Gene expression analysis of pluripotent stem cells

Published on March 5, 2014   25 min
0:00
Hi. My name is Uma Lakshmipathy. I'm with the stem cell R&D at Life Technologies. My research team is involved in the development or reprogramming tools and platforms for stem cell characterization. Today, I would like to share our studies on the use of gene expression data for the identification of novel markers and for rapid characterization and standardization of pluripotent stem cells.
0:26
I'll start off with a brief introduction on pluripotent stem cells followed by current methods used to characterize these cells. I would then like to share two recent studies. The first study describes development of a focus gene expression panel for confirming function and pluripotencies of ESC and iPSC lines. This method relies on monitoring the expression of cell renewal and lineage markers in undifferentiated pluripotent cells and in their corresponding differentiating cells. The second study is focused on identification of CD44 as a negative mark of pluripotent cells.
1:07
Pluripotent stem cells, or PSCs, are cells that have the potential to differentiate into nearly every type of cell found in the body. This potential makes them valuable research tools for many different applications, including drug discovery, disease modeling, and regenerative medicine. Prior to 2006, the primary source of pluripotent stem cells was embryonic stem cells, or ESCs, which are harvested from the inner cell mass of a blastocyst early stage embryo. In 2006, Shinya Yamanaka and colleagues published a landmark paper describing the derivation of induced pluripotent stem cells, or iPSCs, by reprogramming. Reprogramming is a process wherein an adult somatic cell, such as a dermal fibroblast, can be induced to turn into a cell that is pluripotent and is essentially indistinguishable from an ESC.
2:08
Successful reprogramming requires a sustained expression of certain factors for a minimum time period, typically, two to four weeks. This can be accomplished in many different ways, some of which include integrating viruses, non-integrating viruses, non-integrating DNA vectors such as episomal vectors, transcription of mRNA or protein, and small molecules. Retrovirus was the first method used for reprogramming-- a method where the transgene are randomly inserted into the host cell genome. Once iPSCs are established, these viral transgenes are silenced but remain a part of the genome. This is referred to as a footprint. Depending on the intended use of the iPSCs, this footprint may or may not pose a problem. If it does, integrating viruses can be engineered so that the genes they deliver will be exercisable using systems such as Cre-Lox system. There are other reprogramming methods that are inherently footprint-free such as non-integrating virus. Sendai virus is one example of this. In this case, a virus is still used to deliver the reprogramming transgene, but the virus never integrates into the genome and will be eventually diluted out, leaving no footprint behind. Other examples are episomal vector system and mRNA delivery method.
3:32
Once cells are reprogrammed, the next step is to confirm that the colonies obtained are pluripotent. The clones that are selected and expanded need to be shown to differentiate into cell types representative of the three germ layers. The critical and most involved part of the pluripotent stem cell workflow is therefore the characterization of established cells to confirm their identity, purity, and quality. It is estimated that, over the next three years, over 10,000 unique iPSC clones will be generated from diverse patient samples.
4:09
Current characterization methods rely on a combination of cellular methods. Assessment of pluripotent stem cell, via cell, and colony morphology is the least invasive but requires a trained eye and is subjective, depending on the culture conditions. Dyes against differentially expressed enzymes such as alkaline phosphatase is easy to use but not very specific to pluripotent stem cells and hence not definitive. Antibody staining for pluripotent markers is more specific but cost and sterility issues makes it a challenge. In addition to biomarker expression, confirmation of trilineage differentiation requires in vitro differentiation followed by lineage-specific antibody staining. In vivo assays commonly used for murine embryonic stem cells are irrelevant for human cells except for teratoma formation. This is by far the most definitive measure of pluripotency but it's labor intensive, costly, and comes with a high animal testing burden.
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Gene expression analysis of pluripotent stem cells

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