Characterization of mesenchymal stem cells isolated from mouse fetal bone marrow

Mesenchymal stem cells (MSCs) are defined as cells that can differentiate into multiple mesenchymal lineage cells. MSCs have some features (surface molecules and cytokine production, etc.) common to so-called traditional bone marrow (BM) stromal cells, which have the capacity to support hemopoiesis. In the present study, we isolated murine MSCs (mMSCs) from the fetal BM using an anti-PA6 monoclonal antibody (mAb) that is specific for bone marrow stromal cells. The mMSCs, called FMS/PA6-P cells, are adherent, fibroblastic, and extensively expanded and have the ability to differentiate not only into osteoblasts and adipocytes but also into vascular endothelial cells. The FMS/PA6-P cells produce a broad spectrum of cytokines and growth factors closely related to hemopoiesis and show good hemopoiesis-supporting capacity both in vivo and in vitro, suggesting that they are a component of the hemopoietic stem cell niche in vivo. Interestingly, although the FMS/PA6-P cells express a high level of the PA6 molecule, which is reactive with anti-PA6 mAb, they gradually lose their ability to express this molecule during the course of differentiation into osteoblasts and adipocytes, indicating that the PA6 molecule might serve as a novel marker of mMSCs.     

骨桥蛋白对小鼠胚泡黏附和扩展的影响及其机制

目的 探讨骨桥蛋白(OPN)对小鼠胚泡黏附、扩展的影响及机制. 方法 采用纤维蛋白铺板微滴培养法培养胚胎,观察并统计重组小鼠OPN(rmOPN)、OPN抗体及精氨酸-甘氨酸-天冬氨酸多肽(RGD)对胚泡的脱带、黏附和扩展的影响;采用24孔板培养胚胎,酶联免疫吸附测定(ELISA)法检测胚泡培养液基质金属蛋白酶2和9(MMP-2、-9)的浓度. 结果不同浓度的OPN组间胚泡的脱带率、黏附率与对照组相比无显著性差异(P>0.05);但1.0 mg/L和10.0 mg/L OPN组可促进小鼠胚泡提前扩展,72h囊胚扩展率与对照组相比差异有显著性,10.0 mg/L组72h的扩展率显著高于OPN 0.1 mg/L组(P<0.05).OPN抗体和RGD均显著抑制胚泡的脱带、黏附和扩展,与对照组相比,差异有显著性,且随着浓度的增加,抑制作用越明显.OPN促进胚泡分泌MMP-2及MMP-9,且存在时间和剂量依赖性. 结论 OPN在体外可通过促进小鼠胚泡的扩展和分泌MMP-2、-9来调节小鼠早期胚泡着床.     

Effects of gangliosides on the development of selective afferent connections within fetal mouse spinal cord explants

Monosynaptic evoked responses were used to localize dorsal root ganglion (DRG) afferent terminals within organotypic fetal mouse spinal cord explants which had been chronically exposed to purified bovine gangliosides during early development. Ganglioside grown cultures showed a highly significant increase in dorsal cord innervation preferences in comparison with control cultures. The amount of evoked activity was significantly lower than was observed in controls, suggesting that the formation of 'incorrect' functional connections was blocked by specific chemical factors. Exposure to N-acetylgalactosamine, a major ganglioside amino sugar, also resulted in an increased dorsal cord innervation preference by the DRG afferents.     

Proliferation and differentiation of cells from explants of fetal rat stomach

The current understanding of the mechanisms controlling the proliferation and differentiation of the stem cells of the gastric oxyntic glands is limited. The aim of the present study was to develop a method for investigating proliferation and differentiation of undifferentiated cells from fetal rat stomach. Outgrowth of cells was initiated from explants of 16-day-old fetal rat stomachs. At this stage of the fetal development the gastric epithelial cells are undifferentiated. The explants were cultured in DMEM/F-12 medium supplemented with fetal calf serum only, or fetal calf serum combined with either hydrocortisone or pentagastrin. Morphological characterization by means of light microscopy, dye staining and immunostaining was used to identify the growing cells. Both hydrocortisone and pentagastrin accelerated the differentiation towards H,K-ATPase-positive cells, mucus-producing cells and other epithelial cells. H,K-ATPase-positive cells, which were identified by immunostaining with a monoclonal antibody reacting with the α-subunit of the H,K-ATPase, grew on top of the confluent layer of epithelioid and fibroblastoid cells. With this method in vitro investigations of the mechanisms of proliferation and differentiation of gastric mucosal cells are possible. Although by different mechanisms, both hydrocortisone and pentagastrin appear to play a regulatory role in these processes.     

