Differentiation of Human Bone Marrow Mesenchymal Stem Cells to Chondrocytes for Construction of Three-dimensional Cartilage Tissue

A differentiation method of human bone marrow mesenchymal stem cells (MSCs) to chondrocytes was developed for the construction of a three-dimensional (3D) cartilage tissue. The adhesive cells, which were isolated from a human bone marrow aspirate were embedded in type I collagen in a poly-l-lactate-glycolic acid copolymer (PLGA) mesh and cultivated for 4 week together with growth factors. The degree of cellular differentiation was estimated by quantitative RT-PCR of aggrecan and type II collagen mRNAs and by staining with Safranin O. The 3D culture showed a higher degree of differentiation even without growth factors than the conventional pellet culture with growth factors, namely, dexamethasone and transforming growth factor (TGF)-β 3. The 3D culture for 2 week with the combined addition of dexamethasone, TGF-β 3, and insulin-like growth factor (IGF)-I reached a 30% expression of aggrecan mRNA compared with that in primary human chondrocytes, while the aggrecan mRNA expression in the conventional pellet culture was less than 2%. The sequential two-step differentiation cultivation, during which the cells were cultivated in 3D for 1 week after the conventional two-dimensional (2D) culture for 1 week, could markedly accelerate the expression of aggrecan mRNA compared with the 3D cultivation for 2 week.     

Carica papaya (Caricaceae): a case study into the effects of domestication on plant vegetative growth and reproduction

Few studies have quantitatively evaluated the gender specific effects of cultivation on plant growth and reproduction. The availability of cultivated and wild populations of different genders of Carica papaya L. (Caricaceae) on Guam provided an opportunity to study these effects quantitatively. We compared the gender specific allometry of height vs. basal stem diameter (H vs. D), stem slenderness ratio (H/D), and the height at first flowering (H(fl)) of carpellate and staminate plants growing under natural conditions (N = 150 each) with those of carpellate and hermaphroditic plants (N = 250 each) from two cultivars (Sunrise and Tainung 2). These comparisons indicated that (1) wild carpellate and staminate plants are significantly taller than either gender of the two cultivars with equivalent D; (2) the scaling exponent governing the H vs. D relationship of both genders of wild plants is significantly higher than that of either cultivated gender; (3) cultivar type does not affect the H vs. D exponent, but gender expression does; (4) gender expression (but not cultivar-type) also affects H(fl) (cultivation substantially reduces carpellate plant H(fl)); and (5) the onset of sexual maturity is associated with a dramatic reversal in H/D ontogeny. Cultivation therefore has "condensed" patterns of vegetative growth in a gender specific manner, whereas gender expression alters both vegetative and reproductive growth significantly more so than does cultivar-type.     

3 dimensional cell cultures: a comparison between manually and automatically produced alginate beads

Cancer diseases are a common problem of the population caused by age and increased harmful environmental influences. Herein, new therapeutic strategies and compound screenings are necessary. The regular 2D cultivation has to be replaced by three dimensional cell culturing (3D) for better simulation of in vivo conditions. The 3D cultivation with alginate matrix is an appropriate method for encapsulate cells to form cancer constructs. The automated manufacturing of alginate beads might be an ultimate method for large-scaled manufacturing constructs similar to cancer tissue. The aim of this study was the integration of full automated systems for the production, cultivation and screening of 3D cell cultures. We compared the automated methods with the regular manual processes. Furthermore, we investigated the influence of antibiotics on these 3D cell culture systems. The alginate beads were formed by automated and manual procedures. The automated steps were processes by the Biomek^® Cell Workstation (celisca, Rostock, Germany). The proliferation and toxicity were manually and automatically evaluated at day 14 and 35 of cultivation. The results visualized an accumulation and expansion of cell aggregates over the period of incubation. However, the proliferation and toxicity were faintly and partly significantly decreased on day 35 compared to day 14. The comparison of the manual and automated methods displayed similar results. We conclude that the manual production process could be replaced by the automation. Using automation, 3D cell cultures can be produced in industrial scale and improve the drug development and screening to treat serious illnesses like cancer.     

