Format

Send to

Choose Destination
J Vis Exp. 2019 Feb 15;(144). doi: 10.3791/58933.

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

Author information

1
Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center.
2
Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center; Department of Medicine, University of Washington; Department of Pathology, University of Washington.
3
Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center; Department of Medicine, University of Washington; cwpeters@fredhutch.org.

Abstract

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

PMID:
30829324
DOI:
10.3791/58933

Supplemental Content

Full text links

Icon for MyJove Corporation
Loading ...
Support Center