Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY; Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester, Rochester, NY.
Center for Pediatric Biomedical Research, Department of Pediatrics, University of Rochester, Rochester, NY.
Exp Hematol. 2019 Jun;74:19-24.e4. doi: 10.1016/j.exphem.2019.04.001. Epub 2019 Apr 18.
A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.
用于输血治疗的体外红细胞生产的一个主要障碍是培养成分的成本，细胞因子占培养成本的一半以上。细胞培养细胞因子也是体外研究人类红细胞生成的主要费用。HUDEP-2 细胞是一种 E6/E7 永生化红细胞系，用于体外研究人类红细胞生成。与用于研究人类红细胞生成的其他细胞系（如 K562 细胞）相比，HUDEP-2 细胞能够进行终末成熟，包括血红蛋白积累和染色质浓缩。因此，HUDEP-2 细胞是研究不适合原代细胞培养的宝贵资源；然而，依赖于细胞因子干细胞因子（SCF）和促红细胞生成素（EPO）使得 HUDEP-2 培养维持成本非常高。为了降低培养成本，我们使用 CRISPR/Cas9 基因组编辑将一个组成型激活突变引入到 SCF 受体基因 KIT 中，目的是产生能够独立于 SCF 扩增的人类红细胞。生成并表征了三个具有独特 KIT 受体基因型的独立 HUDEP-2 系。所有三条系都能够在没有 SCF 的情况下进行强大的扩增，使培养成本降低了大约一半。重要的是，这些系仍然能够进行终末成熟。这些数据表明，向人类红细胞中引入 c-Kit 激活突变可能有助于降低红细胞培养成本，使体外红细胞生成研究以及最终的体外红细胞生产更具经济可行性。
