26 IN VIVO EXOGENIC ORGAN GENERATION WITH ORGANOGENESIS-DISABLED CLONED PIGS AS A PLATFORM

2014 ◽  
Vol 26 (1) ◽  
pp. 127
Author(s):  
H. Matsunari ◽  
K. Nakano ◽  
T. Kanai ◽  
T. Matsuda ◽  
M. Maehara ◽  
...  

The generation of organs from pluripotent stem cells (PSC) is one of the ultimate goals of regenerative medicine. We have demonstrated that functional organs can be generated in vivo from xenogenic PSC in the body of organogenesis-disabled mice using blastocyst complementation. To apply this principle in generating human organs, a technical platform using large non-rodent mammals is essential. The aim of the present study was to establish a blastocyst complementation system using cloned pig embryos. We generated transgenic-cloned pigs with an apancreatic phenotype via the overexpression of Hes1 (hairy and enhancer of split-1) under the Pdx1 promoter (pancreatic and duodenal homeobox-1). Cloned embryos of apancreatic pigs (host embryos, male) were complemented (i.e. chimerized) by blastomeres of cloned embryos (donor cells, female) with normal developmental competence. Chimeric embryos were cultured for 1 or 2 days before being transferred into the uteri of oestrus-synchronized gilts. The complementation of 292 Pdx1-Hes1 cloned embryos gave rise to 260 (89.0%) blastocysts. The transfer of these blastocysts resulted in 5 male chimeric pigs. Chimerism was confirmed by the detection of host embryo-derived Pdx1-Hes1 and marker transgenes of the donor cells, such as humanized Kusabira-Orange (huKO) or Pdx1-Venus. Chimeric pigs possessed normally formed pancreata entirely derived from the exogenous donor cells. We thus established a blastocyst complementation system in the pig using cloned embryos that would otherwise give rise to apancreatic animals. Chimeric pigs obtained developed normally, maintaining normal serum glucose concentrations up to maturity, and became fertile boars. Mating the chimeric boars with 7 wild-type sows gave rise to 72 fetuses/piglets of which 37 (51.4%) exhibited the apancreatic phenotype. These results indicate that a missing organ can be generated from exogenous cells when functionally normal pluripotent cells chimerize with a cloned dysorganogenetic embryo. Blastocyst complementation using cloned porcine embryos may permit the use of a large animal for the generation of functional organs from xenogenic PSC, including human iPSC. The chimeric boar produced by blastocyst complementation sired fetuses/offspring with the apancreatic phenotype in a Mendelian fashion. Porcine fetuses with an organogenesis-disabled phenotype may provide a useful platform for organ regeneration research. Table 1.Production of chimeric pigs by complementation and of Pdx1-Hes1 cloned embryos

1927 ◽  
Vol 45 (5) ◽  
pp. 807-814 ◽  
Author(s):  
Hobart A. Reimann

It is conceivable that a change from the virulent, non-phagocytable S form of Pneumococcus to the avirulent phagocytable R form may take place in pneumococcus disease, but the experiments here reported do not settle the question whether or not this is an important factor in determining the outcome in natural infection. It has been shown experimentally that the degradation from the S form to the R form actually does take place in cultures of Pneumococcus growing in agar subcutaneously embedded in guinea pigs, in agar enclosed in vials subcutaneously embedded in rabbits, and spontaneously in the blood stream of infected horses. However, it was not possible in any of the experiments here cited to demonstrate the complete change from S to R pneumococci before the bacteria disappeared from the body. When the intermediate or R forms did appear, they were always accompanied and usually exceeded in number by the S forms and all three forms disappeared together. S organisms may disappear entirely without evidence of first going through the intermediate and R stages. On the other hand, contrary to expectations, pure cultures of R forms remained viable in subcutaneous foci for weeks although apparently freely accessible to the action of phagocytes. It seems of some significance that the R forms appeared early in the vials (inoculated with S pneumococci) in immunized and normal rabbits alike, indicating that the presence of demonstrable specific immune bodies was not alone responsible for the variation of the bacteria. Of some importance also is the fact that R forms were never derived from similarly prepared control cultures growing in vitro at the same temperature and immersed in normal serum, although the S forms remained viable and unaltered for 6 weeks. It is likely that variations of pneumococci do not occur readily when S cultures are exposed to normal serum in vitro, especially when growing in closed vials under a diminished oxygen supply, for it has previously been shown (2) that only slight variation occurs even after prolonged (240) transfers in heterologous serum broth in the test-tube. It is possible, therefore, that the variation which occurred among pneumococci growing in agar vials embedded in normal rabbits was actually provoked by unknown influences in the living tissue fluids. Although R forms have been shown to occur in vivo, no positive evidence can be derived from these experiments to prove that recovery from pneumococcus infection depends upon the degradation of the virulent S forms of pneumococci to the avirulent R forms and the subsequent destruction of the latter by phagocytes.


