In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

Effect of chromosome instability on the maintenance and differentiation of human embryonic stem cells in vitro and in vivo

The therapeutic potential of human embryonic stem cells (hESCs) has long been appreciated, and the recent FDA approval of hESC derivatives for cell-based therapy encourages the clinical application of hESCs. Here, using CHA3-hESCs with normal and abnormal karyotypes, we report the importance of maintaining normal chromosomes during in vitro culture and the differentiation of hESCs for minimization of posttransplantation complications. We found that undifferentiated CHA3-hESCs with trisomy chromosome 12 undergo abnormal cell division with multiple spindles in comparison to the bipolar cell division of the karyotypically normal CHA3-hESCs. Transplanted karyotypically abnormal CHA3-hESC derivatives formed a tumor-like tissue 6weeks after transplantation in two out of seven mice tested. Our results demonstrate that the preservation of normal chromosomes is indispensable for maintaining the true properties of hESCs in vitro and abolishing adverse effects posttransplantation. Thus, the development of optimized techniques for stabilizing the chromosome state during in vitro hESC culture is a prerequisite for the therapeutic application of hESCs.     

Lipids promote survival, proliferation, and maintenance of differentiation of rat liver sinusoidal endothelial cells in vitro

Primary rat liver sinusoidal endothelial cells (LSEC) are difficult to maintain in a differentiated state in culture for scientific studies or technological applications. Relatively little is known about molecular regulatory processes that affect LSEC differentiation because of this inability to maintain cellular viability and proper phenotypic characteristics for extended times in vitro, given that LSEC typically undergo death and detachment around 48-72 h even when treated with VEGF. We demonstrate that particular lipid supplements added to serum-free, VEGF-containing medium increase primary rat liver LSEC viability and maintain differentiation. Addition of a defined lipid combination, or even oleic acid (OA) alone, promotes LSEC survival beyond 72 h and proliferation to confluency. Moreover, assessment of LSEC cultures for endocytic function, CD32b surface expression, and exhibition of fenestrae showed that these differentiation characteristics were maintained when lipids were included in the medium. With respect to the underlying regulatory pathways, we found lipid supplement-enhanced phosphatidylinositol 3-kinase and MAPK signaling to be critical for ensuring LSEC function in a temporally dependent manner. Inhibition of Akt activity before 72 h prevents growth of SEC, whereas MEK inhibition past 72 h prevents survival and proliferation. Our findings indicate that OA and lipids modulate Akt/PKB signaling early in culture to mediate survival, followed by a switch to a dependence on ERK signaling pathways to maintain viability and induce proliferation after 72 h. We conclude that free fatty acids can support maintenance of liver LSEC cultures in vitro; key regulatory pathways involved include early Akt signaling followed by ERK signaling.     

Chloroplast alterations in albino plants obtained from in vitro culture

Abstract

Regenerated plants of Lycopersicon esculentum var. Alice were obtained from in vitro culture of cotyledons. Some of them showed different grades of leaf variegation, but a few plants were completely white. Here the chloroplasts of the mesophyll cells had completely failed to differentiate and contained no thylakoids. On the contrary those of the epidermal and stomata guard cells were normally developed. This suggests that in the albino plants a mutation had occurred in the submarginal initial cell responsible for mesophyll formation.     

Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of hydrocortisone and cyclic AMP

