Octacalcium Phosphate for Bone Tissue Engineering: Synthesis, Modification, and In Vitro Biocompatibility Assessment.

Octacalcium phosphate (OCP, Ca8H2(PO4)6·5H2O) is known to be a possible precursor of biological hydroxyapatite formation of organic bone tissue. OCP has higher biocompatibility and osseointegration rate compared to other calcium phosphates. In this work, the synthesis of low-temperature calcium phosphate compounds and substituted forms of those at physiological temperatures is shown. Strontium is used to improve bioactive properties of the material. Strontium was inserted into the OCP structure by ionic substitution in solutions. The processes of phase formation of low-temperature OCP with theoretical substitution of strontium for calcium up to 50 at.% in conditions close to physiological, i.e., temperature 35-37 °C and normal pressure, were described. The effect of strontium substitution range on changes in the crystal lattice of materials, the microstructural features, surface morphology and biological properties in vitro has been established. The results of the study indicate the effectiveness of using strontium in OCP for improving biocompatibility of OCP based composite materials intended for bone repair.

TGF-ß-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-ß), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-ß-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-ß-induced EndMT. Using these techniques, we provide evidence that TGF-ß2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

Simultaneous quantitative assessment of two distinct cell lineages with a nuclear-localized dual genetic reporter.

Genetic lineage tracing has been widely used for studying in vivo cell fate plasticity during embryogenesis, tissue homeostasis, and disease development. Recent applications with multiple site-specific recombinases have been used in complex and sophisticated genetic fate mapping studies. However, the previous multicolor reporters for dual recombinases had limitations of precise in situ quantification of cell number, which is mainly due to the intermingling of cells in condensed tissues. Here, we generated a dual recombinase-mediated nuclear-localized GFP and tdTomato reporter line, which enables clear, simultaneous quantification of two distinct cell lineages in vivo. Combining this dual genetic reporter with Tbx18-Cre and Cdh5-Dre lines, which genetically trace epicardial and endothelial cells, respectively, we obtained high-resolution images for the anatomic distribution of the descendants of these two distinct cell lineages in the valve mesenchyme during development, remodeling, and maturation stages. This new dual genetic reporter is expected to facilitate fate tracing of two cell lineages and their objective quantification in vivo.

Application of a human mesoderm tissue elongation system in vitro derived from human induced pluripotent stem cells to risk assessment for teratogenic chemicals.

Toxic compounds from the mother's diet and medication in addition to genetic factors and infection during pregnancy remain risks for various congenital disorders and misbirth. To ensure the safety of food and drugs for pregnant women, establishment of an in vitro system that morphologically resembles human tissues has been long desired. In this study, we focused on dorsal mesoderm elongation, one of the critical early development events for trunk formation, and we established in vitro autonomous elongating tissues from human induced pluripotent stem cells (hiPSCs). This artificial tissue elongation is regulated by MYOSIN II and FGF signaling, and is diminished by methylmercury or retinoic acid (RA), similar to in vivo human developmental disabilities. Moreover, our method for differentiation of hiPSCs requires only a short culture period, and the elongation is cell number-independent. Therefore, our in vitro human tissue elongation system is a potential tool for risk assessment assays for identification of teratogenic chemicals via human tissue morphogenesis.

Assessment of the nitrofen model of congenital diaphragmatic hernia and of the dysregulated factors involved in pulmonary hypoplasia.

