Background: Sickle Cell Disease (SCD) is a devastating inherited disease, characterized by polymerization of sickle hemoglobin under deoxygenated conditions that can lead to acute pain crises, ischemia, and chronic organ damage. Pharmacologic anti-sickling agents that decrease polymerization are currently under investigation, however there is no consistent in vitro system to study these compounds; whole patient blood, subject to clinical variability and limited supply, is most often used. Human Umbilical Cord Derived Erythroid Progenitor 2 cells (HUDEP2) are an immortalized CD34+ hematopoietic stem cell (HSC) derived erythroid precursor cell line that can differentiate into red blood cells. We have engineered S-HUDEP2 cells to express sickle hemoglobin (HbS) via CRISPR/Cas9 gene editing. We hypothesized that this cell line will sickle under hypoxic conditions, produce dense red blood cells (DRBC, red cells with a density>1.11 mg/mL that are dehydrated and prone to sickling. If these intrinsic, essential SCD RBC properties are found, we propose to use this novel cell line to screen drug compounds for anti-sickling capabilities.
Methods: S-HUDEP cells were cultured as previously described (Kurita et al, 2013). %HbS and %HbA produced by parent HUDEP and S-HUDEP2 cells were measured by high performance liquid chromatography (HPLC). Hypoxia was induced by placing the cells at 2% O2 for four hours. Parent HUDEP and S-HUDEP2 cells were then fixed with glutaraldehyde and Giemsa stained. % sickling estimated at 40x magnification by a pathologist blinded to cell group counting sickle forms out of 1000 cells. The percentage of dense red blood cells (DRBCs) was quantified by an ADVIA hematology analyzer (Siemens). S-HUDEP2 cells were dosed with 0, 2.5 and 5 µM 5-hydroxymethylfurfural (5-HMF) and 75, 150, and 300 µM GBT440, two known anti-sickling agents, on day ten and day 14 of culture for one hour, subjected to hypoxic conditions and % sickling quantified as described above.
Results: S-HUDEP2 cells express 98% HbS. Under hypoxia, 20% of S-HUDEPs sickle at day 10 of differentiation; 30% of S-HUDEP2 cells sickle at Day +14 of differentiation. Parent HUDEP-2 cells, which produce 98% HbA, did not sickle under hypoxic conditions at any stage of differentiation. 70% of S-HUDEP2 cells were determined to be DRBC under hypoxia at Day 10 and 14 time points; parent HUDEP-2 cells did not produce DRBC under hypoxia. Treatment of S-HUDEP2 cells with 5μM of 5-HMF and 150 μM of GBT440 reduced sickling by 40-50% under hypoxic conditions compared with untreated S-HUDEP2 cells (p<0.01), and reduced %DRBC by 30% (p<0.01).
Conclusions: S-HUDEP2 cells express HbS, form DRBC, and sickle under hypoxic conditions, just like erythroid precursors and mature red blood cells from individuals with SCD. Exposure to two anti-sickling agents, one of which is currently in Phase III clinical trials, significantly decreased S-HUDEP2 sickling under hypoxic conditions, and reduced %DRBC, a marker of disease severity. Advantages of S-HUDEPs over patient samples include genetic and phenotypic uniformity, no human pathogens, and availability to groups without access to patient samples. We therefore conclude that S-HUDEP2s have utility in clinical research, may be used to screen anti-sickling and anti-dense cell compounds in vitro, and may lead to identification of new therapeutic options for SCD patients.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.