We have examined the effect of hydrocortisone and cyclic AMP on the maintenance of lipid synthesis in primary cultures of adult rat alveolar type II cells. These hormones were tested in the presence of either 1% or 5% charcoal-stripped rat serum (CS-rat serum). The effect of substratum on responsiveness to these hormones was evaluated by comparing cells cultured for 4 days on tissue culture plastic, on floating type I collagen gels, on rat lung fibroblast feeder layers on floating collagen gels (floating feeder layers), and on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. Type II cells cultured on floating feeder layers in medium containing 1% CS-rat serum and 10(-5) M hydrocortisone plus 0.5 mM dibutyryl cyclic AMP exhibited significantly increased incorporation of [14C]acetate into total lipids (238% of control). The hormone combination also increased the relative percentage of acetate incorporated into phosphatidylglycerol (PG; 7.3% versus 1.9%) and saturated phosphatidylcholine (PC; 43.6% versus 37.6%). The percentage of acetate incorporated into neutral lipids was significantly decreased by the addition of hormones (28.6% versus 70.0%). The addition of hydrocortisone and cyclic AMP to medium containing 5% CS-rat serum resulted in an increase in the relative incorporation of acetate into saturated PC (51.2% versus 46.4%), but had no effect on the relative incorporation of acetate into PG or on the incorporation of acetate into total lipids. Type II cells cultured on EHS gels in medium containing 1% CS-rat serum plus hydrocortisone and cyclic AMP showed increased acetate incorporation into total lipids (204% of control) and a relative decrease in the percentage of acetate incorporated into neutral lipids (16.9% versus 47.0%). The hormone combination also increased the relative incorporation of acetate into PG (4.4% versus 2.5%) and saturated PC (49.9% versus 42.1%). Hydrocortisone and cyclic AMP added to medium containing 5% CS-rat serum concentration increased the relative incorporation of acetate into saturated PC by type II cells on EHS gels, but these additions had no effect on acetate incorporation into PG. No responses to these soluble factors were seen when type II cells were cultured on floating type I collagen gels without feeder layers or on tissue culture plastic. These data indicate that there are positive interactions between substratum, soluble factors and serum in the maintenance of differentiated function of adult rat alveolar type II cells in vitro.     

Chick lens differentiation in vitro

If that portion of a chick embryo destined to form the eye, the lens, part of the brain and the surrounding head region be removed from the chick before the lens has begun to develop, and placed in a liquid culture medium for four days, a structure resembling the lens will form in appropriate proximity to the presumptive retina, and the cells of the lens will synthesize proteins unique to and characteristic of the lens. The time course of morphological development of such explants is here described. In vitro the lens placode dose not invaginate to form a lens vesicle, as it does in ovo. Instead placode cells elongate directly to form fibre cells. The shape of the lens formed in this aberrant manner is remarkably similar to that of normal lens. At least one, and probably all three, of the characteristic crystallins of the lens form in these aberrant lenses, the cytological and biochemical differentiation of which proceeds normally, despite failure of the normal morphogenetic activities of the organ.     

Precardiac mesoderm differentiation in vitro

The differentiation of precardiac mesoderm into beating heart tissue was examined during explant culture. Explanted tissue forms tubular heart-like vesicles and initiates rhythmic contractility within 18-24 h in vitro, a developmental time-course approximating that observed during in vivo development. Electron-microscopic observations reveal that beating heart cells are rich in cytoplasmic myofibrils in varying degrees of order, with some regions containing highly organized myofibrillar arrays. The analysis of actin-isotype biosynthesis, using metabolic labeling with [35S]-methionine and isoelectric-focusing resolution of the synthesized radioactive polypeptides, demonstrates that the initiation of cardiac alpha-actin synthesis and the pattern of transition in the synthesis of alpha-, beta-, and gamma-actin isotypes is equivalent to the initiation time and pattern observed in vivo. A possible collagen involvement in the differentiation process was investigated by assessing the effects of collagen-synthesis inhibitors on the development of the explant cultures. Two different agents, alpha, alpha'-dipyridyl and L-azetidine-2-carboxylic acid, exhibited a dose-dependent ability to inhibit the formation of beating heart tissue. When examined by electron microscopy, the nonbeating tissue exhibited a drastic depression of myofibrillogenesis, but otherwise appeared healthy. Further examination of the effect of L-azetidine-2-carboxylic acid demonstrated that the inhibition of myofibril formation and heartbeat was correlated with a 60% inhibition of native collagen synthesis; however, the time-course and pattern of actin-isotype biosynthesis was completely unaffected. The data suggest a possible involvement in heart differentiation that is necessary for either the synthesis of non-actin cardiac contractile proteins or the assembly of cardiac contractile proteins into myofibrils.     

