Rodent Bone Marrow Research Articles

Genotoxicity assessment of S-equol in bacterial mutation, chromosomal aberration, and rodent bone marrow micronucleus tests

Intervention studies in man have shown that dietary soy isoflavones may provide certain health benefits. One of the possible reasons for this benefit is that the daidzein contained in soy is converted to S-equol. As part of a drug development program for S-equol, three genotoxicity studies were conducted. The studies comprised bacterial mutation, chromosomal aberration, and in vivo bone marrow micronucleus tests conducted according to Good Laboratory Practices (GLP). No evidence of genotoxic activity was observed in the in vitro tests at concentrations up to those associated with cell toxicity. In addition, no evidence of cytotoxicity or genotoxicity was seen in the rat bone marrow micronucleus test in animals dosed at levels up to the standard limit of 2000 mg/kg. It is concluded that S-equol is not active in the standard battery of genotoxicity assays recommended by the International Conference on Harmonisation (ICH) for registration of new pharmaceuticals. The current results support the further development of S-equol.

Historical perspective on the development of the genetic toxicity test battery in the United States

The currently used genetic toxicity testing battery (the Ames Salmonella test, the in vitro mammalian cell mouse lymphoma assay and/or the in vitro mammalian cell chromosome assay, and the rodent bone marrow chromosome aberration or micronucleus assay) had its origins in the early-to-mid 1970s. By the late 1970s, a large number of genetic tests had been proposed or recommended by the US-EPA for identifying germ cell mutagens and carcinogens. After a number of modifications that were primarily directed toward minimizing the number of tests used, the test battery reached its current state in the mid-1980s. This test battery, with some minor modifications in the timing or ordering of the tests is mandated by regulatory authorities worldwide. Although it would be intellectually satisfying to presume that this compendium of tests was developed and selected for regulatory screening based solely on scientific grounds, it was actually based on a combination of scientific data, theoretical considerations, chance, and advocacy, and not always in equal proportions. The evolution of the current genetic toxicity test battery, and some of the activities and considerations that directed this evolution are described.

Differentiation of Rodent Bone Marrow Mesenchymal Stem Cells into Intervertebral Disc-like Cells Following Coculture with Rat Disc Tissue

This study aimed to evaluate whether rat mesenchymal stem cells (rMSCs) could be differentiated in vitro into disc-like cells by coculturing with intervertebral disc tissue. rMSCs were cultured with rodent intervertebral disc for up to 30 days in transwell plates. The differentiation of rMSCs was evaluated by immunostaining, Western blot, real-time RT-PCR, Northern blot, and electron microscopy. The potentials of multilineage differentiation and proteoglycan and collagen synthesis were also investigated. rMSCs underwent morphological changes to form three-dimensional micromasses and expressed collagen-2, aggrecan, and sox-9 at RNA and protein levels after 14 days of coculture. These changes were not detected in the samples of rMSCs cultured alone. Cocultured rMSCs also showed other characteristic features of disc-like cells, including the extracellular matrix formation, and proteoglycan and collagen synthesis. In addition, cellular contact between cocultured rMSCs and disc tissue was observed by electron microscopy. Committed rMSCs still retained their differentiation ability into mesoderm lineages of adipocytes or osteocytes when the local environment was altered. This study supports that MSCs are a promising source for cell therapy and tissue engineering in disc regeneration, and highlights that rMSCs can be induced into nucleus pulposus-like cells in vitro under the direct influence of intact disc tissue.

Tinospora cordifolia, a safety evaluation

Tinospora cordifolia is one of the indispensable medicinal plants used in veterinary folk medicine/Ayurvedic system of medicine for the treatment of diverse diseases and recommended for improving the immune system by means of body resistance. In the current study, we evaluated the genotoxic risk of the aqueous extract of T. cordifolia (TC) in a battery of four different genotoxicity tests viz., Ames, in vitro chromosome aberration (CA), rodent bone marrow micronucleus (MN), and Comet assay. Experimental results confirmed that in Ames test up to 5000 μg/plate of TC did not exhibit any mutagenic effect in Salmonella typhimurium mutant strains (TA97a, TA98, TA100, TA102, and TA1535). In CA assay, TC was not clastogenic to human peripheral blood lymphocytes up to a concentration of 3000 μg/ml. In MN and Comet assays, TC was pre-treated for 7 days at three dose levels (150, 200 and 250 mg/kg body weight) orally to male Balb/c mice. The results showed that TC treatment did not display clastogenicity and DNA damaging effect in bone marrow erythrocytes and peripheral blood lymphocytes respectively.