PURPOSE: To study pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH), investigators have been employing a fetal rat model based on nitrofen administration to dams. Herein, we aimed to: (1) investigate the validity of the model, and (2) synthesize the main biological pathways implicated in the development of PH associated with CDH. METHODS: Using a defined strategy, we conducted a systematic review of the literature searching for studies reporting the incidence of CDH or factors involved in PH development. We also searched for PH factor interactions, relevance to lung development and to human PH. RESULTS: Of 335 full-text articles, 116 reported the incidence of CDH after nitrofen exposure or dysregulated factors in the lungs of nitrofen-exposed rat fetuses. CDH incidence: 54% (27-85%) fetuses developed a diaphragmatic defect, whereas the whole litter had PH in varying degrees. Downregulated signaling pathways included FGF/FGFR, BMP/BMPR, Sonic Hedgehog and retinoid acid signaling pathway, resulting in a delay in early epithelial differentiation, immature distal epithelium and dysfunctional mesenchyme. CONCLUSIONS: The nitrofen model effectively reproduces PH as it disrupts pathways that are critical for lung branching morphogenesis and alveolar differentiation. The low CDH rate confirms that PH is an associated phenomenon rather than the result of mechanical compression alone.

Assessment of intratumor heterogeneity in mesenchymal uterine tumor by an 18F-FDG PET/CT texture analysis.

OBJECTIVE: The aim of this study was to retrospectively evaluate the clinical significance of 18F-FDG PET/CT textural features for discriminating uterine sarcoma from leiomyoma. METHODS: Fifty-five patients with suspected uterine sarcoma based on ultrasound and MRI findings who underwent pretreatment 18F-FDG PET/CT were included. Fifteen patients were histopathologically proven to have uterine sarcoma, 14 patients by surgical operation and one by biopsy, and 40 patients were diagnosed with leiomyoma by surgical operation or in a follow-up for at least 2 years. A texture analysis was performed on PET/CT images from which second- and higher order textural features were extracted in addition to standardized uptake values (SUVs) and other first-order features. The accuracy of PET features for differentiating between uterine sarcoma and leiomyoma was evaluated using a receiver-operating-characteristic (ROC) analysis. RESULTS: The intratumor distribution of 18F-FDG was more heterogeneous in uterine sarcoma than in leiomyoma. Entropy, correlation, and uniformity calculated from normalized gray-level co-occurrence matrices and SUV standard deviation derived from histogram statistics showed greater area under the ROC curves (AUCs) than did maximum SUV for differentiating between sarcoma and leiomyoma. Entropy, as a single feature, yielded the greatest AUC of 0.974 and the optimal cut-off value of 2.85 for entropy provided 93% sensitivity, 90% specificity, and 92% accuracy. When combining conventional features with textural ones, maximum SUV (cutoff: 6.0) combined with entropy (2.85) and correlation (0.73) provided the best diagnostic performance (100% sensitivity, 94% specificity, and 95% accuracy). CONCLUSIONS: In combination with the conventional histogram statistics and/or volumetric parameters, 18F-FDG PET/CT textural features reflecting intratumor metabolic heterogeneity are useful for differentiating between uterine sarcoma and leiomyoma.

An optimized and simplified system of mouse embryonic stem cell cardiac differentiation for the assessment of differentiation modifiers.

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is optimized for investigating factors that affect cardiac differentiation.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

The concept of the onychodermis (specialized nail mesenchyme): an embryological assessment and a comparative analysis with the hair follicle.

BACKGROUND: Recently, an intriguing concept was introduced into the literature that defines the area underlying the nail bed as a specific mesenchymal substructure unique to the nail organ. It has been termed onychodermis. The onychodermis expresses CD10 with remarkable specificity. Herein, we compare adult and fetal human hair follicles with fetal nail organs in an attempt to draw analogies for the mesenchyme associated with both adnexal structures. METHODS: We examined immunohistochemically samples from adult and fetal hair follicles for the expression of CD10, CD34 and the mesenchymal stem cell marker nestin and compared the antigen profile with that of the fetal nail organ. RESULTS: The CD10-positive/CD34-negative onychodermis is prominently visible at the end of the second trimester. A corresponding follicular structure was not identified, either in the adult or in the developing hair follicle. Nestin staining does not define the onychodermis. CONCLUSIONS: The concept of the onychodermis is equally valid in the developing nail organ where it is also defined by its expression for CD10. Its function may be related to the anchorage of the overlying nail bed but may also involve a more dynamic role in the induction of hard keratins in the latter, contributing to the formation of the nail plate.

