MHC-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity in pigs.

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

DNA methylation profiles provide a viable index for porcine pluripotent stem cells.

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor-intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)-specific hypomethylated loci (EShypo-T-DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso-4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso-4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso-622-14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum-free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs.

Generating porcine chimeras using inner cell mass cells and parthenogenetic preimplantation embryos.

BACKGROUND: The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. METHODOLOGY/SIGNIFICANT PRINCIPAL FINDINGS: In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4-8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4-8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. CONCLUSION/SIGNIFICANCE: Our findings indicate that the aggregation method using parthenogenetic morulae or 4-8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.

Generation of naive-like porcine-induced pluripotent stem cells capable of contributing to embryonic and fetal development.

In pluripotent stem cells (PSCs), there are 2 types: naive and primed. Only the naive type has the capacity for producing chimeric offspring. Mouse PSCs are naive, but human PSCs are in the primed state. Previously reported porcine PSCs appear in the primed state. In this study, putative naive porcine-induced pluripotent stem cells (iPSCs) were generated. Porcine embryonic fibroblasts were transduced with retroviral vectors expressing Yamanaka's 4 genes. Emergent colonies were propagated in the presence of porcine leukemia inhibitory factor (pLIF) and forskolin. The cells expressed pluripotency markers and formed embryoid bodies, which gave rise to cell types from all 3 embryonic germ layers. The naive state of the cells was demonstrated by pLIF dependency, 2 active X chromosomes (when female), absent MHC class I expression, and characteristic gene expression profiles. The porcine iPSCs contributed to the in vitro embryonic development (11/24, 45.8%) as assessed by fluorescent markers. They also contributed to the in utero fetal development (11/71, 15.5% at day 23; 1/13, 7.7% at day 65). This is the first demonstration of macroscopic fluorescent chimeras derived from naive-like porcine PSCs, although adult chimeras remain to be produced.

A Simplified In Vitro Teratoma Assay for Pluripotent Stem Cells Injected Into Rodent Fetal Organs.

Teratoma formation assays are established methods for evaluating the pluripotency of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. Teratoma formation in immunodeficient mice takes approximately 2 months. Here, we have developed a novel assay system for developing teratomas in vitro from ES cells and iPS cells in a short period. In vitro culture of ES, iPS, and mesenchymal stem cells (MSCs) in fetal rat metanephroi for 1 week resulted in distinct cell-dependent distribution patterns: Pluripotent cells (ES and iPS cells) formed aggregated masses, whereas MSCs showed disseminated distribution. The aggregated masses that had developed from ES cells and iPS cells after 2 weeks of culture comprised teratomas, though they were largely composed of immature components. Furthermore, in vitro organ culture for 1 week followed by relay transplantation into immunodeficient mice resulted in considerably rapid growing teratomas (teratomas developed in 4 weeks) having similar pathological features as of the teratomas developed using conventional 7-week in vivo teratoma formation assays. In addition, the initial cell number required in the in vitro assay was 1 × 103 cells, which was about 1% of the number of cells required in the conventional in vivo teratoma formation assays. These results suggest that the in vitro teratoma assay is a rapid and convenient screening system and might be an alternative method for developing teratomas for investigating the pluripotency of ES cells and iPS cells.

ERK1/2 phosphorylate GEF-H1 to enhance its guanine nucleotide exchange activity toward RhoA.

Rho GTPases play an essential role in the regulation of many cellular processes. Although various guanine nucleotide exchange factors (GEFs) are involved in the activation of Rho GTPases, the precise mechanism regulating such activity remains unclear. We have examined whether ERK1/2 are involved in the phosphorylation of GEF-H1, a GEF toward RhoA, to modulate its activity. Expression of GEF-H1 in HT1080 cells with constitutive ERK1/2 activation induced its phosphorylation at Thr(678), which was totally abolished by treating the cells with PD184352, an ERK pathway inhibitor. Stimulation of HeLa S3 cells with 12-O-tetradecanoyl-phorbol-13-acetate induced the phosphorylation of GEF-H1 in an ERK-dependent manner. ERK1/2-mediated Thr(678)-phosphorylation enhanced the guanine nucleotide exchange activity of GEF-H1 toward RhoA. These results suggest that the ERK pathway, by enhancing the GEF-H1 activity, contributes to the activation of RhoA to regulate the actin assembly, a necessary event for the induction of cellular responses including proliferation and motility.

Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44.

Elevated expression of matrix metalloproteinases (MMPs) is associated with increased metastatic potential in many tumor cells. As activation of the ERK pathway has been linked to the expression of MMP-9, we examined a possible correlation between ERK activation, MMP-9 expression, and invasive phenotype in human tumor cells. Activation state of the ERK pathway in tumor cells was well correlated with the invasive phenotype, which was determined by the ability of cells to invade through reconstituted extracellular matrix. Elevated expression of MMP-9 as well as of MMP-3, MMP-14, and CD44 was observed in tumor cells in which constitutive activation of the ERK pathway is detected. Blockade of the ERK pathway by treatment with PD184352, a specific and powerful inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), suppressed the expression of MMP-3, MMP-9, MMP-14, and CD44, and inhibited markedly the invasiveness of tumor cells. These results imply that, in addition to anti-proliferative effects, specific blockade of the ERK pathway is expected to result in anti-metastatic effects in tumor cells.