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Rh D blood group conversion using transcription activator-like effector nucleases.

Kim YH, Kim HO, Baek EJ, Kurita R, Cha HJ, Nakamura Y, Kim H - Nat Commun (2015)

Bottom Line: Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs).After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained.Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, South Korea [2] Department of Pharmacology, Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Yonsei University College of Medicine, Seoul 120-752, South Korea.

ABSTRACT
Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

No MeSH data available.


The RHD mutation does not affect the differentiation and function of HiDEP-1.(a,b) At the indicated times after the induction of erythrocyte differentiation, differentiation of each HiDEP-1 cell line was quantified using microscopy after Wright–Giemsa staining. (a) Morphological analysis of differentiation. The sum of two independent experiments is shown. (b) Cell size during differentiation. ANOVA showed that the cell sizes were the same in the parental and RHD-mutant clones. Error bars represent the s.e.m. n=3. (c) Oxygen equilibrium curves of parental and RHD-mutant clones (E1_B, E4_B and E4_M). Human adult peripheral blood (PB) cells were used as the control.
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f7: The RHD mutation does not affect the differentiation and function of HiDEP-1.(a,b) At the indicated times after the induction of erythrocyte differentiation, differentiation of each HiDEP-1 cell line was quantified using microscopy after Wright–Giemsa staining. (a) Morphological analysis of differentiation. The sum of two independent experiments is shown. (b) Cell size during differentiation. ANOVA showed that the cell sizes were the same in the parental and RHD-mutant clones. Error bars represent the s.e.m. n=3. (c) Oxygen equilibrium curves of parental and RHD-mutant clones (E1_B, E4_B and E4_M). Human adult peripheral blood (PB) cells were used as the control.

Mentions: We evaluated whether TALEN-induced RHD knockout affects erythrocyte generation from the erythroid progenitor cells or the oxygen-carrying functions. Parental and E1_B cells were induced for differentiation into erythrocytes as previously reported by our group19. Morphological analysis showed that erythrocyte differentiation was comparable among parental, E1_B, E4_B and E4_M clones (Fig. 7a), suggesting that TALEN-induced RHD knockout does not affect this process. Furthermore, the average cell size during differentiation was similar among the parental and three mutant clones (Fig. 7b), supporting that RHD knockout does not influence differentiation. Moreover, flow cytometry showed that, after the induction of differentiation, the expression levels of erythroblast markers such as glycophorin A and CD71 were similar between the parental and three mutant clones (Supplementary Fig.10), corroborating that RHD knockout does not affect differentiation. For a functional comparison between the parental line and three mutant clones, we measured their oxygen-carrying abilities. The oxygen binding and dissociation curves obtained were similar for each of these cell types (Fig. 7c), indicating that the TALEN-induced RHD mutation does not affect red blood cell function. Taken together, these results indicate that erythrocyte differentiation and oxygen-carrying functions are not affected by the TALEN-induced RHD mutation.


Rh D blood group conversion using transcription activator-like effector nucleases.

Kim YH, Kim HO, Baek EJ, Kurita R, Cha HJ, Nakamura Y, Kim H - Nat Commun (2015)

The RHD mutation does not affect the differentiation and function of HiDEP-1.(a,b) At the indicated times after the induction of erythrocyte differentiation, differentiation of each HiDEP-1 cell line was quantified using microscopy after Wright–Giemsa staining. (a) Morphological analysis of differentiation. The sum of two independent experiments is shown. (b) Cell size during differentiation. ANOVA showed that the cell sizes were the same in the parental and RHD-mutant clones. Error bars represent the s.e.m. n=3. (c) Oxygen equilibrium curves of parental and RHD-mutant clones (E1_B, E4_B and E4_M). Human adult peripheral blood (PB) cells were used as the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4490371&req=5

f7: The RHD mutation does not affect the differentiation and function of HiDEP-1.(a,b) At the indicated times after the induction of erythrocyte differentiation, differentiation of each HiDEP-1 cell line was quantified using microscopy after Wright–Giemsa staining. (a) Morphological analysis of differentiation. The sum of two independent experiments is shown. (b) Cell size during differentiation. ANOVA showed that the cell sizes were the same in the parental and RHD-mutant clones. Error bars represent the s.e.m. n=3. (c) Oxygen equilibrium curves of parental and RHD-mutant clones (E1_B, E4_B and E4_M). Human adult peripheral blood (PB) cells were used as the control.
Mentions: We evaluated whether TALEN-induced RHD knockout affects erythrocyte generation from the erythroid progenitor cells or the oxygen-carrying functions. Parental and E1_B cells were induced for differentiation into erythrocytes as previously reported by our group19. Morphological analysis showed that erythrocyte differentiation was comparable among parental, E1_B, E4_B and E4_M clones (Fig. 7a), suggesting that TALEN-induced RHD knockout does not affect this process. Furthermore, the average cell size during differentiation was similar among the parental and three mutant clones (Fig. 7b), supporting that RHD knockout does not influence differentiation. Moreover, flow cytometry showed that, after the induction of differentiation, the expression levels of erythroblast markers such as glycophorin A and CD71 were similar between the parental and three mutant clones (Supplementary Fig.10), corroborating that RHD knockout does not affect differentiation. For a functional comparison between the parental line and three mutant clones, we measured their oxygen-carrying abilities. The oxygen binding and dissociation curves obtained were similar for each of these cell types (Fig. 7c), indicating that the TALEN-induced RHD mutation does not affect red blood cell function. Taken together, these results indicate that erythrocyte differentiation and oxygen-carrying functions are not affected by the TALEN-induced RHD mutation.

Bottom Line: Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs).After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained.Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate School of Biomedical Science and Engineering/College of Medicine, Hanyang University, Seoul 133-791, South Korea [2] Department of Pharmacology, Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Yonsei University College of Medicine, Seoul 120-752, South Korea.

ABSTRACT
Group O D-negative blood cells are universal donors in transfusion medicine and methods for converting other blood groups into this universal donor group have been researched. However, conversion of D-positive cells into D-negative is yet to be achieved, although conversion of group A or B cells into O cells has been reported. The Rh D blood group is determined by the RHD gene, which encodes a 12-transmembrane domain protein. Here we convert Rh D-positive erythroid progenitor cells into D-negative cells using RHD-targeting transcription activator-like effector nucleases (TALENs). After transfection of TALEN-encoding plasmids, RHD-knockout clones are obtained. Erythroid-lineage cells differentiated from these knockout erythroid progenitor cells do not agglutinate in the presence of anti-D reagents and do not express D antigen, as assessed using flow cytometry. Our programmable nuclease-induced blood group conversion opens new avenues for compatible donor cell generation in transfusion medicine.

No MeSH data available.