The rat bone marrow micronucleus test--study design and statistical power

Although the rodent bone marrow micronucleus test has been in routine use for over 20 years, little work has been published to support its experimental design and all this has used the mouse rather than the rat. When it was decided to change the strain of rat routinely used in this laboratory to the Han Wistar, a preliminary study was performed to investigate the possible factors influencing experimental variability and to use statistical tools to examine possible study designs. Subsequently, a historical database comprising of vehicle controls accumulated from 65 studies was used to establish test acceptance criteria and a strategy for analysing equivocal results. The following conclusions were made: (i) no statistically significant differences were observed in experimental variability within or between control animals; although not statistically significant, the majority of experimental variability seen was found to be between separate counts on the same slide, with minimal differences found between duplicate slides from the same rat or between individual rats; (ii) power analyses showed that, if an equivocal result is obtained after scoring 2000 immature erythrocytes (IE), it is appropriate to re-code the slides and score an additional 4000 IE, i.e. analysing a total of 6000 IE; no meaningful increase in statistical power is gained by scoring >6000 IE; this is consistent with the variability observed between separate counts on the same slide; (iii) there was no significant difference between the control micronucleated immature erythrocyte (MIE) values at 24 and 48 h after dosing or between males and females; therefore, if an unusually low control value at either time point results in apparent small increases in MIE in a treated group, it is valid to pool control values from both time points for clarification and (iv) similar statistical power can be achieved by scoring 2000 IE from seven rats or 4000 IE from five rats, respectively. However, this is based only on control animals and does not consider possible differences in responses between animals to treatment with a potential genotoxin. In order to minimize the possible influence of responders and non-responders, the preferred study design in this laboratory is to score 2000 IE from groups of seven rats. Study data obtained over time confirmed observations made in the control study. Also from an ethical viewpoint, clarifying equivocal responses by combining control data from the 24- and 48-h time points and/or increasing the number of IE scored per animal has minimized the numbers of repeat studies necessary to determine the genotoxic status of a novel compound. However, before any laboratory can use these procedures, experimental data must be generated to demonstrate their validity.

Avaliação do efeito mutagênico do extrato hidroalcoólico bruto, por meio de bioensaios in vivo e prospecção fitoquímica de Cecropia glaziovii Sneth (embaúba), Cecropiaceae

O gênero Cecropia é conhecido popularmente por "embaúba" e apresenta várias espécies medicinais, sendo que estudos com a espécie C. glaziovii Sneth indicam que o extrato aquoso apresenta efeitos broncodilatador, anti-hipertensivo e antidepressivo, provavelmente, atribuídos às catequinas, procianidinas e flavonóides. O presente estudo objetivou avaliar os potenciais efeitos tóxico, citotóxicos, clastogênicos e aneugênicos do extrato bruto hidroalcoólico de folhas de C. glaziovii, por meio da dose letal média (DL50), da análise de micronúcleo em células de medula óssea de roedores e do teste com Allium cepa e realizar a prospecção fitoquímica. Os resultados demonstraram que as concentrações testadas não causaram toxicidade aguda, nem apresentaram atividade clastogênica e aneugênica, sugerindo que o extrato de C. glaziovii não interferem na divisão celular. A prospecção fitoquímica indicou a presença de taninos, flavonóides, fenóis, antraquinonas, cumarinas, catequinas, proteínas, açúcares redutores, depsídeos/depsidonas e triterpenos. Mesmo assim, esses resultados não prescrevem o consumo terapêutico da espécie, sem continuidade da sua avaliação e do cumprimento de todas as etapas pré-clínicas e clínicas.

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward β-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic β cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.

The micronucleus test and erythropoiesis: Effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2- O-dibutyladenosine-3′,5′-cyclic monophosphate into adenosine 3′,5′-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose–response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously . The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.

Safety evaluation to support first-in-man investigations II: Toxicology studies

Toxicology (single dose, range-finding, repeat dose and genotoxicity) data available in 34 Investigator’s Brochures used to support First-In-Man clinical trials over a 10 year period have been evaluated to give an insight into the types of study designs used and how these have changed over the period analysed (1997–2006). Study packages had single dose toxicity studies in the rodent (although there has been a recent trend to reduce the number of these studies), range-finding toxicity studies in the rodent and non-rodent (with only small numbers of the latter used) and key 2–4 week repeat dose toxicity studies in rodent (usually rat) and non-rodent (both dog and monkey). The majority of the latter studies established No Observed Adverse Effect Levels, showed the rodent to be generally less sensitive to target organ toxicity than the non-rodent and showed the liver and then the kidney to be the most common target organs. Genotoxicity assessment included 2 in vitro assays (a reverse mutation bacteria and either a chromosome aberration or mouse lymphoma assay) and commonly, an in vivo rodent bone marrow micronucleus test. Considerations for general toxicology and genotoxicity study designs are discussed along with the use of appropriate information to help set the clinical starting dose.

