Gene regulation by morpholines and piperidines in the cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) is an in vitro embryotoxicity screen which uses cardiomyocyte formation as the main differentiation route. Studies are ongoing into whether an improved specification of the biological domain can broaden the applicability of the test, e.g. to discriminate between structurally similar chemicals by measuring expression of dedicated gene transcript biomarkers. We explored this with two chemical classes: morpholines (tridemorph; fenpropimorph) and piperidines (fenpropidin; spiroxamine). These compounds cause embryotoxicity in rat such as cleft palate. This malformation can be linked to interference with retinoic acid balance, neural crest (NC) cell migration, or cholesterol biosynthesis. Also neural differentiation within the ESTc was explored in relation to these compounds. Gene transcript expression of related biomarkers were measured at low and high concentrations on differentiation day 4 (DD4) and DD10. All compounds showed stimulating effects on the cholesterol biosynthesis related marker Msmo1 after 24 h exposure and tridemorph showed inhibition of Cyp26a1 which codes for one of the enzymes that metabolises retinoic acid. A longer exposure duration enhanced expression levels for differentiation markers for cardiomyocytes (Nkx2-5; Myh6) and neural cells (Tubb3) on DD10. This readout gave additional mechanistic insight which enabled previously unavailable in vitro discrimination between the compounds, showing the practical utility of specifying the biological domain of the ESTc.

Prenatal exposure to environmental chemical contaminants and asthma and eczema in school-age children.

BACKGROUND: Emerging evidence suggests that prenatal or early-life exposures to environmental contaminants may contribute to an increased risk of asthma and allergies in children. We aimed to the explore associations of prenatal exposures to a large set of environmental chemical contaminants with asthma and eczema in school-age children. METHODS: We studied 1024 mother-child pairs from Greenland and Ukraine from the INUENDO birth cohort. Data were collected by means of an interview-based questionnaire when the children were 5-9 years of age. Questions from the ISAAC study were used to define asthma, eczema, and wheeze. We applied principal components analysis (PCA) to sixteen contaminants in maternal serum sampled during pregnancy, including perfluoroalkyl substances (PFASs), metabolites of diethylhexyl (DEHP) and diisononyl (DiNP) phthalates, PCB-153, and p,p'-DDE. Scores of five principal components (PCs) explaining 70% of the variance were included in multiple logistic regression models. RESULTS: In a meta-analysis that included both populations, the PC2 score, reflecting exposure to DiNP, was negatively associated with current eczema (OR 0.71, 95% CI 0.52-0.96). Other associations were not consistent between the two populations. In Ukrainian children, the PC3 score (DEHP) was positively associated with current wheeze (adjusted OR 1.56, 95% CI 1.03-2.37), whereas the PC5 score, dominated by perfluorooctanoic acid (PFOA), was inversely associated with current wheeze (OR 0.64, 0.41-0.99). In Greenlandic children, a negative association of PC4 (organochlorines) with ever eczema (OR 0.78, 0.61-0.99) was found. CONCLUSIONS: We found limited evidence to support a link between prenatal exposure to environmental chemical contaminants and childhood asthma and eczema.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn).

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.

Endoderm and mesoderm derivatives in embryonic stem cell differentiation and their use in developmental toxicity testing.

Embryonic stem cell differentiation models have increasingly been applied in non-animal test systems for developmental toxicity. After the initial focus on cardiac differentiation, attention has also included an array of neuro-ectodermal differentiation routes. Alternative differentiation routes in the mesodermal and endodermal germ lines have received less attention. This review provides an inventory of achievements in the latter areas of embryonic stem cell differentiation, with a view to possibilities for their use in non-animal test systems in developmental toxicology. This includes murine and human stem cell differentiation models, and also gains information from the field of stem cell use in regenerative medicine. Endodermal stem cell derivatives produced in vitro include hepatocytes, pancreatic cells, lung epithelium, and intestinal epithelium, and mesodermal derivatives include cardiac muscle, osteogenic, vascular and hemopoietic cells. This inventory provides an overview of studies on the different cell types together with biomarkers and culture conditions that stimulate these differentiation routes from embryonic stem cells. These models may be used to expand the spectrum of embryonic stem cell based new approach methodologies in non-animal developmental toxicity testing.

Genome-wide expression screening in the cardiac embryonic stem cell test shows additional differentiation routes that are regulated by morpholines and piperidines.

