Document Type

Dissertation

Date of Degree

Fall 2014

Degree Name

PhD (Doctor of Philosophy)

Degree In

Molecular and Cellular Biology

First Advisor

Dana N. Levasseur

Abstract

The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another.

First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types .

Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model.

In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.

Public Abstract

Many human diseases are linked to cellular malfunction. In their search for ways to replace cells, scientists use skin cells to generate “induced” stem cells, which, under the right conditions, can mature into a variety of cell types (e.g., pancreas, liver, eye, brain, and blood). Our goal is to generate high quality and quantity blood cells that have direct clinical applications.

To generate blood cells, we used two approaches with skin cells taken from mice. We used blood-related genes to convert skin cells directly into blood cells. We have yet to identify the most effective genetic factors, but we have narrowed the search. We used skin cells to make induced pluripotent stem cells (iPSCs) that can be coaxed to become blood cells. We employed fluorescent proteins to track iPSC formation and to characterize reprogrammed colonies with or without exogenous genes. iPSCs that lack fluorescence after upregulation of the endogenous genes are more efficient in differentiating to other cell types, thus fluorescent protein usage should be considered for studies gearing toward “patient” cell-based therapy. Finally, to track the reprogramming process of skin cells and stem cells into blood cells, we have identified a blood reporter that was effective in marking blood cells exclusively. This is important to researchers studying the safety of blood cells generated from pluripotent stem cells in therapeutic applications.

Although much research remains before we will be able to replace malfunctioning cells in patients, our research on cell generation produces key findings that lay the foundation for disease treatment.

Keywords

publicabstract, bone marrow transplantation, cellular reprogramming, hematopoietic, induced pluripotent stem cells (iPSCs), Lentivirus production and transduction, stem cells

Pages

xv, 190 pages

Bibliography

Includes bibliographical references (pages 175-190).

Copyright

Copyright 2014 Khanh L. Duong

Included in

Cell Biology Commons

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