Marrow Stromal Cells as Universal Donor Cells for Myocardial Regenerative Therapy: Their Unique Immune Tolerance

Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. To confirm these findings in human cells, we tested whether human MSCs are also immune tolerant, such that they can be used as universal donor cells for myocardial regenerative therapy. Immunocompetent female rats underwent coronary ligations (n = 90). In group I, lacZ-labeled male human MSCs were implanted into the peri-infarcted area. In groups II, III, and IV, isogeneic rat MSCs, culture medium, or human fibroblasts were injected, respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, fluorescent in situ hybridization, and polymerase chain reaction to examine MSCs survival and differentiation. Human MSCs survived within the rat myocardium for more than 8 weeks without immunosuppression. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated left ventricular remodeling. No cellular infiltration characteristic of immune rejection was noted in contrast to group IV. Human MSCs survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as universal donor cells for xenogeneic or allogeneic cell therapy, as they can be prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional.

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Amended Final Report on the Safety Assessment of Glyceryl Dilaurate, Glyceryl Diarachidate, Glyceryl Dibehenate, Glyceryl Dierucate, Glyceryl Dihydroxystearate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Diricinoleate, Glyceryl Dipalmitate, Glyceryl Dipalmitoleate, Glyceryl Distearate, Glyceryl Palmitate Lactate, Glyceryl Stearate Citrate, Glyceryl Stearate Lactate, and Glyceryl Stearate Succinate1

Failure of antimony trioxide to induce micronuclei or chromosomal aberrations in rat bone-marrow after sub-chronic oral dosing

Antimony trioxide (Sb 2O 3, CAS 1309-64-4) is widely used as a flame retardant synergist in a number of household products, as a fining agent in glass manufacture, and as a catalyst in the manufacture of various types of polyester plastics. It does not induce point mutations in bacteria or mammalian cells, but is able to induce chromosomal aberrations (CA) in cultured cells in vitro. Although no CA or micronuclei (MN) have been induced after acute oral dosing of mice, repeated oral dosing for 14 or 21 days resulted in increased CA in one report, but did not result in increased MN in another. In order to further investigate its in vivo genotoxicity, Sb 2O 3 was dosed orally to groups of rats for 21 days at 250, 500 and 1000 mg/kg day. There were no clinical signs of toxicity in the Sb 2O 3-exposed animals except for some reductions in body-weight gain in the top dose group. Toxicokinetic measurements in a separate study confirmed bone-marrow exposure, and at higher levels than would have been achieved by single oral dosing. Large numbers of cells were scored for CA (600 metaphases/sex group) and MN (12,000 PCE/sex group) but frequencies of CA or MN in Sb 2O 3-treated rats were very similar to controls, and not biologically or statistically different, at all doses. These results provide further indication that Sb 2O 3 is not genotoxic to the bone marrow of rodents after 21 days of oral administration at high doses close to the maximum tolerated dose.

Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests : I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards

In vivo genotoxicity tests play a pivotal role in genotoxicity testing batteries. They are used both to determine if potential genotoxicity observed in vitro is realised in vivo and to detect any genotoxic carcinogens that are poorly detected in vitro. It is recognised that individual in vivo genotoxicity tests have limited sensitivity but good specificity. Thus, a positive result from the established in vivo assays is taken as strong evidence for genotoxic carcinogenicity of the compound tested. However, there is a growing body of evidence that compound-related disturbances in the physiology of the rodents used in these assays can result in increases in micronucleated cells in the bone marrow that are not related to the intrinsic genotoxicity of the compound under test. For rodent bone marrow or peripheral blood micronucleus tests, these disturbances include changes in core body temperature (hypothermia and hyperthermia) and increases in erythropoiesis following prior toxicity to erythroblasts or by direct stimulation of cell division in these cells. This paper reviews relevant data from the literature and also previously unpublished data obtained from a questionnaire devised by the IWGT working group. Regulatory implications of these findings are discussed and flow diagrams have been provided to aid in interpretation and decision-making when such changes in physiology are suspected.

