The isolation and characterization of putative mesenchymal stem cells from the spiny mouse

The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). MSCs are capable of differentiating into tissues of the three primary lineages and have the potential to enhance repair in damaged organs through the principals of regenerative medicine. Given the ease with which MSCs may be isolated from different species the aim of this study was to isolate and characterize putative bone marrow derived MSCs from the spiny mouse, Acomys cahirinus. MSCs were isolated from the spiny mouse in a traditional manner, and based on plastic adherence, morphology, colony forming unit-fibroblast assays and functional assessment (adipogenic, osteogenic and chondrogenic differentiation potential) a population of putative mesenchymal stem cells from the compact bone of the spiny mouse have been isolated and characterized. Such methodological approaches overcome the lack of species-specific antibodies for the spiny mouse and could be employed for other species where the cost of generating species-specific antibodies is not warranted.     

Separation of mouse spermatogenic cells by sedimentation velocity. A morphological characterization.

A method utilizing sequential enzymatic incubation in collagenase (1 mg/ml) and trypsin (2.5 mg/ml) has been developed for the dissociation of the seminiferous epithelium. A significant advantage of this method is that, following collagenase incubation and washings in an enriched Krebs-Ringer bicarbonate buffer solution, isolated seminiferous tubules are obtained which are free of interstitial cells. The “purified” seminiferous epithelium is then dissociated with trypsin. A further advantage of this dissociation technique has been a reduction in the number of symplasts (multinucleate cells) which form by the opening up of the intercellular bridges that occur between synchronously differentiating clusters of germ cells. Both the elimination of the interstitial cells and the reduction in the number of symplasts have made possible the recovery of more highly enriched germ cell fractions. The homogeneity of the cell fractions was determined by light and electron microscopy. Integrity of the isolated cells was verified by Trypan blue exclusion and measurement of oxygen consumption.     

Human endometrial stem cells: High-yield isolation and characterization

Background

Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium.

Circulating tumor cells: approaches to isolation and characterization

Circulating tumor cells (CTCs) shed from primary and metastatic cancers are admixed with blood components and are thus rare, making their isolation and characterization a major technological challenge. CTCs hold the key to understanding the biology of metastasis and provide a biomarker to noninvasively measure the evolution of tumor genotypes during treatment and disease progression. Improvements in technologies to yield purer CTC populations amenable to better cellular and molecular characterization will enable a broad range of clinical applications, including early detection of disease and the discovery of biomarkers to predict treatment responses and disease progression.     

Spermatogenic effects of male-fertile translocations in the mouse

Four male-fertile translocations, T(2;4)13H, T(2.8)26H, T(7;18)50H and T(1;13)70H were crossed to the inbred strains CBA/H and C57BL/6J. F1 heterozygotes were compared with wild-type litter-mates for signs of spermatogenic impairment, in view of previous reports that the C57BL strain had this effect in the T(14;15)6Ca translocation. There was a general tendency for body-weights to be slightly reduced in translocation carriers vs. wild-type. Mean testis weights were significantly reduced on the C57BL background with all four translocations as compared to wild-type, but also significantly increased in T26H on CBA. Sperm counts were also reduced on the C57BL background in T13H, T50H and T70H (significantly so in the last two) but were significantly increased in T13H on a CBA background. Only in T50H did the frequency of sperm-head abnormalities show any marked change in the translocation heterozygotes, being approximately doubled with both CBA and C57BL backgrounds although still remaining at a low level. It was concluded that the deleterious effects of C57BL strains on spermatogenesis in translocation heterozygotes were not confined to T6Ca but were probably widespread. Some inconclusive evidence suggested that this might be because some genetic factors associated with C57BL tended to reduce chiasma frequencies in translocation heterozygotes.     

Rapid isolation and lipid characterization of plasma membranes from normal and malignant lymphoid cells of mouse

A rapid isolation method was developed for plasma membranes from mouse lymphoid cells such as lymph node lymphocytes, thymocytes, radiation-induced thymoma cells and L1210 cells. Lysates of these lymphoid cells were prepared by Dounce homogenization under hypotonic conditions and directly layered on sucrose step density gradients containing 2 mM CaCl₂ and 5 mM MgCl₂, and centrifuged at $\text{52 000 ×}\text{g}$ for 1 h. Plasma membrane fractions appeared at the interface between 20 and 42% sucrose in the gradients. The procedure permitted purified membranes from cells to be obtained within 3 h, and the preparations appeared to be uniform by electron microscopy. Specific activities of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATpase}$, Mg²⁺-ATPase and 5′-nucleotidase of the isolated plasma membranes were enriched 23- to 61-fold, 12- to 15-fold and 18- to 34-fold, respectively, in comparison with those of the corresponding cell homogenates. Cholesterol content of the malignant cell membranes was lower than that of the normal membranes and the molar ratio of cholesterol to phospholipid of the malignant cell membranes was also lower than that of the normal membranes. A decreased plasmalogen content was observed in the malignant plasma membranes, together with a higher percentage of phosphatidylethanolamine and a lower percentage of phosphatidylserine. In the normal cell membranes, thymocytes contained a higher percentage of phosphatidylcholine and a lower percentage of sphingomyelin than those of the lymph node lymphocytes. At all temperature ranges (5 to 40°C) the plasma membranes of the malignant cells had lower microviscosity than those of the normal cells.     

