HLA Engineering of Human Pluripotent Stem Cells

Abstract

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells

“…In addition to the direct contribution of arteriogenesis, other mechanisms maybe also involved. For example, AECs expressed high levels of DLL4, which can activate the Notch pathway and promote cardiac regeneration (40)(41)(42)(43)(44)(45). Another mechanism may be high NO production as NO plays a pivotal role in ischemic protection (46).…”

Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy

“…1a), can be eliminated by either disrupting the TCR, using target site–specific nucleases after T-cell differentiation 33 , or by generating T-iPSCs from virus-specific T cells, which due to their recognition of a pathogen-derived antigen, are less likely to cause graft-versus-host disease 34 . Allorejection of CAR-iPSC-T cells (which express HLA molecules) can be minimized by generating iPSCs from common HLA haplotypes (to ensure their histocompatibility with matched unrelated recipients 35 ) or by repressing HLA expression through additional genetic modification 36 . Finally, the risk of insertional oncogenesis secondary to gene transfer can be decreased by integrating the CAR cDNA and other genes, such as suicide genes and noninvasive imaging reporters 37 , at genomic safe harbor sites 38 .…”

Toward eliminating HLA class I expression to generate universal cells from allogeneic donors

“…9 As an alternative, the Cre-LoxP system can be deployed to disrupt the b 2 -microglobulin locus, and thus HLA class I expression, but this requires removal of antibiotic-resistant genes by Cre recombinase, which may introduce unwanted recombination events. 10 We and others have previously attempted to downregulate HLA class I expression by introducing small interfering RNA targeting HLA heavy chains or b 2 -microglobulin. [11][12][13] Although these posttranscriptional approaches reduce antigen levels, they require sustained transgene expression and, moreover, reduce but do not completely eliminate HLA expression.…”