BME 695-Engineering Nanomedical Systems-Final Project 2010 1 Proposal for Magnetic Labeling of Stem Cells for Subsequent Reprogramming to a Primitive State Lisa M. Reece 11/29/2010 ABSTRACT This study presents a proposed novel nanomedical systems based approach to the reprogramming of mature stem cells (SC) isolated from human peripheral blood, into induced pluripotent stem cells (iPSC) utilizing the OCT4 gene as a possible gene therapy for cancer. This oncogene is a known POU family transcription factor expressed in human embryonic stem cells and tumor cells, but not in normal differentiated tissues (Tai, Chang et al. 2005) and is thus used as a marker for undifferentiated cells (MacDougall 2008). Peripheral blood-derived iPSC are comparable to primitive stem cells with respect to morphology, expression of surface antigens, and activation of endogenouse pluripotency genes (Staerk, Dawlaty et al. 2010). OCT4 has been shown to be expressed in some human tumors but not normal somatic tissues (Tai, Chang et al. 2005) leading researchers to believe that this gene may have greater potential for therapeutic targeting in cancer treatment. In this study, we will be harvesting pooled samples of freshly collected human peripheral blood in order to identify, target, sort, and reprogram the adult SC into iPSC for subsequent biodistribution in a nude mouse model. The delivery vehicles for the OCT4 gene will be superparamagnetic iron oxide nanoparticles (SPION) constructed to have a CD34 targeting antibody to identify adult SC. The bound SPION will be taken up by the SC for the delivery of the OCT4 gene sequence into the nucleus for transcription. Further, because the SPIO NP contain a magnetic core, the bound SPIO NP-SC complexes will be sorted from the blood via the Quadrupole Magnetic Cell Sorter for molecular analysis. Proof of the gene transcription and translation of the OCT4 protein will be used to signify the reprogramming of the sorted adult SC into the more primitive iPSC. To confirm that OCT4 transgenes are silenced in the blood-derived SC, qRT-PCR via primers specific for endogenous and total transcripts of the reprogramming factors shall be performed. Once iPSC reprogramming has been confirmed, these cells will be bound to SPION programmed to target SKBR3 cancer cells and introduced into a mouse model for a biodistribution study. This new approach for gene therapy for cancer medicine is cheaper than the current chemotherapeutic and radiation therapies, and is designed to target specific cells in the body that can regulate the metastasis of tumors without the debilitating side effects of said cancer treatments. Keywords: SPION, stem cells, iPSC, OCT4, CD34, nanoparticles, nanomedical systems 1. INTRODUCTION It is a belief that human induced pluripotent stem cells (iPSC) hold great promise for modeling human diseases. Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow for the creation of patient- and disease-specific stem cells that would be used in regenerative medical techniques. It has been shown that there have been studies where the derivation of iPSC from peripheral blood mononuclear cells (PBMC) are similar to human embryonic stem cells when comparing morphology, expressions of surface
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antibodies are used to quantify and purify hematopoietic progenitor stem cells for research and
for clinical bone marrow transplantation. The CD34 protein is a member of a family of
transmembrane proteins that show expression on early hematopoietic and vascular-associated
tissue The streptavidin (SAv) molecules bound to the –COOH groups (see Fig. 3) are able to
bind to the biotinylated ends of the CD34 antibodies. This leaves the variable heavy chain
portions of anti-CD34 to bind to the CD34 membrane receptors on adult SC or iPSC.
2.5.1. Binding of LTVSPWY to SPION
The purified SKBr3 targeting peptide is bound to the SPION using passive adsorption according to standardized protocol Technote 204 (Bangs Labs, Fishers, IN). The appropriate amount of purified ligand is dissolved in adsorption buffer. The SPION suspension is added next to the appropriate volume of dissolved protein, and mixed gently for 1-2 hours. The solution is then incubated overnight at 4˚C, with constant mixing. The following day, the suspension is centrifuged, the supernatant removed, and the microsphere pellet is resuspended in storage buffer to desired storage concentration (often10 mg/ml). Absorbance of the suspension and separate adsorption buffer is measured with a UV/VIS spec at 280 nm.
2.6. Generation of PMAO-PEG Outer Layer
For both SPION types, the simplified process for generating the biocompatible iron oxide NP
will be done according to Yu et al (Yu, Chang et al. 2006). The outermost layer rendering the
SPION biocompatible is coated by PMAO-PEG. PMAO has a monomeric unit that can easily
react with PEG. The PMAO is mixed with PEG methyl ether in chloroform at room temperature
overnight (molar ratio of PMAO:PEG is 1:30) as shown in Figure 7.
Figure 8 illustrates how PMAO-PEG polymers form in chloroform through the anhydride
group and the amine (-NH2) group. The reaction leaves COOH groups available for
bioconjugation with biomolecules if needed. The formation of PMAO-PEG is verified using
Fourier Transform Infrared Spectroscopy (FTIR) as seen in Fig. 8.
Nanorods and nanotubes have longer circulation time in other studies and the binding chemistry
is not that different than what has been proposed in this paper. Also, these shapes provide
greater surface area with a very small volume that will allow for binding of several biomolecules.
Finally, this type of approach, once optimized, will allow cancer treatments to become less
invasive, less costly, and provide the patient with a higher quality of life. These nanomedical
devices do not only have the potential to kill tumors or reprogram cells into ones that will
differentiate and apoptose properly, but with the gene thereapy/drug they deliver precisely to
their target, patients will experience far less side effects that can only add to that higher quality
of life and allow their immune system to recover much more quickly. This is the medical
treatment of the future and shows great promise as being a cheaper, cleverer, a more reliable,
and more available method of treatment that now currently exists.
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