Derivation of induced pluripotent stem cells from rat primary cells utilising the cre-lox lentiviral delivery system

Smith, Michael James (2012). Derivation of induced pluripotent stem cells from rat primary cells utilising the cre-lox lentiviral delivery system. University of Birmingham. M.Res.

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Abstract

Somatic cells can be reprogrammed to an embryonic stem cell-like state, regaining the ability to differentiate into any cell in the body. Induced pluripotent stem cells (iPSCs) can be derived through the delivery and expression of exogenous transcription factors; Oct4, Sox2, Klf4 and c-Myc being the original and most commonly used. The project utilised the STEMCCA virus, a lentivirus delivery system comprising of all four reprogramming factors in a single cassette. Different media and culturing conditions were used in an attempt to derive rat iPSCs from adiposeand dental pulp derived stem cells (ADSCs and DPSCs, respectively). Staining for alkaline phosphatase, which is highly expressed in ESCs and iPSCs, was used to demonstrate whether colonies observed were potentially composed of iPSCs. Initial attempts using iPSC media, a feeder layer or ascorbic acid and lithium chloride supplementation, were unsuccessful in generating iPSC colonies. Medium supplemented with two inhibitors, CHIR-99021 and PD- 0325901, was successful in generating iPSC colonies with an ESC-like morphology from rat embryonic fibroblasts. iPSC colonies failed to form using the same culture conditions with the ADSCs and DPSCs.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

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