Steve K.W. Oh

Whole-Genome Mapping of Histone H3 Lys4 and 27 Trimethylations Reveals Distinct Genomic Compartments in Human Embryonic Stem Cells

Epigenetic modifications are crucial for proper lineage specification and embryo development. To explore the chromatin modification landscapes in human ES cells, we profiled two histone modifications, H3K4me3 and H3K27me3, by ChIP coupled with the paired-end ditags sequencing strategy. H3K4me3 was found to be a prevalent mark and occurred in close proximity to the promoters of two-thirds of total human genes. Among the H3K27me3 loci identified, 56% are associated with promoters and the vast majority of them are comodified by H3K4me3. By deep-transcript digital counting, 80% of H3K4me3 and 36% of comodified promoters were found to be transcribed. Remarkably, we observed that different combinations of histone methylations are associated with genes from distinct functional categories. These global histone methylation maps provide an epigenetic framework that enables the discovery of novel transcriptional networks and delineation of different genetic compartments of the pluripotent cell genome.

Hyper-stimulation of monoclonal antibody production by high osmolarity stress in eRDF medium.

An earlier study (Chua et al., 1994) showed that hybridoma 2HG11 cultivated in a basal medium called eRDF, which is enriched in amino acids, enabled higher immunoglobulin (Ig) production with and without serum, when compared to two other traditional media RPMI and DMEM/F12. A further enhancement of Ig productivity was achieved when the osmolarity of the culture medium was increased from 300 mOsm to 350 mOsm (Oh et al., 1993). To determine whether the eRDF media was indeed better, three other cell lines, two IgG producers (TB/C3 and I13/17) and an IgM producer (B10), were tested. The results showed that maximum viable cell densities in eRDF medium were up to 3-times higher than in RPMI and maximum Ig titres were 2-8-times higher than in DMEM/F12 and RPMI. The three cell lines were similarly subjected to osmotic increases from 300 mOsm to 350 and 400 mOsm by addition of NaCl. There was an increase in Ig titres of between 30% to 100% compared to the control medium, although cell growth was reduced. Thus, hyper-stimulation by osmolarity stress was found to be generally effective in eliciting higher Ig production; the extent of enhancement being more pronounced for certain cell lines. Other osmolytes such as sucrose and KCl demonstrated similar effects of increasing Ig productivity. Study on the mechanism of action of osmotic stress on hybridoma 2HG11 revealed that hyper-stimulation of Ig productivity was fundamentally related to a greater availability of amino acids to cells as the cells actively accumulated more salt and amino acids to compensate for the higher medium osmolarity. Uptake of the amino acid analogues 14C-aminoisobutyric acid and 3H-methylaminoisobutyric acid into cells increased to 2.34 x 10(3) cpm per cell per min and 6.35 x 10(3) cpm per cell per min, respectively, under osmotic stress. This corresponds to an 85% increase in uptake via the Na(+)-dependent symport and a 50% increase in uptake via the Na(+)-independent and Na(+)-dependent symports. In the 350 mOsm medium, hybridomas also demonstrated an increase in metabolic activities of 5-10% compared to the control. This, together with the reduced specific growth rate in cells under osmotic stress, suggests that more energy was channelled into the biosynthetic pathway of Ig production.

Enhanced IgG production in eRDF media with and without serum: A comparative study

The performance of three basal media RPMI, DMEM/F12 (DF) and eRDF (enhanced RDF, RPMI:DMEM:F12 in 2:1:1) were evaluated in cultures with and without serum with respect to cell proliferation, metabolism and monoclonal antibody (Mab) productivity. Based on the ease of adaptation, growth rate, maximum cell density and Mab production, the media were ranked as follows: eRDF > DF > RPMI. This was true for serum-free (SF) and serum supplemented (SS) media in static and shaker cultures. Growth performances in static and shaker cultures were consistently 20-50% lower in all three SF media compared to the corresponding SS conditions. Antibody titres in DF/SF and RPMI/SF cultures, irrespective of the culture condition, were generally similar or slightly lower than their SS counterparts. However, eRDF/SF medium yielded a much higher Mab titre (193 mg l-1) compared to eRDF/SS medium (145 mg l-1). This was also six times higher than the lowest titre of 30 mg l-1 in RPMI/SF medium. Hybridomas in eRDF/SF were further adapted to media without bovine serum albumin (eRDF/SF-BSA). Maximum cell densities in these cultures improved with scale up, from 1.1 x 10(6) ml-1 in static, to 1.9 x 10(6) ml-1 in shaker flasks, to 2.5 x 10(6) ml-1 in bioreactors. However, Ig levels remained between 100-130 mg l-1 which were much lower than in eRDF/SF medium. Thus BSA appears to be necessary for Ig production. The manufacturing cost (excluding purification) of Ig using eRDF was calculated to be between 17-50% of the price of the other two media and therefore this is regarded as the best medium for Ig production.