Modulation of differentiation in vitro

Summary Myogenic cells of the L₆ line proliferate and fuse in culture to form myotubes that actively synthesize muscle-specific proteins such as myosin. We show that the expression of the differentiated phenotype can be influenced by the electrical charges of the substratum on which the cells were grown. Negatively charged surfaces did not influence the developmental program of the cells although positively charged ones interfered with myogenesis. The interaction operates primarily by interfering with the mitotic cycle, which is slowed down, with fusion which is blocked, and with myosin synthesis, which is reduced. Our results show that growth of the cells on positively charged surfaces prevents the switching of a large fraction of the population from a proliferative state to a differentiating program. We postulate that this interference might operate through the slowdown in DNA replication. The cell culture method described represents a good model for studying the different steps involved in the differentiation of L₆ cells. This work was supported by the American Muscular Dystrophy Association.     

Spermatogenic cell differentiation in vitro

Culture conditions that support the in vitro development of many spermatogenic stages from the frog Xenopus laevis are described. Spermatogenic cells were dissociated with collagenase and preelongation stages aseptically isolated by density gradient centrifugation in Metrizamide. The cells were then cultured in modified forms of defined nutrient oocyte medium (DNOM). The development of spermatogenic cells was affected significantly by changes in fetal calf serum concentration, cell density, energy sources, and NaCl concentration. Optimum in vitro spermatid development was obtained when spermatogenic cells were cultured at relatively high densities (3–7 × l0⁷ cells/25 cm²) in DNOM modified to contain 10% heat-inactivated, dialyzed fetal calf serum, 2 mM 1-glutamine, 0.1 % glucose, 15 mM HEPES buffer (pH 7.4), and 38.3–48.3 mM NaCl. These culture conditions also supported the differentiation of preelongation spermatids and spermatocytes isolated by density-gradient centrifugation in Metrizamide and subsequent unit gravity sedimentation in gradients of bovine serum albumin. Approximately 95 % of such isolated spermatids and spermatocytes continued differentiating in vitro for 14 days at in vivo rates. Phase-contrast and electron microscopy of the cultured cells demonstrated that in vitro differentiation was morphologically normal between the leptotene and elongate spermatid stages. Autoradiographic studies of preleptotene development demonstrated that spermatogonia proliferated and preleptotene spermatocytes developed to zygotene in 12-day cultures. The results suggest that many spermatogenic stages in Xenopus can develop independent of Sertoli cells, and demonstrate that spermatogenic cell cultures can now be used for in vitro studies of spermatogenesis.     

Nerve cell differentiation in vitro

The differentiation of cultured neurons from 4- and 8-day chick spinal ganglia has been studied when the nerve cell bodies rest on plastic, gelatin, glial cells, and heart fibroblasts, and when conditioned media are present. The percentage of neurons differentiated, time of axon initiation, total axonal length, rate of axon elongation, and level of ³H-leucine incorporation are employed as parameters of neuronal differentiation. Glia and heart fibroblasts increase the percentage of neuronal differentiation. Gelatin increases the percentage of neurons differentiated, as well as the rate of axon elongation and protein synthesis. Conditioned media also enhance the percentage of neuronal differentiation and the length of axon formed, but their effects are not equivalent to those of gelatin.     

Stem cells and pancreatic differentiation in vitro

Cell therapy using pancreatic islets would be a promising therapy to treat diabetes. But, because of the limited supply of human donor islets, other cellular sources have to be considered. Stem cells characterized by extensive proliferation and differentiation capacity may be a valuable source for the in vitro generation of islets. Insulin-producing cells derived from embryonic stem (ES) cells have been shown to reverse experimentally induced diabetes in animal models. However, the oncogenic properties of ES cells are critical in the context of clinical applications and efficient cell-lineage selection systems need to be established. Future studies have to demonstrate whether somatic stem cells residing in adult tissues, such as bone marrow, pancreatic ducts, intestine or liver may provide alternatives to generate functional pancreatic endocrine cells.     