Knockdown of IKK1/2 promotes differentiation of mouse embryonic stem cells into neuroectoderm at the expense of mesoderm.

Activation of nuclear factor kappa B (NF-κB) is accomplished by a specific kinase complex (IKK-complex), phosphorylating inhibitors of NF-κB (IκB). In embryonic stem cells (ESCs), NF-κB signaling causes loss of pluripotency and promotes differentiation towards a mesodermal phenotype. Here we show that NF-κB signaling is involved in cell fate determination during retinoic acid (RA) mediated differentiation of ESCs. Knockdown of IKK1 and IKK2 promotes differentiation of ESCs into neuroectoderm at the expense of neural crest derived myofibroblasts. Our data indicate that RA is not only able to induce neuronal differentiation in vitro but also drives ESCs into a neural crest cell lineage represented by differentiation towards peripheral neurons and myofibroblasts. The NC is a transiently existing, highly multipotent embryonic cell population generating a wide range of different cell types. During embryonic development the NC gives rise to distinct precursor lineages along the anterior-posterior axis determining differentiation towards specific derivates. Retinoic acid (RA) signaling provides essential instructive cues for patterning the neuroectoderm along the anterior-posterior axis. The demonstration of RA as a sufficient instructive signal for the differentiation of pluripotent cells towards NC and the involvement of NF-κB during this process provides useful information for the generation of specific NC-lineages, which are valuable for studying NC development or disease modeling.

Replication timing: a fingerprint for cell identity and pluripotency.

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify "replication timing fingerprints" unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization.

Computational modeling of epithelial-mesenchymal transformations.

An epithelial-mesenchymal transformation (EMT) involves alterations in cell-cell and cell-matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell-substrate adhesion than by a decrease in cell-cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.

Monte Carlo analysis of an ODE Model of the Sea Urchin Endomesoderm Network.

BACKGROUND: Gene Regulatory Networks (GRNs) control the differentiation, specification and function of cells at the genomic level. The levels of interactions within large GRNs are of enormous depth and complexity. Details about many GRNs are emerging, but in most cases it is unknown to what extent they control a given process, i.e. the grade of completeness is uncertain. This uncertainty stems from limited experimental data, which is the main bottleneck for creating detailed dynamical models of cellular processes. Parameter estimation for each node is often infeasible for very large GRNs. We propose a method, based on random parameter estimations through Monte-Carlo simulations to measure completeness grades of GRNs. RESULTS: We developed a heuristic to assess the completeness of large GRNs, using ODE simulations under different conditions and randomly sampled parameter sets to detect parameter-invariant effects of perturbations. To test this heuristic, we constructed the first ODE model of the whole sea urchin endomesoderm GRN, one of the best studied large GRNs. We find that nearly 48% of the parameter-invariant effects correspond with experimental data, which is 65% of the expected optimal agreement obtained from a submodel for which kinetic parameters were estimated and used for simulations. Randomized versions of the model reproduce only 23.5% of the experimental data. CONCLUSION: The method described in this paper enables an evaluation of network topologies of GRNs without requiring any parameter values. The benefit of this method is exemplified in the first mathematical analysis of the complete Endomesoderm Network Model. The predictions we provide deliver candidate nodes in the network that are likely to be erroneous or miss unknown connections, which may need additional experiments to improve the network topology. This mathematical model can serve as a scaffold for detailed and more realistic models. We propose that our method can be used to assess a completeness grade of any GRN. This could be especially useful for GRNs involved in human diseases, where often the amount of connectivity is unknown and/or many genes/interactions are missing.

Lung cancer and chronic obstructive pulmonary disease: needs and opportunities for integrated research.

