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.

Figure 1

Derivation of HLA-homozygous ESCs. (a) Diagram showing the strategy for obtaining HLA-homozygous clones by targeting the HMGA1 gene centromeric to the HLA locus, then selecting for cells that had lost the HyTK gene by mitotic recombination with ganciclovir. The red and blue chromosomes represent the two copies of chromosome 6 in each cell, with the clusters of HLA class I, II, and III genes indicated. (b) Map of the AAV2-HMGA1-HyTKpA targeting vector and HMGA1 gene, showing the locations of the Southern blot probes and restriction enzyme sites (K, Kpn I). (c) Southern blot analysis of Kpn I–digested genomic DNA of parental H1 ESCs, two clones targeted at the HMGA1 gene (c4 and c5), and three subclones obtained by ganciclovir selection (c4A, c4B, and c5A), probed with HMGA1 and HyTK probes. Asterisks indicate the locations of the two HyTK-hybridizing fragments derived from the targeted allele. The faint HyTK-hybridizing band in clone c5A and H1 represents trace signal from the hygromycin-resistant mouse embryonic fibroblast feeder cells. Subclones c4A and c4B contain a novel HyTK-hybridizing fragment demonstrating a rearrangement of the HyTK gene that can account for ganciclovir resistance in these clones. (d) HLA typing results for parental H1 ESCs, gene-targeted clone c5, and ganciclovir-resistant subclone c5A. (e) Copy number analysis of SNP data. Loss of heterozygosity in clone c5A telomeric to the PRIM2A gene on chromosome 6 is shown by an increase in copy number of one allele and decrease of the other relative to the parental cell line (H1). GCV-R, ganciclovir-resistance; HLA, human leukocyte antigens.

Figure 2

B2M targeting and transgene removal. (a) The structures of the AAV3-B2M-ETKNpA– and AAV3-EHyTKpA–targeting vectors, and the human B2M locus are shown before targeting, and after two rounds of targeting and transgene removal by Cre. The locations of Southern blot probes and enzymes (X, Xba I) are indicated. (b) Diagram of the wild-type, targeted, and transgene-deleted alleles of the B2M gene with the locations of PCR primers, and the results obtained from an analysis of 45 Cre-treated clones. The gel below shows a representative PCR analysis of Cre-transduced clones with the different primer combinations, and the resulting allele designations are shown. DNA from the parental H1 cells (+/+), and clones targeted in one (+/TKNeo) or both (HyTK/TKNeo) B2M alleles were used as controls. (c) Southern blot analysis of Xba I–digested genomic DNAs of parental H1 cells (+/+), one clone targeted at one B2M allele (+/TKNeo), one clone targeted at both B2M alleles (HyTK/TKNeo), and four clones obtained after removal of the transgenes with Cre (loxP/loxP c1–4), probed with B2M- or TK-specific probes. Positions of the different possible B2M fragments are shown at the left.

Figure 3

Gene expression in B2M-targeted clones. (a) Flow cytometry analysis showing surface expression of B2M and HLA class I heavy chains (antibody W6/32 binds HLA-A, -B, and -C) on undifferentiated parental H1 ESCs (+/+), ESCs targeted at one B2M allele (+/TKNeo), both B2M alleles (HyTK/TKNeo), or after Cre-mediated transgene removal (loxP/loxP). Mean fluorescence intensity is indicated in each case for the specific antibody (blue numbers) and isotype controls (black numbers). (b) Western blot analysis of total cellular protein extracts from parental H1 ESCs (+/+), ESCs targeted at one B2M allele (+/TKNeo), or both B2M alleles after Cre-mediated transgene excision (loxP/loxP) performed with the indicated antibodies. Signal ratios for B2M or HLA class I heavy chains are shown after normalization to the wild-type (+/+) sample. (c) Transcriptional array analysis. The relative mRNA expression levels for B2M, HLA class I heavy chains, and other B2M-binding protein genes (CD1A-D, FCGRT, HFE, and MR1) are shown for two B2M^−/− clones (loxP/loxP c1 and c2) and two independent cultures of parental H1 ESCs (+/+). Values are expressed as log2, after normalization and background subtraction. (d) Sample relation between two duplicate cultures of parental H1 cells (H1A and H1B) and two B2M^−/− clones (loxP/loxP c1 and c2) based on a global expression analysis of 18,174 genes with SD/mean >0.1. HLA, human leukocyte antigens.

Figure 4

Immune responses to B2M-targeted cells. (a) MLR results showing ³[H]-thymidine incorporation by PBMC responder cells mixed with irradiated, undifferentiated B2M^+/+ or B2M^loxP/loxP ESCs (left panel) or with day 15 EB-derived cells (right panel) at a ratio of 1:1. Two independent PBMC responder cell preparations were used when analyzing EB cells. Controls included unstimulated PBMCs, and PBMCs stimulated by allogeneic PBMCs. (b) Flow cytometry analysis of day 15 B2M^+/+ or B2M^loxP/loxP EB cells showing the surface expression of CD34, HLA class I (HLA-A, -B, and -C), and HLA class II (HLA-DR), with isotype controls. All preparations were labeled with 7-amino-actinomycin D (7AAD) to improve gating and remove dead cells. (c) Intracellular cytokine staining of HLA-A*0201/NLV-CMVpp65-specific T cells stimulated with an equal number of B2M^+/+ or B2M^loxP/loxP EB cells, HLA class I–deficient K562 cells, or HLA-A*0201–expressing K562-A2 cells, with or without prior pulsing with NLVPMVATV (NLV) peptide from the CMV pp65 protein. HLA, human leukocyte antigens.

Figure 5

NK cells do not lyse B2M^−/− EB cells. Flow cytometry analysis of CD107a expression on CD56⁺ NK effector cells when incubated with B2M^+/+ or B2M^loxP/loxP day 15 EB cells at a ratio of 1:1. Controls included unstimulated NK cells, and NK cells incubated with class I–negative K562 cells. NK, natural killer.