Search results

(1 - 1 of 1)

Title

REGENERATIVE POTENTIAL OF HUMAN CD34+ STEM CELLS MOBILIZED AND ENRICHED FROM BONE MARROW

Creator

Cohen, Amy

Date

2014, 2014-12

Description

This thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating...
Show moreThis thesis provides an in-depth investigation of the heterogeneous population of CD34+ cells obtained through granulocyte-colony stimulating factor (G-CSF) mobilization, apheresis, and an immuno-magnetic selection process. The results provide a better understanding of the phenotypic characteristics, functional migratory capabilities and paracrine secretory performance when exposed to normal and low-oxygen environments in vitro. The overall composition of the population acquired through the cells selection process and the CD34+ cells were characterized for maturity and commitment to various cell types as well as their potential for migration, adhesion and proliferation. The detailed phenotypic characteristics included positivity for more primitive, undifferentiated hematopoietic stem cells (HSCs) as well as markers indicating enrichment of long term HSCs. A functional quantitative bioassay was used to measure the migratory ability of the CD34+ cells. On average, 18.7% of the CD34+ cells migrated after 3 hours of incubation in the presence of 200ng/mL of Stromal Cell-Derived Factor One alpha (SDF-1α) as compared to 0.6% of the CD34+ cells incubated without chemoattractant. The migration was successfully neutralized by using monoclonal antibodies to CXCR4 and a CXCR4 antagonist, AMD3100. An evaluation of the migratory cell population showed that the cells exhibited an enhanced commitment to a monocyte or lymphocyte lineage and a larger percentage were CXCR4+. CD34+ cells were incubated in normoxic and hypoxic conditions and the proliferation, viability, immunophenotypic profile, migratory potential and secreted cytokines were evaluated and compared to incubation in normoxic conditions. The CD34+ cells proliferated in both the normoxic and hypoxic conditions over three days of incubation. The proliferation was higher in the hypoxic condition after one day of incubation, but after three days of incubation the normoxic conditions resulted in more proliferation. An increase in CD164 and a decrease in CD99 in the Day 3 hypoxic condition suggest that hypoxia was stimulating an adhesion pathway and suppressing the functional migration of the CD34+ cells. The three hour hypoxia incubation resulted in a significant (63%) reduction in the migration of the CD34+ cells as compared to the 3 hour normoxia condition, but many of the cells continued to migrate in hypoxia over the 24 hour period while the normoxic cells continued to migrate, but at a much slower rate. Culture media from CD34+ stem cells that had been incubated in normoxic or hypoxic conditions for one day and three days was assayed for sixty cytokine proteins known for possessing angiogenic qualities. Twenty five angiogenesis cytokines were increased in the culture media incubated with CD34+ cells, including tissue inhibitor of metalloproteinases-1 and 2 (TIMP-1 and TIMP-2), angiopoietin-1 (ANG-1) and insulin-like growth factor-1 (IGF-1). Thirteen angiogenesis cytokines were elevated in hypoxia as compared to normoxia, including TIMP-1, IGF and growth-regulated oncogene-β (Gro-β).
Ph.D. in Biological and Chemical Sciences, December 2014
Show less