Effect of tunicamycin, an inhibitor of protein glycosylation, on testicular cord organization in fetal mouse gonadal explants in vitro

The effect of tunicamycin (TM) on testicular cord organization in the fetal mouse was examined in vitro at light and electron microscopic levels, with special reference to the glycoprotein functions during Sertoli cell differentiation. In testicular explants treated with TM, testicular cord organization was inhibited. TM treatment affected basal lamina formation by Sertoli cells, resulting in a discontinuous basal lamina or none at all in certain areas. The disorganized Sertoli cells were amorphous in shape, exhibited poor epithelial polarity, and were irregularly arranged in the testicular parenchyma. Extracellular matrix and collagen fibers were often observed in the intercellular spaces between the disorganized Sertoli cells. Lectin histochemical observation revealed that the number of wheat germ agglutinin binding sites on the plasma membrane and basal lamina of disorganized Sertoli cells was significantly decreased by TM treatment. However, junctions were normally observed in the plasma membrane between disorganized Sertoli cells. Leydig cells showed a normal differentiation in the testicular parenchyma in the presence of TM. These observations suggest that basal lamina formation of Sertoli cells and/or the expression of their cell surface glycoconjugates may be crucial for the establishment of Sertoli cell polarity and/or the Sertoli-Sertoli cell interactions required for proper testicular cord formation. Sertoli cell organization into testicular cords and Leydig cell differentiation may be controlled by different regulatory mechanisms.     

Lumbar spinal cord explants from neonatal rat display age-related decrease of outgrowth in culture

Monosodium glutamate was administered subcutaneously to male neonate rats, and the effects on cell number and cytoarchitecture of third-layer pyramidal neurons from the prefrontal cerebral cortex were studied in the adult. Monosodium glutamate treatment (4 mg/g of body weight, on post-natal days 1, 3, 5 and 7) resulted in fewer neurons, and shorter and less ramified dendritic processes, than those observed in control animals. Both density and proportional shapes of dendritic spines were not modified. We propose a dual effect of neonatal exposure to glutamate: an excitotoxic effect leading to cell death, and; a secondary neuroprotective effect, arising from the proliferation of glial cells and their subsequent uptake of glutamate, that favors the survival of the remaining neurons, and leads to a further hypotrophic effect on their dendritic processes.     

Morphology of osteoclasts in resorbing fetal rat bone explants: Effects of PTH and AIF in vitro

Osteoclastic bone resorption was studied using ⁴⁵Ca-labeled fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH revealed significant increases in the numbers and activity of osteoclasts, whereas bones cultured in the presence of AIF showed significant decreases in numbers of osteoclasts and altered cell features including the loss of osteoclast contact with bone surfaces. These structural modifications were evaluated with ⁴⁵Ca release data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured bones and morphological expressions of osteoclastic activity.     

Effect of hashish compounds on mouse peritoneal macrophages.

Light microscopy reveals an induction of extensive vacuolation in the macrophage after exposure to either delta 1-tetrahydrocannabinol or cannabidiol. Numerous small vacuoles appear in the cell periphery as early as 15 minutes after exposure (at 37degrees C.) to either of the compounds in 20 per cent newborn calf serum-Dulbecco's modified Eagle medium. The small lucent vacuoles coaleasce and yield enormous vacuoles which dominate the the cytoplasm. At approximately 3 hours, many of the vacuoles seem to burst with a concomitant expulsion of cell interior. The effect of hashish compounds on macrophages is essentially irreversible; exposure for 15 minutes to 10(-5) m of delta 1-THC or cannabidiol and a thorough wash in 20 per cent serum-medium, suffices to trigger the sequence of vacuolation and total cell death in the culture. Two major processes involving early reorganization of cellular membranes have been observed using electron microscopy. One relates to the formation of numerous autophagic vacuoles full of organelles, the other relates to the appearance of cytoplasmic inclusions representing extensive destruction of intracellular constituents. Both types of cytoplasmic change have been observed in alveolar macrophages of hashish smokers. Thus, the conditions in the in vitro studies are similar to conditions in people exposed to hashish smoke.     

Effect of dexamethasone on the fetal mouse small intestine in organ culture

The formation of intestinal villi (organogenesis phase) may be studied in organ culture with a completely synthetic medium in 15-day fetal mouse duodenal explants. However, in these explants absorptive cells remained poorly differentiated with all the hormones studied except with epidermal growth factor. In order to elucidate the role of hormones and other factors on the maturation of absorptive cells (maturation phase) in the fetal rodent in organ culture, we have taken the explants after the organogenesis phase. We have studied different culture conditions and have found that 17-day mouse duodenal explants can be cultured during 48 hours with Leibovitz L-15 medium in a 95% O2-5% CO2 atmosphere provided that the explants are relatively large (5 X 2 mm). With this method, dexamethasone (Dx) has been shown to have a direct effect on the maturation of the fetal duodenal mucosa. The addition of Dx (300 ng/ml) to the completely synthetic medium 1) improves the morphology of the explants, 2) induces a significant increase in maltase activity in the tissues, and 3) reduces significantly the labeling index of the duodenal explants after 48 hours of culture. Direct action of Dx on the duodenal mucosa is shown for the first time in organ culture using a completely synthetic medium. This method will permit us to study the effects of other intrinsic and extrinsic factors on the regulation of enzymatic maturation in fetal small intestine.     

Morphology of osteoclasts in resorbing fetal rat bone explants: effects of PTH and AIF in vitro.

