Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics

Daniels, Deborah E.; Ferguson, Daniel; Griffiths, Rebecca E.; Trakarnsanga, Kongtana; Cogan, Nicola; MacInnes, Katherine A.; Mordue, Kathryn E.; Andrienko, Tatyana N; Ferrer-Vicens, Iván; Jiménez, Daniel Ramos; Lewis, Philip A.; Wilson, Marieangela C.; Canham, Maurice A.; Kurita, Ryo; Nakamura, Yukio; Anstee, David J.; Frayne, Jan

doi:10.1016/j.omtm.2021.06.002

Cited by 16 publications

(24 citation statements)

References 53 publications

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“…BEL-A cells 66 , 67 , established in the lab of Deborah E. Daniels and Jan Frayne, were cultured in StemSpan™ SFEM (Stemcell Technologies) containing 50 ng/mL SCF, 3 U/mL EPO, 1 μM dexamethasone, and 1 µg/mL doxycycline. To induce erythroid differentiation, expanding cells were transferred to primary medium supplemented with 1 µg/mL doxycycline for 4 days, and for a further 4 days without doxycycline.…”

Section: Methodsmentioning

confidence: 99%

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

et al. 2021

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Within the bone marrow microenvironment, endothelial cells (EC) exert important functions. Arterial EC support hematopoiesis while H-type capillaries induce bone formation. Here, we show that BM sinusoidal EC (BM-SEC) actively control erythropoiesis. Mice with stabilized β-catenin in BM-SEC (Ctnnb1OE-SEC) generated by using a BM-SEC-restricted Cre mouse line (Stab2-iCreF3) develop fatal anemia. While activation of Wnt-signaling in BM-SEC causes an increase in erythroblast subsets (PII–PIV), mature erythroid cells (PV) are reduced indicating impairment of terminal erythroid differentiation/reticulocyte maturation. Transplantation of Ctnnb1OE-SEC hematopoietic stem cells into wildtype recipients confirms lethal anemia to be caused by cell-extrinsic, endothelial-mediated effects. Ctnnb1OE-SEC BM-SEC reveal aberrant sinusoidal differentiation with altered EC gene expression and perisinusoidal ECM deposition and angiocrine dysregulation with de novo endothelial expression of FGF23 and DKK2, elevated in anemia and involved in vascular stabilization, respectively. Our study demonstrates that BM-SEC play an important role in the bone marrow microenvironment in health and disease.

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Section: Methodsmentioning

confidence: 99%

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

et al. 2021

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“…Recent progress supports the translatability of such an approach, including work by clinical stage companies to engineer donor human CD34 + hematopoietic stem cells via lentiviral transduction to generate therapeutic RBCs and engineered GPA-based eRBCs . Others have generated immortalized CD34 + cell lines for the production of human erythrocytes from bone marrow, cord blood, and peripheral blood. , Together, these research avenues are exciting potential advances in eRBC technology that could be enabled by the foundations established in this study.…”

Section: Discussionmentioning

confidence: 67%

Building Synthetic Biosensors Using Red Blood Cell Proteins

Dolberg,

Gunnels,

Ling

et al. 2024

ACS Synth. Biol.

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As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.

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“…In this study, we revealed that immortalized erythroid cells are damaged via γ‐irradiation and almost eliminated by leukoreduction filters, but these methods are not completely safe. HPV16 E6/E7 ‐transduced erythroid cell lines have abnormal karyotypes 15 ; thereby, increasing the risk of tumorigenesis if even one transduced cell persists. A recent study found that the double filtration method for blood products can reduce residual leukocyte levels.…”

Section: Discussionmentioning

confidence: 99%

Irradiation is not sufficient to eradicate residual immortalized erythroid cells in in vitro‐generated red blood cell products

Kim

Park

et al. 2023

Transfusion

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Background The generation of immortalized erythroid progenitor cell lines capable of producing enough red blood cells (RBCs) for blood transfusion typically requires the overexpression of oncogenes in stem cells or progenitor cells to permanently proliferate immature cells. It is essential that any live oncogene‐expressing cells are eliminated from the final RBC products for clinical use. Study Design and Methods It is believed that safety issues may be resolved by using a leukoreduction filter or by irradiating the final products, as is conventionally done in blood banks; however, this has never been proven to be effective. Therefore, to investigate whether immortalized erythroblasts can be completely removed using γ‐ray irradiation, we irradiated the erythroblast cell line, HiDEP, and the erythroleukemic cell line, K562 that overexpress HPV16 E6/E7. We then analyzed the extent of cell death using flow cytometry and polymerase chain reaction (PCR). The cells were also subjected to leukoreduction filters. Results Using γ‐ray irradiation at 25 Gy, 90.4% of HiDEP cells, 91.6% of K562‐HPV16 E6/E7 cells, and 93.5% of non‐transduced K562 cells were dead. In addition, 5.58 × 107 HiDEP cells were passed through a leukoreduction filter, and 38 intact cells were harvested, revealing a filter removal efficiency of 99.9999%. However, both intact cells and oncogene DNA were still detected. Discussion Irradiation cannot induce total cell death of oncogene‐expressing erythroblasts and leukocyte filter efficiency is not 100%. Therefore, our findings imply that for clinical applications, safer methods should be developed to completely remove residual nucleated cells from cell line‐derived RBC products.

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Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics

Cited by 16 publications

References 53 publications

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation

Building Synthetic Biosensors Using Red Blood Cell Proteins

Irradiation is not sufficient to eradicate residual immortalized erythroid cells in in vitro‐generated red blood cell products

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