In vitro modeling of liver fibrosis with 3D co-culture system using a novel human hepatic stellate cell line

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC-1), and applied it to various types of three-dimensional (3D) co-culture systems with differentiated HepaRG cells. Furthermore, we compared LSC-1 with a commercially available HSC line on conventional monolayer culture. LSC-1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast-like or transdifferentiated state. However, activated states reverted to a quiescent-like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC-1 exerted an overall positive effect on co-cultured differentiated HepaRG, which significantly increased hepatic functionality upon long-term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC-1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix-related protein expression mechanism. Therefore, the LSC-1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.     

Cooperative effects of gamma-interferon and 1 alpha, 25-dihydroxyvitamin D3 on in vitro differentiation of the blast cells of RAEB and RAEB-T

We added recombinant human gamma-interferon (gamma-IFN) and 1 alpha, 25-dihydroxyvitamin D3 (1 alpha, 25 (OH)2D3) to the bone marrow cells from six patients with RAEB or RAEB-T in liquid suspension cultures. After cultivation for 7 to 9 days, numerical, morphological and functional changes of the cells were assessed. gamma-IFN and 1 alpha, 25 (OH)2D3 additively suppressed cell growth, especially the number of blast cells decreased. The expression of alpha-naphthylbutyrate esterase (NBE) activity appeared to be promoted but that of naphthol AS-D chloroacetate esterase (NAE) activity was apparently suppressed by the addition of gamma-IFN and/or 1 alpha, 25 (OH)2D3. The percentage of NBT reduction-positive cells and latex-phagocytizing cells was only slightly increased by both agents. These results indicate that gamma-IFN and 1 alpha, 25 (OH)2D3 cooperate to induce monocytoid differentiation of the patients' blast cells. Combination therapy with both agents merits further study.     

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.     

Tri-dimensional prostate cell cultures in simulated microgravity and induced changes in lipid second messengers and signal transduction

The high aspect rotating-wall vessel (HARV) was designed to cultivate cells in an environment that simulate microgravity. We studied previously the effects of HARV cultivation on DU-145 human prostate carcinoma cells. We determined that HARV cultivation produced a less aggressive, slower growing, less proliferative, more differentiated and less pliant cell than other cell cultivation methods. The result was a 3-dimensional (3D) growth model of prostate cancer which mimics in vivo tissue growth. This work examines the signal transduction-second messenger pathways existing temporarily in these HARV cells and correlates these features with the special properties in growth and 3D spheroid formation. We found an initial very active ceramide, a diacylglycerol increase together with increases in PI-PLC and PLA₂ a central defect in PLD (no phosphatic acid or phosphatidylethanol at any time during 15 days of HARV cultivation). There is a cross-talk between ceramide and PI3K pathways with activation of PI3K, after 6 days of HARV growth concomitant with down-regulation of ceramide. At this time, there is also an increase of cAMP (seen by increases in arachidonic acid). Taken together these results can explain the 3D organoidlike growth. We therefore developed a model for growth in HARV prostate cancer cells which involve temporal "switches" between second messengers, activation and cross-talk between multiplicity of signaling pathways and a central defect in PLD pathways. Essential to the late slow growth, and 3D organotypic formation are the apoptotic, anti-survival, anti-proliferation and differentiation pathways in the first days of HARV, with growth of "new" different types of prostate cancer cells which set-up for later "switch" in ceramide-PI3K to survival and proliferation.     

Pellet culture elicits superior chondrogenic redifferentiation than alginate‐based systems

