Figure 1. General HemaTox™ Kit Procedure

*The cell isolation step may be omitted if pre-enriched CD34⁺ cells are used.

Table 1. Antibody Staining Panel for Immunophenotyping by Flow Cytometry

*Recommended flurochromes are shown; however, users should select the fluorochrome that is appropriate for their instrument and analysis.

Figure 2. Flow Cytometry Plots Showing Cells Produced After Culture of CD34⁺ HSPCs with HemaTox™ Erythroid, Myeloid and Megakaryocyte Kits

(A) Human CB CD34⁺ cells were cultured with (B) HemaTox™ Erythroid, (C) Myeloid and (D) Megakaryocyte Kits using the protocol above. After the appropriate culture period, cells were harvested and stained for cell surface proteins expressed on erythroid (CD71 and GlyA), myeloid (CD13 and CD15) or megakaryocytic (CD45 and CD41) cells, respectively.

Figure 3. Concentration- Dependent Inhibition of Hematopoietic Progenitor Cell Outgrowth with HemaTox™ Erythroid, Myeloid and Megakaryocyte Kits

Dose-response curves were produced for a selection of 6 drugs using the HemaTox™ Erythroid, Myeloid and Megakaryocyte Kits. These curves can be used to calculate IC₅₀ and IC₉₀ values for each drug. Cell numbers at each drug concentration were normalized to the percentages (%) of maximum cell growth. Two topoisomerase inhibitors (Topotecan [A,B,C] and Irinotecan [not shown]), two tyrosine kinase inhibitors (Sunitinib [D,E,F] and Imatinib [not shown]) and two anti-metabolites (Cisplatin [G,H,I] and 5-Fluorouracil [5-FU, not shown]) were evaluated using human BM CD34⁺ cells according to the protocol for each kit (see PIS or protocol on page 2). Each data point represents the average of 3 - 6 replicate cultures (vertical bars = standard error of the mean).

Figure 4. Reproducibility of HemaTox™ Kit Results Between Experiments and Using Different CD34⁺ Cell Preparations

Dose-response curves were generated from titrations of 5-FU added to human CB CD34⁺ cells isolated from 3 donors and cultured with the HemaTox™ (A,D) Erythroid, (B,E) Myeloid and (C,F) Megakaryocyte Kits. Three to five separate experiments were performed with cells from each donor. In each assay similar IC₅₀ values were obtained with cells from different donors and in different experiments with cells from the same donor. Shown are values normalized to the percentages (%) of maximum cell growth without the drug. Despite differences in absolute cell counts, curves are reproducible when normalized within each experiment. (D,E,F) Tables showing IC₅₀ values generated for 5-FU in culture with each kit including standard deviation (SD) and the coefficient of variation (% CV) across experiments.

Figure 5. Correlation Between IC₅₀ Values for Six Drugs Measured Using the CFU-GM Assay and the 96-Well Plate Liquid Culture-Based HemaTox™ Myeloid Kit

Human BM CD34⁺ cells were cultured in colony-forming unit - granulocyte/ macrophage (CFU-GM) assays with MethoCult™ medium and in liquid culture with the HemaTox™ Myeloid Kit. IC₅₀ values generated using each assay were plotted on the X and Y axes and shown to be highly correlated with a coefficient of determination (R²) of 0.91.

Figure 6. Lineage-Specific Differences in Hematotoxicity Identified with HemaTox™ Erythroid, Myeloid and Megakaryocyte Kits

Results show average IC₅₀ values for each drug tested on human BM CD34⁺ cells using the HemaTox™ Erythroid (grey), Myeloid (gold) and Megakaryocyte (orange) Kits. Most drugs show similar toxicity for each lineage but some, such as Sunitinib, are ~100-fold more toxic for erythroid than for megakaryocyte progenitor differentiation with intermediate toxicity for myeloid progenitor differentiation. Vertical lines indicate standard error of the mean (SEM) (n = 4 - 8). See Table 2 for specific IC₅₀ values determined using the CFU-GM assay vs. HemaTox™ kits for Sunitinib.

Table 2. Comparison of IC₅₀ Values for Sunitinib as Determined Using CFU Assay and HemaTox™ Kits

IC₅₀ values were generated in CFU assays for lineage-specific progenitor cells by plating human BM CD34⁺ cells in MethoCult™ media that supports the growth of erythroid progenitor cells (BFU-E: burst-forming unit - erythroid) or myeloid progenitor cells (CFU-GM: colony-forming unit - granulocyte/macrophage), or in MegaCult™-C medium that supports the growth of megakaryocyte progenitor cells (CFU-Mk: colony-forming unit - megkaryocyte). Cells were also cultured with the HemaTox™ Erythroid, Myeloid and Megakaryocyte Kits. IC₅₀ values were generated for all 6 assays. These results show that the erythroid lineage is ~100- fold more sensitive to Sunitinib than the megakaryocyte lineage. Additionally, the IC₅₀ measured with the HemaTox™ Erythroid Kit was ~10-fold lower than the IC₅₀ measured in the CFU assay for erythroid progenitor cells, BFU-E. This indicates that for certain drugs the HemaTox™ kits may identify toxicities that are less apparent when using classical CFU assays.