2012 ◽  
Vol 24 (1) ◽  
pp. 119
Author(s):  
Z. Mei-Ling ◽  
Z. Yun-Hai ◽  
T. Yong ◽  
L. Ya ◽  
C. Hong-Guo ◽  
...  

The objective of present study was to investigate the effects of treatments to donor cells with fresh digestion (FD), cryopreservation/thawing (CT), trichostatin A (TSA) and durations of culture using TSA-CR1aa medium on in vitro development of dairy cow cloned embryos. In addition, some somatic cell cloned embryos were transferred to surrogates in heat to evaluate the in vivo developmental competence. The results (Table 1) showed that pretreatment of donor cells using TSA could significantly increase both cleavage and blastocyst rates of embryos (P < 0.05) compared with FD and CT group, whereas no significant difference was found between FD and CT group. When cloned embryos were subjected to TSA treatment in CR1aa for different times (0, 24, 48 and 60 h), the results showed that the blastocyst rate in the 60-h group was the highest (36.11 ± 1.78%) compared with the other groups (P < 0.05). Whereas the reconstructed embryos derived from donor cells treated with TSA for 24 h were continually cultured in TSA for different times (24, 48 and 60 h), the results showed that the blastocyst rate (37.39 ± 1.78%) in the 60-h group was significantly higher than that of the 24-h (25.48 ± 1.34%) group (P < 0.05). Finally, when the cloned embryos from different groups were respectively transferred to 40 natural oestrus recipients, no significant difference in terms of pregnancy rate among groups was found; however, a viable cloned calf was successfully obtained from TSA-treated donor cells and cloned embryo. Therefore, cloned embryos treated with optimized methods can develop to term. Table 1.Pregnancy results established from embryos of different origins


2016 ◽  
Vol 28 (2) ◽  
pp. 142
Author(s):  
N. L. Selokar ◽  
P. Sharma ◽  
D. Kumar ◽  
R. K. Sharma ◽  
P. S. Yadav

Selection of the donor cell type for somatic cell NT is very important based on its capability to be reprogrammed by the oocyte cytoplasm. A very wide variety of donor cells of different origin have been used for somatic cell NT, having differences in the overall efficiency. The aim of this study was to compare the cloning efficiency of donor cells derived from the ventral side of origin of tail skin and seminal plasma of a buffalo bull (age: 3 years old). Somatic cells from skin and seminal plasma were isolated and cultured as described by Selokar et al. (2014 PLOS ONE 9(3), e90755). Cultured seminal plasma cells had classic epithelial morphology, grew in clusters, were hexagonal in outline shape, and were positive for immunocytochemical detection of keratin marker, indicating that they were of epithelial origin, whereas tail-derived cells were spindle in shape and found positive for vimentin expression, indicating the fibroblast origin. To determine their reprogramming potential, these cells between passages 5 to 8 were used for the production of buffalo cloned embryos by handmade cloning as per the method described by Selokar et al. (2012 Theriogenology 78, 930–936). In brief, oocytes were isolated from slaughter-house ovaries and matured in vitro. After 21 h of maturation, cumulus cell mass and zona pellucida were removed by enzymatic treatment, hyaluronidase and pronase, respectively. Zona-free buffalo oocytes were enucleated on the basis of protrusion cone. A single somatic cell was attached to an enucleated oocyte with addition of phytohemagglutinin, followed by sandwich type of electrofusion between the somatic cell-bearing oocyte and enucleated oocyte using BTX electrofusion machine. Fused oocytes were activated by 4 μM calcium ionophore for 5 min and incubated in 2 mM 6-DMAP for 4 h and were cultured in K-RVCL-50® medium for 7 days on a flat surface in a 4-well dish in an incubator (5% CO2 and 38.5°C temperature). The total numbers of embryos reconstructed from tail-derived cells and semen-derived cells were 132 and 158, respectively. Cleavage and blastocyst rate were calculated from total embryos cultured, and data were analysed by Student’s t-test. We found no significant effect on both cleavage (89.30 ± 2.1 v. 94.1 ± 0.6) and blastocyst rate (40.7 ± 4.0 v. 43.1 ± 9.6) for the embryos produced from cells derived from tail and seminal plasma. To study the in vivo developmental competence of embryos derived from the 2 donor cell types, one embryo of each cell type was transferred into 6 recipient animals. Pregnancies were confirmed by ultrasonography at 30 to 35 days after transfer and monitored regularly at 15-day intervals up to 90 days. Three pregnancies were found for tail-derived cells, whereas no pregnancy was obtained for semen-derived cells. Out of 3 pregnancies obtained, 1 embryonic death was observed before 45 days, and 2 are continuing at advance stage. In conclusion, tail-derived cells are the better donor cell choice for buffalo somatic cell NT research. Currently, our focus is on epigenetic reprogramming behaviour of these 2 different cell types to elucidate the possible reprogramming mechanism.