Microtubule-associated proteins and in vitro astrocyte differentiation

Primary cultures of mouse brain astrocytes have been used to identify the microtubule-associated proteins (MAPs) present in this cell type at different stages of in vitro differentiation. The MAPs of the astrocyte have been identified by polyacrylamide gel electrophoresis and immunological detection. Two antisera were raised against two brain MAPs, tau and MAP-2. These antisera were also used to label the microtubular network in the intact astrocytes at different stages of the culture. The mature astrocyte contains a variety of MAP-like proteins. Anti-MAP-2 serum detected several proteins of high molecular weight (380,000, 260,000, 205,000 and 165,000 mol wt) and one microheterogeneous peak of 83,000 mol wt. Anti-tau also detected high molecular weight components (380,000 to approximately 200,000 mol wt) but not the 165,000-mol-wt peak; in addition two microheterogeneous peaks of 83,000 and 62,000 mol wt were detected by the anti-tau serum. The 62,000-mol-wt peak was therefore detected only by the anti-tau serum whereas the 83,000-mol-wt component cross-reacted with both antisera. At early stages of the culture the immature cell contained about two times less immunoreactive material than at mature stages. Qualitative changes of the high molecular weight components were also observed. In the intact cell both antisera revealed a dense fibrous network. At early stages of the culture the astroblasts were stained by the antisera but the reaction was very diffuse in the cytoplasm; few fibrous cells were intensively stained. Morphological differentiation, which began after serum deprivation and which was accelerated by forskolin (a drug that induces cyclic AMP accumulation), led to high labeling of both the cell body and the cellular processes. In the presence of colchicine the staining regressed, the processes shortened, and the cell returned to a less-apparently differentiated state.     

Chloroplast ultrastructure of Hypericum perforatum plants regenerated in vitro after cryopreservation

The ultrastructure of leaf mesophyll cells of in vitro cultured Hypericum perforatum L. plants regenerated after cryopreservation was studied. Electron microscopy analysis revealed that the chloroplasts in plants pretreated with abscisic acid and regenerated after cryopreservation were round, with increased amount of starch, rather small volume of the thylakoid system, and destroyed envelope. Plants pretreated with 0.3 M mannitol and cooled at rates of 0.1 or 0.3 °C min^?1 possessed chloroplasts with high starch content that resulted in a reduction of a membrane system. However, the pretreatment with 0.3 M mannitol and cooling at a rate of 0.2 °C min^?1 was the best as chloroplast ultrastructure resembled the controls regenerated without cryopreservation.     

Chloroplast phosphofructokinase in the green alga, Dunaliella marina: partial purification and kinetic and regulatory properties

