Elevated NTCP expression by an iPSC-derived human hepatocyte maintenance medium enhances HBV infection in NTCP-reconstituted HepG2 cells

Background The sodium taurocholate cotransporting polypeptide (NTCP) is a functional receptor for hepatitis B virus (HBV). NTCP-reconstituted human hepatoma cells support HBV infection, but the infection is suboptimal and no apparent HBV spread has been observed in this system. Results We found that NTCP-reconstituted HepG2 cells were highly susceptible to HBV infection after cells were cultured in a commercial human inducible pluripotent stem cell (iPSC)-derived hepatocyte maintenance medium (HMM). The enhanced HBV infection coincided with increased NTCP expression, and was observed in six different clones of HepG2-NTCP cells. Promoter assays indicated that HMM activated the cytomegalovirus immediate-early (IE) promoter that drives the NTCP expression in the HepG2-NTCP cells. RNA-Seq analysis revealed that HMM upregulated multiple metabolic pathways. Despite highly upregulated NTCP expression by HMM, no obvious HBV spread was observed even in the presence of PEG 8000. Conclusions Our data suggest that this particular medium could be used to enhance HBV infection in NTCP-reconstituted hepatocytes in vitro.

Direct shoot regeneration from cotyledon, leaf and root of Citrus jambhiri Lush

ABSTRACTCitrus jambhiri (Rough lemon) is popularly preferred for rootstock for cultivated species of Citrus. Tissue culture is an appreciable technique for mass-multiplication of plant propagules. In this communication direct regeneration of plantlets of Citrus jambhiri Lush. were obtained from cotyledons, roots and leaves. Most of the cotyledon (96%) enlarged on medium supplemented with 50 mg/L of casein hydrolysate. Few of those enlarged cotyledons responded to direct regeneration of shoots. Maximum shoot per responded cotyledon was 32. Conversely, the health of the plantlets were poor with semi-cylindrical leaves. Most of them dried on maintenance medium or on rooting medium ad died. Plantlets regenerated on medium supplemented with IAA in combination with IBA were healthy and they established on maintenance medium and rooted on rooting medium. Direct regeneration was also obtained from leaf on MS medium supplemented with 0.50 mg/L of dicamba. Our finding concluded that tissue culture tools may be used for direct regeneration of plantlets from different explants of C. jambhiri to obtained true-to-type plant propagules.

THE MAINTENANCE OF MANDAILINGNESE LANGUAGE IN TANJUNGBALAI

This study deals with the language maintenance of Mandailingnese in Tanjungbalai. It usesqualitative research design. It is conducted descriptively. This study aims to examine the types of language maintenance by Mandailingnese people in Tanjungbalai, find out the ways in maintaining the Mandailingnese language in Tanjungbalai and find out the reasons why the speakers of Mandailingnese people maintain their language in Tanjungbalai. The subjects were 10teenagers (boys and girls) of Mandailingnese. The use of instruments in this study were observation and interview. The observation were used to examine the types of language maintenance by Mandailingnese people in Tanjungbalai in order to describe the ways of Mandailingnese language maintenance. The interview was used to gain the reasons of Mandailingnese language maintainancein Tanjungbalai. The theories of Fasold, Fishman and Holmes are used in this study. The data were analyzed by Miles, Huberman and Saldana (2014). There are four types of language maintenancein Tanjungbalai namely; low maintenance, medium maintenance, strong maintenance and extreme maintenance. There are two ways to maintain Mandailingnese language in Tanjungbalai namely; join the community and teach the language to younger sister/brother. There are four reasons why they maintain their language namely; to express their identity as Mandailingnese people, to achieve self pride as Mandailingnese people, to tell certain issue and to creat closer relationship. Analysis of data clearly indicates that Mandailingnese language maintenance were done in Tanjungbalai.Keyword : language maintenance, reasons for language maintenance, Mandailingnese in Tanjungbalai

Enhanced Adipogenic Differentiation of Human Adipose-Derived Stem Cells in an in vitro Microenvironment: The Preparation of Adipose-Like Microtissues Using a Three-Dimensional Culture