2005 ◽  
Vol 17 (2) ◽  
pp. 166
Author(s):  
S.K. Cho ◽  
M.R. Park ◽  
D.N. Kwon ◽  
E.K. Lee ◽  
S.J. Kang ◽  
...  

The present study was conducted to investigate the developmental competence of male and female somatic cell derived nuclear transfer (NT) porcine embryos and also the production and survival efficiency of cloned male and female piglets. Maturation of porcine COCs was accomplished by incubation in NCSU-23 medium supplemented with 0.6 mM cysteine, 10% porcine follicular fluid, 1 mM dibutyryl cyclic adenosine monophosphate, and 0.1 IU/mL human menopausal gonadotrophin for 20 h and then culture without dbcAMP and hMG for another 18 to 24 h. Fetal cells were isolated from a male fetus and two female fetuses, and cultured in ES-DMEM medium containing 10% FCS. Enucleated oocytes were fused with fetal fibroblasts (passage 4 to 15). Reconstructed embryos were cultured in NCSU-23 with 4 mg/mL BSA under mineral oil at 39°C in 5% CO2 in air for up to 6 days. NT eggs that had been activated with electric pulses and cultured for 1 or 2 days were transported to the experimental station in modified NCSU-23 with antibiotics. NT embryos were surgically transferred into the oviducts of recipients between Day 27 and Day 30; pregnancy was determined by ultrasound. The potential of NT embryos to develop into blastocysts was not different among donor cells of different origins. However, the mean cell number of in vivo female and male blastocysts (83.8 ± 46.2 to 99.2 ± 55.7) was higher than in in vitro culture of NT groups (31.4 ± 8.29 to 33.2 ± 10.15). A total of 11,535 NT embryos (1- to 8-cell stage) were surgically transferred into 66 surrogate gilts. Among fourteen pregnant gilts, four recipients aborted during the period of conception. Five pregnant gilts delivered fifteen female piglets, 1.28 ± 0.33 kg (0.48∼1.83 kg) in female piglets and 0.84±0.25 kg (0.45∼1.25 kg) in male piglets. Nine live cloned female (60.0%) and four male piglets (18.2%) were produced. According to these results, survival rates and birth weights of female cloned piglets were higher than those of cloned male piglets (P < 0.05). This study suggests that use of female, compared with male, fetal fibroblast cells as nuclear donors may increase cloning outcomes. This work was supported in part by a grant program from RDA(Biogreen21) and Cho-A, Republic of Korea.


2015 ◽  
Vol 27 (1) ◽  
pp. 110
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
G. A. Kim ◽  
H. N. Suh ◽  
Y. K. Jo ◽  
...  

Although dog cloning technology has been applied to conservation of endangered canids, propagation of elite dogs and production of transgenic dogs, the efficiency of cloning is still very low. To help overcome this problem, we evaluated the effect of treating donor cells with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACi), on dog cloning efficiency. Relative mRNA expression of the bax1, bcl2, and Dnmt1 in fibroblasts treated with different concentrations (0, 1, 10, 50 μM) of SAHA and durations (0, 20, 44 h) were assessed using real-time polymerase chain reaction. After determining an optimum concentration and duration, histone acetylation levels (H3K9, H4K5/K8/K12/K16) of SAHA-treated cells were analysed using immunostaining. The SAHA-treated cells were used as donor cells for somatic cell nuclear transfer, and activated reconstructed embryos were transferred to recipients. Pregnancy diagnosis was performed by ultrasonography at least 29 days after the embryo transfer. All experiments were repeated more than 3 times and the data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). An unpaired t-test was used to compare transcripts levels and fluorescence intensities. A chi-squared test was used to compare the implantation rates. The bax1/bcl2 ratio of the 1 μM SAHA group was similar to that of control but significantly increased in the 10 μM and 50 μM groups. Expression of Dnmt1 was decreased in the 1 μM SAHA group, and the 10 μM and 50 μM groups showed the lowest expression compared with the control group. Although the bax1/blc2 ratio was not affected by the SAHA treatment duration, 20-h treatment group showed significantly decreased Dnmt1 levels compared with control group. As a pan-HDAC inhibitor, 1 μM for 20 h of SAHA treatment significantly increased acetylation of H3K9, H4K5, H4K8, and H4K16. For control and SAHA groups, a total of 76 and 64 cloned embryos were produced and transferred to 7 and 5 recipients, respectively. Three fetuses were diagnosed in both groups but there was no significant difference in the pregnancy rate. In conclusion, although SAHA treatment as used in this study significantly decreased bax/bcl2 and Dnmt1 transcripts of donor nuclei, as well as increased H3 and H4 acetylation, it would not enough to increase in vivo developmental competence of cloned dog embryos.This study was supported by RDA (#PJ008975022014), IPET (#311062–04–3SB010), Research Institute for Veterinary Science and the BK21 plus program.


