Biomedical subjects

Andre Choo

Publications and source records attributed to Andre Choo.

4 recordsLinked to original sources

PubMed2006

Establishing efficient siRNA knockdown in mouse embryonic stem cells.

FAM-labeled oligo dT (FAMdT) was utilized as a means to gauge efficient transfection of small nucleic acids into mouse embryonic stem cell (mESC) colonies. Using colonies grown overnight, transfection was restricted largely to the periphery of the colonies with only a 40% decrease in Oct-3/4 RNA transcript levels following cognate Oct-3/4, small interfering RNA (siRNA) delivery. However, transfection of mESC 4 h after seeding gave greater than 90% cells being successfully transfected based on quantitative real-time PCR detection of approximately 90% Oct-3/4 RNA transcript knockdown. This method provides an economical and efficient means by which to determine effective transfection conditions, and establish efficient siRNA knockdown of reportedly difficult to transfect cell lines such as mESC.

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PubMed2006

TGF-beta2 allows pluripotent human embryonic stem cell proliferation on E6/E7 immortalized mouse embryonic fibroblasts.

In this study we report observations that mouse embryonic fibroblasts (MEF) capable of supporting expansion of pluripotent, human embryonic stem cells (hESC) fail to support after immortalization using E6/E7 oncogenes in serum conditions; however this can be reversed following addition of exogenous TGF-beta2. Microarray analysis of immortalized and non-immortalized MEF revealed differential gene expression of several TGF-beta related genes. By supplementing TGF-beta2 into E6/E7 immortalized MEF cultures, this enabled proliferation of undifferentiated, pluripotent hESC as demonstrated by marker expression (Oct-4, SSEA-4, alkaline phosphatase) and teratoma formation representing three germ layers following hESC injection into immuno-deficient mice. Subsequent investigation using quantitative real-time PCR highlighted differential gene expression of several extracellular matrix related transcripts in primary and immortal (+/-TGF-beta2) feeder cells including the induction of osteopontin following addition of TGF-beta2. Our results demonstrate that TGF-beta2 and its related genes in MEF play a role in the support of pluripotent hESC expansion.

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PubMed2005

Perfusion cultures of human embryonic stem cells.

Human embryonic stem cells (hESC) are self-renewing pluripotent cells capable of differentiating into cells representative of all three embryonic germ layers. Hence, they hold great potential for regenerative medicine. However, significant cell numbers are required to fulfill their potential therapeutic applications. In this study, perfusion with supplemented conditioned media (SCM), produced by mouse embryonic fibroblasts (MEF), was adopted to improve cell densities of hESC cultures. Perfusion enhanced hESC numbers by 70% compared to static conditions, on both organ culture dish (OCD) and petri dish cultures. All cultures maintained healthy expression of the pluripotent marker, Oct-4 transcription factor. In vivo, perfused hESC formed teratomas in severe combined immunodeficiency (SCID) mice models that represent the three embryonic germ layers. When SCM was produced with lower concentrations of MEF, hESC densities and Oct-4 levels were reduced. Hence, perfusion with SCM is a potential feeding method for scale-up production of hESC.

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PubMed2005

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions.

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.

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