Improved Culture System Research Articles

Inducible Pluripotent Stem Cells to Model and Treat Inherited Degenerative Diseases of the Outer Retina: 3D-Organoids Limitations and Bioengineering Solutions.

Inherited retinal degenerations (IRD) affecting either photoreceptors or pigment epithelial cells cause progressive visual loss and severe disability, up to complete blindness. Retinal organoids (ROs) technologies opened up the development of human inducible pluripotent stem cells (hiPSC) for disease modeling and replacement therapies. However, hiPSC-derived ROs applications to IRD presently display limited maturation and functionality, with most photoreceptors lacking well-developed outer segments (OS) and light responsiveness comparable to their adult retinal counterparts. In this review, we address for the first time the microenvironment where OS mature, i.e., the subretinal space (SRS), and discuss SRS role in photoreceptors metabolic reprogramming required for OS generation. We also address bioengineering issues to improve culture systems proficiency to promote OS maturation in hiPSC-derived ROs. This issue is crucial, as satisfying the demanding metabolic needs of photoreceptors may unleash hiPSC-derived ROs full potential for disease modeling, drug development, and replacement therapies.

Hypertrophied human adipocyte spheroids as in vitro model of weight gain and adipose tissue dysfunction.

Adipocyte enlargement is a key feature of obesity and associated with insulin resistance and metabolic disease The cause and consequences of adipocyte enlargement have remained hard to study in vitro due to a lack of human cell models with representative morphology This paper provides an easily set up spheroid culture method, HUVAS (human unilocular vascularized adipocyte spheroids), for the differentiation and culturing of human adipocytes with a more unilocular morphology We show that providing adipocyte progenitors with a vascular differentiation niche is key for achieving in vitro differentiated adipocytes with large lipid droplets Lipid treatment of the HUVAS spheroids can further adipocyte enlargement and induce cellular dysfunction, mimicking the in vivo effects of weight gain The model will allow a wider research community to perform mechanistic studies of the factors impacting on human adipocyte differentiation and growth, increasing our understanding of how obesity develops and why it has such detrimental consequences on whole body metabolism ABSTRACT: The rise in obesity prevalence has created an urgent need for new and improved methods to study human adipocytes and the pathogenic effects of weight gain in vitro. Despite the proven advantage of culturing adipocyte progenitors as 3D structures, the majority of studies continue to use traditional 2D cultures which result in small, multilocular adipocytes with poor representability. We hypothesized that providing differentiating pre-adipocytes with a vascular growth niche would mimic in vivo adipogenesis and improve the differentiation into unilocular adipocytes. Here we present HUVAS (human unilocular vascularized adipocyte spheroids), a simple, easily applicable culture protocol that allows for the differentiation of human adipocytes with a more unilocular morphology and larger lipid droplets than previous protocols. Moreover, we offer a protocol for inducing adipocyte enlargement in vitro, resulting in larger lipid droplets and development of several key features of adipocyte dysfunction, including altered adipokine secretion, impaired lipolysis and insulin resistance. Taken together, our HUVAS model offers an improved culture system for studying the cellular and molecular mechanisms causing metabolic dysfunction and inflammation in human adipose tissue during weight gain.

Optimization of donor cell cycle synchrony, maturation media and embryo culture system for somatic cell nuclear transfer in the critically endangered Vietnamese Ỉ pig

Our aim was to establish an efficient culture system to produce embryos by SCNT of the endangered Vietnamese Ỉ pig. Reducing the serum concentration from 10.0% to 0.2% during culture efficiently synchronized Ỉ pig fibroblasts used as donor cells at the G0/G1 stage. Oocyte maturation in a defined porcine oocyte medium (POM) supplemented with EGF and gonadotrophins resulted in higher cleavage and blastocyst rates compared with a non-defined POM containing pig follicular fluid (but without EGF) and both the defined and non-defined variants of NCSU-37. For embryo culture PZM3 and PZM5 media were superior to NCSU-37, in terms of the percentage of cleaved embryos. Addition of serum to PZM3 medium on Day 5 of culture (Day 0 = SCNT) improved blastocyst development. When SCNT embryos were transferred at the blastocyst stage, 7 of 11 recipients became pregnant. However, live offspring were not obtained. In conclusion, we established a system for the production of Ỉ pig embryos by SCNT and achieved blastocyst production rate at 26.4% by improving culture systems for donor cells, oocytes and embryos culture. Transfer of embryos resulted in pregnancies; however, live offspring were not obtained.