Although pellet culture and encapsulation of chondrocytes into gel‐like biomaterials have lead to major advances in cartilage tissue engineering, a quantitative comparative characterization of cellular differentiation behavior during those cultivation procedures has not yet been performed. Our study therefore aimed at answering the following question: is the redifferentiation pathway of chondrocytes altered by slight changes in the type of alginate biomaterial (pure alginate, alginate‐fibrin, alginate‐chitosan) and how do the cells behave in comparison to biomaterial‐free (pellet) three‐dimensional culturing? Monolayer‐expanded chondrocytes from healthy adult porcine knee joints were cultivated in alginate, alginate‐chitosan, alginate‐fibrin beads and as pellets up to 4 weeks. Quantitative PCR and Immunohistology were used to assess chondrogenic markers. Alginate‐fibrin—encapsulated chondrocytes behaved almost like monolayer chondrocytes. Alginate‐ and alginate‐chitosan encapsulation lead to a low chondrogenic marker gene expression. Although all 3D‐cultured chondrocytes showed a considerable amount of Sox9 expression, only pellet cultivation lead to a sufficient Collagen II expression. This puts the usage of alginate‐cultivated cartilage tissue engineering constructs under question. Fibrin addition is not beneficial for chondrogenic differentiation. Sox9 and Collagen II behave differently, depending upon the surrounding 3D‐environment. © 2009 American Institute of Chemical Engineers AIChE J, 2009     

Reduction of PDC1 expression in S. cerevisiae with xylose isomerase on xylose medium

Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.     

Vitamin D3 inhibits fatty acid synthase expression by stimulating the expression of long-chain fatty-acid-CoA ligase 3 in prostate cancer cells

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.     

饲料脂肪水平对长养殖周期异育银鲫“中科3号”生长和脂代谢的影响

实验以初重为(11.33±0.03) g的异育银鲫(Carassius auratus gibelio)为研究对象,分别投喂脂肪水平为4%(L4)、8%(L8)、12% (L12)、16% (L16)和20% (L20)的5种等氮饲料进行为期340d的长养殖周期实验,以探究饲料脂肪水平对长养殖周期异育银鲫生长性能、消化酶活性和脂代谢的影响。期间共取样5次,生长阶段分为63d(D63,幼鱼期)、110d(D110,养成前期)、223d(D223,越冬期)、275d(D275,越冬后)和340d(D340,养成中后期)。实验结果显示,以增重率为评价指标,幼鱼期D63的异育银鲫适宜脂肪水平为8%,养成前期D110的异育银鲫适宜脂肪水平为12%,而其他生长阶段饲料脂水平对增重率无显著影响。饲料脂肪水平对幼鱼期异育银鲫肠道消化酶活性有显著影响,脂肪酶活性随脂肪水平的升高呈现先降低后升高的趋势,幼鱼期(D63)异育银鲫肠道胰蛋白酶和淀粉酶活性高于越冬期和养成中后期的异育银鲫,表明幼鱼期的异育银鲫对脂肪的利用较低。幼鱼期(D63)异育银鲫脂肪合成相关基因pparγ和fas的表达量饲料脂肪水平升高呈先升高后下降的趋势,且在L8组表达量最高,而脂解基因lpl和cpt1a的表达量在低脂组L4显著低于其他各组。pparγ和cpt1a在越冬后期(D275)的表达量随饲料脂肪水平的升高呈现先上升后下降,而fas表达量在L4组显著高于其他组,表明不同生长阶段异育银鲫对饲料脂肪摄入的响应策略不一致,摄入过高或过低均会导致代谢紊乱。适宜的脂水平(8%—12%)可促进幼鱼期和养成前期异育银鲫的生长,增强脂肪利用率和脂代谢能力,而较大规格的异育银鲫对脂肪的变化不敏感。     

The extensive commercial cultivation of Dunaliella salina

Betatene Ltd has established an extensive system for cultivation of D. salina for β-carotene production at Whyalla in South Australia.The operation, believed to be the largest area under man-made algal cultivation in the world, covers over 300 hectares in area, divided into three major cultivation lakes. A necessary requirement of the extensive system is to have a cost-effective harvesting system to handle the relatively large culture volumes. The extraction plant at the lakeside is designed to handle up to one million litre per hour in four parallel modules using a proprietary purely physical harvesting system.Current products are a crystalline β-carotene preparation for food colour applications, a high solubility preparation dispersed in soya bean oil for dietary supplementation and a dried powdered algal extract as an animal feed ingredient.     

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.     

