iPS Cell-Based Model for MAPT Haplotype as a Risk Factor for Human Tauopathies Identifies No Major Differences in TAU Expression

The H1 haplotype of the microtubule-associated protein tau (MAPT) gene is a common genetic risk factor for some neurodegenerative diseases such as progressive supranuclear palsy, corticobasal degeneration, and Parkinson’s disease. The molecular mechanism causing the increased risk for the named diseases, however, remains unclear. In this paper, we present a valuable tool of eight small molecule neural precursor cell lines (smNPC) homozygous for the MAPT haplotypes (four H1/H1 and four H2/H2 cell lines), which can be used to identify MAPT-dependent phenotypes. The employed differentiation protocol is fast due to overexpression of NEUROGENIN-2 and therefore suitable for high-throughput approaches. A basic characterization of all human cell lines was performed, and their TAU and α-SYNUCLEIN profiles were compared during a differentiation time of 30 days. We could identify higher levels of conformationally altered TAU in cell lines carrying the H2 haplotype. Additionally, we found increased expression levels of α-SYNUCLEIN in H1/H1 cells. With this resource, we aim to fill a gap in neurodegenerative disease modeling with induced pluripotent stem cells (iPSC) for sporadic tauopathies.

Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis

Background/Aims: Hepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro. Methods: Human embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells. Results: All differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group. Conclusion: Retaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis.

T Cells Bearing Anti-CD19- and/or Anti-CD38-Chimeric Antigen Receptors Effectively Abrogate Primary Double-Hit Lymphoma Cells

Patients with B-cell lymphomas bearing MYC translocation combined with an additional translocation involving other genes, such as BCL2, BCL3, or BCL6, whose category is defined as double-hit lymphoma (DHL), have dismal prognosis, because these cells are refractory to conventional immunochemotherapy. Recent studies have expanded the concept of a such disease entity to include double-expressing lymphomas (DEL) that co-overexpress MYC protein with those proteins, which have also poor prognosis. An adoptive T-cell immunotherapy with a chimeric antigen receptor (CAR) is clinically shown to have a powerful cytotoxicity in refractory neoplasias. Especially, several results showed that T cells transduced with an anti-CD19-CAR have successfully worked well in patients with refractory acute B-cell lymphoblastic leukemia and B-cell lymphoma as well as chronic B-cell lymphocytic leukemia. Thus, CAR T-cell therapy is a clinically promising tool for various refractory hematopoietic disorders. Accordingly, we examined whether anti-CD19- and/or anti-CD38-CAR-T cells, both of which we previously developed, could abrogate DHL cells. Here, we revealed that the remarkable cytotoxicity of anti-CD19- and/or anti-CD38-CAR T cells against DHL cells from patients. Firstly, DHL cell line cells (KPU-H1, a generous gift form Dr. Junya Kuroda, Kyoto Prefectural University of Medicine) were co-cultured with anti-CD19- and/or anti-CD38-CAR T cells at an effector (E) target (T) ratio of 1: 2 for three days. Cells harvested and stained with anti-CD19 and/or anti-CD38 antibody-APC or -PE were subjected to flow cytometry. Flow cytometric analysis showed that anti-CD19- or anti-CD38-CAR T cells almost killed KPU-H1 cells, respectively (specific cytotoxicity was >90%). Intriguingly, T cells expressing anti-CD19-CAR exerted a collaborative cytotoxicity against KPU-H1 cells with anti-CD38-CAR T cells in vitro. CD38-specific T cells were co-cultured with cytogenetic DHL (n=3) or DEL (n=2) cells from five patients carrying a poor prognosis for 3 days. We examined whether T cells retrovirally transduced with anti-CD19- and/or anti-CD38-CAR vector could show cytotoxicity against primary DHL cells obtained from patients. Anti-CD19 and/or ant-CD38-CAR T cells were co-cultured with primary DHL cells at an E: T ratio of 1: 2 for 3 days. Interestingly, anti-CD19 and anti-CD38-CAR T cells completely abolished these DHL cells from patients, respectively. Additionally, anti-CD19- and anti-38-CAR T cells were synergistically effective to eliminate DHL cells. These results showed that DHL cells, which are refractory or resistant to existing chemotherapeutic agents, can be efficiently abrogated by a clinical use of T cells with anti-CD19- and/or anti-CD38-CAR. These results might warrant adoptive immunotherapy with autologous T cells transduced with anti-CD19 and anti-CD38-CAR for patients with refractory DHL. Disclosures No relevant conflicts of interest to declare.

Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Interferon-γ and Tumor Necrosis Factor-α Induce An Immunoinhibitory Molecule, B7-H1, Via NfκB Activation in Blasts of Myelodysplastic Syndromes.

Abstract 2766 Poster Board II-742 (Introduction) B7-H1 (CD274) is a costimulatory/coinhibitory molecule that plays a crucial role in T cell regulation in various immune responses. B7-H1 expression is detected not only on professional antigen-presenting cells but also on some tumor cells. It has been reported that B7-H1 molecules on tumor cells inhibit the proliferation of tumor-specific cytotoxic T-lymphocytes and are associated with poor prognosis in patients with some tumors such as renal and breast cancers. In this study, we investigated whether functional B7-H1 molecules are expressed on blasts from myelodysplastic syndromes (MDS) and, if so, are associated with pathophysiology in specific immune evasion in MDS. (Materials and methods) (1) First, we analyzed the expression of B7-H1 mRNA and protein in MDS cell lines (OIH-1, SKM-1, and F36P) using reverse transcription-PCR and flow cytometry (FCM), respectively. Although F36P cells alone expressed B7-H1 molecules at mRNA and protein levels, interferon (IFN)-γ and more efficiently a combination of IFN-γ and tumor necrosis factor (TNF)-α enhanced or induced B7-H1 expression in all of these cell lines. Notably, the combination of IFN-γ and TNF-α induced nuclear factor (NF) γB activation of MDS cell lines, and the blocking of NFκB activation by pyrrolidine dithiocarbamate (PDTC) or NFκB inhibitor peptide turned off the B7-H1 induction. (2) To investigate whether B7-H1+ MDS blasts have a proliferative advantage, we compared the cell cycle and colony formation between B7-H1+ and B7-H1– cell fractions in F36P cells. B7-H1+ cells had fewer G0/G1-phase cells and more G2/M-phase cells compared with B7-H1– cells. Furthermore, purified B7-H1+ cells formed more colonies than B7-H1– cells in the colony-forming assay using the methylcellulose. (3) We investigated the immunomodulatory effects of B7-H1+ blasts on T cells. When normal CD3+ T cells were cultured with or without irradiated B7-H1+ F36P cells, the B7-H1+ F36P cells had markedly increased percentages of T cells showing apoptosis. The blocking antibody for B7-H1 or PD-1 inhibited the T-cell apoptosis induced by B7-H1+ cells, decreased the percentage of caspase-3+ cells in T cells, and increased T-cell proliferation. (4) We then examined B7-H1 expression on blasts from 41 MDS patients, 20 patients with acute myeloid leukemia transformed from MDS (AML-MDS), and 10 hematologically normal individuals using FCM. B7-H1+ cases, in whom more than 5% of blasts expressed B7-H1 molecules, were observed in 6 of 14 (43%) patients with refractory anemia with excess blasts (RAEB), 6 of 12 (50%) with RAEB in transformation (RAEB-t), and 4 of 20 (20%) AML-MDS patients, but in none of 15 RA patients and normal individuals. Similar to the data obtained using MDS cell lines, B7-H1 expression on blasts purified from MDS patients was induced by stimulation with IFN-γ or TNF-α, and more strikingly by their combination. PDTC inhibited the induction of B7-H1 expression. The percentages of G0/G1-phase cells were lower and those of G2/M-phase cells higher in B7-H1+ blasts than in B7-H1– blasts from patients. Furthermore, in allogeneic coculture, in which B7-H1+ blasts from an MDS patient and CD3+ T cells from healthy volunteers were cultured, T-cell proliferation was significantly augmented by blocking B7-H1 molecules. (5) Finally, we investigated the expression of PD-1, a counterreceptor of B7-H1, on circulating T cells in 34 MDS patients. PD-1 expression on CD3+, CD4+, and CD8+ T cells in MDS patients was significantly higher than that in healthy volunteers. (CONCLUSIONS) Taking the results together, in MDS: 1) B7-H1 expression on blasts occurs more often in the advanced disease stages; 2) B7-H1 expression on blasts may be induced by cytokines derived from the bone marrow environment via NFκB activation; 3) cell cycle and proliferation were more active in B7-H1+ blasts than in B7-H1– blasts; and 4) B7-H1 molecules on blasts suppress T-cell immunity. These findings indicate that B7-H1 molecules on blasts may be involved in the pathophysiology of MDS. Disclosures: No relevant conflicts of interest to declare.