Cyclosporin A and FGF signaling support the proliferation/survival of mouse primordial germ cell-like cells in vitro†.

Primordial germ cells (PGCs) are the founding population of the germ cell lineage that undergo a multistep process to generate spermatozoa or oocytes. Establishing an appropriate culture system for PGCs is a key challenge in reproductive biology. By a chemical screening using mouse PGC-like cells (mPGCLCs), which were induced from mouse embryonic stem cells, we reported previously that forskolin and rolipram synergistically enhanced the proliferation/survival of mPGCLCs with an average expansion rate of ~20-fold. In the present study, we evaluated other chemicals or cytokines to see whether they would improve the current mPGCLC culture system. Among the chemicals and cytokines examined, in the presence of forskolin and rolipram, cyclosporin A (CsA) and fibroblast growth factors (FGFs: FGF2 and FGF10) effectively enhanced the expansion of mPGCLCs in vitro (~50-fold on average). During the expansion by CsA or FGFs, mPGCLCs comprehensively erased their DNA methylation to acquire a profile equivalent to that of gonadal germ cells in vivo, while maintaining their highly motile phenotype as well as their transcriptional properties as sexually uncommitted PGCs. Importantly, these mPGCLCs robustly contributed to spermatogenesis and produced fertile offspring. Furthermore, mouse PGCs (mPGCs) cultured with CsA ex vivo showed transcriptomes and DNA methylomes similar to those of cultured mPGCLCs. The improved culture system for mPGCLCs/mPGCs would be instructive for addressing key questions in PGC biology, including the mechanisms for germ cell migration, epigenetic reprogramming, and sex determination of the germline.

Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.

Time-Course Changes and Role of Autophagy in Primary Spinal Motor Neurons Subjected to Oxygen-Glucose Deprivation: Insights Into Autophagy Changes in a Cellular Model of Spinal Cord Ischemia.

Spinal cord ischemia is a severe clinical complication induced by thoracoabdominal aortic surgery, severe trauma, or compression to the spinal column. As one of the most important functional cells in the spinal cord, spinal motor neurons (SMNs) suffer most during the process since they are vulnerable to ischemic injury due to high demands of energy. Previous researches have tried various animal models or organotypic tissue experiments to mimic the process and get to know the pathogenesis and mechanism. However, little work has been performed on the cellular model of spinal cord ischemia, which has been hampered by the inability to obtain a sufficient number of pure primary SMNs for in vitro study. By optimizing the isolation and culture of SMNs, our laboratory has developed an improved culture system of primary SMNs, which allows cellular models and thus mechanism studies. In the present study, by establishing an in vitro model of spinal cord ischemia, we intended to observe the dynamic time-course changes of SMNs and investigate the role of autophagy in SMNs during the process. It was found that oxygen-glucose deprivation (OGD) resulted in destruction of neural networks and decreased cell viability of primary SMNs, and the severity increased with the prolonging of the OGD time. The OGD treatment enhanced autophagy, which reached a peak at 5 h. Further investigation demonstrated that inhibition of autophagy exacerbated the injury, evidencing that autophagy plays a protective role during the process.

Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle.

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity.

A simple, efficient and economical method for isolating and culturing human umbilical cord blood‑derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) hold broad therapeutic potential in various diseases, however, it is difficult to produce sufficient numbers of MSCs for clinical application, therefore, improved culture systems are required. The present study aimed to develop a novel method for isolating and culturing human umbilical cord blood‑derived mesenchymal stromal cells (hUCB‑MSCs). A sequential culture method was developed that uses two types of culture media to optimize the isolation and culture of hUCB‑MSCs. First, DMEM supplemented with mesenchymal stem cell growth supplement was used to improve the colony formation and primary culture success rates of hUCB‑MSCs. Then, after removing the heterogeneous cell population, ordinary DMEM was used from the fourth passage. This method obtained hUCB‑MSCs with high culture efficiency and at a greatly reduced cost. The optimal culture conditions were determined and the hUCB‑MSCs were phenotypically characterized after passaging. Taken together, this simple, efficient and economical method can produce a large number of high‑quality hUCB‑MSCs in <1month, therefore facilitating the future clinical applications of hUCB‑MSCs.