Frog stomach enteric plexuses in culture: isolation, morphological characterization and bioelectrical recordings

We have succeeded in the isolation, culture and morphological characterization of Rana ridibunda stomach enteric plexuses. We have furthermore obtained intra and extracellular bioelectric recordings from the explants in culture. The culture medium used (Eagle MEM), the collagenase digestion and the general culture conditions followed are similar to those applied to mammal enteric plexus explant cultures. The most striking difference is that the solutions were diluted to 70% in order to maintain the osmolar conditions required by the amphibian cells. Acetylcholinesterase, osmium tetroxide-zinc iodide- and para-formaldehyde-induced fluorescence methods reveal similar morphological images from the perivascular fibre plexuses. The different cell types observed by phase contrast light microscopy from the myenteric explants in culture have been identified by comparison with those revealed by the acetylcholinesterase method. The prevailing neurons show piramidal somas; other neurons are bipolar with oval somas and a third type shows oval somas tightly aligned, following sinusoidal courses. The intra and extracellular bioelectric recordings from the explants in culture show that the culture conditions we have applied preserve the electrophysiological properties of the neuronal membranes. These preliminary recordings will allow us to undertake the synaptic characterization of the gastrointestinal neurotransmitters in frogs.     

Alpha-1-antitrypsin from mouse serum isolation and characterization

Alpha-1-antitrypsin (alpha-1-protease inhibitor) was isolated from mouse serum by a series of electrophoretic and chromatographic steps. We found it to be a glycoprotein of a mass ratio of 57.7 Kd. The extinction coefficient was E1%1cm,280=4.74. It inhibits bovine trypsin, human granulocytic and porcine pancreatic elastase. Its concentration in serum differs between inbred strains. Of those tested the concentration in C57BL/6J males was lowest with 5.58 +/- 0.71 mg/ml (females: 3.02 +/- 0.39) and that in DBA/2J was highest: 8.5 +/- 0.87 mg/ml (females: 4.09 +/- 0.51). The concentration of alpha-1-antitrypsin in male serum was almost twice as high as that in females of all strains tested.     

Thymic nurse cells in culture: morphological and antigenic characterization

Epithelial monolayers were derived from thymic nurse cells (TNC), and were seeded onto collagen-coated dishes immediately after their isolation from young adult C3H-murine thymuses. Different media and supplements were tested in order to obtain cultures that were as pure as possible. Primary cultures were enriched in epithelial cells but always contained non-epithelial components among which fibroblasts predominated. Immunodetection of keratins, and repeated light- and electron-microscopic observations established the epithelial nature of the elongated cells derived from TNC; these elongated cells were cortical reticular cells, and were different from medullary globular cells that immediately adopted a mosaic pattern in vitro. At the beginning of the culture, the necrosis of cortical lymphocytes appeared to be toxic for epithelial cells; when epithelial cells survived, they showed a temporary lipid accumulation. After a 5-day culture, they still synthesized DNA but lost this capacity thereafter and dedifferentiated. The lympho-epithelial symbiosis appeared to be necessary to maintain some epithelial characteristics of the cultured cells, such as the clear vesicles and the expression of la antigens. In sub-cultures, the monolayers were almost purely epithelial in nature but growth was no longer observed. The cells remained reticular in shape, as they were in vivo, but their cytoplasm and their nucleus became larger and numerous cells were multinucleated. Confluence was not obtained with classical media even after mitogenic stimulation. The frequent observation of strongly keratinized areas suggested a process of terminal differentiation; this could not be avoided by using low serum concentration.     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

The isolation and characterization of mouse liver glyoxalase I.

The purification of glyoxalase I (S-lactoyl-glutathione methylglyoxal-lyase (isomerizing) EC 4.4.1.5) from DBA/1J mouse liver employing ion exchange and affinity chromatography is described. The enzyme was purified 1140-fold and it exhibits a specific activity of 2200 units/mg of protein. The activity was determined to be homogeneous by sedimentation velocity and sedimentation equilibrium ultracentrifugation and by polyacrylamide electrophoresis. The molecular weight is approimately 43 000 and the sedimentation coefficient is 3.4 S. Kinetic data are consistent with a one-substrate (hemimercaptal) reaction mechanism but do not rule out alternate branches at low substrate and free glutathione concentrations.     