Knockdown of the Fanconi Anemia Gene FANCD2 Directly Affects Hematopoiesis in Human Embryonic Stem Cells.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by the progressive bone marrow failure, developmental anomalies and cancer susceptibility. Twelve distinct FA complementation groups have been identified (A–C, D1, D2, E–G, I, J, L, M) and 11 cDNAs cloned. A current working model proposes that eight FA proteins (A, B, C, E, F, G, L and M) assemble to form a multi-protein nuclear complex, involved the repair of damaged DNA. The FA complex facilitates the monoubiquitination of the FANCD2 protein following exposure to DNA damaging agents. Ubiquinated FANCD2 co-localizes with the key DNA repair proteins RAD51, BRCA2 (know as FANCD1), BRCA1, and FANCJ (BACH1) to promote homologous recombination. The hallmark of FA patients is marrow failure due to defective hematopoietic stem cells. The study of human FA stem cell biology is severely restricted due to the few CD34+ cells that can be isolated from FA patients compared with normal individuals. We generated human embryonic stem cells (hESC) exhibiting FA phenotype by introducing small double strand RNA species to ablate FANCD2 gene function in hESC. Human ES cells were then differentiated into hematopoietic cells to investigate FA hematopoiesis. FANCD2-specific small hairpin RNAs (shRNAs) were designed and cloned into a self-inactivated lentiviral vector with a GFP cDNA. High titer virus (2×108 iu/ml) was used for transduction of the hESC line H1. More than 90% of H1 cells were transduced with lentivirus that was observed by GFP expression. The FANCD2 protein expression was analyzed by Western blotting; FANCD2 shRNA targeted cells expressed 1–3% of the FANCD2 protein compared with control H1 cells. Functional assessment of the H1-FANCD2 was performed by incubation with DNA cross-linking agent, mitomycin C (MMC). The H1-FANCD2 cells were nearly 10 times more sensitive to MMC than untreated or scrambled shRNA ES cells. The effective dose 50 (ED50) of MMC required to induce apoptosis was only 4 ng/ml in H1-FANCD2 cells compared with 30 ng/ml in mock-infected of scrambled shRNA controls. Cytogentic abnormalities are the hallmark of Fanconi anemia. When H1-FANCD2 cells were exposed to MMC, 57% of cells had abnormal cytogenetics compared with 2% of scrambled shRNA transduced H1 cells. In addition, 75.34±0.69% of H1-FANCD2 cells were arrested at the G2/M phase of the cell cycle after MMC exposure compared with 60.63±2.5% of H1-scramble cells. MMC hypersensitivity, cell cycle defects and chromosomal abnormalities of H1-FANCD2 confirmed the FA phenotype. Differentiation of H1 and H1-scramble cells through a blastocyst intermediate produced a discrete number of KDR+/CD31+ hemangioblast cells that give rise to erythroid and myeloid hematopoietic colonies in methylcellulose culture. H1-FANCD2 ES cells produced blastocyst and hemangioblast cells without significant development of end-stage hematopoietic lineages. In conclusion, we successfully converted, using shRNAs, hESC H1 cells that exhibit the FA phenotype including abnormal hematopoiesis. Generation of mutant FA hES cells will be a valuable model to study FA pathophysiology and treatment.

