Culture conditions for human embryonic stem cells

Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.

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Isolation and culture of pluripotent cells from in vitro produced porcine embryos

Zygote ◽

10.1017/s0967199404002679 ◽

2004 ◽

Vol 12 (1) ◽

pp. 43-48 ◽

Cited By ~ 39

Author(s):

Ming Li ◽

Yong-Hai Li ◽

Yi Hou ◽

Xiao-Fang Sun ◽

Qingyuan Sun ◽

...

Keyword(s):

Fetal Bovine Serum ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Es Cell ◽

Embryonic Fibroblasts ◽

Feeder Layers ◽

Edta Solution ◽

Fetal Bovine

The present study was designed to examine whether in vitro produced porcine embryos can be used to establish an embryonic stem (ES) cell line. Porcine embryos were produced by in vitro maturation and in vitro fertilization. Embryos at the 4-cell to blastocyst stages were cultured in an ES medium containing 16% fetal bovine serum with mouse embryonic fibroblasts as a feeder layer. It was found that ES-like colonies were derived only from blastocysts. When these ES-like colonies were separated in 0.25% trypsin–0.02% EDTA solution and cultured again, ES-like colonies were further observed in the subsequent culture until the fourth passage. The cells from ES-like colonies showed positive alkaline phosphatase activity. Some cells from the colonies differentiated into several types of cells in vitro when they were cultured in the medium without feeder layers and leukemin inhibitory factor. Embryoid bodies were also formed when the cells were cultured in a suspension status. These results indicate that porcine ES-like cells can be derived from in vitro produced porcine blastocysts and these ES-like cells are pluripotent. The culture system used in the present study is useful to isolate and culture ES cells from in vitro produced porcine embryos.

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293 EMBRYONIC STEM-LIKE CELLS DERIVED FROM PORCINE INNER CELL MASS CELLS ISOLATED BY DIFFERENT METHODS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab293 ◽

2008 ◽

Vol 20 (1) ◽

pp. 226

Author(s):

X. A. Wolf ◽

M. A. Rasmussen ◽

K. Schauser ◽

P. Maddox-Hyttel

Keyword(s):

Inner Cell Mass ◽

Cell Mass ◽

Calf Serum ◽

Embryonic Stem ◽

Culture Conditions ◽

Embryonic Fibroblasts ◽

Serum Replacement ◽

Sox2 Expression ◽

Differentiated Cells

The aim was to examine isolation methods and culture conditions for the establishment of embryonic stem-like cells derived from the inner cell mass (ICM) of porcine embryos. A total of 83 zona pellucida-enclosed (ZPE) and 88 hatched (ZPH) porcine in vivo Days 5–7 blastocysts were assigned for ICM isolation by: (A) manual dissection by needles (ZPE: n = 10; ZPH, n = 15), (B) immunosurgical isolation (ZPE: n = 30; ZPH: n = 10), (C) immunosurgery and manual cleaning (ZPE: n = 11; ZPH: n = 40), and (D) culture of whole blastocysts (ZPE: n = 42; ZPH: n = 23). Culture was done on mouse embryonic fibroblasts (MEF) at 5% O2 (for A–C) and 20% O2 (for D) in DMEM with fetal calf serum (FCS), serum replacement, and leukemia inhibitory factor (LIF). Outgrowth colonies (OC) were evaluated by phase contrast and subjected to either (1) physical passage and RT-PCR for Oct-4, Nanog, and Sox2; or (2) immunocytochemical localization of Oct-4 at Days 6–7. Five OC categories were defined: (I) epiblast-like colonies (multilayered ICM-like with homogeneous nuclear Oct-4 staining), (II) ES-like colonies with few surrounding cells (ES-like cells with homogeneous nuclear Oct-4 staining surrounded by few cells), (III) clearly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4 staining and clear demarcation to many differentiated cells), (IV) poorly delineated ES-like colonies (ES-like cells with homogeneous or heterogeneous nuclear Oct-4-staining and poor demarcation to many differentiated cells), and (V) differentiated colonies (heterogeneous cell populations lacking nuclear Oct-4 staining). Oct-4 staining was supported by expression of Oct-4, often associated with Nanog and Sox2. The attachment rates were similar for methods A, B, and C, being higher for ZPH (75–80%) than for ZPE (40–50%) blastocysts. Method D gave 42% attachment for ZPE, but only 23% for ZPH blastocysts at 5% O2, whereas the figures at 20% O2 were 63% and 80%, respectively. Methods B and C gave the highest proportion of OCs in categories II–IV (ES-like cell-containing), and the ZPE-derived OCs exhibited a more homogeneous nuclear Oct-4 staining than the ZPH-derived. Method D gave the highest proportion of category II colonies. Passage was performed from OC categories II–IV (with ES-like cells). For method C, 24 OCs resulted in 18 (75%) passage 1 (P1) colonies. In 11, 6, 3, and 2 cases, they were passed to P2, P3, P4, and P5, respectively, maintaining ES-like morphology before they went into quiescence or differentiation. However, most colonies attained cytoplasmic Oct-4 staining and lost Oct-4, Nanog, and Sox2 expression at P1 or P2; only a single colony maintained Nanog and Sox2 expression up to P3. For isolation method D, 10 OCs resulted in 3 (30%) P1 colonies, but only one continued to P2 before differentiation. In conclusion, all methods consistently gave ES-like OCs. Whole blastocysts at 20% O2 gave the highest attachment rates. However, immunosurgery, eventually followed by manual cleaning, tended to result in the highest proportion of OCs presenting ES-like cells, with the ZPE-derived OCs exhibiting the most homogeneous Oct-4 staining. When subjected to passage, a few colonies maintained ES-like morphology up to P5, but expression of pluripotency markers was lost during the initial passages.

