A Novel 3D Extracellular Matrix Model Enriching Human Acute Myeloid Leukemia Stem Cells

Background and objective: Acute myeloid leukemia (AML) develops from leukemia stem cells (LSCs), a small subset of leukemia cells possessing both self-renewal and multilineage differentiation potential similar to normal hematopoietic stem cells. The stem cell niche of the bone marrow microenvironment protects LSCs from chemotherapy, resulting in subsequent leukemia relapse. The study of AML LSCs in vitro is limited because of the lack of an ideal culture system mimicking the protective bone marrow microenvironment. The bone marrow stem cell niche is mainly composed of stromal cells, soluble cytokines and growth factors, as well as extracellular matrix (ECM). We therefore developed a 3-dimensional ECM model using decellularized Wharton's jelly from human umbilical cords to better characterize AML LSCs in vitro. Previously we have shown that leukemia cells grown in DWJM changed morphology to become spindle shaped and maintained viability but had decreased proliferation as measured by Alamar blue assay. Herein, we further characterize leukemia cells cultured in DWJM.

Methods: Wharton's jelly decellularization process included multiple osmotic shock cycles using hypertonic and hypotonic solutions, a non-ionic detergent Triton-x, an anionic detergent sodium lauroyl succinate, and an enzyme digestion with recombinant endonuclease Benzonase™. We examined three human leukemia cell lines: HL60, Kasumi I and MV411. We characterized leukemia cell proliferation by CellTrace proliferation assay and phenotype by flow cytometry for stem cell markers. Serial colony forming unit (CFU) assays were used to test the self-renewal of leukemia cells.

Results: CellTrace proliferation assay showed that, compared to cells in suspension, cells cultured in DWJM divided less frequently. To assess for LSCs properties, we measured the ALDH+ population by Aldefluor assay and found that the ALDH+ cells from Kasumi I and HL60 increased significantly in DWJM compared to suspension (p<0.05). Lastly, serial CFU showed significantly increased colony forming units in both primary and secondary plating in MV411 and HL60 cells cultured in DWJM versus suspension (P<0.05). CFU also increased in Kasumi-I cells cultured in DWJM versus suspension, though the increase was not statistically significant (P=0.08). Taken together, our findings suggest that leukemia cells cultured in DWJM demonstrated an increased self-renewal ability due to the enrichment of LSCs rather than progenitors.

Conclusion: Decellularized Wharton's jelly matrix may serve as a practical in vitro ECM model to enrich for LSCs and study the ECM-LSC interactions.