Horizontal cell morphology in nocturnal and diurnal primates: A comparison between owl-monkey (Aotus) and capuchin monkey (Cebus)

Horizontal cell morphology was studied in the retina of the nocturnal owl-monkey,Aotus, and compared with that of its diurnal, close relative, the capuchin monkey,Cebus. Cells were initially labeled with DiI and the staining was later photoconverted in a stable precipitated using DAB as chromogen. The sizes of cell bodies, dendritic fields, and axon terminals, number of dendritic clusters, intercluster spacing, and intercone spacing were measured at increasing eccentricities. Two distinct morphological classes of horizontal cells were identified, which resembled those of H1 and H3 cells described in diurnal monkeys. A few examples of a third class, possibly corresponding to the H2 cells of diurnal monkeys, were labeled. Both H1 and H3 cells increased in size and had increasing numbers of dendritic clusters with eccentricity. H3 cells were larger and had a larger number of dendritic clusters than H1 cells. Owl-monkey H1 cells had larger dendritic fields than capuchin monkey H1 cells at all quadrants in the central and midperipheral retinal regions, but the difference disappeared in the far periphery. Owl-monkey and capuchin monkey H1 cells had about the same number of dendritic clusters across eccentricity. As owl-monkey H1 cells were larger than capuchin monkey H1 cells, the equal number of clusters in these two primates was due to the fact that they were more spaced in the owl-monkey cells. H1 intercluster distance closely matched intercone spacing for both the owl-monkey and capuchin monkey retinas. On the other hand, H3 intercluster distance was larger than intercone spacing in the retina of both primates. Owl-monkey H1 axon terminals had 2–3 times more knobs than capuchin monkey H1 axon terminals in spite of having about the same size and, consequently, knob density was 2–3 times higher for owl-monkey than capuchin monkey H1 axon terminals across all eccentricities. The differences observed between owl-monkey and capuchin monkey horizontal cells, regarding the morphology of their dendritic trees and axon terminals, may be related to the differences found in the cone-to-rod ratio in the retina of these two primates. They seem to represent retinal specializations to the nocturnal and diurnal life styles of the owl-monkey and capuchin monkey, respectively.

In Vivo and in Vitro Screening for Illegitimate Receptors in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Causing Cushing’s Syndrome: Identification of Two Cases of Gonadotropin/Gastric Inhibitory Polypeptide-Dependent Hypercortisolism

In ACTH-independent macronodular adrenal hyperplasia (AIMAH) causing Cushing’s syndrome, cortisol production can be controlled by illegitimate membrane receptors. The aim of the present study was to evaluate in vivo and in vitro the sensitivity of AIMAH to various regulatory factors to detect the expression of illegitimate receptors by the tissues. Four consecutive patients with AIMAH and hypercortisolism (H1–H4) preoperatively underwent a series of pharmacological and/or physiological tests. After adrenalectomy, in vitro studies were conducted to investigate the cortisol responses of cultured cells, derived from hyperplastic tissues, to various membrane receptor ligands. The adrenal tissues of the two patients who responded in vivo to food intake (H2 and H4) were stimulated in vitro by gastric inhibitory polypeptide. GnRH and human chorionic gonadotropin, but not FSH, stimulated cortisol secretion in patients H2 and H4. In these two cases, human chorionic gonadotropin but not GnRH stimulated cortisol production from cultured adrenocortical cells. Cisapride induced a significant increase in cortisol levels in patient H1. In addition, serotonin (5-HT) was more efficient to stimulate cortisol production in H1 cells than in normal adrenocortical cells. Upright stimulation test provoked an increase in cortisol levels in patients H1, H2, and H3. H1 and H2 cells were more sensitive to the stimulatory action of angiotensin II than normal cells. Similarly, arginine vasopressin (AVP) more efficiently activated steroidogenesis in H1 cells than in normal cells. In H1 tissue, immunohistochemical studies revealed the presence of 5-HT- and AVP-like immunoreactivities within clusters of steroidogenic cells, suggesting that these two factors acted through an autocrine/paracrine mechanism to stimulate cortisol secretion. The present study provides the first demonstration of primary adrenal Cushing’s syndrome dependent on both gonadotropin and gastric inhibitory polypeptide. Our data also show a hyperresponsiveness of hyperplastic adrenal tissues to 5-HT, angiotensin II, and AVP. Finally, they reveal for the first time the presence of paracrine regulatory signals in adrenal hyperplasia tissues.