Lung cancer and chronic obstructive pulmonary disease (COPD) are leading causes of morbidity and mortality in the United States and worldwide. They share a common environmental risk factor in cigarette smoke exposure and a genetic predisposition represented by the incidence of these diseases in only a fraction of smokers. The presence of COPD increases the risk of lung cancer up to 4.5-fold. To investigate commonalities in disease mechanisms and perspectives for disease chemoprevention, the National Heart, Lung, and Blood Institute (NHLBI) and the National Cancer Institute (NCI) held a workshop. The participants identified four research objectives: 1) clarify common epidemiological characteristics of lung cancer and COPD; 2) identify shared genetic and epigenetic risk factors; 3) identify and validate biomarkers, molecular signatures, and imaging-derived measurements of each disease; and 4) determine common and disparate pathogenetic mechanisms. These objectives should be reached via four research approaches: 1) identify, publicize, and enable the evaluation and analysis of existing datasets and repositories of biospecimens; 2) obtain phenotypic and outcome data and biospecimens from large studies of subjects with and/or at risk for COPD and lung cancer; 3) develop and use animal and other preclinical models to investigate pathogenetic links between the diseases; and 4) conduct early-phase clinical trials of potential chemopreventive agents. To foster much needed research interactions, two final recommendations were made by the participants: 1) incorporate baseline phenotyping and outcome measures for both diseases in future longitudinal studies of each disease and 2) expand collaborative efforts between the NCI and NHLBI.

The assessment of cell cycle genes in the rat mandibular condyle.

OBJECTIVES: We aimed to investigate the expression profile of cell cycle genes in the mandibular condyle on mechanical strain and natural growth, and quantify their expression intensity. METHODS: Three hundred and fifty 35 days old Sprague-Dawley rats were randomly divided into experimental groups fitted with bite-jumping appliances and control groups. Groups were sacrificed at days 1, 3, 7, 9, 14, 30, and 33. Then, condyles were dissected and total RNA was extracted for microarray analysis. RESULTS: Thirty-nine known cell cycle genes were present in the condyle, where Cyclin D1, PCNA, and Wnt5a were differentially expressed. Reverse transcriptase-PCR confirmed that Wnt5a showed a 2-fold increase on experimental day 1, Cyclin D1 showed a 2-fold increase on experimental day 1 and a 3-fold increase on experimental day 14, while PCNA shows 2.2-fold increase both on experimental days 9 and 30. PCNA, Cyclin D1, and Wnt5a were all expressed by cells in both the proliferative layer and erosive zone. CONCLUSION: Mandibular advancement leads to the expression of Cyclin D1 that accelerates entry to the S phase. The increased level of PCNA indicates increased DNA replication of MSC. Then, elevated level of Wnt5a indicates the commitment of MSC to the chondrogenic lineage.

Assessment of pluripotency and multilineage differentiation potential of NTERA-2 cells as a model for studying human embryonic stem cells.

Embryonal carcinoma cells are pluripotent stem cells derived from teratocarcinomas and are considered to be the malignant counterparts of human embryonic stem cells. As there are few reliable experimental systems available to study the molecular mechanisms governing normal embryogenesis, well-characterized human embryonal carcinoma stem cell lines may provide a robust and simple model to study certain aspects of pluripotency and cellular differentiation. Here, we have analysed NTERA-2 cL.D1 cells at molecular and cellular levels during expansion and differentiation, via formation of cell aggregates similar to embryoid bodies in embryonic stem cells. Thus, human embryonal carcinoma cells may provide a valuable insight into cell fate determination, into the embryonic ectoderm, mesoderm and endoderm and their downstream derivatives.

Hepatic mesenchymal hamartoma mimicking hemangioma on multiple-phase gadolinium-enhanced MRI.

Mesenchymal Hamartoma [MH] is a cystic benign liver mass occurring in children. Diagnostic confusion with hepatoblastoma may arise when alpha-feto-protein is elevated, and with hemangioendothelioma when imaging shows centripetal filling. We demonstrate one such case with progressive centripetal enhancement of a liver mass with Gadolinium enhanced multiple phase gradient-echo MRI, in a child with raised alpha-feto protein.