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Application of Multiplex PCR for Characterization of Human Embryonic Stem Cells (hESCs) and Its Differentiated Progenies

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110370211 ◽

2010 ◽

Vol 15 (6) ◽

pp. 630-643 ◽

Cited By ~ 6

Author(s):

Murali Krishna Mamidi ◽

Rajarshi Pal ◽

Ramesh Bhonde ◽

Zubaidah Zakaria ◽

Satish Totey

Keyword(s):

Gene Expression ◽

Embryoid Body ◽

Embryonic Stem ◽

Cost Effective ◽

Turnaround Time ◽

Mouse Embryonic Fibroblast ◽

Gene Markers ◽

Real Time Quantitative Pcr ◽

Hesc Lines

Techniques to evaluate gene expression profiling, including real-time quantitative PCR, TaqMan® low-density arrays, and sufficiently sensitive cDNA microarrays, are efficient methods for monitoring human embryonic stem cell (hESC) cultures. However, most of these high-throughput tests have a limited use due to high cost, extended turnaround time, and the involvement of highly specialized technical expertise. Hence, there is a paucity of rapid, cost-effective, robust, yet sensitive methods for routine screening of hESCs. A critical requirement in hESC cultures is to maintain a uniform undifferentiated state and to determine their differentiation capacity by showing the expression of gene markers representing all germ layers, including ecto-, meso-, and endoderm. To quantify the modulation of gene expression in hESCs during their propagation, expansion, and differentiation via embryoid body (EB) formation, the authors developed a simple, rapid, inexpensive, and definitive multimarker, semiquantitative multiplex RT-PCR (mxPCR) platform technology. Among the 15 gene primers tested, 4 were pluripotent markers comprising set 1, and 3 lineage-specific markers from each ecto-, meso-, and endoderm layers were combined as sets 2 to 4, respectively. The authors found that these 4 sets were not only effective in determining the relative differentiation in hESCs, but were easily reproducible. In this study, they used the HUES-7 cell line to standardize the technique, which was subsequently validated with HUES-9, NTERA-2, and mouse embryonic fibroblast cells. This single-reaction mxPCR assay was flexible and, by selecting appropriate reporter genes, can be designed for characterization of different hESC lines during routine maintenance and directed differentiation.

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383 IDENTIFICATION OF PLURIPOTENCY MARKERS IN SWINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab383 ◽

2010 ◽

Vol 22 (1) ◽

pp. 348

Author(s):

F. R. O. de Barros ◽

M. D. Goissis ◽

M. G. Marques ◽

M. I. Giassetti ◽

F. F. Paula-Lopes ◽

...

Keyword(s):

Stem Cells ◽

In Vitro Culture ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Culture Conditions ◽

Rabbit Serum ◽

Embryonic Fibroblasts ◽

Pluripotency Markers

Embryonic stem cells (ESC) are a useful tool for studying embryonic development, cell differentiation, and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral, and religious considerations by scientists and laymen alike. Therefore, an animal model such as the pig (Sus scrofa) is valuable in overcoming such hurdles because this species holds physiologic parameters similar to humans. In spite of the great biomedical potential of ESC, many difficulties have been faced in maintaining these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro- and in vivo-produced swine blastocysts; (2) to compare 2 in vitro culture conditions to maintain isolated inner cell masses (ICM), murine embryonic fibroblasts (MEF), or Matrigel; and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2, and FoxD3 at ESC and in vitro- and in vivo-produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vivo-produced blastocysts were obtained by superovulation followed by AI of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing 5 days after ovulation. In vitro- and in vivo-produced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution, and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. Resultant ICM (14 and 66 ICM from in vitro- and in vivo-produced blastocysts, respectively) were cultured in stem cells media (GMEM added by 15% FCS, 0.1 mM β-mercaptoethanol, 1% nonessential amino acids, and 4 ng mL-1 of basic fibroblast growth factor) over monolayer of irradiated mouse embryonic fibroblasts (MEF) or Matrigel for 3 weeks. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro- and in vivo-produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis was performed only with whole in vitro- and in vivo-produced blastocysts. Results showed that Nanog and Sox2 were less expressed in in vitro-produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Because no ESC lineage was obtained in swine until now, we believe this species has different requirements compared with murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC. Acknowledgments are given to FAPESP (processes 06/58507-0 and 07/51732-0).

