Biomedical subjects

Andre B H Choo

Publications and source records attributed to Andre B H Choo.

6 recordsLinked to original sources

PubMed2006

Deciphering the importance of three key media components in human embryonic stem cell cultures.

Development of a serum free, feeder-free (SFFF) culture platform for human embryonic stem cells (hESC) will be important for the expansion of hESC for future cell therapy applications. However, currently, culture of hESC consists of a combination of basal media, basic fibroblast growth factor (bFGF), serum replacer (SR) and conditioned media (CM) from feeders, and it is unclear which components of the mixture are absolutely critical in the maintenance of hESC. To evaluate the relative contributions of these media components in the development of SFFF culture, each was systematically eliminated and pluripotency assayed by dual embryonic stem cell markers, Oct-4 and TRA-1-60. We concluded that SR was the most critical component in the platform, followed by bFGF and CM produced by feeders, where down-regulation of Oct-4 occurred after 2, 5 and 5 passages, respectively, upon their withdrawal from the complete media.

Cell Culture Techniques↗

PubMed2005

High density cultures of embryonic stem cells.

Embryonic stem cells (ESC) have the unique ability to differentiate into a variety of tissue types. However, the realization of regenerative medicine will require the production of large quantities of ESC which subsequently have to be differentiated into the final phenotype. Thus, we sought to develop a simple and scaleable bioprocess to increase densities of ESC to achieve this goal. Using mouse embryonic stem cells (mESC) as a model, by combining automated feeding and culture of mESC on petriperm dishes, cell densities were enhanced up to 6.4 x 10(6) cells/cm2 compared to conventional petri dish culture which only reached 0.2 to 1.4 x 10(6) cells/cm2. It was found that mESC from all experiments maintained excellent viability, pluripotency, and genetic stability after growing for 6 days in petriperm cultures with automated feeding. The expression of Oct-4 transcription factor was observed in all cultures, mESC formed embryoid bodies in differentiated cultures and teratomas in SCID mice, confirming their pluripotency, and karyotype of the cultures was normal. This culture method was stable for routine passaging and a second mESC cell line was shown to perform in a similar manner on petriperm with automated feeding. This work represents an important step towards achieving high density cultures of ESC.

Alkaline Phosphatase↗

PubMed2004

Single chain antibody fragments for the selective targeting of antigens to dendritic cells.

In order to target antigens (Ags) selectively to dendritic cells (DC), we derived single chain antibody fragments (scFvs) from NLDC-145 and N418, two monoclonal antibodies binding the mouse dendritic cell-restricted surface molecules DEC-205 and CD11c. Recombinant hexahistidine-tagged forms of the scFvs (scNLDC and scN418) were efficiently produced in a baculovirus expression system. Both scFvs bound DEC-205(+) Langerhans cells and CD11c(+) fetal skin-derived dendritic cells (FSDCs) comparably to their parental antibodies. Immunization of C57BL/6 mice with a DNA vaccine encoding a model protein antigen fused to scNLDC stimulated specific immune responses in both the humoral and cellular compartments, in contrast to DNA vaccines expressing scN418-targeted or untargeted antigen. Our results show that antigen targeting to DCs via a DEC-205 binding scFv leads to enhanced immunogenicity. Further, this work suggests that scFvs fused to protein antigens and delivered as DNA vaccines may provide a generic means for delivering vaccinal molecules to selected cell populations.

Animals↗

PubMed2004

Expansion of pluripotent human embryonic stem cells on human feeders.

Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications.

Animals↗

PubMed2002

Soluble expression of a functional recombinant cytolytic immunotoxin in insect cells.

We have previously described the production of a recombinant melittin-based cytolytic immunotoxin (IT), scFv-mel-FLAG, in bacterial cells. While the IT exhibited specific cytotoxicity for a human lymphoblastoid cell line, HMy2, yields from expression were low. Here, we describe a baculovirus expression system for the overexpression and secretion of scFv-mel-FLAG. A novel snake phospholipase A2 inhibitor signal peptide was used to aid in the secretion of the immunotoxin. Sf21 insect cells infected with the recombinant virus secreted soluble scFv-mel-FLAG into the culture medium from which it was purified directly on an affinity column. The final yield of scFv-mel-FLAG was estimated at 3-5 mg/L, which was an improvement of 30-fold compared to expression in the prokaryotic system. The cell binding characteristics of the recombinant IT were assessed by flow cytometry using the antigen expressing cell line HMy2. ScFv-mel-FLAG bound specifically to HMy2 cells in direct binding assays and this binding was completely inhibited in the presence of an excess of soluble antigen. Significant cytotoxicity for HMy2 cells, measured by leakage of cytosolic LDH, was also observed for the IT at a concentration of 60 pmol/10(4) cells. Cytotoxicity was concentration dependent and was specific for antigen-positive cells. Thus the baculovirus expression system, under the control of a novel secretion signal, can be used for the production of soluble and functional recombinant cytolytic immunotoxins. To our knowledge, this is the first report of expression of a recombinant immunotoxin in the baculovirus expression vector system.

Amino Acid Sequence↗

PubMedDate not supplied

Human embryonic stem cells: technological challenges towards therapy.

1. Human embryonic stem cells (hESC) hold promise for overcoming many diseases because they provide a potential source for many of the slow-growing cell types needed for effective tissue repair, such as the dopaminergic neural cells for Parkinson's disease or the pancreatic islet cells needed to relieve diabetic patients of their daily insulin injections. 2. Human embryonic stem cells can be characterized by several surface antigen markers, transcription factors and enzymes, as well as their ability to differentiate into cells representative of the three germ layers, both in vivo and in vitro. 3. Significant progress has been made in defining the feeder-free and serum-free conditions needed for the culture of hESC. The fibroblast growth factor-2 and transforming growth factor-b signalling pathways appear to be important in maintaining self-renewal and preventing differentiation, respectively. 4. Several important quality controls, including karyotyping, immunogenicity and murine viral assays, will have to be established to monitor the production of hESC for therapeutic purposes. 5. Methods of expansion and differentiation of hESC are still in their infancy and the efficiency of these processes needs to be significantly enhanced.

Cell Culture Techniques↗