Gene knock-outs in human CD34+ hematopoietic stem and progenitor cells and in the human immune system of mice

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protoco...

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Bibliographic Details
Published in:PloS one 2023-06, Vol.18 (6), p.e0287052-e0287052
Main Authors: Kuppers, Daniel A, Linton, Jonathan, Ortiz Espinosa, Sergio, McKenna, Kelly M, Rongvaux, Anthony, Paddison, Patrick J
Format: Article
Language:English
Subjects:
Analysis
Animals
Antigens, CD34
Biology and Life Sciences
Biotechnology
CD34 antigen
Cell culture
Cells (biology)
Computer and Information Sciences
CRISPR
Efficiency
Engineering and Technology
Flow cytometry
Gene Knockout Techniques
Genes
Genomes
Genomics
Health aspects
Hematopoietic Stem Cell Transplantation - methods
Hematopoietic Stem Cells
Hemopoiesis
Humans
Immune System
Immunodeficiency
Lab Protocol
Medical research
Mice
Mice, SCID
Progenitor cells
Protocol
Research and Analysis Methods
Scientific equipment and supplies industry
Technology application
Transfection
Transplantation
Transplants & implants
Xenotransplantation
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Summary:Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate "humanized mice". To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and transplant these modified cells in immunodeficient mice with high engraftment levels and multilineage hematopoietic differentiation. The result is a humanized mouse from which we knocked out a gene of interest from their human immune system.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0287052