Genetically Engineered In Vitro Erythropoiesis

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the US blood supply for transfusion treatments. Engineered blood produced from hematopoietic stem cell (HSC) derived red blood cells in a laboratory is possible, but critical barriers exist to the production of clinically relevant quantities of red blood cells required to create a unit of blood. Erythroblasts have a finite expansion capacity and there are many negative regulatory mechanisms that inhibit in vitro erythropoiesis. In order to overcome these barriers and enable mass production, the expansion capacity of erythroblasts in culture will need to be exponentially improved over the current state of art. This work focused on the hypothesis that genetic engineering of HSC derived erythroblasts can overcome these obstacles. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis efficiency from human adult stem cell derived erythroblasts utilizing genetic engineering. The ultimate goal is to enable the mass production of engineered blood. METHODS: HSCs were isolated from blood samples and cultured in a liquid media containing growth factors. Cells were transfected using a Piggybac plasmid transposon. RESULTS: Cells transfected with SPI-1 continued to proliferate in a liquid culture media. Fluorescence-activated cell sorting (FACS) analysis on culture day 45 revealed a single population of CD71+CD117+ proerythroblast cells. The results of this study suggest that genetically modified erythroblasts could be immortalized in vitro by way of a system modeling murine erythroleukemia. CONCLUSION: Genetic modification can increase erythroblast expansion capacity and potentially enable mass production of red blood cells.

Exogenous c-Myc Blocks Differentiation and Improves Expansion of Human Erythroblasts In vitro

BACKGROUND: Engineered blood has the greatest potential to combat a predicted future shortfall in the blood supply for transfusion treatment. The production of red blood cells from hematopoietic stem cells in the laboratory is possible but the mass production of red blood cells to the level present in a blood transfusion unit is currently not possible. The proliferation capacity of the immature red blood cell will need to be increased to enable mass production. This work focused on the hypothesis that exogenous c-Myc can delay the differentiation process of highly proliferative immature erythroblasts, and increase the proliferation capacity of erythroblast cell cultures. OBJECTIVES: The objective of this research effort was to improve in vitro erythropoiesis from stem cells without gene transfection with the eventual goal of producing blood for transfusion treatment in a manner that could be easily translated into clinical medicine. METHODS: The hematopoietic stem cell containing mononuclear cell fraction of venous blood samples was cultured in a liquid media containing erythroblasts growth factors with and without exogenous c-Myc combined with a cell-penetrating peptide. The cells were maintained in the liquid culture media for 23 days. Viable cells were counted and analyzed with flow cytometry. RESULTS: Our results show a 4 fold increase in expansion of the erythroblasts grown in the c-Myc containing growth media compared to the control. Eighty percent of these cells retained the CD117 surface receptor, indicating immature cells. CONCLUSION: Exogenous c-Myc blocks the differentiation and improves in vitro expansion of human erythroblasts.