Development of the mouse retina: emerging morphological diversity of the ganglion cells

The time course and regulatory mechanisms of dendritic development are subjects of intense interest. We approached these problems by investigating dendritic morphology of retinal ganglion cells (RGCs) at four early postnatal stages. The RGCs develop from a diffusely stratified and poorly differentiated group at birth (P0), to 16 distinct, morphologically well-defined subtypes before eye opening (P13). Even before bipolar cells make synaptic contacts with the RGCs (P8), most adultlike RGC subtypes are already present. Similar to previous studies in other mammalian species, our results indicate that the initiation of the RGC morphological maturation is independent of light stimulation and of formation of glutamatergic synapses. This study narrowed down the window of RGCs morphological maturation and highlighted a few early postnatal events as potential factors controlling the developmental process. Because mouse is the most popular mammalian model for genetic manipulation, this study provided a foundation for further exploring regulatory mechanisms of RGC dendritic development.     

Nuclear proteoglycans from mouse liver cells: isolation and identification]

Proteoglycans (PG) have been isolated from mouse liver nuclei and identified. Nuclear PG are represented by various classes: i) PG containing dermatan sulfate (DS) chains; ii) PG containing heparan sulfate (HS) chains and, apparently, iii) mixed PG whose protein core contains both DS and HS chains.     

Thymic nurse cells: morphological study during their isolation from murine thymus

Summary Thymic nurse cells (TNC), which are multicellular complexes composed of epithelial cells and thymocytes, were obtained from C3H-mice thymuses. They were described by means of light and electron microscopy. The morphology of epithelial cells forming isolated TNC compared to that of small tissue fragments obtained by enzymatic digestion revealed that TNC could be derived from all parts of the thymus: cortex, corticomedullary junction and medulla, the cortex being their principal source. This variety of origin, the presence of several epithelial cells inside a single TNC, the presence of non-lymphoid cells, and the various locations of eleaved desmosomes confirmed that their aspect in vitro as round and sealed structures can be considered to be an artifact due to the isolation technique used. Indeed, during this procedure, they are formed by a process of wrapping of the epithelial cytoplasm around the tightly associated thymocytes. All three epithelial cell types: cortical reticular cells, medullary reticular cells, and medullary globular cells can form TNC.A portion of this work was presented at the first Thymus Workshop. Rolduc, Netherlands, April, 1988     

Geographic isolation facilitates the evolution of reproductive isolation and morphological divergence

Geographic isolation is known to contribute to divergent evolution, resulting in unique phenotypes. Oftentimes morphologically distinct populations are found to be interfertile while reproductive isolation is found to exist within nominal morphological species revealing the existence of cryptic species. These disparities can be difficult to predict or explain especially when they do not reflect an inferred history of common ancestry which suggests that environmental factors affect the nature of ecological divergence. A series of laboratory experiments and observational studies were used to address what role biogeographic factors may play in the ecological divergence of Hyalella amphipods. It was found that geographic isolation plays a key role in the evolution of reproductive isolation and divergent morphology and that divergence cannot be explained by molecular genetic variation.     

An improved method for isolation of mutator mutants from mouse FM3A cells and their characterization

An improved method to select mutator mutants was developed. By this new method, mutator mutants were isolated efficiently, and 7 mutants were obtained from cultured mouse FM3A cells. These mutator mutants have an elevated rate of spontaneous mutation at 3 genetic loci (resistance to ouabain, blasticidin S, and tunicamycin). The sensitivity of these mutants to aphidicolin and arabinofuranosylcytosine was the same as in the wild-type cells. Determination of the size of the cellular dNTP pool revealed that there was no large imbalance in the precursor pool in the mutator mutants. These results suggested that the mutator character may be due to alteration in some factor(s) correlated directly to DNA replication. Also, there was no change in the sensitivity of all these mutator mutants to DNA damaging agents.     

Spermatogenic stage sensitivity to 6-mercaptopurine in the mouse

Hybrid (101 × C3H)F₁ male mice were given [³H]thymidine intraperitoneally, and 1 h later 150 mg/kg 6-mercaptopurine in 0.03 N NaOH. Autoradiography of testis sections showed that the rate of spermatogenesis was not altered, and the time of appearance of labeled spermatozoa in the ejaculate indicated that 6-mercaptopurine also had no effect on minimum sperm transport time. Labeled spermatozoa persisted in the ejaculate for a longer interval in 6-mercaptopurine-treated males than in controls, most likely as a result of oligospermia. Combined treatment with 150 R X-rays and 150 mg/kg 6-mercaptopurine gave an additive effect and demonstrated conclusively that the peak incidence of dead implants observed at 30.5–35.5 days can be attributed to cells treated as preleptotene spermatocytes and must result from genetic damage that is not cytologically detectable; previous work has shown that chromatid and isochromatid breaks at diakinesis-metaphase I occurred only on days 14 and 15 after 150 mg/kg 6-mercaptopurine. From the present experiments it is clear that these aberrations are not related to the dominantly lethality at 30.5–35.5 days.