Publication Date

2010-12-16

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

April 2010

First Committee Member

Walter Scott - Committee Chair

Second Committee Member

Terace Fletcher - Committee Member

Third Committee Member

Wayne Balkan - Committee Member

Fourth Committee Member

Paul Schiller - Mentor

Fifth Committee Member

Ian McNiece - Outside Committee Member

Abstract

Adult stem cells currently represent the most promising cell type for cell therapy approaches in regenerative medicine. To obtain a thorough understanding of their properties is essential for successful stem cell therapy approaches. We previously identified a unique population of human marrow-isolated adult multilineage inducible (MIAMI) cells isolated from males and females 3 to 72 years of age. Although their numbers decrease with age, their molecular profile is sustained, suggesting that these cells self-renew during the lifetime of the individual. Understanding the mechanisms of in vitro self-renewal will allow us to expand these cells to numbers suitable for cell therapy without undermining their biological and physiological properties. Oxygen tension plays a key role in modulating the self-renewal of MIAMI cells. The focus of this study is to delineate the molecular mechanisms by which oxygen tension regulates their self-renewal. We have shown that low oxygen tension may assist in the maintenance of an immature stem cell phenotype and possibly make MIAMI cells capable of self-renewal by activating the canonical Wnt and Notch signaling pathways. Our data show that low (3%) oxygen tension promotes proliferation and increases the expression of the stem cell self-renewal markers Oct4, Sox2,and Nanog while selectively increasing expression of key mediators in the canonical Wnt and Notch pathways, such as Wnt3a, LRP5, beta catenin, Cyclin D1, and the Notch intracellular domain (NICD). In addition, we have shown the low oxygen increased accumulation of the proteins beta catenin, Oct4, Cyclin D1 and NICD in the nucleus as well as nuclear localization of hypoxia inducible factor (HIF)-1", a key transcriptional mediator of the oxygen sensing pathway. Treatment of MIAMI cells with Wnt3a increased nuclear accumulation of Oct4a and increased proliferation at 3% and 21% oxygen, suggesting it did not mediate the low oxygen effects. In contrast, siLRP5-mediated inhibition of LRP5 decreased Oct4a and Sox2 transcripts and nuclear accumulation of Oct4a, suggesting it may participate in mediating the low oxygen effects. Our data suggest that expanding human stromal cells, such as MIAMI cells, at low oxygen tension contributes to maintaining the cells in a developmentally immature state by modulating the Wnt and Notch signaling pathways.

Keywords

Multipotent; Repairative; Regeneration

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