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Evaluation of a Domestic Steam Disinfector-Dryer Device for Disinfection of Health Care Workers’ Identification Lanyards

Workplace Health & Safety ◽

10.1177/21650799211012653 ◽

2021 ◽

pp. 216507992110126

Author(s):

Beverley C. Millar ◽

John E. Moore

Keyword(s):

Health Care ◽

Health Care Workers ◽

Culture Conditions ◽

Broth Culture ◽

Culture Methods ◽

Simple Method ◽

Diverse Range ◽

Nosocomial Pathogens ◽

Care Workers ◽

Test Organisms

Background Fabric lanyards are commonly worn by health care workers (HCWs) and are known to harbor infectious organisms and contribute to the transmission of infection to HCWs and patients. A diverse range of nosocomial pathogens have been found on lanyards, but there are very few studies describing how to successfully disinfect lanyards to break the chain of transmission. Recently, a steam disinfector-dryer device has come on the market, which performs rapid disinfection against nosocomial pathogens and also dries the contents of the device. It was the aim of this study to evaluate steam disinfection-drying as a method to eliminate pathogens from lanyards. Methods Thirty-eight strips of new, unused, and autoclaved polyester neck lanyards (4 × 2 cm) were inoculated with 30 (12 Gram-positive + 18 Gram-negative) bacteria and one yeast organism. The inoculated lanyard fabric (five organisms per lanyard strip) was placed into a steam disinfector-dryer device and disinfected for 5 minutes and dried for 30 minutes, in accordance with the manufacturer’s instructions. Following disinfection and drying, the presence of viable organisms on lanyard fabric was evaluated using enhanced microbiological broth culture methods for 48 hours. Control lanyard strips were treated with organisms and left at room temperature without undergoing disinfection and drying procedures. Findings Steam disinfection-drying eradicated all test organisms from treated lanyards, with no culturable organisms detected following disinfection-drying, even when employing enhanced bacteriological culture conditions. All test organisms remained viable on the control lanyards. Conclusion/Application to Practice Steam disinfection-drying offers a simple method of decontaminating lanyards, producing dry lanyards for immediate reuse. Occupational health practitioners and hospitals should consider assessing the feasibility of adopting this method in their settings to aid in breaking the chain of transmission of nosocomial pathogens via contaminated lanyards.

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Acid-fast bacilli smear test of a blood culture sample for the diagnosis of disseminated Mycobacterium genavense infection: A case report

International Journal of STD & AIDS ◽

10.1177/0956462420972224 ◽

2021 ◽

Vol 32 (5) ◽

pp. 483-485

Author(s):

Yo Murata ◽

Nobuaki Mori ◽

Narito Kagawa ◽

Kentaro Okuma ◽

Shinji Yoshida ◽

...

Keyword(s):

Blood Culture ◽

Culture Conditions ◽

Nontuberculous Mycobacterium ◽

Sequencing Analysis ◽

Culture Methods ◽

Smear Test ◽

Acid Fast Bacilli ◽

Mycobacterium Genavense ◽

Old Japanese ◽

Immunosuppressed Patients

Mycobacterium genavense, a nontuberculous Mycobacterium, is found in immunosuppressed patients, particularly in those with HIV. Mycobacterium genavense incubation under standard culture conditions is difficult, and its identification is challenging using routine culture methods. Herein, we report the case of a 40-year-old Japanese man with HIV presenting with disseminated M. genavense infection. An analysis using an automated blood culture system did not show positive signals during 6 weeks of incubation. However, an acid-fast bacilli smear of his blood sample was positive for the bacterium. Mycobacterium genavense was identified using sequencing analysis, targeting the heat shock protein 65 gene. The patient recovered from the infection, following antibiotic therapy for 18 months. Under suspicion of disseminated M. genavense infection and the absence of bacterial growth in blood culture samples, an acid-fast bacilli smear test of the sample may be useful for timely diagnosis.

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Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

International Journal of Molecular Sciences ◽

10.3390/ijms22095011 ◽

2021 ◽

Vol 22 (9) ◽

pp. 5011

Author(s):

Daehwan Kim ◽

Sangho Roh

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Stem Cell Research ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Culture Conditions ◽

Human Diseases ◽

Induced Pluripotent

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

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Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Chemosensors ◽

10.3390/chemosensors9060139 ◽

2021 ◽

Vol 9 (6) ◽

pp. 139

Author(s):

Wiktoria Blaszczak ◽

Zhengchu Tan ◽

Pawel Swietach

Keyword(s):

Real Time ◽

Cell Lines ◽

Acid Production ◽

Metabolic Regulation ◽

Cost Effective ◽

Culture Conditions ◽

Metabolic Inhibitors ◽

Metabolic Phenotype ◽

Ductal Adenocarcinoma ◽

Oxygen Utilization

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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