Insect cell line dependent gene expression of recombinant human tumor necrosis factor-β

The expression and secretion of recombinant human tumor necrosis factor-β (TNF-β lymphotoxin) was examined in seven different insect host cell lines, namely, Spodoptera frugiperda (Sf) 9-ATCC, Sf9-IXP, Sf21, Trichoplusia ni (BTI-TN-5B1-4), Mamestra brassicae (IZD-MB-0503), IPLB-HZ1075, and Antheraea moth ovarian cells. The highest yield of secreted recombinant human TNF-β was obtained by using the Sf21 cells; the maximum protein productivity achieved was 71.7 μg 1−1 × 106Sf21 cells. This is an increase of 100% over the yields obtained from the Sf9-ATCC cells. In addition, a new cell clone, Sf9-IXP, was derived which was larger in cell volume and had a 76% higher TNF-β expression and secretion in comparison to the native original Sf9 cells. Sf9-ATCC cells infected with recombinant baculoviruses secreted TNF-β at 32.1 μg 1−1 × 106 cells. The lowest productivity was seen for IPLB-HZ1075 cells which attained 7 μg 1−1 × 106 cells. There is a proportional relationship between increased protein production and the increase in cell volume and granularity. Recombinant protein expression and secretion was also shown to be influenced by the intracellular metabolic activities of the cells and it was observed that the recombinant human TNF-β expressed and secreted by the different insect cells has the same molecular weight.

Expansion of Undifferentiated Human Embryonic Stem Cells

Human embryonic stem cells (hESC) are pluripotent cells derived from the inner cell mass of blastocysts. They have the potential to proliferate indefinitely in culture, but still retain their capacity for differentiation into a wide variety of cells. hESC are currently maintained either on feeder layers or on matrigel with conditioned-medium (CM) from primary mouse embryonic fibroblasts (MEFs). We describe our groups strategy and results to date in establishing defined culture conditions for the continuous expansion of undifferentiated hESC in a feeder-free and serum-free culture system. This includes the development of a robust hESC passaging method based on enzymatic dissociation and culture on immortalized mouse and human feeders for the generation of consistent batches of conditioned-media, and the development of an alternative serum free culture platform based on sphingosine-1-phosphate and platelet derived growth factor.

Measurement of human embryonic stem cell in the growing cycle

A measurement and imaging system has been developed for in-line continuous measurement of live, unmodified, human embryonic stem cells (hESC). The measurement will not affect cell growth, structure, sterility and suitability for clinical use. The stem cell imaging system (SCIS) can be used to support the optimization of automated stem cell growth for invitro study and for high-volume bio-manufacture. This paper present the experimental and analysis for the optimization of system parameters. A non-linear lighting is developed to obtain a clear images. The individual cluster can be traced from day one to day two. The whole system is calibrated with measurement microscope and haemacytometer. The measurement accuracy is better than 90%.

A fast assay for developing serum free media for cord blood haematopoietic cells

One of the bottlenecks in haematopoietic cell expansion is the lack of a defined serum free media. We describe a fast method of monitoring live haematopoietic cell numbers, using fluorescein diacetate, to facilitate the development of such a media. Several carbohydrates (fructose at 500μg/ml and pyruvate at 300μg/ml) and lipids (cholesterol and lecithin, both at 10μg/ml) were tested and could replace serum in cord blood haematopoietic cell cultures.

Overproduction Of Monoclonal Antibodies In High Salt Medium In The Presence of Butyrate

Hybridomas were hyper-stimulated to produce industrial-like concentrations of Ig levels by suppressing cell growth and increasing culture longevity by adaptation to higher osmolarity medium and addition of sodium butyrate. Cells produced 265 mg/l of Ig in 350 mOsm medium, compared to 140 mg/l in the control (300 mOsm). There was a noticeable suppression of cell growth rate and maximum cell numbers. Cell sizes were larger at higher osmotic pressures as measured by flowcytometry. Treatment with butyrate elevated Mab levels further to 350 mg/l, while maximum cell numbers were again reduced. Environmental manipulation with salt and butyrate increased Mab levels by 2.3-fold. A similar technique could be used to enhance Mab production of low secreting human hybridomas and immortalised B-cells.

BIOSYNTHETIC RESPONSES AND SCANNING ELECTRON MICROSCOPIC STUDIES OF MURINE HYBRIDOMAS SUBJECTED TO HYDRODYNAMIC STRESSES

ABSTRACT In previous work, we showed that DNA synthesis was inhibited under conditions of intense hydrodynamic stress1; however, cellular metabolic activity was increased. Here, we report on the initial biosynthetic responses of cells during batch cultivation subjected to high hydrodynamic stresses. During the first few hours of exposure to sparging or high agitation entraining a vortex, hybridomas showed increased DNA and protein synthesis as measured by 3H thymidine and 35S methionine incorporation. Scanning electron microscopy showed different cell morphological changes depending on whether hybridomas were subjected to high agitation or sparging. The morphological responses was related to the form and intensity of energy dissipation. Sparging caused immediate cell destruction by complete dismemberment of the cellular structures. Agitation, in contrast, led to loss of integrity of the cells through partial disruption of the plasma membrane. Cells are therefore seen as non-viable but intact in normal counting procedures.

Adaptation to Stirred Culture - A Study of a Moderately Shear Sensitive Cell Line (PQXB1/2).

A hybridoma was shown to be sensitive to low levels of agitation. Hybridoma PQXBI/2 (from ICI, Pharmaceuticals Division) normally grew in RPMI with 5% FCS in static culture, however it was shown that even at speeds as low as 15 or 40 rpm, growth performances were severely reduced. Both growth rate and viability index were reduced at these speeds but these effects were ameliorated by increasing the serum levels.