Int J Stem Cells.  2016 May;9(1):53-59. 10.15283/ijsc.2016.9.1.53.

Genetically Engineered In Vitro Erythropoiesis

• Geiler C
• Andrade I
• Clayton A
• Greenwald

• KMID: 2164161
• DOI: http://doi.org/10.15283/ijsc.2016.9.1.53

Abstract

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.

Keyword

Hematopoietic Stem Cells; Erythroblasts; Erythropoiesis; Adult Stem Cells; Erythroleukemia

MeSH Terms

Adult Stem Cells
Culture Media
Erythroblasts
Erythrocytes
Erythropoiesis*
Flow Cytometry
Genetic Engineering
Hematopoietic Stem Cells
Humans
Intercellular Signaling Peptides and Proteins
Leukemia, Erythroblastic, Acute
Plasmids
Culture Media
Intercellular Signaling Peptides and Proteins

Figure

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  Fig. 1 Stages in the development of erythroblasts. (A) Proerythroblast is the earliest committed stage in erythropoiesis. It is rather large cell (12~20 μm), up to three times a normal erythrocyte. Proerythroblast have large nucleus, and blue cytoplasm that forms a thin rim around the nucleus. The chromatin is granular and stripped. The nucleus have multiple nucleoli. Proerythroblast have small pale area adjacent to the nucleus that corresponds to the Golgi apparatus and have a characteristic pale perinuclear halo. (B) Polychromatophilic Normoblast is smaller (12~15 μm) then the proerythroblast. Hemoglobin in the cytoplasm reduces the basophilia of the cytoplasm. The chromatin shows a greater degree of clumping and irregular dense areas of staining are seen in the nucleus. (C) Orthochromatophilic Normoblast is smaller (8~12 μm) than the polychromatophilic normoblast The cytoplasm has the same color as a mature erythrocyte. The orthochromatophilic normoblast is the nucleated erythroid precursor. Scale bar 10 μm.

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  Fig. 2 Fluorescence micrographs of HSC derived erythroblasts after electroporation. (A) 48 hours after electroporation. (B) 96 hours after electroporation. (C) 96 hours after electroporation.

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  Fig. 3 Flow cytomtery analysis. Serial phenotypic analysis by FC anaylsis of HSC derived erythroblast of a control culture compared to genetically enhanced HSC derived erythroblast. By day 41, almost all of the detectable cells (95.9%) analyzed appeared to be CD71+ CD117+, in a pattern that could be consistent with one population of very similar early proerythroblasts. (D) CD117 is the Stem Cell Factor receptor and expressed at the earliest pro-erythroblast stage. CD71 is the transferrin receptor and is strongly expressed in all cells erythroid precursors. (A) Erythroblast culture day 10. (B) Erythroblast culture day 14, control. (C) Genetically enhanced erythroblasts culture day 14, and 3 days after transfection with PU.1/SPI-1 gene. (D) Genetically enhanced erythroblasts culture day 41, and 30 days after transfection with PU.1/SPI-1 gene.

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  Fig. 4 (A) Erythroblast culture post, transfection day #3, culture day #14, (B) Erythroblast culture control, transfection day #30, culture day #41.

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  Fig. 5 (A) Erythroblast culture post, transfection day #3, culture day #14, (B) Erythroblast culture control, transfection day #30, culture day #41.

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  Fig. 6 Erythroblast cells count average of three experiments.

Reference

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