Actinomycin D-induced change of mating type in the ciliate Dileptus anser

The effects of actinomycin D on the expression and inheritance of mating types (MTs) were studied in mature laboratory clones of the ciliate Dileptus anser. In these ciliates, each mature clone isolated from the natural population belongs to one of three complementary MTs, i.e., MT I, MT II, or MT III. In the course of further cultivation of the clone under laboratory conditions, in a series of vegetative generations, its MT remained unchanged. However, treatment with actinomycin D (15 μg/ml, 3 days) causes these clones to transition to a state that is hereditarily unstable for their MTs. At weekly testing for MT for at least 15 weeks after this treatment (which corresponds to more than 100 cell divisions), many subclones of the treated clone were observed to reversibly exchange their MT for another one; a temporary state of immaturity and/or partial maturity was also revealed. These data confirm our hypothesis about epigenetic MT determination in D. anser. Taking into account that actinomycin D also induces the inheritable destabilization of some characters in amoebas Amoeba proteus, which obviously has an epigenetic nature, this antibiotic might be considered an epimutagen.     

Functional interaction between dopamine receptor subtypes for the regulation of c-fos expression

Dopaminergic drugs increase the expression of the proto-oncogene, c-fos, in the brain, which is involved in the coordination of neurobiological changes caused by repeated cocaine or amphetamine use. This study examined the roles of five dopamine receptor subtypes on the c-fos promoter activity. D(1)R or D(5)R significantly increased the expression of c-fos promoter by activating protein kinase A. However, D(2)R, D(3)R, or D(4)R did not show any noticeable effects. The co-expression of D(1)R/D(3)R or D(1)R/D(2)R synergistically activated the basal and agonist-induced expression of the c-fos promoter, respectively. The Ral guanine-nucleotide-dissociation-stimulator-like, which was found to interact with the 3rd cytoplasmic loop of D(3)R, mediated the inhibitory activity of D(3)R in c-fos expression. In summary, the expression of the c-fos promoter was increased by the D1-like receptors and enhanced synergistically by the D2-like receptors via the modulation of cellular cAMP. D(3)R inhibited the expression of the c-fos promoter through an interaction with RGL.     

Improved Production of a Bioadhesive Precursor Protein by Fed-Batch Cultivation of a Recombinant Escherichia coli with a pLysS Vector

In batch cultivation, growth of a recombinant Escherichia coli with an inducible T7 expression system and maximum expression of a bioadhesive precursor (BP) protein was similar in the strains with and without the plasmid vector, pLysS. In fed-batch cultivation, however, the strain harboring pLysS grew slower and had a lower level of BP protein expression than that obtained with the strain without pLysS. This suggests that the presence of pLysS in the T7 expression system strongly affects the cell growth and expression of BP protein in high cell density cultivation.     

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

Optimization of expression conditions for soluble protein by using a robotic system of multi-culture vessels

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columns x six rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.     

Combined effect of cyclin D3 expression and abrogation of cyclin D1 prevent mouse skin tumor development

We have previously demonstrated that ras-mediated skin tumorigenesis depends on signaling pathways that act preferentially through cyclin D1 and D2. Interestingly, the expression of cyclin D3 inhibits skin tumor development, an observation that conflicts with the oncogenic role of D-type cyclins in the mouse epidermis. Here, we show that simultaneous up and downregulation of particular members of the D-type cyclin family is a valuable approach to reduce skin tumorigenesis. We developed the K5D3/cyclin D1^-/- compound mouse, which overexpresses cyclin D3 but lacks expression of cyclin D1 in the skin. Similar to K5D3 transgenic mice, keratinocytes from K5D3/cyclin D1^-/- compound mice show a significant reduction of cyclin D2 levels. Therefore, this model allows us to determine the effect of cyclin D3 expression when combined with reduced or absent expression of the remaining two members of the D-type cyclin family in mouse epidermis. Our data show that induced expression of cyclin D3 compensates for the reduced level of cyclin D1 and D2, resulting in normal keratinocyte proliferation. However, simultaneous ablation of cyclin D1 and downregulation of cyclin D2 via cyclin D3 expression resulted in a robust reduction in ras-mediated skin tumorigenesis. We conclude that modulation of the levels of particular members of the D-type cyclin family could be useful to inhibit tumor development and, in particular, ras-mediated tumorigenesis.