Generation of Rh D-negative blood using CRISPR/Cas9

Area of Science:

• Regenerative Medicine
• Hematology
• Gene Editing

Background:

• Global blood supply shortages, particularly for rare blood types, pose a significant challenge to healthcare systems.
• Induced pluripotent stem cells (iPSCs) offer a promising avenue for in vitro blood cell manufacturing.
• The development of universal red blood cells (RBCs) is a key goal in transfusion medicine.

Purpose of the Study:

• To generate O-type Rh D-negative human iPSCs (hiPSCs) using CRISPR/Cas9 gene editing.
• To differentiate these modified hiPSCs into universal RBCs for potential clinical application.
• To address the critical need for universal donor blood products.

Main Methods:

• Utilized homology-directed repair (HDR)-based CRISPR/Cas9 to knock out the RHD gene in hiPSCs.
• Selected hiPSCs with hematopoietic differentiation preferences for gene modification.
• Optimized an erythroid differentiation protocol and culture conditions (oxygen concentration) for RHD-knockout hiPSCs.

Main Results:

• Successfully generated an RHD-knockout hiPSC line (HuAiPSC-A1-RHD-/-).
• Differentiated hiPSCs into erythrocytes expressing RBC markers (CD71, CD235a) but lacking the D antigen.
• Demonstrated that the generated erythrocytes did not agglutinate with anti-Rh D reagents, confirming their universal type.

Conclusions:

• The study successfully produced O-type Rh D-negative universal erythrocytes from RHD-knockout hiPSCs.
• The combination of hematopoietic-preferring hiPSCs, HDR-based CRISPR/Cas9, and optimized differentiation is effective.
• This efficient production method holds significant potential for clinical transfusion medicine applications.