COS-7-based model: methodological approach to study John Cunningham virus replication cycle

John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

A Refined Culture System for Human Induced Pluripotent Stem Cell-Derived Intestinal Epithelial Organoids.

SummaryGut epithelial organoids are routinely used to investigate intestinal biology; however, current culture methods are not amenable to genetic manipulation, and it is difficult to generate sufficient numbers for high-throughput studies. Here, we present an improved culture system of human induced pluripotent stem cell (iPSC)-derived intestinal organoids involving four methodological advances. (1) We adopted a lentiviral vector to readily establish and optimize conditioned medium for human intestinal organoid culture. (2) We obtained intestinal organoids from human iPSCs more efficiently by supplementing WNT3A and fibroblast growth factor 2 to induce differentiation into definitive endoderm. (3) Using 2D culture, followed by re-establishment of organoids, we achieved an efficient transduction of exogenous genes in organoids. (4) We investigated suspension organoid culture without scaffolds for easier harvesting and assays. These techniques enable us to develop, maintain, and expand intestinal organoids readily and quickly at low cost, facilitating high-throughput screening of pathogenic factors and candidate treatments for gastrointestinal diseases.

Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs.MethodshUCB-MSCs were maintained under hypoxic conditions (3% O2) with 1.8 mM Ca2+ during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs.ResultsThe proliferation of Ca2+/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca2+/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca2+/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs.ConclusionsThese findings suggest that a Ca2+/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.

Secondary Metabolite Production from Industrially Relevant Bacteria is Enhanced by Organic Nanofibers.

Streptomycetes are exploited for the production of a wide range of secondary metabolites, including antibiotics. Therefore, both academic and industrial research efforts are focused on enhancing production of these precious metabolites. So far, this has been mostly achieved by classical or recombinant genetic techniques, in association with process optimization for either submerged or solid state fermentation. New cultivation approaches addressing the natural mycelial growth and life cycle would allow the biosynthetic potential of filamentous strains to be much better exploited. We developed a cultivation system for antibiotic-producing microorganisms which involves electrospun organic nanofibers deposited onto agar plates or immersed in liquid media. Dense filamentous networks of branched hyphae formed by bacterial colonies were found to wrapped around the fibers. We analyzed the effects of fibers on growth and antibiotic production in Streptomyces lividans, and found that the actinorhodin, undecylprodigiosin and calcium dependent antibiotic productions were positively modulated, with a two- to sixfold enhancement compared to standard culture conditions. Highlighting the secondary metabolism-promoting role of nanofibers in bacterial cultures, these results open a route to the design of improved culture systems for microorganisms based on organic nanostructures.

Dosage effects of ZP2 and ZP3 heterozygous mutations cause human infertility.

The zona pellucida (ZP) is an extracellular matrix universally surrounding mammalian eggs, which is essential for oogenesis, fertilization, and pre-implantation embryo development. Here, we identified two novel heritable mutations of ZP2 and ZP3, both occurring in an infertile female patient with ZP-abnormal eggs. Mouse models with the same mutations were generated by CRISPR/Cas9 gene editing system, and oocytes obtained from female mice with either single heterozygous mutation showed approximately half of the normal ZP thickness compared to wild-type oocytes. Importantly, oocytes with both heterozygous mutations showed a much thinner or even missing ZP that could not avoid polyspermy fertilization, following the patient's pedigree. Further analysis confirmed that precursor proteins produced from either mutated ZP2 or ZP3 could not anchor to oocyte membranes. From these, we conclude that ZP mutations have dosage effects which can cause female infertility in humans. Finally, this patient was treated by intracytoplasmic sperm injection (ICSI) with an improved culture system and successfully delivered a healthy baby.

