Robust erythroid differentiation system for rhesus hematopoietic progenitor cells allowing preclinical screening of genetic treatment strategies for the hemoglobinopathies

• Published in: Cytotherapy (Oxford, England) Vol. 20; no. 10; pp. 1278 - 1287
• Main Authors: DEMIRCI, SELAMI, BHARDWAJ, SAURABH K, UCHIDA, NAOYA, HARO-MORA, JUAN J, RYU, BYOUNG, BLOBEL, GERD A, TISDALE, JOHN F
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.10.2018
• Subjects: Advanced Basic Science; Animals; Cell Culture Techniques - methods; Cell Differentiation; chromatin looping; Chromatography, High Pressure Liquid - methods; DNA-Binding Proteins - genetics; erythroid differentiation; gamma-Globins - analysis; gamma-Globins - genetics; gene therapy; Genetic Therapy - methods; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - metabolism; Hemoglobinopathies - therapy; Hemoglobins - analysis; Humans; lentiviral vector; LIM Domain Proteins - genetics; Macaca mulatta; non-human primate; Other; Promoter Regions, Genetic; Recombinant Proteins - genetics; Tandem Mass Spectrometry; Transcription Factors - genetics; Transduction, Genetic; Zinc Fingers - genetics; gene therapy; non-human primate; chromatin looping; lentiviral vector; erythroid differentiation
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2018.07.002

Background aims γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. Methods We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. Results Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels ( P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media ( P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups ( P < 0.01). Discussion In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.