Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity.

BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch. METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous ß-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed. RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo. CONCLUSION: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

Neurotrophic effect of citrus auraptene: neuritogenic activity in PC12 cells.

The activation of extracellular signal-regulated kinases (ERK) leads to a number of cellular changes associated with the development of long-term memory. Using cultured cortical neurons, we previously showed that the n-hexane extract prepared from the peels of Citrus grandis (Kawachi bankan) induces the activation of ERK1/2 and that one of the compounds with this ability in the extract is 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), a Citrus polymethoxyflavone. In fact, we found that HMF has the ability to rescue mice from drug-induced learning impairment. This hexane extract contains auraptene (AUR), a coumarin derivative with a monoterpene unit, together with HMF. The present study was designed to investigate the effect of AUR in vitro. Our results show that 1) AUR had the ability to induce the activation of ERK1/2 in not only cortical neurons but also the rat pheochromocytoma cell line (PC12 cells), which is a model system for studies on neuronal proliferation and differentiation; and 2) AUR had the ability to promote neurite outgrowth from PC12 cells.

Isolation and characterization of activators of ERK/MAPK from citrus plants.

Extracellular signal-regulated kinases 1/2 (ERK1/2), components of the mitogen-activated protein kinase (MAPK) signaling cascade, have been recently shown to be involved in synaptic plasticity and in the development of long-term memory in the central nervous system (CNS). We therefore examined the ability of Citrus compounds to activate ERK1/2 in cultured rat cortical neurons, whose activation might have a protective effect against neurodegenerative neurological disorders. Among the samples tested, extracts prepared from the peels of Citrus grandis (Kawachi bankan) were found to have the greatest ability to activate ERK1/2. The active substances were isolated by chromatographic separation, and one of them was identified to be 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF). HMF significantly induced the phosphorylation of cAMP response element-binding protein (CREB), a downstream target of activated ERK1/2, which appears to be a critical step in the signaling cascade for the structural changes underlying the development of long-term potentiation (LTP). In addition, the administration of HMF into mice treated with NMDA receptor antagonist MK-801 restored the MK-801-induced deterioration of spatial learning performance in the Morris mater-maze task. Taken together, these results suggest that HMF is a neurotrophic agent for treating patients with memory disorders.

Evaluation of the reproducibility and positive controls of cellular immortality test for the detection of immortalized cellular impurities in human cell-processed therapeutic products.

Introduction: Contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic/immortalized cellular impurities is a major concern in the manufacturing and quality control of hCTPs. The cellular immortality test based on cell growth analysis is a method for detecting tumorigenic/immortalized cellular impurities in hCTPs. However, the performance of the cellular immortality test has not yet been well characterized. In this study, we examined the reproducibility of the cellular immortality test in detecting HeLa cells as a model of tumorigenic cellular impurities, as well as the applicability of other models of cellular impurities with different tumorigenicity to the cellular immortality test. Methods: Using HeLa cells as a model for cellular impurities, we measured the growth rate of human mesenchymal stem cells (hMSCs) supplemented with HeLa cells at concentrations ranging from 0.01 to 0.0001% at each passage in three laboratories and evaluated the reproducibility of the detection of immortalized cellular impurities. In addition, HEK293 cells (another immortalized cell line) and MRC-5 cells (a non-immortalized cell line) were employed as cellular impurity models that exhibit different growth characteristics from HeLa cells, and the ability of the cellular immortality test to detect these different impurities when mixed with hMSCs was examined. Results: In the multisite study, the growth rate of hMSCs supplemented with 1 and 10 HeLa cells (0.0001% and 0.001%) significantly increased and reached a plateau in all three laboratories, whereas those of hMSCs alone eventually decreased. Moreover, when hMSCs were supplemented with 10 and 100 HEK293 and MRC-5 cells (0.001% and 0.01%), the growth rate significantly increased. The growth rate of hMSCs supplemented with HEK293 cells increased with passage and remained high, whereas that of hMSCs supplemented with MRC-5 cells eventually decreased, as in the case of hMSCs alone. Conclusions: These results indicate that the cellular immortality test is reproducible and can detect immortalized (i.e., potentially tumorigenic) cells such as HEK293 cells with a lower growth rate than HeLa cells by discriminating against normal cells, which could contribute to ensuring the safety and quality of hCTPs.