Development of an improved in vitro culture system for Cryptosporidium parvum

The protozoan parasite Cryptosporidium parvum is a leading cause of pediatric diarrhea in developing countries, yet effective treatments for the disease are lacking due to the difficulty of studying Cryptosporidium in the laboratory. Specifically, current culturing methods rely on adenocarcinoma cell lines that do not support continual growth of C. parvum in vitro, hindering the development of reagents such as stage‐specific antibodies and genetic tools for this parasite. To better mimic the native intracellular niche of C. parvum, we have utilized primary intestinal epithelial cell (IEC) monolayers as an improved system for the study of C. parvum. We have found that human and mouse IECs support enhanced growth of C. parvum compared to adenocarcinoma cell lines. Importantly, parasite growth in mouse IECs, the first mouse cell line to support robust asexual C. parvum growth, enabled us to generate a unique panel of monoclonal antibodies against the intracellular life cycle stages of the parasite. We have also developed quantitative PCR assays to measure expression of genes associated with sexual‐stage development of the parasite independent of absolute parasite replication. With the development of these new tools and the enhanced culture system, we are now able to screen culture conditions mimicking the hypoxic, reducing environment of the small intestine to identify conditions that further improve C. parvum growth and/or complete development of the life cycle in vitro. Additionally, we are also screening a panel of gut microbial metabolites for their effects on C. parvum growth to gain insight into the complex community interactions that may contribute to C. parvum infection in vivo. In all, these new tools and improved culture system will facilitate the study of Cryptosporidium in vitro, allowing us to gain a better understanding of the parasite's biology as a means to more effective therapeutics for the disease in the future.Support or Funding InformationThis work is supported by the Bill and Melinda Gates Foundation.

Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications.

554. Efficient Scalable Purification of rAAV1 Using Ion-Exchange and Gel-Filtration Chromatography To Avoid Ultracentrifugation Procedure

[Background]: Recombinant adeno-associated virus vectors (rAAVs) are attractive tools for gene transfer and are evaluated for use in human gene therapies. However, the current production and purification of rAAVs based on CsCl or iodixanol density ultracentrifugation are not suitable for large-scale production in GMP (Good Manufacturing Practice) and inadequate to meet SOP (Standard Operating Procedure). In this study, we elaborate the novel methods for production and purification of type1 AAV vector (rAAV1) using chromatography steps (ion-exchange and gel-filtration chromatography). We study here for the chromatography-based purification of rAAV1, because Glybera, the first rAAV-based drug with the official approval of the European Medicines Agency (EMA), was produced as the rAAV1.[Methods]: rAAV1 (scAAV1-CBA-EGFP) was produced by triple-transfection of HEK293 cells in serum free medium with PEI (polyethyleneimine). The transfected cells were cultured for five days without the medium replacement. The cultured medium was subjected to ultrafiltration and concentration by the Hollow Fiber (exclusion limit is 750 kDa). After reducing protein debris by 1/3-saturated ammonium sulfate precipitation, rAAV1 was precipitated in 1/2-saturated ammonium sulfate solution. Subsequently, rAAV1 was dissolved in PBS (with 3mM MgCl2) and dialyzed against 20mM Tris-HCl (pH8.0) buffer containing 50mM NaCl and 3mM MgCl2 for 3-4 hours. Next, the sample was dialyzed against 20mM Tris-HCl (pH8.0) buffer containing 5mM NaCl and 3mM MgCl2 for 20 hours with a buffer replacement. After the sample was diluted with dH2O to adjust conductivity around 1.0mSV/cm, it was loaded at a rate of 1ml/min to tandem dual cation-exchange (Mustang SXT) and anion-exchange (Mustang QXT) high-capacity membranes. After removing the Mustang SXT, rAAV1 was eluted from the Mustang QXT by stepwise NaCl gradient and the vector-containing fraction was eluted by a buffer containing 140mM NaCl. After ultrafiltration with Ultracel 30K, rAAV1 was finally purified by gel-filtration chromatography with Superdex 200 HR using AKTA Explorer 100 HPLC system. The physiological and biological properties of the resultant rAAV samples were characterized by qPCR, electron micrograph, fluorescent micrograph and SDS-PAGE.[Results]: The purified rAAV1 represented three major bands (VP1, VP2, VP3) on SDS-PAGE and more than 90% of rAAV1 particles were fully packaged virions by electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 4.6×1013 vg/ml from the 2×109 of HEK293 cells. This system would be scalable up to 1015 with our improved culture system and membrane capsule.[Conclusion]: We refined rAAV1-production protocol with high purity with minimum empty particles. This steamlined method with high performance scalable ion-exchange membrane would be useful for the large scale production of rAAV1 to facilitate clinical studies.