The cardiac embryonic stem cell test (ESTc) is a well-studied non-animal alternative test method based on cardiac cell differentiation inhibition as a measure for developmental toxicity of tested chemicals. In the ESTc, a heterogenic cell population is generated besides cardiomyocytes. Using the full biological domain of ESTc may improve the sensitivity of the test system, possibly broadening the range of chemicals for which developmental effects can be detected in the test. In order to improve our knowledge of the biological and chemical applicability domains of the ESTc, we applied a hypothesis-generating data-driven approach on control samples as follows. A genome-wide expression screening was performed, using Next Generation Sequencing (NGS), to map the range of developmental pathways in the ESTc and to search for a predictive embryotoxicity biomarker profile, instead of the conventional read-out of beating cardiomyocytes. The detected developmental pathways included circulatory system development, skeletal system development, heart development, muscle and organ tissue development, and nervous system and cell development. Two pesticidal chemical classes, the morpholines and piperidines, were assessed for perturbation of differentiation in the ESTc using NGS. In addition to the anticipated impact on cardiomyocyte differentiation, the other developmental pathways were also regulated, in a concentration-response fashion. Despite the structural differences between the morpholine and piperidine pairs, their gene expression effect patterns were largely comparable. In addition, some chemical-specific gene regulation was also observed, which may help with future mechanistic understanding of specific effects with individual test compounds. These similar and unique regulations of gene expression profiles by the test compounds, adds to our knowledge of the chemical applicability domain, specificity and sensitivity of the ESTc. Knowledge of both the biological and chemical applicability domain contributes to the optimal placement of ESTc in test batteries and in Integrated Approaches to Testing and Assessment (IATA).

Cell differentiation in the cardiac embryonic stem cell test (ESTc) is influenced by the oxygen tension in its underlying embryonic stem cell culture.

Oxygen (O2) levels in the mammalian embryo range between 2.4% and 8%. The cardiac embryonic stem cell test (ESTc) is a model for developmental toxicity predictions, which is usually performed under atmospheric O2 levels of 20%. We investigated the chemical sensitivity of the ESTc carried out under 20% O2, using embryonic stem cells (ESC) cultured under either 20% O2 or 5% O2. ESC viability was more sensitive to valproic acid (VPA) but less sensitive to flusilazole (FLU) when cultured under 5% versus 20% O2. For beating cardiomyocyte differentiation, lower ID50 values were found for FLU and VPA when the ESCs had been cultured under 5% versus 20% O2. At differentiation day 4, gene expression values were primarily driven by the level of O2 during ESC culture instead of exposure to FLU. In addition, using ESCs cultured under 5% O2 tension, VPA enhanced Nes (ectoderm) expression. Bmp4 (mesoderm) was enhanced by VPA when using ESCs cultured under 20% O2. At differentiation day 10, using ESCs cultured under 5% instead of 20% O2, Nkx2.5 and Myh6 (cardiomyocytes) were less affected after exposure to FLU or VPA. These results show that O2 tension in ESC culture influences chemical sensitivity in the ESTc. This enhances awareness of the standard culture conditions, which may impact the application of the ESTc in quantitative hazard assessment of chemicals.

Molecular neural crest cell markers enable discrimination of organophosphates in the murine cardiac embryonic stem cell test.

The cardiac embryonic stem cell test (ESTc) originally used the differentiation of beating cardiomyocytes for embryotoxicity screenings of compounds. However, the ESTc consists of a heterogeneous cell population, including neural crest (NC) cells, which are important contributors to heart development in vivo. Molecular markers for NC cells were investigated to explore if this approach improved discrimination between structurally related chemicals, using the three organophosphates (OP): chlorpyrifos (CPF), malathion (MLT), and triphenyl phosphate (TPP). To decrease the test duration and to improve the objective quantification of the assay read-out, gene transcript biomarkers were measured on study day 4 instead of the traditional cardiomyocyte beating assessment at day 10. Gene expression profiling and immunocytochemistry were performed using markers for pluripotency, proliferation and cardiomyocyte and NC differentiation. Cell proliferation was also assessed by measurements of embryoid body (EB) size and total protein quantification (day 7). Exposure to the OPs resulted in similar patterns of inhibition of beating cardiomyocyte differentiation and of myosin protein expression on day 10. However, these three chemically related compounds induced distinctive effects on NC cell differentiation, indicated by changes in expression levels of the NC precursor (Msx2), NC marker (Ap2α), and epithelial to mesenchymal transition (EMT; Snai2) gene transcripts. This study shows that investigating NC markers can provide added value for ESTc outcome profiling and may enhance the applicability of this assay for the screening of structurally related test chemicals.