The aim of this work was to study the pathway(s) of sugar phosphate metabolism in chloroplasts of the unicellular green alga, Dunaliella marina (Volvocales). Phosphofructokinase, detectable in crude cell extracts, copurifled with intact chloroplasts on sucrose density gradients. In isolated chloroplasts, phosphofructokinase activity displayed latency to the same degree as chloroplast marker enzymes. From the quantitative distribution of enzyme activities in fractionated cells, it is concluded that there is an exclusive localization of phosphofructokinase in chloroplasts. In addition, no separation into multiple forms could be achieved. For the study of regulatory properties, chloroplast phosphofructokinase was partially purified by ammonium sulfate fractionation followed by DEAE-cellulose chromatography. The pH optimum of the enzyme activity was 7.0 and was not altered with varying concentrations of substrates or low-molecular-weight effectors. Fructose 6-phosphate showed a sigmoidal saturation curve whose shape was further changed with varying protein concentrations of the preparation. The second substrate, ATP, gave a hyperbolic saturation curve with a Michaelis constant of 60 μm. At a Mg²⁺ concentration of 2.5 mm, ATP concentrations exceeding 1 mm inhibited the enzyme in a positive cooperative manner. The same type of inhibition was observed with other phosphorylated intermediates of carbon metabolism, the most efficient being phosphoenolpyruvate, glycolate 2-phosphate, glycerate 3-phosphate, and glycerate 2-phosphate. Inorganic phosphate was the only activator found for phosphofructokinase. With nonsaturating fructose 6-phosphate concentrations, P_i activated in a positive cooperative fashion, while no activation occurred with saturating fructose 6-phosphate concentrations. In the presence of either an activator or an inhibitor, the sigmoidal shape of the fructose 6-phosphate saturation curve was altered. Most notably, the activator P_i could relieve the inhibitory action of ATP, phosphoenolpyruvate, glycerate 3-phosphate, glycerate 2-phosphate, and glycolate 2-phosphate. Based on these experimental findings, the regulatory properties of D. marina chloroplast phosphofructokinase are discussed with respect to its playing a key role in the regulation of chloroplast starch metabolism during a light/dark transition. All available evidence is compatible with the interpretation that phosphofructokinase is active only in the dark thus channeling starch degradation products into glycolysis.     

Defects in telomere maintenance molecules impair osteoblast differentiation and promote osteoporosis

Osteoporosis and the associated risk of fracture are major clinical challenges in the elderly. Telomeres shorten with age in most human tissues, including bone, and because telomere shortening is a cause of cellular replicative senescence or apoptosis in cultured cells, including mesenchymal stem cells (MSCs) and osteoblasts, it is hypothesized that telomere shortening contributes to the aging of bone. Osteoporosis is common in the Werner (Wrn) and dyskeratosis congenita premature aging syndromes, which are characterized by telomere dysfunction. One of the targets of the Wrn helicase is telomeric DNA, but the long telomeres and abundant telomerase in mice minimize the need for Wrn at telomeres, and thus Wrn knockout mice are relatively healthy. In a model of accelerated aging that combines the Wrn mutation with the shortened telomeres of telomerase (Terc) knockout mice, synthetic defects in proliferative tissues result. Here, we demonstrate that deficiencies in Wrn^−/– Terc^−/– mutant mice cause a low bone mass phenotype, and that age-related osteoporosis is the result of impaired osteoblast differentiation in the context of intact osteoclast differentiation. Further, MSCs from single and Wrn^−/– Terc^−/– double mutant mice have a reduced in vitro lifespan and display impaired osteogenic potential concomitant with characteristics of premature senescence. These data provide evidence that replicative aging of osteoblast precursors is an important mechanism of senile osteoporosis.     

Niche differentiation and maintenance of genetic identity in cohabiting Eucalyptus species

Differences in niche utilization between two cohabiting eucalypt species (Eucalyptus signata and E. umbra ssp. umbra) on North Stradbroke Island, Qld., are documented, including differences in morphology, growth patterns, seasonality, insect attack and a three month difference in peak seasonal uptake of most essential elements. The significance of these niche differences in aiding the coexistence of competing eucalypt species is supported by results using recent theoretical models from niche theory. It is noted that the presence of significant niche complementarity amongst cohabiting pairs of eucalypts will place hybrid offspring at a disadvantage relative to homozygous strains. This phenomenon helps explain the scarcity or absence of hybrids in a number of communities where potentially interbreeding species of Eucalyptus coexist. Co-occurrence of species pairs which are capable of interbreeding will not be favored by environmental selection both because of the reduced production of homozygous offspring, and because of the reduced niche separation exhibited by taxonomically closely-related species. Such differences help explain the scarcity of co-occurrence of interbreeding pairs of species of Eucalyptus in relatively unperturbed natural communities.