Redifferentiation of dedifferentiated chondrocytes in a novel three-dimensional microcavitary hydrogel.

Although chondrocytes exist in native cartilage all over the body, it is still a challenge to use them as therapeutic cells for cartilage tissue engineering (TE) because of their easy dedifferentiation in in vitro culture. An improved culture system to maintain the characteristics of chondrocytes or recover their chondrocytic phenotype should be developed. In this study, we have set up an innovative microcavitary alginate hydrogel in an easy way. We compared this culture system with the conventional hydrogel and found that the microcavitary hydrogel exhibited outstanding superiorities in helping the dedifferentiated chondrocytes recover the capability for synthesizing cartilaginous extracellular matrix. In addition, we explored the correlation between chondrocyte redifferentiation in microcavitary hydrogels and changes in p38 and Erk1/2 activity. Our findings indicated that this microcavitary hydrogel would be a promising culture system to provide sufficient competent cells for cartilage regeneration and TE.

Robust, microfabricated culture devices with improved control over the soluble microenvironment for the culture of embryonic stem cells

The commercial use of stem cells continues to be constrained by the difficulty and high cost of developing efficient and reliable production protocols. The use of microfabricated systems combines good control over the cellular microenvironment with reduced use of resources in process optimization. Our previously reported microfabricated culture device was shown to be suitable for the culture of embryonic stem cells but required improvements to robustness, ease of use, and dissolved gas control. In this report, we describe a number of improvements to the design of the microfabricated system to significantly improve the control over shear stress and soluble factors, particularly dissolved oxygen. These control improvements are investigated by finite element modeling. Design improvements also make the system easier to use and improve the robustness. The culture device could be applied to the optimization of pluripotent stem cell growth and differentiation, as well as the development of monitoring and control strategies and improved culture systems at various scales.

Impact of In-Pond Nursery of Shrimp (&lt;i&gt;Penaeus monodon&lt;/i&gt;) Post Larvae on the Survival Rate and Production Under Modified Improved Culture System

To observe the effectiveness of short time in-pond nursery of post larvae (PL) on the survival rate and production of shrimp (Penaeus monodon) under modified improved culture system, an experiment was conducted in earthen ponds at Brackishwater Station of Bangladesh Fisheries Research Institute, Khulna. Results of the experiment revealed that short time in-pond nursery of PL provided the significantly higher (p&lt;0.05) survival rate (58%) than direct stocking of PL (47.81%) in the rearing pond. In-pond nursery of PL provided 9.95% better survival rate than direct stocking. The production of shrimp was 661 kg/ha under in-pond nursery of PL and it was 534 kg/ha under direct stocking of PL in rearing ponds. The production of shrimp was also significantly higher (p&lt;0.05) under in-pond nursery system than direct stocking of PL in rearing ponds. Final weight gain and Food Conversion Ratio (FCR) values were similar in both the treatments. Water quality parameters in both stocking systems were insignificant and it was within the range of shrimp culture. In this study, the increment in production under in-pond nursery system was mainly due to the increase of survival rate in the respective treatment was ascertained.DOI: http://dx.doi.org/10.3329/pa.v19i2.16958 Progress. Agric. 19(2): 167 - 175, 2008

The impact of culture on epigenetic properties of pluripotent stem cells and pre-implantation embryos

Cultured pluripotent stem cells hold great promise for regenerative medicine. Considerable efforts have been invested into the refinement and definition of improved culture systems that sustain self-renewal and avoid differentiation of pluripotent cells invitro. Recent studies have, however, found that the choice of culture condition has a significant impact on epigenetic profiles of cultured pluripotent cells. Mouse and human ESCs (embryonic stem cells) show substantial epigenetic differences that are dependent on the culture condition, including global changes to DNA methylation and histone modifications and, in female human ESCs, to the epigenetic process of X chromosome inactivation. Epigenetic perturbations have also been detected during culture of pre-implantation embryos; limited research undertaken in mouse suggests a direct effect of the invitro environment on epigenetic processes in this system. Widespread epigenetic changes induced by the culture condition in stem cells thus emphasize the necessity for extensive research into both immediate and long-term epigenetic effects of embryo culture during assisted reproductive technologies.