Date of Award

Fall 9-23-2025

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Jiwang Zhang

Abstract

The relapse and chemotherapy resistance are the major challenges of acute myeloid leukemia (AML) treatment, driven by a rare population of leukemia stem cells (LSCs). LSCs are unique types of cells which evade standard chemotherapies and can regenerate AML post-treatment. Targeting LSC therapy remains limited, in part due to the lack of reliable in vitro AML culture systems to grow LSCs for drug discovery. This study aimed to engineer a chemically defined, serum-free 2D in vitro culture condition that supports either the expansion of leukemic blasts (LBs) or primary human LSCs by optimizing current protocols for hematopoietic stem cells (HSCs) expansion and AML cell growth. This study was divided into two parts for short-term (7 days) and long-term (28 days) cultures. In the short-term protocol, ex vivo AML mononuclear cells (MNCs) were cultured in Iscove’s Modeified Dulbecco Medium (IMDM) and supplemented with polyvinyl alcohol (PVA) or Soluplus, PI3K-Akt and MPL agonists (SCF+TPO or 740Y-P+butyzamide), under physioxia (5% O2) to promote AML blast maintenance and expansion (Aim 1). In the long-term protocol, cells were cultured in hypoxic (1-3% O2) conditions, supplemented with PVA or Soluplus, and standard cytokines were replaced with stem- and quiescence-promoting and cell death-inhibiting factors (adiponectin AdipoRon, Necrostatin-1s) to preserve a stem-like phenotype and promote LSC expansion (Aim 2). Cell viability, immunophenotyping, functional colony forming assay, and morphological analysis were preformed to instruct the optimization of each condition. As a control, cells were also cultured in 20% O2 (normoxia) in parallel. Development of a protocol that can promote AML cell proliferation for at least one week could allow for clinicians to screen drugs for induction therapy. A long-term culture protocol for LSC expansion could help grow a large library of LSCs from different subtypes of AML cases and facilitate high-throughput screening to accelerate the discovery of novel targeted LSC therapies to overcome AML relapse.

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