock. The complex operational issues raised by circulatory insufficiency at the systemic, as well as the local tissue level, during the shock syndrome, are used as a framework to keep in perspective the impact of advances in methodology, ultrastructure, and in recent years in molecular biology on microvascular research. In view of the current continuing need to integrate the newly emerging body of in vitro information into specific aspects of the operation of the peripheral vascular tree, emphasis is placed on the advances in dealing with data provided by in vivo microscopy of selected tissues. This overall review had made it clear that a top priority remains the need to separate the effects of systemic and locally derived readjustments, and to identify the basis for the substantial differences in cellular behavior exhibited by the vascular and perivascular components of the various organs and tissues of the body that can serve as predisposing factors in regional pathophysiology.

Defective Low-Density Lipoprotein Metabolism in Cultured, Normal, Transformed, and Malignant Keratinocytes

To obtain more information on differences in cellular behavior during the differentiation process, a number of types of epithelial cells with and without a defect in cornified envelope formation were compared as to the regulation of intracellular cholesterol synthesis by low-density lipoprotein (LDL) and the uptake and degradation of [125I]LDL. The following cells were cultured: normal skin fibroblasts (F), normal (K) and SV 40 transformed (SVK14) keratinocytes, and a number of squamous carcinoma cell (SCC) lines in which the defective terminal differentiation was found to occur in the following order of intensity: SCC-12F2 less than SCC-25 approximately equal to SCC-15 less than SCC-12B2 less than SCC-4. Compared with normal human fibroblasts, most of the cells under study showed a defective response to changes of the extracellular serum LDL concentration. The degree of inducibility of cholesterol synthesis after the cells were deprived of extracellular sources of cholesterol as well as the degree of the LDL-induced suppression of the intracellular cholesterol synthesis in cells preincubated in medium supplemented with lipoprotein-deficient serum decreased in the following order: F greater than SCC-4 greater than SCC-15 approximately equal to SCC-25 greater than SCC-12B2 congruent to SCC-12F2 greater than SVK14 approximately equal to K. A defect in LDL metabolism was found to be responsible for the partial or complete failure of LDL to regulate the cholesterol metabolism, because when sterol was delivered to all cell types in artificial nonlipoprotein form (i.e., as 25-hydroxycholesterol) a marked suppression of cholesterol synthesis was observed. For all SCC lines tested except SCC-12B2 good correlation was found between the degree of LDL-induced suppression of cholesterol synthesis and the decreasing ability of cells to differentiate into squames or cornified envelope-forming cells. The transformation of keratinocytes by SV 40 virus did not lead to any change in the response of the cells to changes in the extracellular LDL concentration since both the normal and the transformed keratinocytes showed the same response to LDL (i.e., no response).

Cellular changes in cultured lenses.

Observations were made on the frog lens epithelium after culture of the entire lens or of capsular explants. General deviations from normal lens structure as well as specific changes in two media were studied. DNA synthesis and mitosis were induced in the central epithelial cells. Disruption of the orderly, single, epithelial layer that is characteristic of normal lenses was accompanied by the appearance of multilayered plaques of epithelial cells and invasion of vacuolated regions of the lens fibers by epithelial cells. Cells that are fibroblast-like in appearance were observed in regions of the capsule depleted of cells and at the free edges of epithelial sheets in cell culture. Epithelial cells were surrounded by capsule-like material even situated in the lens interior. Nuclie derived from central epithelial cells of lenses cultured in L-15 medium and medium 199 had served as donors in previous nuclear transfer experiments in this laboratory. In our current observation of L-15-cultured lenses, cells were sparsely distributed on the capsule and nuclei were abnormally shaped; in 199-cultured lenses, cells were more densely distributed and nuclei resembled those of normal lenses. Medium 199 without serum could better maintain normal lens structure than L-15 medium without serum. In addition, the percentage of epithelial explants demonstrating cellular outgrowth was greater in medium 199. The differences in cellular behavior were shown not to be the result of different sugars, pH, or the presence of CO2. The nuclear transfer results may reflect the structural changes in the epithelium after lens culture in the two media.

THE GROWTH CHARACTERISTICS IN VITRO OF NORMAL EPITHELIUM, DYSPLASIA, AND CARCINOMA IN SITU OF THE UTERINE CERVIX

Normal cervical epithelium and epithelium containing only dysplasia and carcinoma- in-situ have been grown in vitro as “pure” cell types without contamination by fibroblasts or squamous epithelium of a significantly different histological type. Excellent growth was obtained from all types of tissues explanted, and the morphology and pattern of growth were compared. Individual differences in growth pattern between the different cases of dysplasia and carcinoma- in-situ were noted. These were distinctive enough so that the cases could be consistently identified by their morphology without reference to identifying labels. No striking differences in growth rates were noted among the three epithelia, and, although there were remarkable differences in cellular behavior and morphology between the normal and abnormal epithelia, no striking differences were observed between dysplasia and carcinoma- in-situ .