Generation of HIV-Resistant Macrophages from IPSCs by Using Transcriptional Gene Silencing and Promoter-Targeted RNA

Abstract

Highly active antiretroviral therapy (HAART) has markedly prolonged the prognosis of HIV-1 patients. However, lifelong dependency on HAART is a continuing challenge, and an effective therapeutic is much desired. Recently, introduction of short hairpin RNA (shRNA) targeting the HIV-1 promoter was found to suppress HIV-1 replication via transcriptional gene silencing (TGS). The technology is expected to be applied with hemato-lymphopoietic cell transplantation of HIV patients to suppress HIV transcription in transplanted hemato-lymphopoietic cells. Combination of the TGS technology with new cell transplantation strategy with induced pluripotent stem cell (iPSC)-derived hemato-lymphopoietic cells might contribute to new gene therapy in the HIV field. In this study, we evaluated iPSC-derived macrophage functions and feasibility of TGS technology in macrophages. Human iPSCs were transduced with shRNAs targeting the HIV-1 promoter region (shPromA) by using a lentiviral vector. The shPromA-transfected iPSCs were successfully differentiated into functional macrophages, and they exhibited strong protection against HIV-1 replication with alteration in the histone structure of the HIV-1 promoter region to induce heterochromatin formation. These results indicated that iPS-derived macrophage is a useful tool to investigate HIV infection and protection, and that the TGS technology targeting the HIV promoter is a potential candidate of new gene therapy.

Keywords: HIV-1; NF-κB; induced pluripotent stem cells; macrophage; siRNA; transcriptional-gene-silencing.

Figure 1

Generation of iPSCs Transduced with shRNA Targeting HIV Promoter (A) Map of the short hairpin RNA (shRNA) in SIN lentivirus vector. SIN vector includes a central polypurine tract (cPPT), U6 promoter (U6 P), shRNA, ubiquitin C promoter (Ubc), and EGFP. WPREmt stands for mutant woodchuck promoter response element. Lack of the entire enhancer-promoter of the U3 region allows viral genome integration, but not expression. The sense, hairpin, and anti-sense sequence were inserted downstream of the U6 promoter sequence. Alignment of the shRNA targeting the NF-κB sites (shPromA) and the two-base mismatch control (shpromM2) is shown below. The red text in the alignment of shPromM2 highlights nucleic acids that differ from the shPromA sequence. (B) The iPSC lines were observed by alkaline phosphatase (ALP) staining or fluorescence microscopy after transduction with shPromA (PromA), shPromA-M2 (M2), and control (wild-type [WT]). Scale bar, 200 μm. (C) Flow cytometric analysis of human SSEA-4 and TRA-1-60 on unstained untransduced iPSCs, untransduced iPSCs, and iPSCs transduced with shPromA and with shPromA-M2 (unstained WT, WT, PromA, and M2, respectively). (D) iPSC lines were fixed by ethanol, stained by anti-OCT3/4 antibodies, and analyzed by fluorescence microscopy for OCT3/4 expression after transduction with shPromA (PromA), shPromA-M2 (M2), and control (WT). (E) iPSCs transduced with shPromA (PromA) and shPromA-M2 (M2) were analyzed for EGFP expression by using flow cytometer (black). Untransduced iPSCs are shown as negative controls (white).

Figure 2

Differentiation of Human iPSCs into HSCs and Macrophages In Vitro (A) In vitro macrophage differentiation protocol. (B) May-Giemsa staining of macrophages derived from shPromA (PromA), shPromA-M2 (M2), or original iPSCs (wild-type [WT]) with an image of monocyte-derived macrophage (MDM). (C) iPSCs transduced with shPromA (PromA), shPromA-M2 (M2), and original iPSCs (WT) were differentiated into macrophages. EGFP expression was analyzed using fluorescence microscopy. (D) Macrophages derived from iPSCs transduced with shPromA (PromA) and shPromA-M2 (M2) were analyzed for EGFP expression by using flow cytometer (black). Macrophages derived from wild-type iPSCs are shown as negative controls (white).

Figure 3

Characterization of Macrophages Generated from iPSCs (A) Flow cytometric analysis of human CD45, CD86 on macrophages derived from peripheral monocytes, control iPSCs, and iPSCs transduced with shPromA or shPromM2 (MDM, WT, PromA, and M2, respectively). (B) Flow cytometric analysis of human CD11b, CD11c, HLA-DR, and CCR5 on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively). (C) Macrophages (green) derived from iPSCs transduced with shPromA (upper) and shPromA-M2 (lower) were incubated with Alexa Flour 594 Escherichia coli (red). The cells were microscopically observed after incubation for 1 hr. BioParticles were co-localized in macrophages (yellow in merged pictures). Scale bar, 50 μm. (D) Global gene expression of macrophages derived from iPSCs or MDM was analyzed. Heatmaps show the correlation coefficients between samples.

Figure 4

Suppression of Viral Replication in iPS-Derived Macrophages Expressing shPromA Macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively) were challenged with an R5-tropic HIV-1 Ba-L virus. (A) Real-time PCR of the gag region confirms similar levels of integrated HIV-1 provirus presented in all samples. However, HIV-1 replication was inhibited in PromA, as determined by (B) viral mRNA levels normalized to GAPDH and (C) viral reverse transcriptase activity. Two-way ANOVA revealed statistically significant interaction between transduction of shRNA and viral mRNA levels or reverse transcriptase activity (p < 0.01).

Figure 5

shPromA Suppressed Viral Replication through TGS Chromatin immunoprecipitation assays were performed on macrophages derived from iPSCs transduced with shPromA or shPromM2, and control iPSCs (PromA, M2, and WT, respectively) at day 4. DNA fragments from whole-cell extracts were co-precipitated against (A) histone 3 lysine 9 acetylation (H3K9Ac) and (B) histone 3 lysine 27 trimethylation (H3K27me3). Each value shown is the relative enrichment normalized to the value obtained from mock transfection. *p < 0.05; **p < 0.01.

Figure 6

RNA Sequence of the 86 NF-κB-Driven Genes, Including the IFN Genes Scatterplot of log transformation of the relative expression of mRNA from 86 NF-κB-driven genes. No significant difference was noted in the relative expression (A) between macrophages derived from untransduced iPSCs (wild-type [WT]) and those derived from iPSCs transduced with shPromA (PromA); (B) between macrophages derived from iPSCs transduced with shPromA (PromA) and those derived from iPSCs transduced with shPromA-M2 (M2); and (C) between macrophages derived from iPSCs transduced with shPromA-M2 (M2) and those derived from untransduced iPSCs (WT).