Osteoclastic bone resorption was studied using 45Ca-labeled fetal rat bones cultured in the presence of parathyroid hormone (PTH) and an anti-invasion factor (AIF) derived from bovine hyaline cartilage which is enriched in a collagenase inhibitor. The specific morphological expressions of osteoclasts cultured in PTH and AIF were observed in both light and electron microscopy and analyzed cytometrically. Stimulation of bone resorption with PTH revealed significant increases in the numbers and activity of osteoclasts, whereas bones cultured in the presence of AIF showed significant decreases in numbers of osteoclasts and altered cell features including the loss of osteoclast contact with bone surfaces. These structural modifications were evaluated with 45Ca release data derived from matched-pair explants of fetal rat bones, revealing the existence of a relationship between resorptive states of the cultured bones and morphological expressions of osteoclastic activity.     

Effect of mobile telephony on blood-brain barrier permeability in the fetal mouse brain

AIMS: To study the effect of mobile telephone exposure on blood-brain barrier (BBB) permeability in the immature brain. METHODS: Using a purpose-designed exposure system at 900 MHz, pregnant mice were given a single, far-field, whole body exposure at a specific absorption rate of 4 W/kg for 60 min/day from day 1 to day 19 of gestation. Pregnant control mice were sham-exposed or freely mobile in a cage without further restraint and a positive control group with cadmium-induced BBB damage was also included. Immediately prior to parturition on gestational day 19, fetal heads were collected, fixed in Bouin's fixative and paraffin embedded. Disruption of BBB integrity was detected immunohistochemically using endogenous albumin as a vascular tracer in cerebral cortex, thalamus, basal ganglia, hippocampus, cerebellum, midbrain and medulla. RESULTS: No albumin extravasation was found in exposed or control brains. CONCLUSION: In this animal model, whole of gestation exposure to global system for mobile communication-like radiofrequency fields did not produce any increase in vascular permeability in the fetal brain regions studied using endogenous albumin as a light microscopic immunohistochemical marker.     

Tumor-initiating cells of various tumor types exhibit differential angiogenic properties and react differently to antiangiogenic drugs

Tumor-initiating cells (TICs) are a subtype of tumor cells believed to be critical for initiating tumorigenesis. We sought to determine the angiogenic properties of TICs in different tumor types including U-87MG (glioblastoma), HT29 (colon), MCF7 (breast), A549 (non-small-cell lung), and PANC1 (pancreatic) cancers. Long-term cultures grown either as monolayers ("TIC-low") or as nonadherent tumor spheres ("TIC-high") were generated. The TIC-high fractions exhibited increased expression of stem cell surface markers, high aldehyde dehydrogenase activity, high expression of p21, and resistance to standard chemotherapy in comparison to TIC-low fractions. Furthermore, TICs from U-87MG and HT29 but not from MCF7, A549, and PANC1 tumor types possess increased angiogenic activity. Consequently, the efficacy of vascular endothelial growth factor-A (VEGF-A) neutralizing antibody is limited only to those tumors that are dependent on VEGF-A activity. In addition, such therapy had little or reversed antiangiogenic effects on tumors that do not necessarily rely on VEGF-dependent angiogenesis. Differential angiogenic activity and antiangiogenic therapy sensitivity were also observed in TICs of the same tumor type, suggesting redundant angiogenic pathways. Collectively, our results suggest that the efficacy of antiangiogenic drugs is dependent on the angiogenic properties of TICs and, therefore, can serve as a possible biomarker to predict antiangiogenic treatment efficacy. Stem Cells2012;30:1831-1841.     

Comparative radiosensitivity of CFU-s from mouse bone marrow and fetal liver,forming 7- and 11-day colonies

Central Institute of Hematology and Blood Transfusion, Ministry of Health of the USSR, Moscow. Research Institute of Medical Radiology, Academy of Medical Sciences of the USSR, Obninsk. (Presented by Academician of the Academy of Medical Sciences of the USSR A. I. Vorob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 93–95, January, 1989.     

Culture and in vitro bromodeoxyuridine-labelling of mouse kidney explants

Summary A method is described for extended mouse renal organ culture and is evaluated for the study of cell proliferation in 1 mm³ explants of renal cortex. Explants were maintained for up to 14 days with cellular viability being sustained at 20% of the original tissue block from day 2. Labelling indices in tuft, capsular and tubular cells ranged from 0 to approximately 20% over the culture period, following in vitro bromodeoxyuridine uptake. This technique offers potential for the study of local effects of inhibitors of proliferation.     

Translation of messenger RNA from fetal and adult mouse kidney

Polyadenylated mRNA was prepared from fetal, adult, and regenerating adult mouse kidneys. The polyadenylated portion is 2% of total RNA in the fetus, falls abruptly to 1% after birth, and returns to about 2% in the adult. In vitro translation of these mRNA's showed great similarities between 17 and 20 day fetal samples and between the normal and regenerating adult samples, but few similarities between the fetal and adult samples. The data do not support the hypothesis that renal growth following contralateral nephrectomy may entail the de-repression of genes active in the fetus but not active in the normal adult.