The application of stem cells for cell therapy has been extensively studied in recent years. Among the various types of stem cells, human adipose tissue-derived stem cells (ASCs) can be obtained in large quantities with relatively few passages, and they possess a stable quality. ASCs can differentiate into a number of cell types, such as adipose cells and ectodermal cells. We therefore focused on the in vitro microenvironment required for such differentiation and attempted to induce the differentiation of human stem cells into microtissues using a microelectromechanical system. We first evaluated the adipogenic differentiation of human ASC spheroids in a three-dimensional (3D) culture. We then created the in vitro microenvironment using a 3D combinatorial TASCL device and attempted to induce the adipogenic differentiation of human ASCs. The differentiation of human ASC spheroids cultured in maintenance medium and those cultured in adipocyte differentiation medium was evaluated via Oil red O staining using lipid droplets based on the quantity of accumulated triglycerides. The differentiation was confirmed in both media, but the human ASCs in the 3D cultures contained higher amounts of triglycerides than those in the 2D cultures. In the short culture period, greater adipogenic differentiation was observed in the 3D cultures than in the 2D cultures. The 3D culture using the TASCL device with adipogenic differentiation medium promoted greater differentiation of human ASCs into adipogenic lineages than either a 2D culture or a culture using a maintenance medium. In summary, the TASCL device created a hospitable in vitro microenvironment and may therefore be a useful tool for the induction of differentiation in 3D culture. The resultant human ASC spheroids were “adipose-like microtissues” that formed spherical aggregation perfectly and are expected to be applicable in regenerative medicine as well as cell transplantation.

Maintenance culture medium and inoculum based on peach palm leaves for Pleurotus spp. production

ABSTRACT: This study evaluated the use of immersion water from peach palm leaves (PPLDA) as a component of the culture medium for the maintenance of Pleurotus spp. and the use of agricultural waste, peach palm leaves, as inoculum support for the fungi. The performance of the inoculum based on peach palm leaves (PPL) for the production of Pleurotus spp. fruiting bodies was compared with that using wheat grains (WG) as inoculum support. PPLDA culture medium (immersion water of peach palm leaves, dextrose, and agar) showed a lower radial velocity of mycelial growth for both fungi than that obtained with the culture medium WDA (wheat extract, dextrose and agar), commonly used as maintenance medium for Pleurotus spp. However, the type of inoculum support does not significantly influence the linear velocity of P. ostreatus mycelial growth, reaching 6.71 mm/day on wheat grains and 6.18 mm/day on peach palm leaves. Thus, when the inoculum based on peach palm leaves is utilized, the immersion water used for preparing this support can be used for preparing the PPLDA maintenance culture medium, diminishing the production costs of Pleurotus mushrooms. Data also showed that when Pleurotus sajor-caju was cultivated on peach palm leaves, using PPL as inoculum support, the fruiting bodies production parameters (Y = 47%, BE = 3% and Pr = 0.2 g/day) did not differ from that obtained using WG.

Pre-Adipocytes Differentiate Into Megakaryocytes (MK) by a Paracrine Thrombopoietin (TPO) Loop

Abstract 4742 The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p>0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (>15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p<0.05). These observations support that pre-adipocytes differentiate into MKs using endogenous TPO stimulation via c-mpl. We next examined the ability to release platelets from these treated pre-adipocytes in each experimental condition (pre-adipocytes and 3T3-L1 cells, each ± exogenous TPO). Infusion of 2 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MKs into irradiated thrombocytopenic mice (7 days post-2.0Gy exposure) led to a time-dependent appearance of CFSE+/CD41+ platelet-sized particles with a peak at 4 hours after the infusion reaching ∼2.5% of the total circulating platelet population (∼30 platelets/infused MK) under all tested experimental conditions. For platelet function, blood samples from each of these mice were perfused over a collagen-coated chip for 10 minutes, and the incorporation of CFSE+ particles into thrombi on the chip was determined (Total Thrombus-formation Analyzing System). A similar degree of platelet incorporation was observed under all experimental conditions and each was with an efficiency similar to that seen when CFSE+ control platelets were infused. These findings demonstrate that pre-adipocytes differentiate into MKs and subsequent platelets by an endogenous TPO release and a paracrine loop involving c-mpl. We propose that such pre-adipocytes could be used as a model of a continuously replicating cell line that upon switching media simultaneous expresses TPO and its receptor c-mpl to establish a paracrine loop leading to terminal differentiation into functional MKs. The basis of why this media switch induces this change needs to be clarified to further develop this pre-adipocyte strategy as a donor-independent source for platelet transfusion as well as for studying mechanism underlying megakaryopoiesis and thrombopoiesis. Disclosures: Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.