Human and rodent bone marrow mesenchymal stem cells that express primitive stem cell markers can be directly enriched by using the CD49a molecule

Bone marrow (BM) from human and rodent species contains a population of multipotential cells referred to as mesenchymal stem cells (MSCs). Currently, MSCs are isolated indirectly by using a culture step and then the generation of fibroblast colony-forming units (CFU-fs). Unprocessed or native BM MSCs have not yet been fully characterised. We have previously developed a direct enrichment method for the isolation of MSCs from human BM by using the CD49a protein (alpha1-integrin subunit). As the CD49a gene is highly conserved in mammals, we have evaluated whether this direct enrichment can be employed for BM cells from rodent strains (rat and mouse). We have also studied the native phenotype by using both immunodetection and immunomagnetic methods and have compared MSCs from mouse, rat and human BM. As is the case for human BM, we have demonstrated that all rodent multipotential CFU-fs are contained within the CD49a-positive cell population. However, in the mouse, the number of CFU-fs is strain-dependent. Interestingly, all rat and mouse Sca-1-positive cells are concentrated within the CD49a-positive fraction and also contain all CFU-fs. In human, the colonies have been detected in the CD49a/CD133 double-positive population. Thus, the CD49a protein is a conserved marker that permits the direct enrichment of BM MSCs from various mammalian species; these cells have been phenotyped as true BM stem cells.

Transient Differentiation of Adult Human Bone Marrow Cells into Neuron-like Cells in Culture: Development of Morphological and Biochemical Traits Is Mediated by Different Molecular Mechanisms

Studies on rodent bone marrow stromal cells (MSCs) have revealed a capacity, for at least a portion of cells, to express neuron-like traits after differentiation in culture. Little, however, is known about the ability of human MSCs in this regard. We show here that incubation with certain differentiation cocktails, particularly those that include reagents that increase cellular cAMP levels, produces a rapid (1-4 h) and transient (24-48 h) transformation of nearly all hMSCs into neuron-like cells displaying a complex network of processes using phase or scanning electron microscopic optics. In addition, differentiated human (h) MSCs express increased quantities of neuron-[beta-tubulin III, neurofilament (NF), neuronal-specific enolase (NSE)] and glial- [glial fibrillary acidic protein (GFAP)] specific proteins and mRNAs, which are also expressed in low levels in undifferentiated MSCs. In contrast, the mesenchymal marker, fibronectin, which is highly expressed in the undifferentiated state, is reduced following differentiation. These biochemical changes, but not the acquisition of a neuron-like appearance, are partially inhibited by incubation of hMSCs with protein (cycloheximide) and mRNA (actinomycin D) synthesis inhibitors with differentiating reagents. Only incubation with 100 ng/ml colchicine, which disrupts the microtubular cytoskeleton, prevents the conversion of hMSCs into neuron- like cells. These results demonstrate that hMSCs acquire the morphological appearance and the biochemical makeup typical of neurons by independently regulated mechanisms.

Swine Bone Marrow Derived Multipotent Adult Progenitor Cells.

