title: Human CD34(+) progenitor hematopoiesis in liquid culture for in vitro assessment of drug-induced myelotoxicity.
abstract: Utilization of validated CFU-GM assays for myelotoxicity screening is hampered by its labor-intensive and low-throughput nature. Herein, we transformed the defined CFU-GM assay conditions and IC90 endpoint into a higher throughput format. Human CD34(+) hematopoietic progenitors were cultured in a 96-well plate for 14 days with the same cytokine (rhGM-CSF) used in the CFU-GM assay. Expansion and differentiation toward myeloid lineages were manifested by characteristic changes in nuclear and cytoplasmic morphology and by temporal expression patterns of CD34, CD11b and CD13 markers. Inhibition of CD34(+) cell myelopoiesis by 12 anticancer drugs known to induce myelotoxicity in the clinic was quantifiable using either general cytotoxicity endpoints (cell growth area or total nucleus count) or lineage specific readouts (count of cells expressing CD11b and/or CD13). The IC50 and IC90 values derived from the concentration-response curves of 14-day drug exposure in CD34(+) cell culture were highly correlated with those from the international validation study of the CFU-GM assay, demonstrating capability to assess general cytotoxicity, cell proliferation and myelopoiesis simultaneously. These results suggest that this human CD34(+) hematopoietic progenitor cell assay can be used as a direct replacement for the validated, low throughput CFU-GM assay, and could expand application of in vitro myelotoxicity testing.
authors.affiliation: Laboratory of Investigative Toxicology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: liang.guo@fnlcr.nih.gov.
authors.forename: Liang
authors.initials: L
authors.lastname: Guo
authors.affiliation: Laboratory of Investigative Toxicology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: john.hamre@fnlcr.nih.gov.
authors.forename: John
authors.initials: J
authors.lastname: Hamre
authors.affiliation: Laboratory of Investigative Toxicology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: davismillinm@mail.nih.gov.
authors.forename: Myrtle
authors.initials: M
authors.lastname: Davis
authors.affiliation: Laboratory of Investigative Toxicology, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. Electronic address: parchmentr@mail.nih.gov.
authors.forename: Ralph E
authors.initials: RE
authors.lastname: Parchment
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000633:toxicity*; D001681:Biological Assay; D039481:CD11b Antigen / Q000378:metabolism; D018826:CD13 Antigens / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D055014:Granulocyte-Macrophage Progenitor Cells; D006410:Hematopoiesis; D006801:Humans; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D000970:Antineoplastic Agents; D039481:CD11b Antigen; C509295:ITGAM protein, human; D018826:CD13 Antigens
keywords: CD11b/CD13; CD34(+) progenitors; FACS analysis; Hematotoxicity; High content analysis (HCA); High throughput screening (HTS); Microplate culture