Predicting the safety of medicines in pregnancy: A workshop report.

The framework for developmental toxicity testing has remained largely unchanged for over 50 years and although it remains invaluable in assessing potential risks in pregnancy, knowledge gaps exist, and some outcomes do not necessarily correlate with clinical experience. Advances in omics, in silico approaches and alternative assays are providing opportunities to enhance our understanding of embryo-fetal development and the prediction of potential risks associated with the use of medicines in pregnancy. A workshop organised by the Medicines and Healthcare products Regulatory Agency (MHRA), "Predicting the Safety of Medicines in Pregnancy - a New Era?", was attended by delegates representing regulatory authorities, academia, industry, patients, funding bodies and software developers to consider how to improve the quality of and access to nonclinical developmental toxicity data and how to use this data to better predict the safety of medicines in human pregnancy. The workshop delegates concluded that based on comparative data to date alternative methodologies are currently no more predictive than conventional methods and not qualified for use in regulatory submissions. To advance the development and qualification of alternative methodologies, there is a requirement for better coordinated multidisciplinary cross-sector interactions coupled with data sharing. Furthermore, a better understanding of human developmental biology and the incorporation of this knowledge into the development of alternative methodologies is essential to enhance the prediction of adverse outcomes for human development. The output of the workshop was a series of recommendations aimed at supporting multidisciplinary efforts to develop and validate these alternative methodologies.

Neural crest related gene transcript regulation by valproic acid analogues in the cardiac embryonic stem cell test.

In vivo, neural crest (NC) cells contribute critically to heart formation. The embryonic stem cells in the cardiac Embryonic Stem cell Test (ESTc) differentiate into a heterogeneous cell population including non-cardiomyocyte cells. The use of molecular biomarkers from different mechanistic pathways can refine quantitative embryotoxicity assessment. Gene expression levels representing different signalling pathways that could relate to beating cardiomyocyte formation were analysed at different time-points. Immunocytochemistry showed NC cells were present in the ESTc and RT-qPCR showed upregulation of NC related gene expression levels in a time-dependent manner. NC related genes were sensitive to VPA and its analogues 2-ethylhexanoic acid (EHA) and 2-ethylhexanol (EHOL) and indicated VPA as the most potent one. STITCH ('search tool for interactions of chemicals') analysis showed relationships between the examined signalling pathways and suggested additional candidate marker genes. Biomarkers from dedicated mechanistic pathways, e.g. NC differentiation, provide promising tools for monitoring specific effects in ESTc.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Use of the rat postimplantation embryo culture to assess the embryotoxic potency within a chemical category and to identify toxic metabolites.

The implementation of in vitro alternatives in the safety evaluation of chemicals in the animal intensive area of reproductive toxicity testing is highly desirable, but has been limited by issues around predictivity and applicability domains. The validation of alternatives may gain from a category approach, in which, rather than validating a test for the universe of chemicals, its predictive value is assessed for each class of chemicals for which the test represents relevant end point(s). We studied the embryotoxicity in rodent postimplantation whole embryo culture (WEC) of a series of phthalates and their metabolites. Phthalate diesters are widely applied industrial chemicals, their monoester derivatives being considered as their embryotoxic metabolites. The relative in vitro potency of three out of four monophthalates was found to mimick that of corresponding diphthalates tested in vivo. The phthalate that deviated from this ranking, monoethylhexylphthalate (MEHP), showed a relatively high in vitro toxicity as compared to in vivo data. This deviation could be explained through kinetic differences among phthalates, as shown between MEHP and monobutylphthalate. In addition, in vitro testing of specific secondary MEHP metabolites showed that they were all less potent than MEHP. This finding confirmed that MEHP in vitro embryotoxicity is most likely the best correlate to DEHP in vivo embryotoxicity. This study shows that a category approach in the assessment of the validation of in vitro alternatives is feasible, and can be improved when kinetic considerations are taken into account.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Estrogenic effects of mixtures of phyto- and synthetic chemicals on uterine growth of prepubertal rats.