Overexpression of Nodal promotes differentiation of mouse embryonic stem cells into mesoderm and endoderm at the expense of neuroectoderm formation.

Understanding how to direct the fate of embryonic stem (ES) cells upon differentiation is critical to their eventual use in therapeutic applications. Clues for controlling ES cell differentiation may be found in the early embryo because mouse ES cells form derivatives of all three embryonic germ layers upon injection into blastocysts. One promising candidate for influencing the differentiation of ES cells into the embryonic germ layers is the transforming growth factor-beta (TGF-beta) growth factor, Nodal. Nodal null mouse mutants lack mesoderm, and injection of Nodal mRNA into nonmammalian embryos induces mesodermal and endodermal tissues. We find that overexpression of Nodal in mouse ES cells leads not only to up-regulation of mesodermal and endodermal cell markers but also to downregulation of neuroectodermal markers. These findings demonstrate the importance of Nodal's influence on the differentiation of pluripotent cells to all three of the primary germ layers. Accordingly, altering expression of factors responsible for cell differentiation in the intact embryo provides an approach for directing ES cell fates in vitro toward therapeutically useful cell types.

Electrochemical and cytocompatibility assessment of NiTiNOL memory shape alloy for orthodontic use.

Orthodontic arcs and wires are mostly realised from alloys and constitute the motor of dental shifting. Ti-base alloys rapidly replaced the formerly used stainless steel wires due to their excellent corrosion resistance, their high mechanical characteristics and their increased biocompatibility. NiTiNOL shape memory alloys add to these advantages their ability of deforming force. NiTiNOL, highly pure Nickel (hp-Ni) and commercially pure titanium (cp-Ti) were tested by electrochemical assays in artificial saliva and in vitro biological tests with L132 cells and HEPM cells. All tests gave concordant results: the electrochemical assays, the proliferation test, the colony forming method, and the inflammatory test clearly show, that nickel is a corrosive and a cytotoxic material. Ti and NiTiNOL are cytocompatible and in particular corrosion resistant. No significant differences are observed for both materials on the electrochemical and the biological level as well. The NiTiNOL shape memory alloy is a master trump for dental practitioners to repair occlusal defects by shifting teeth under optimal biological conditions. In spite of its high Ni-content, it is biocompatible. It considerably reduces the tune of therapeutic treatment, facilitate the occlusal concept and leads to a result of high clinical quality.

Absolute numbers versus proportions in the assessment of differentiation of beta-cells in the embryonic avian pancreas.

Environmental factors may influence the proliferation and differentiation of embryonic pancreatic endocrine cells, creating a need for the quantification of such effects. The explanted dorsal pancreatic bud (DPB) of the 5-day chick embryo is a useful in vitro model. Since all explants cannot be assumed to have the same number of endocrine cells at the start of culture, the proportion of beta-cells with respect to alpha-cells may be a more meaningful measure than absolute numbers. This study aimed to establish baseline values for the proportion of beta-cells in both intact and mesoderm-depleted DPBs before culture. Buds were excised from 12 chick embryos and the surrounding mesoderm was removed from 6 buds following collagenase treatment. All the buds were freeze-dried, fixed in parabenzoquinone vapour, embedded in resin and sectioned at 1 micro m. alpha- and beta-cells were detected by an indirect immunoenzyme method. alpha-cells outnumbered beta-cells in 9 of the 12 buds. The proportion of beta-cells in the intact buds varied from 16% to 64% (mean 39.5%) and in the mesoderm-depleted buds from 17% to 66% (mean 39%). There was no significant difference between the absolute numbers or the proportions of cells in either case. The proportions of beta-cells in the 5-day DPBs were higher than those in buds cultured in previous studies for 7 days under various conditions. This result may reflect the role of apoptosis in response to the culture conditions.