Silver staining of an esterase compatible with activity and mass spectrometry analysis after separation using non-denaturing two-dimensional electrophoresis.

BACKGROUND: Enzyme activity is normally lost when formaldehyde is used as a reductant for silver staining after separation by electrophoresis. Hydrolytic activity of esterases can be examined on membranes without impairing enzyme activity when another reductant such as glucose is used for silver staining of the enzyme after separation by non-denaturing two-dimensional electrophoresis (2-DE) and subsequent transfer. METHODS: The hydrolysis of lipids in human high density lipoprotein (HDL) by esterases first separated on a polyvinylidene fluoride membrane using non-denaturing 2-DE and silver stained using glucose as a reductant was examined. RESULTS: Esterase activity was retained after glucose was used as a silver reductant for silver staining after separation using non-denaturing 2-DE. Lipids of HDL were removed by the esterases retained on the membrane after esterases were separated by 2-DE. CONCLUSION: The results indicated that hydrolytic enzyme activity is retained after separation, staining and immobilization.

Heptamethoxyflavone, a citrus flavonoid, enhances brain-derived neurotrophic factor production and neurogenesis in the hippocampus following cerebral global ischemia in mice.

In the present study using a transient global ischemia mouse model, we showed that (1) a citrus flavonoid 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF) induced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) in the hippocampus after ischemia; (2) HMF increased the expression of brain-derived neurotrophic factor (BDNF), a representative neurotrophic factor in the central nervous system, in the hippocampal dentate gyrus, and most BDNF-positive cells were also stained with anti-glial fibrillary acidic protein (one of the major intermediate filament proteins of mature astrocytes) and (3) HMF increased doublecortin positive neuronal precursor cells in the dentate gyrus subventricular zone or subgranular zone. These results suggest that HMF has the ability to induce BDNF production in astrocytes and enhance neurogenesis after brain ischemia, which may be mediated by activation of ERK1/2 and CREB.

Hydrocephalus and abnormal subcommissural organ in mice lacking presenilin-1 in Wnt1 cell lineages.

Presenilin-1 (PS1) is a transmembrane protein that is in many cases responsible for the development of familial Alzheimer's disease. PS1 is widely expressed in embryogenesis and is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. To further investigate the role of PS1 in the brain, we inactivated the PS1 gene in Wnt1 cell lineages using the Cre-loxP recombination system. Here we show that conditional inactivation of PS1 in Wnt1 cell lineages results in congenital hydrocephalus and subcommissural organ abnormalities, suggesting a possible role of PS1 in the regulation of cerebrospinal fluid homeostasis.

Restricted growth and insulin-like growth factor-1 deficiency in mice lacking presenilin-1 in the neural crest cell lineage.

Presenilin-1 (PS1) is a transmembrane protein that is in many cases responsible for the development of early-onset familial Alzheimer's disease. PS1 is essential for neurogenesis, somitogenesis, angiogenesis, and cardiac morphogenesis. We report here that PS1 is also required for maturation and/or maintenance of the pituitary gland. We generated PS1-conditional knockout (PS1-cKO) mice by crossing floxed PS1 and Wnt1-cre mice, in which PS1 was lacking in the neural crest-derived cell lineage. Although the PS1-cKO mice exhibited no obvious phenotypic abnormalities for several days after birth, reduced body weight in the mutant was evident by the age of 3-5 weeks. Pituitary weight and serum insulin-like growth factor (IGF)-1 level were also reduced in the mutant. Histologic analysis revealed severe atrophy of the cytosol in the anterior and intermediate pituitary lobes of the mutant. Immunohistochemistry did not reveal clear differences in the expression levels of thyroid-stimulating hormone, adrenocorticotropic hormone, or prolactin in the mutant pituitary. In contrast, growth hormone expression levels were reduced in the anterior lobe of the mutant. PS1 was defective in the posterior lobe, but not the anterior or intermediate lobes, in the mutant pituitary. These findings suggest that PS1 indirectly mediates the development and/or maintenance of the anterior and intermediate lobes in the pituitary gland via actions in other regions, such as the posterior lobe.