Through the diet humans are exposed to many weak estrogenic phytochemicals (PCs) and synthetic chemicals (SCs), but most experimental studies used individual compounds rather than mixtures. Estrogenic effects were determined in the rat juvenile uterotrophic assay using a predefined phytochemical mixture (PCmix) containing coumestrol, genistein, naringenin, (+,-)catechin, (-,-)epicatechin and quercetin, and a predefined synthetic chemical mixture (SCmix) containing nonyl-, and octylphenol, beta-hexachlorocyclohexane, methoxychlor, bisphenol A and dibutylphthalate. The mixture composition was based on human dietary uptake and actual ratios in serum. 17beta-Estradiol and genistein were also tested individually. It was found that combinations of phytoestrogens and exogenous 17beta-estradiol act additive. In contrast SCmix, inactive by itself even at high dose levels relative to human exposure, caused no synergistic or antagonistic uterotrophic effect with E(2) and/or the PCmix. Based on ED(05) and ED(01) values of the PCmix the margin of exposure in regular human diet for a uterotrophic effect is estimated many orders of magnitude. However, food supplements with phytochemicals might bring individual exposure around ED(05) and ED(01) values of the PCmix. Based on the results of our study the contribution of SCs to total estrogenicity in human diet can probably be neglected.

Mixture effects of estrogenic compounds on proliferation and pS2 expression of MCF-7 human breast cancer cells.

Humans are exposed to a variety of food-borne phytochemicals (PC) as well as synthetic chemicals (SC). Some of these compounds have been reported to have estrogenic or anti-estrogenic properties and are therefore suspected endocrine disruptors. Until now it remains unclear if non-additive effects occur in combinations with endogenous estrogens, such as 17beta-estradiol (E(2)). To investigate such interactions, several PC and SC were tested individually, in mixtures and as combinations of mixtures with E(2) for effects on ERalpha receptor mediated cell proliferation and estrogen regulated pS2 expression level in MCF-7(bus) cells. PCs (coumestrol, genistein, naringenin, catechin, epicatechin, quercetin) or SCs (4-nonylphenol, octylphenol, beta-hexachlorocyclohexane, bisphenol A, methoxychlor, dibutyl phthalate) were mixed (PCmix and SCmix) either in concentrations reflecting human serum concentrations or at equipotent concentrations for estrogenicity. EC(50) values were applied in two approaches of the concentration-addition model (the method of isoboles and the cumulative estrogen equivalency method) to assess mixture effects. In both models PCmix and SCmix or combinations of the mixtures with E(2) showed no departure from additivity. In conclusion, the tested PCs and SCs appeared to act as (full) agonists for the estrogen receptor and interacted in mixtures and with estradiol in an additive way. In addition, it is concluded that the possible contribution of food-borne PCs to the estrogenic effect of xenobiotics is likely to be more significant than that caused by food-borne SCs.

Differentiation of aggregated murine P19 embryonal carcinoma cells is induced by a novel visceral endoderm-specific FGF-like factor and inhibited by activin A.

Aggregation of P19 embryonal carcinoma cells in the presence of a factor, secreted by the visceral endoderm-like cell line END-2, induces differentiation to cell types including visceral endoderm, mesoderm-derived muscle tissue and neurons. This factor is different from activin A, type beta transforming growth factors (TGF beta) and fibroblast growth factors (FGF) although its acid- and heat-lability and its stability in the presence of reducing agents resemble the properties of the FGFs. The END-2 factor is completely inhibited in its action by activin A. This inhibitory effect of activin A is not specific for the END-2 factor as retinoic acid (RA)-induced differentiation of aggregated P19 EC cells into neurons (10(-8) M RA) or mesoderm-derived muscle tissue (10(-9) M RA) is also completely inhibited by activin A. The results of this study suggest that the END-2 activity and activin A are intimately involved in the induction and regulation, respectively, of early differentiation processes in vertebrate embryogenesis.

Radiation damage to femoral hemopoietic stroma measured by implant regeneration and quantitation of fibroblastic progenitors.

Radiation damage to femoral hemopoietic stroma in mice was measured 6 weeks after irradiation and reconstitution with syngeneic bone marrow by quantitation of fibroblastic progenitor cells (CFUF), and by assaying hemopoietic progenitors and CFUF numbers in regenerated subcutaneous femur implants. A dose-dependent effect of radiation on both preimplantation CFUF numbers and implant regeneration was observed. Changes in bone marrow cell (BMC) graft size did not alter these stromal parameters. Therefore, transplanted CFUF, which were present in the BMC graft, did not alter either the CFUF content of femurs or their regenerative capacity. The strong correlation between CFUF numbers and implant regenerative capacity after irradiation, leads us to suggest a function for CFUF in femoral implant stromal regeneration.

Regulation of in vitro myelopoiesis by a hemopoietic stromal fibroblastic cell line.

