In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells ¹. The whole embryonic organ can be cultured at multiple stages of development ^2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.     

In vitro propagation of Catalpa ovata G. Don

Plants of C. ovata were regenerated in vitro from shoot tips and nodal explants as well as from cotyledon-derived calluses. For shoot proliferation from shoot tips and nodal segments, Schenk and Hildebrandt (1972) or Lloyd and McCown (1980) basal media, supplemented with 6-benzyladenine (2.2–22.2 μM) alone or in combination with indole-3-acetic acid (0.6 μM), were used. Shoot regeneration through organogenesis was achieved by culturing cotyledons on Schenk and Hildebrandt medium containing indole-3-acetic acid (0.6 μM) and 6-benzyladenine (4.4 μM) or zeatin (22.8 μM). TLC and HPLC analysis showed that the multiple shoots and micropropagated plants exhibited similar iridoid patterns as those of the leaves of original plants of C. ovata. The highest levels of catalpol and catalposide (8.2 and 2.4 % of dry weight, respectively) were found in aerial parts of three-month-old in vitro regenerated plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Flower Formation from Citrus unshiu Buds Cultured In Vitro

Flowers were formed in vitro when buds of Satsuma mandarin (Citrusunshiu Mare) from the summer flush of growth harvested duringthe winter rest period before the onset of flower initiation,were grown on a solid Murashige and Skoog medium supplementedwith sucrose and a cytokinin. Flower development was dependentupon illumination, and was enhanced when a piece of stem wasattached to the bud. The percentage of flowering explants wasalways lower than the percentage of naturally flowering budsin spring, but treatments such as ringing which increase floweringin vitro, increased the number of explants flowering int vitroas well. Citrus unshiu Marc, ‘Satsuma’ mandarin, in vitro flowering, ringing     

Photo- and Gravitropic Bending of Potato Plantlets Obtained In Vitro from Single-Node Explants

Etiolated and light-grown plantlets obtained from potato shoot cultures were shown to perform vigorous tropic movements. Unilateral blue irradiation actively induced phototropic curvature of the shoots toward the light source, although etiolated plantlets required ten times longer stimulation than the light-grown plantlets to achieve a 90° angle. The fluence requirements for induction of second positive phototropism (PT) of light-grown plantlets spanned almost five orders of magnitude (~30–1.7?×?10⁵ μmol/m²). Upon responding to unilateral blue light by curving, plantlets entered the process of straightening after irradiation ended. Straightening also occurred in plantlets placed on a clinostat but it was of lower magnitude. Compared to early-morning and day-time hours, plantlets exhibited a significantly lower PT response in the late afternoon (5 p.m.) and gravitropic (GT) response at the end of the day (11 p.m.), suggesting that these responses may be under the control of circadian rhythms. GT was also recorded for both light-grown and etiolated plantlets. Ninety-degree stimulation, used to induce GT in etiolated plantlets, needed to be 50?% longer than stimulation used for light-grown plantlets to induce a similar response. Straightening was also recorded for the shoots that exhibited GT but was smaller when plantlets were placed on a clinostat compared to straightening exhibited by those plantlets left standing in an upright position for 2?h.     

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.     

濒危黄杜鹃的离体快速繁殖体系研究

以濒危野生黄杜鹃(Rhododendron molle G.Don)茎尖为试材,比较研究3种不同基本培养基和9个激素浓度组合对不定芽增殖的影响,以建立黄杜鹃的快速繁殖体系.结果表明:3种不同基本培养基对野生黄杜鹃不定芽增殖的效果差异显著;相同激素种类与浓度下,WPM培养基上的不定芽增殖系数最大,达10.5.激素种类与浓度对黄杜鹃不定芽的增殖影响差异很大,以单独使用1.0 mg/L ZT最佳,再生率达100%,不定芽增殖系数为10.5.综合分析显示,黄杜鹃不定芽增殖的最佳培养条件为WPM+ZT 1.00 mg/L;健壮、整齐一致的不定芽在1/2 WPM+0.05 mg/L NAA上生根,40 d后生根率可达86.7%,驯化成活率达92.86%.     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

Morphogenic Potential of Cultured Floral Explants of Crocus sativus L. for the In Vitro Production of Saffron