We have previously reported that multipotent adult progenitor cells (MAPCs) can be isolated from the bone marrow of rodents and humans. Here we describe isolation of similar cells, fetal swine MAPCs (fsMAPCs) from fetal swine bone marrow (BM). BM was obtained from 5 fetal swine donors. BM mononuclear cells (BMMNCs) were plated into fibronectin-coated T150 flasks at 2x104 cells/cm2 in 58% LG-DMEM and 40% MCDB-201, supplemented with ITS, LA-BSA, 10−8 M dexamethasone, 10−4 M ascorbic acid 2-phosphate, 2% fetal bovine serum (FBS), 10ng/ml EGF and 10ng/ml PDGF-BB. Small clusters of cells appeared by day 5, were detached with 0.25% trypsin-EDTA on day 7 and sub-cloned at 1 cell/well in 96 well plates. Once populations of small cells appeared (40% of wells), they were culture expanded by plating at 500 cells/cm2. Cells were replated at 500cells/cm2 once they divided 2–3 times. Population doubling (PD) time was 24h for the initial 30–40 PDs, and 48 h at higher PDs. 3/5 fetal swine populations have been maintained for more than 100 PDs without cytogenetic abnormalities. For two of the populations, telomere length was evaluated on day-0, 40PDs and 80PDS, and no telomere shortening was seen. The phenotype of cultured cells before 30 PDs is CD45 and MHC class I negative, CD44 and MHC class II positive, while the phenotype after 60 PDs was CD44, CD45, MHC class I and II negative. FsMAPCs, cultured on Matrigel with fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), differentiated into epitheloid cells that expressed cytokeratin 18 (CK18), hepatocyte nuclear factor-1 (HNF-1), albumin protein on day 14–28. Cells differentiated for 21 days secreted 10pg/cell/hour urea (Urea Production Test), as is seen for primary hepatocytes. More functional tests for hepatocytes are ongoing, including LDL uptake, glycogen storage, cytochrome-p450 activity. When treated with vascular endothelial growth factor (VEGF), fsMAPCs differentiated into endothelium-like cells that expressed CD31, CD34, VEGF-Receptor, vascular endothelial cadherin (VE-Cadherin) and von willebrand factor (VWF) protein and mRNA at day 14–28, formed vascular tube on extracellular matrix and took up LDL. Neuroectoderm differentiation studies are ongoing. Finally, we have preliminary evidence that fsMAPCs may differentiate into cells with cardiomyocyte markers. FsMAPCs, plated at 1,500 cells/cm2, were cultured with dickkopf-1 (Dkk-1) and 0.75% dimethyl sulfoxide (DMSO) for 1 day, followed by bone morphogenetic protein-2 (BMP-2), FGF-8, FGF-4, Dkk-1 for 21 days. Cells acquired muscle morphology after d6. By day 21, cells expressed cardiac Troponin T, cardiac myosin heavy chain (cMHC), phospholamban protein, and Nkx2.5, Cardiac Troponin T, cMHC, GATA-4, atrial natriuretic peptide (ANP) mRNA. Although suggestive that the cells may differentiate into cardiomyocytes, they did not beat regularly. Electrophysiology studies are ongoing to determine whether the cells have functional characteristics of cardiomyocytes.

Induction of bone marrow stromal cells to neurons: differentiation, transdifferentiation, or artifact?

Differentiation of stem cells toward a neuronal lineage normally involves a gradually progressive restriction in developmental potential and is regulated by a diverse set of specific and temporally precise genetic events. However, recent studies have indicated that both rodent and human bone marrow stromal cells (MSCs) can be rapidly (within minutes to hours) induced to differentiate into neurons in vitro by relatively simple chemical means (using beta-mercaptoethanol [BME] or dimethylsulfoxide [DMSO] and butylated hydroxyanisol [BHA]; Woodbury et al. [ 2000] J. Neurosci. Res. 61:364-370). The ability to transdifferentiate an easily accessible cell source into neurons could have substantial potential for promoting neural repair. We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC-12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial "neuron-like" pyramidal cell morphology with fine-cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time-lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite-like processes. To determine whether the chemically induced transdifferentiation resulted from simple cellular toxicity, MSCs were exposed to various stressors, including detergents, high-molarity sodium chloride, and extremes of pH. In all cases, cellular shrinkage and adoption of pseudoneuronal morphology were observed. Concomitantly with cellular shrinkage, apparent increases in immunolabeling for the neuronal markers NSE and NeuN were detected in the cell soma that could not be confirmed by RT-PCR. Furthermore, blockade of protein synthesis with cycloheximide did not prevent cells from adopting "neuron-like" morphology after chemical induction. Thus, morphological changes and increases in immunolabeling for certain cellular markers upon "chemical induction" of MSCs are likely the result of cellular toxicity, cell shrinkage, and changes in the cytoskeleton and do not represent regulated steps in a complicated cellular differentiation process.

A crucial role for thiol antioxidants in estrogen-deficiency bone loss.

The mechanisms through which estrogen prevents bone loss are uncertain. Elsewhere, estrogen exerts beneficial actions by suppression of reactive oxygen species (ROS). ROS stimulate osteoclasts, the cells that resorb bone. Thus, estrogen might prevent bone loss by enhancing oxidant defenses in bone. We found that glutathione and thioredoxin, the major thiol antioxidants, and glutathione and thioredoxin reductases, the enzymes responsible for maintaining them in a reduced state, fell substantially in rodent bone marrow after ovariectomy and were rapidly normalized by exogenous 17-beta estradiol. Moreover, administration of N-acetyl cysteine (NAC) or ascorbate, antioxidants that increase tissue glutathione levels, abolished ovariectomy-induced bone loss, while l-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, caused substantial bone loss. The 17-beta estradiol increased glutathione and glutathione and thioredoxin reductases in osteoclast-like cells in vitro. Furthermore, in vitro NAC prevented osteoclast formation and NF-kappaB activation. BSO and hydrogen peroxide did the opposite. Expression of TNF-alpha, a target for NF-kappaB and a cytokine strongly implicated in estrogen-deficiency bone loss, was suppressed in osteoclasts by 17-beta estradiol and NAC. These observations strongly suggest that estrogen deficiency causes bone loss by lowering thiol antioxidants in osteoclasts. This directly sensitizes osteoclasts to osteoclastogenic signals and entrains ROS-enhanced expression of cytokines that promote osteoclastic bone resorption.

