Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.
Generation of 'designer erythroblasts' lacking one or more blood group systems from CRISPR/Cas9 gene-edited human-induced pluripotent stem cells.
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从CRISPR/Cas9基因编辑的人诱导多能干细胞产生缺乏一种或多种血型系统的“设计师成红细胞”。
DOI：
10.1111/jcmm.16872
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发表时间：
2021-10
影响因子：
5.3
通讯作者：
Denomme GA
中科院分区：
文献类型：
--
作者：
Pandey P;Zhang N;Curtis BR;Newman PJ;Denomme GA
尽管输血医学最近取得了进展，但对于多胎妇女和长期输血患者来说，红细胞（RBC）同种异体免疫仍然是一个挑战。有时，诊断实验室依赖于难以获得的稀有试剂红细胞来鉴定这些受试者的不同同种异体抗体。我们利用CRISPR/Cas9基因编辑人类诱导多能干细胞（hiPSC）亲本系（OT1‐1）的血型系统基因（RHAG、GYPB和XK），开发了具有定制表型（Rh null、GPB null和Kx null/Kell low）的红母细胞，解决了这一问题。将引导rna克隆到Cas9‐嘌呤霉素表达载体中，并转染到OT1‐1中。对耐嘌呤霉素的hiPSC KOs进行基因分型。CRISPR/Cas9基因编辑成功产生了三个KO系，RHAG KO， GYPB KO和XK KO。OT1‐1细胞系以及3个KO hiPSC细胞系分化为CD34+CD41+CD235ab+造血祖细胞（HPCs），随后分化为红母细胞。原生OT1‐1红母细胞阳性表达Rh、MNS、Kell和H血型系统。RHAG KO、GYPB KO和XK KO的分化分别导致Rh null、GPB null和Kx null/Kell低的红母细胞形成。OT1‐1以及三种KO红母细胞红细胞标记物cd71和BAND3仍然呈阳性。红母细胞多处于多色/正色分化阶段。观察到造血干细胞衍生的红母细胞增加了约400倍。从CRISPR/Cas9基因编辑的hiPSC中产生的定制红细胞的可用性应该是目前用于检测临床重要的红细胞同种抗体的工具的有用补充。
Despite the recent advancements in transfusion medicine, red blood cell (RBC) alloimmunization remains a challenge for multiparous women and chronically transfused patients. At times, diagnostic laboratories depend on difficult‐to‐procure rare reagent RBCs for the identification of different alloantibodies in such subjects. We have addressed this issue by developing erythroblasts with custom phenotypes (Rh null, GPB null and Kx null/Kell low) using CRISPR/Cas9 gene‐editing of a human induced pluripotent stem cell (hiPSC) parent line (OT1‐1) for the blood group system genes: RHAG, GYPB and XK. Guide RNAs were cloned into Cas9‐puromycin expression vector and transfected into OT1‐1. Genotyping was performed to select puromycin‐resistant hiPSC KOs. CRISPR/Cas9 gene‐editing resulted in the successful generation of three KO lines, RHAG KO, GYPB KO and XK KO. The OT1‐1 cell line, as well as the three KO hiPSC lines, were differentiated into CD34+CD41+CD235ab+ hematopoietic progenitor cells (HPCs) and subsequently to erythroblasts. Native OT1‐1 erythroblasts were positive for the expression of Rh, MNS, Kell and H blood group systems. Differentiation of RHAG KO, GYPB KO and XK KO resulted in the formation of Rh null, GPB null and Kx null/Kell low erythroblasts, respectively. OT1‐1 as well as the three KO erythroblasts remained positive for RBC markers—CD71 and BAND3. Erythroblasts were mostly at the polychromatic/ orthochromatic stage of differentiation. Up to ~400‐fold increase in erythroblasts derived from HPCs was observed. The availability of custom erythroblasts generated from CRISPR/Cas9 gene‐edited hiPSC should be a useful addition to the tools currently used for the detection of clinically important red cell alloantibodies.
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影响因子：
3.7
作者：
Kurita R;Suda N;Sudo K;Miharada K;Hiroyama T;Miyoshi H;Tani K;Nakamura Y
通讯作者：
Nakamura Y
影响因子：
5.9
作者：
ERICKSON, N;QUESENBERRY, PJ
通讯作者：
QUESENBERRY, PJ
影响因子：
2.9
作者：
Kikuchi, Go;Kurita, Ryo;Satake, Masahiro
通讯作者：
Satake, Masahiro
影响因子：
4
作者：
Dias, Jessica;Gumenyuk, Marina;Slukvin, Igor I.
通讯作者：
Slukvin, Igor I.
影响因子：
7.5
作者：
Heshusius, Steven;Heideveld, Esther;van den Akker, Emile
通讯作者：
van den Akker, Emile