Selami Demirci1, Saurabh K Bhardwaj2, Naoya Uchida3, Juan J Haro-Mora1, Byoung Ryu4, Gerd A Blobel2, John F Tisdale1. 1. Sickle Cell Branch, National Heart Lung and Blood Institutes/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. 2. Department of Pediatrics, Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA, USA. 3. Sickle Cell Branch, National Heart Lung and Blood Institutes/National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. Electronic address: uchidan@nhlbi.nih.gov. 4. Experimental Hematology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Abstract
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. Published by Elsevier Inc.
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. Published by Elsevier Inc.
Authors: Selami Demirci; Bjorg Gudmundsdottir; Quan Li; Juan J Haro-Mora; Tina Nassehi; Claire Drysdale; Morgan Yapundich; Jackson Gamer; Fayaz Seifuddin; John F Tisdale; Naoya Uchida Journal: Mol Ther Methods Clin Dev Date: 2020-04-18 Impact factor: 6.698