Prostaglandin E2-mediated anabolic effect of a novel inhibitor of phosphodiesterase 4, XT-611, in the in vitro bone marrow culture.

The mechanism of osteoblast formation by a novel PDE4 inhibitor, XT-611, was studied in the in vitro bone marrow culture system. The compound potentiated the osteoblast differentiation through accumulation of cyclic AMP after autocrine stimulation of EP4 receptor by PGE2 in pro-osteoblastic cells. We previously reported that inhibitors of phosphodiesterase (PDE)4 isoenzyme increase osteoblast formation in an in vitro bone marrow culture system and inhibit bone loss in animal osteoporosis models. Here we investigated the mechanism of the effect of a novel PDE4 inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611), on osteoblast formation in the in vitro bone marrow culture system. Rodent bone marrow cells were cultured in the presence of 0.2 mM ascorbic acid phosphate ester, 1 mM beta-glycerophosphate, and 10 nM dexamethasone for 10 days. Drug treatments were done for 24 h on day 3 of culture. PDE4 inhibitors, including XT-611, but not PDE3 and PDE5 inhibitors, increased mineralized nodule formation in rat and mouse bone marrow cell cultures. During culture of the bone marrow cells, prostaglandin E2 (PGE2) production increased with a peak on day 4, but the increase was completely inhibited by indomethacin, an unselective cyclo-oxygenase (COX) inhibitor. Spontaneous and XT-611-induced mineralized-nodule formation was also inhibited by indomethacin and COX-2 inhibitors, in a similar potential. Alkaline phosphatase-positive nodule formation in the absence or presence of XT-611 was inhibited by an antagonist of EP4 receptor, AH23848B, and synergistically potentiated by 11-deoxy-PGE1, but it was not influenced by other EP antagonists and agonists examined. The expression of PDE4 and EP4 mRNAs was observed in bone marrow cells. The effect of XT-611 was also confirmed to involve an increase of cyclic AMP and the cyclic AMP-dependent protein kinase pathway. These results suggest that PGE2 stimulates differentiation of osteoblast progenitor cells through the EP4 receptor in an autocrine manner, and the PDE4 inhibitor potentiates the differentiation by inhibiting hydrolysis of cyclic AMP in the cells.

Neural differentiation and incorporation of bone marrow-derived multipotent adult progenitor cells after single cell transplantation into blastocyst stage mouse embryos.

Previously we reported the characterization of multipotent adult progenitor cells (MAPCs) isolated from the bone marrow of rodents. In that study, single murine MAPCs derived from ROSA-26, beta-galactosidase (beta-Gal)-positive transgenic mice were injected into E3.5 C57/B16 mouse blastocysts. The resultant chimeric blastocysts were then implanted into pseudopregnant females and were allowed to develop naturally through birth and into adulthood. Chimeric mice were sacrificed 6 to 20 weeks after birth, and were processed for histological analysis. Beta-galactosidase activity was identified in all organs and tissues examined, and tissue-specific differentiation and engraftment was confirmed by colabeling with antibodies that recognize beta-Gal and tissue-specific markers. In the present study we have examined neural engraftment derived from the clonal expansion of a single MAPC during rodent development, and characterized the neural phenotype of MAPCs in the resultant chimeric animals. Donor cell-derived beta-Gal activity was evident throughout the brain. Double and triple immunofluorescent labeling studies revealed MAPC-derived neurons (NeuN/beta-Gal) and astrocytes (GFAP/beta-Gal) in the cortex, striatum, medial septal nucleus, hippocampus, cerebellum, substantia nigra, and thalamus. More specifically, donor-derived neurons contributed to each of the cellular layers of the cortex; the pyramidal and granule cell layers, as well as the hilus, of the hippocampus; Purkinje and granule cell layers in the cerebellum; and GABAergic cells in the caudate and putamen. This study characterizes the potential for MAPCs to differentiate into specific neuronal and glial phenotypes, and to integrate normally during development, after implantation into blastocysts, and provides additional evidence that MAPCs exhibit properties similar to embryonic stem cells.