Explants of immature ovaries, stigmas, anthers and petals ofCrocus sativus were cultured on White's media supplemented witheither NAA and zeatin or 2,4-D and BAP in various combinations.The formation of stigma-like structures occurred on the explantsor on the callus derived from the explants, but this was onlyobserved when NAA and zeatin were used. Formation of stigma-likestructures were observed on anthers, petals, stigmas and half-ovaries,with the best result being obtained on explants consisting ofhalf-ovaries cultured on medium containing NAA at 40 mg dm^–3and zeatin at 4.0 mg dm^–3. These stigmas developed anintense orange pigment and grew to 3.0 cm in length and hada strong saffron aroma. A preliminary comparison using thinlayer chromatography of the yellow pigments produced by thestigma-like structures grown in vitro and those grown naturallyshowed the pigment composition to be similar. Key words: Crocus sativus L., explants, NAA, zeatin, saffron     

In Vitro Antibacterial and Antioxidant Studies of Croton roxburghii L., from Similipal Biosphere Reserve

In vitro antibacterial activities of acetone, ethanol, methanol and water extracts of leaves and bark of Croton roxburghii L. studied against ten human pathogenic bacterial strains showed significantly higher activity in acetone extract and least activity in case of aqueous. Minimal inhibitory concentration (MIC) values of all extracts ranged between 0.62 and 10 mg/ml, while minimal bactericidal concentration (MBC) values ranged from 1.25 to values greater than 10 mg/ml. The antioxidant assays viz. DPPH, hydrogen peroxide scavenging, iron reducing and iron chelating assays along with total phenol and ascorbic acid content were carried out with aqueous extracts of leaves and bark. While the total phenol contents in leaves and bark extracts were 0.766 ± 0.014 and 0.735 ± 0.028% respectively their ascorbic acid contents were found to be 0.252 ± 0.019 and 0.431 ± 0.013% respectively. DPPH activities in both (leaves and bark) extracts increased with the increase in concentrations. Iron chelating capacity of leaves extract is significantly higher than that of the bark. Leaves extract showed an increase in percentage of scavenging property with the increase in concentrations. Plant extracts showed low amount of iron reducing property at all concentrations. Hydrogen peroxide scavenging properties of bark was low than that of the leaves.     

Antidiabetogenic oligostilbenoids and 3-ethyl-4-phenyl-3,4-dihydroisocoumarins from the bark of Shorea roxburghii

A methanol extract of the bark of Shorea roxburghii (Dipterocarpaceae) was found to inhibit plasma glucose elevation in sucrose-loaded mice. From the extract, three new 3-ethyl-4-phenyl-3,4-dihydroisocoumarins, 1'S-dihydrophayomphenol A(2) (1) and phayomphenols B(1) (2) and B(2) (3), were isolated together with 24 known compounds including 20 stilbenoids and oligostilbenoids. The structures of 1-3 were determined on the basis of their spectroscopic properties as well as of chemical evidences. Among the isolates, (-)-hopeaphenol (6), hemsleyanol D (8), (+)-α-viniferin (15), and (-)-balanocarpol (18) showed inhibitory activity against plasma glucose elevation in sucrose-loaded rats at doses of 100-200mg/kg, p.o. To clarify the mode of action of the antihyperglycemic property, effects of these oligostilbenoids on gastric emptying in mice, those on glucose uptake in isolated intestinal tissues as well as inhibitory activities against rat intestinal α-glucosidase and rat lens aldose reductase were examined.     

A Study on Comparative Morphology of Normal and Hyperhydric Stems and leaves from Sophora japonica Plantlets Cultured in Vitro

A large numar of plantlets were obtained from cotyledon explants of Sophom japonica L. cultured in vitro. They could be classified into 3 kinds according to their morphological characteristics, viz. the normal plantlets, the hyperhydric planfiets,and the intermediate state between the two or the sub-hyperhydric type. The free water content was more than 79% in the hyperhydric shoots,and 70% in the sub-hyperhydric shoots,while less than 50% in the normal shoots. The surface anatomy of normal, sub-hyperhydric and hyperhydric stems and leaves of the plantlets were compared by scanning electron microscopy. The surface structure of the normal plantlets was similar to those found in field-grown plants,but great change occurred in that of hyperhydric and the sub-hyperhydric plantlets. The stems and leaves surface of the hyperhydric and sub-hyperhydric plantlets appeared to be uneven, wrinkled, brittle and translucent and besides the leaves were thick, curled with a reduced surface area. There was little or no epicuticular wax on the surface of epidermal cells which had irregular shapes and patterns. All leaves were amphistomatic and the stomatal density, size and degree of opening were obviously bigger in the sub-hyperhydric and hyperhydric leaves than in the normal ones. Normal stomata had kidney-shaped guard cells and resembled closely those found in the feild-grown plants, whereas abnormal stomata had deformed guard cells. All of the morphological characteristics mentioned above indicated that the sub-hyperhydric and hyperhydric shoots bended to lose their water easily and resulted in desiccation, which might be one of the major causes of failure to transfer sub-hyperhydric and hyperhydric plantlets to soil.     