An adherent cell line (AP63) derived from murine spleen was characterized as fibroblastic, and several of its properties distinguished it from other adherent cells (i.e., macrophages and endothelial cells). The ability of the AP63 cells to regulate in vitro myelopoiesis was investigated. Medium conditioned by the cell line (CM) induced granulocyte-macrophage (GM) colonies, thus demonstrating the production of colony-stimulating activity by AP63 cells. A relatively large proportion of these colonies had a "tight" morphology and contained many early myeloid cells and cells capable of secondary cluster and colony formation. CM also contained a prostaglandinlike inhibitor of colony formation. Furthermore, AP63 cells inhibited GM colony formation by bone marrow cells in their immediate vicinity, whereas colony formation was stimulated at greater distances. These observations may reflect in vivo regulatory properties of hemopoietic stromal fibroblasts with respect to proliferation and differentiation of GM progenitor cells.

Hemopoiesis on purified bone-marrow-derived reticular fibroblast in vitro.

Murine bone-marrow-derived reticular fibroblast cultures were tested for the ability to support hemopoiesis and release hemopoietic growth factors in vitro. The reticular fibroblast cultures employed in these studies were 95%-100% pure on the basis of Mac-1, F4/80, MIV-51, T200, antifactor VIII and ER-TR7 monoclonal antibody staining. Further support for the fibroblastic nature of these cells was obtained from collagen and laminin analyses. Addition of stromal cell-depleted bone marrow cell suspensions to flasks of confluent reticular fibroblasts resulted in production and release of granulocytes, monocyte-macrophages, and granulocyte-monocyte progenitors from the adherent layer for 4-8 weeks. Pluripotent spleen colony-forming units were detected during the first four weeks. Assay of reticular fibroblast conditioned medium for hemopoietic growth factors demonstrated production of granulocyte-macrophage colony-stimulating activity and stem-cell-activating factor. We did not detect any erythroid burst-promoting activity. These results suggest that reticular fibroblasts may play a role in the maintenance of pluripotent stem cells and in the proliferation and differentiation of cells committed to the granulocyte-monocyte lineage.

Characterization of fibroblastic stromal cells from murine bone marrow.

Several properties of fibroblastic colony-forming units (CFU-F) from murine bone marrow and their in vitro progeny were evaluated. CFU-F had a high buoyant density relative to total bone marrow cells; they were noncycling in situ and adhered to nylon wool. The fibroblastic cells stained positively for fibronectin, lipid, alkaline phosphatase, and nonspecific esterase, while phagocytosis assays were negative, and ultrastructural analysis failed to reveal desmosomes. These properties contrasted bone-marrow-derived fibroblastic cells to both endothelial cells and macrophages. Fibroblastic cells derived from several hemopoietic organs and skin were screened for antigenic determinants present on hemopoietic cells using monoclonal antibodies. Mac-1 and B220 were absent from all fibroblastic cells studied, whereas the Forsmann and Pgp-1 antigens were always present. Thy-1 was not detected on bone-marrow-derived fibroblasts, but was present on fibroblastic cells derived from other sources. T200 was found on all hemopoietic organ-derived fibroblastic cells, but not on those derived from blood and skin. Thus, analysis of antigenic determinants allowed distinction between fibroblastic cells from different organs.

Recovery of hemopoietic stromal progenitor cells after lethal total-body irradiation and bone marrow transplantation in mice.

The recovery of fibroblastic colony-forming units (CFU-F) in murine bone marrow hemopoietic stroma was studied during eighteen months after 9 Gy lethal total-body irradiation and reconstitution with syngeneic bone marrow cells. After an initial depletion, CFU-F numbers increased from 10% of normal values at three months to 40% at 18 months after treatment, irrespective of graft size and presence of CFU-F in the graft. Fourteen months after treatment 35% of all CFU-F present in the recipients' bone marrow was donor-derived independent of graft size. When mice were treated with high-dose lipopolysaccharide-W three months after irradiation and bone marrow transplantation, CFU-F numbers decreased to hardly detectable levels within one day, and then recovered to normal numbers four weeks later--whereas radiation control mice still had low CFU-F numbers. These data suggest that after lethal total-body irradiation the stroma still contained viable fibroblastic cells that had lost their in vitro colony-forming capacity as a result of radiation damage. In consequence there was no need for replacement of these fibroblastic cells by donor-derived or host-derived CFU-F. Only depletion of CFU-F from the bone marrow, as was induced with lipopolysaccharide, stimulated repopulation of the stroma with colony-forming fibroblastic cells.