2018 ◽  
Vol 30 (1) ◽  
pp. 152
Author(s):  
G. Maeng ◽  
X. Pan ◽  
S. Das ◽  
K.-D. Choi ◽  
N. Koyano-Nakagawa ◽  
...  

In transplantation medicine, the embryo complementation method has been introduced as a possible means to produce an organ derived from the desired cells. Previously, our laboratory demonstrated that the transcription factor Ets variant 2 (Etv2) regulates hematoendothelial lineage differentiation. In this study, ETV2-null pig fibroblasts were generated using the CRISPR/Cas9 system, and these cells were utilised as donor cells for porcine somatic cell nuclear transfer (SCNT) to produce mutant embryos. After transplantation of these mutant embryos into 4 surrogate gilts, 12 fetuses were found in 2 gilts at E-18. Eight of those embryos lacked hematoendothelial lineages, were nonviable, and lacked ETV2 by PCR analysis. To rescue the hematoendotheilal phenotypes, the blastomeres were collected from green fluorescent protein (GFP)-expressing embryos, which were generated by SCNT with the pig GFP-fibroblasts. Then, the GFP-blastomeres were injected into the ETV2-null SCNT embryos (4 GFP-blastomeres per a complementation on average) at the morula stage. These complemented embryos were transferred into 4 surrogate gilts. Two surrogate gilts were not pregnant, but 2 pregnant gilts harbored complemented fetuses. Complemented fetuses were evaluated at E26 and had intact and fully complemented hematoendothelial lineages, which were confirmed by CFU-assays, fluorescence-activated cell sorting (FACS), qPCR, and immunohistochemistry. Importantly, the hematoendotheilal lineages completely expressed GFP. In the complemented fetuses, the GFP-positive cells were observed throughout the body at more than 70%. These studies provide a platform for the in vivo production of functional hematoendothelial tissues from pluripotent stem cells (such as human pluripotent cells) using the embryo complementation technique.


2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.


Author(s):  
А.А. Раецкая ◽  
С.В. Калиш ◽  
С.В. Лямина ◽  
Е.В. Малышева ◽  
О.П. Буданова ◽  
...  