Dual Appearances of Pigment Cells from In Vitro Cultured Embryonic Cells of Japanese Flounder: An Implication for a Differentiation–Associated Clock

The cells dissociated from developing embryos of Japanese flounder (Paralichthys olivaceus) are cultured in vitro to examine the developmental fate of their pigment cells in relation to establishment of bilaterally asymmetric integumental coloration in vivo. When neurula embryos are dissociated using trypsin–EDTA in Dulbecco's modified Ca²⁺–, Mg²⁺–free phosphate buffered saline and then cultured in vitro using L–15–based fetal calf serum–supplemented growth medium at 20°C, numerous pigment cells appear twice in the same culture with an interval of approximately 1 month even under similar culture conditions. The first group of pigment cells, which is relatively larger in cell size (about 70 μm wide) and lower in cell density, emerges within 12 hr after plating, whereas the second, which is far smaller in cell size (about 30 μm) and overwhelmingly higher in cell density than the first, does so about 1 month after plating. The timing of their appearances in vitro is in good accordance, respectively, with that observed for the larvae under normal development in vivo; the first group appears at the period corresponding to hatching, whereas the second at the period corresponding to the completion of metamorphosis. Light microscopic examinations disclose that each group of pigment cells is composed of black melanophores and reflecting leucophores, and that the population density of melanophores and leucophores in the first group at the climax of appearance is approximated as 1:4. Typical xanthophores that are distributed in the skin of the larvae of this species are scarcely observed in culture in vitro. Because of their dual synchronous appearances with about 1 month interval under the similar culture conditions, and because of their low proliferative activity during the periods from the first appearance to the second, it is presumed that both groups of pigment cells are installed with a clock set differently for their differentiation. Light and electron microscopic immunocytochemistry on cultured cells using the HNK–I antibody, which marks avian migratory neural crest cells, both disclose that the antibody cross–reacts with all these pigment cells, and that a certain number of immunoreactive unpigmented cells exist even at the time of the second appearance of pigment cells. These findings would imply that the second group of pigment cells served in a form of undifferentiated propigment cells up to metamorphosis, at which they start to differentiate under control of a clock presumably set during neurulation.     

胚胎干细胞体外分化为多巴胺能神经元

近年来,胚胎干细胞在体外分化为多巴胺能神经元方面取得了重大突破,这对神经发生的基础性研究和神经细胞移植具有重要意义。现对胚胎干细胞体外定向诱导分化为多巴胺能神经元的方法、相关细胞因子及检测鉴定等方面进行了分析和比较,并探讨了当前存在的问题和今后发展的方向。     

In Vitro Production of Stigma-like Structures from Stigma Explants of Crocus sativus L.

Stigma-like structures (TC stigmas) were produced in tissuecultures from stigma explants of Crocus sativus under definedconditions. MS medium supplemented with NAA (10 mg dm^–3)+BA (1.0 mg dm^–3) induced the optimum response. NAA wasfound to be an important addendum to achieve a good response.The TC stigma regeneration response as a function of explantage showed significant differences (except between stage 1 andstage 4). A culture temperature of 20 °C seems to be betterthan 25 °C with reference to all parameters. Crocin andpicrocrocin pigments, responsible for colour and bitter taste,respectively, were extracted, identified and quantified fromthe TC stigmas. Safranal was not detected in fresh samples. Key words: Crocus sativus, stigma-like structures, organ regeneration, crocin, picrocrocin, safranal     

In Vitro Induction of Haploid Plantlets from Unpollinated Young Ovaries of Lily and Its Embryo logical Observations

Unpollinated young ovaries of lily (Lilium davidii var. willmottiae (Wilson) Roffill) were inoculated on modified MS medium and N6 medium. Ovary cultures were incubated at 25–28℃, and illuminated with a fluorescent light of about 800–1200 Lux. Cultured ovaries gradually became thicker and longer after 10 days. The calli (about 6–12 mm in size) were produced after 40 days. The calli were then transferred to the differentiation medium. After 50 days, regeneration plantlets were formed. Embryoids were directly produced from some ovaries, which then developed into plantlets. Observation of chromosome number of regeneration plants shows: 65.71% regeneration plants are haploid plants, 34.29% are diploid. Embryological observation of ovary culture shows that haploid plants are from megaspore tetrad, while diploid plants are probably from nucellus cell.     

In Vitro Germ Cell Differentiation from Cynomolgus Monkey Embryonic Stem Cells

Background

Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro.

Methods and Findings

To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene.

Conclusion

VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.