The Development of T7 Phage Based Imaging Probe and the Nature of Floating Cells in Human Embryonic Stem Cell Culture

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Description

Target specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached... Show moreTarget specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached to the particles via metalchelator interactions. Due to the nature of bio-conjugation process, the number of the chelator molecules and the geometry of the affinity reagent are hard to control. To overcome these limitations, a new approach of constructing an imaging probe was developed. In specific, T7 phage was used as a vehicle to synthesize a particle based imaging probe by displaying peptide that had both metal chelating (6 histidine) and targeting (RGD domains on the phage surface. It was demonstrated that the attachment of copper ions to the metal chelating domain does not interfere with target binding. Furthermore, by reducing the metal ions to metal, we were able to generate a very stable peptide templated metal hybrid T7 phage particle as potential target specific imaging probe. The behavior of the probes against both normal and tumor cells was investigated. The presence of floating cells is a common phenomenon in human embryonic stem (hES) cell cultures. Current assumption for the source of floating cells is that they are from apoptosis / senescence, cellular differentiation and other unavoidable imperfections in culture conditions. Inspired by recent studies on mitotic activities in stem cell colony, we believe the existence of floating cells is resulting from essential events required for hES cells proliferation. It was discovered that not all floating cells are dead and the percentage of floating cells over the attached cells was significantly increased with culture time possibly due to the space limitation for cells in the central part of the colonies. By providing more horizontal or vertical space through colony cutting and overlaid membrane insert in the Z direction, the percentages of floating cells were significantly reduced. All these results indicate that continuous cell division across the colonies is responsible for the emergence of floating cells during hES cell culture.
Ph.D. in Biology, May 2011 Show less

Details
Department

BIOL / Biology

Title

The Development of T7 Phage Based Imaging Probe and the Nature of Floating Cells in Human Embryonic Stem Cell Culture

Institution

Illinois Institute of Technology

Creator(s)

Dasa, Siva Sai Krishna

Advisor(s)

Mehta, Rajendra

Date

2011-05-16
2011-05

Description

Target specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and... Show moreTarget specific nanoparticle based nuclear imaging probes have attracted significant attention recently. In most of the cases, these probes were synthesized by conjugating both affinity reagent and chelator molecules on the particle surface chemically. Subsequently, the isotope ions were attached to the particles via metalchelator interactions. Due to the nature of bio-conjugation process, the number of the chelator molecules and the geometry of the affinity reagent are hard to control. To overcome these limitations, a new approach of constructing an imaging probe was developed. In specific, T7 phage was used as a vehicle to synthesize a particle based imaging probe by displaying peptide that had both metal chelating (6 histidine) and targeting (RGD domains on the phage surface. It was demonstrated that the attachment of copper ions to the metal chelating domain does not interfere with target binding. Furthermore, by reducing the metal ions to metal, we were able to generate a very stable peptide templated metal hybrid T7 phage particle as potential target specific imaging probe. The behavior of the probes against both normal and tumor cells was investigated. The presence of floating cells is a common phenomenon in human embryonic stem (hES) cell cultures. Current assumption for the source of floating cells is that they are from apoptosis / senescence, cellular differentiation and other unavoidable imperfections in culture conditions. Inspired by recent studies on mitotic activities in stem cell colony, we believe the existence of floating cells is resulting from essential events required for hES cells proliferation. It was discovered that not all floating cells are dead and the percentage of floating cells over the attached cells was significantly increased with culture time possibly due to the space limitation for cells in the central part of the colonies. By providing more horizontal or vertical space through colony cutting and overlaid membrane insert in the Z direction, the percentages of floating cells were significantly reduced. All these results indicate that continuous cell division across the colonies is responsible for the emergence of floating cells during hES cell culture. Show less

Type

Dissertation

File type

application/pdf

Access

Restricted Access

Rights

In Copyright
http://rightsstatements.org/page/InC/1.0/

Identifier

http://hdl.handle.net/10560/2501

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