Цель исследования. Доказательство гипотезы, что репрограммированные in vitro на М3 фенотип макрофаги при введении в организм будут существенно ограничивать развитие солидной карциномы in vivo . Методика. Рост солидной опухоли инициировали у мышей in vivo путем подкожной инъекции клеток карциномы Эрлиха (КЭ). Инъекцию макрофагов с нативным М0 фенотипом и с репрограммированным M3 фенотипом проводили в область формирования солидной КЭ. Репрограммирование проводили с помощью низких доз сыворотки, блокаторов факторов транскрипции STAT3/6 и SMAD3 и липополисахарида. Использовали две схемы введения макрофагов: раннее и позднее. При раннем введении макрофаги вводили на 1-е, 5-е, 10-е и 15-е сут. после инъекции клеток КЭ путем обкалывания макрофагами с четырех сторон область развития опухоли. При позднем введении, макрофаги вводили на 10-е, 15-е, 20-е и 25-е сут. Через 15 и 30 сут. после введения клеток КЭ солидную опухоль иссекали и измеряли ее объем. Эффект введения макрофагов оценивали качественно по визуальной и пальпаторной характеристикам солидной опухоли и количественно по изменению ее объема по сравнению с группой без введения макрофагов (контроль). Результаты. Установлено, что M3 макрофаги при раннем введении от начала развития опухоли оказывают выраженный антиопухолевый эффект in vivo , который был существенно более выражен, чем при позднем введении макрофагов. Заключение. Установлено, что введение репрограммированных макрофагов M3 ограничивает развитие солидной карциномы в экспериментах in vivo . Противоопухолевый эффект более выражен при раннем введении М3 макрофагов. Обнаруженные в работе факты делают перспективным разработку клинической версии биотехнологии ограничения роста опухоли, путем предварительного программирования антиопухолевого врожденного иммунного ответа «в пробирке». Aim. To verify a hypothesis that macrophages reprogrammed in vitro to the M3 phenotype and injected into the body substantially restrict the development of solid carcinoma in vivo . Methods. Growth of a solid tumor was initiated in mice in vivo with a subcutaneous injection of Ehrlich carcinoma (EC) cells. Macrophages with a native M0 phenotype or reprogrammed towards the M3 phenotype were injected into the region of developing solid EC. Reprogramming was performed using low doses of serum, STAT3/6 and SMAD3 transcription factor blockers, and lipopolysaccharide. Two schemes of macrophage administration were used: early and late. With the early administration, macrophages were injected on days 1, 5, 10, and 15 following the injection of EC cells at four sides of the tumor development area. With the late administration, macrophages were injected on days 10, 15, 20, and 25. At 15 and 30 days after the EC cell injection, the solid tumor was excised and its volume was measured. The effect of macrophage administration was assessed both qualitatively by visual and palpation characteristics of solid tumor and quantitatively by changes in the tumor volume compared with the group without the macrophage treatment. Results. M3 macrophages administered early after the onset of tumor development exerted a pronounced antitumor effect in vivo , which was significantly greater than the antitumor effect of the late administration of M3 macrophages. Conclusion. The observed significant inhibition of in vivo growth of solid carcinoma by M3 macrophages makes promising the development of a clinical version of the biotechnology for restriction of tumor growth by in vitro pre-programming of the antitumor, innate immune response.


2020 ◽  
Author(s):  
Johannes Karges ◽  
Shi Kuang ◽  
Federica Maschietto ◽  
Olivier Blacque ◽  
Ilaria Ciofini ◽  
...  

<div>The use of photodynamic therapy (PDT) against cancer has received increasing attention overthe recent years. However, the application of the currently approved photosensitizers (PSs) is somehow limited by their poor aqueous solubility, aggregation, photobleaching and slow clearance from the body. To overcome these limitations, there is a need for the development of new classes of PSs with ruthenium(II) polypyridine complexes currently gaining momentum. However, these compounds generally lack significant absorption in the biological spectral window, limiting their application to treat deep-seated or large tumors. To overcome this drawback, ruthenium(II) polypyridine complexes designed in silico with (E,E’)-4,4´-bisstyryl 2,2´-bipyridine ligands showed impressive 1- and 2-Photon absorption up to a magnitude higher than the ones published so far. While non-toxic in the dark, these compounds were found phototoxic in various 2D monolayer cells, 3D multicellular tumor spheroids and be able to eradicate a multiresistant tumor inside a mouse model upon clinically relevant 1-Photon and 2 Photon excitation.</div>


Author(s):  
Pavani C H

Hyperlipidemia is the immediate results of the excessive fat intake in food. This results in the elevated levels of cholesterol and triglycerides in the blood. This leads to heart conditions like CAD, hypertension, congestive heart failure as risk factors which can be lethal. There are many drugs to treat and control the lipids levels in the body. These drugs are either designed to prevent LDL accumulation and VLDL synthesis. Some drugs also lower the elevated levels of saturated lipids in the body. But many drugs are known to cause side effects and adverse effects; therefore, alternatives to the drugs are the subjects for current investigations. Herbs and medicinal plants are used as treatment sources for many years. They have been used in the Indian medical systems like Ayurveda, Siddha etc. As the application of herbs in the treatment is growing, there is an urgent need for the establishment of Pharmacological reasoning and standardization of the activity of the medicinal plants. Chloris paraguaiensis Steud. is Poyaceae member that is called locally as Uppugaddi. Traditionally it is used to treat Rheumatism, Diabetes, fever and diarrhoea. The chemical constituents are known to have anti-oxidant properties and most of the anti-oxidants have anti-hyperlipidemic activity too. Since the plant has abundant flavonoid and phenol content, the current research focusses on the investigation of the anti-hyperlipidemic activity of the plant Chloris extracts. Extracts of Chloris at 200mg/kg showed a comparably similar anti hyperlipidemia activity to that of the standard drug. The extracts showed a dose based increase in the activity at 100 and 200mg/kg body weight.


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