Gelatin-coated Dishes Research Articles

Fasudil and viscosity of gelatin promote hepatic differentiation by regulating organelles in human umbilical cord matrix-mesenchymal stem cells

BackgroundHuman mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods.MethodsTo effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs.ResultsFasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin.ConclusionsThis study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.

Production of Cardiac Extracellular Matrix from Adult Human Fibroblasts for Culture Dish Coating.

The myocardium is composed of cardiomyocytes and an even greater number of fibroblasts, the latter being responsible for extracellular matrix production. From the early stages of heart development throughout the lifetime, in both normal and pathological conditions, the composition of the extracellular matrix changes and influences myocardium structure and function. The purpose of the method described here is to obtain the substrate for the culture of cardiac cells in vitro (termed cardiac ECM), mimicking the myocardial extracellular matrix in vivo. To this end, fibroblasts isolated from the adult human heart were cultured to confluence on gelatin-coated dishes to produce the myocardium-specific extracellular matrix. The subsequent removal of cardiac fibroblasts, while preserving the deposited cardiac ECM, produced the substrate for studying the influence of the myocardium-specific extracellular matrix on other cells. Importantly, the composition of the fibroblast-derived coating of the culture dish changes according to the in vivo activity of the fibroblasts isolated from the heart, allowing subsequent studies of cell-matrix interactions in different normal and pathological conditions.

Abstract 11256: Endothelial-to-Mesenchymal Transition (endMT) is a Driver of Endothelial Cell Heterogeneity in Human in vitro and ex vivo Atherosclerotic Plaques

Atherosclerosis begins with endothelial cell (EC) dysfunction. However, the relationship between single-cell (sc) EC profiles in vitro and in human atherosclerotic plaques is unexplored. Herein, we exploited sc EC transcriptomes from human plaques and related them to in vitro models of inflammation and angiogenesis, which we expected to be relevant. To curate a gene expression profile of ECs found in human atherosclerotic plaques, we performed a meta-analysis of three publicly available human scRNA-seq datasets comprised of 17 coronary and carotid specimens. In parallel, we generated a Multiome in vitro dataset composed of 6 primary human aortic ECs (HAECs), under three experimental conditions: (1) untreated control, and (2) IL-1B-treated (10ng/mL for 1 hour) HAECs on typical gelatin-coated plastic dishes; and (3) untreated HAECs grown on Matrigel to mimic angiogenesis. Five clusters represented the HAECs from all three in vitro conditions. Pathway enrichment analysis revealed that Matrigel-grown ECs (cluster 1) were largely distinguished by Signaling by Rho GTPases, Miro GTPases and RHOBTB3 (R-HSA-9716542; Log10(P) -45.96), while untreated and IL-1B-treated ECs (clusters 0 and 2) were distinguished by regulation of cell adhesion (GO:0030155; Log10(P) -39.92) and Cell Cycle (R-HSA-1640170; Log10(P) -32.2). Of note, clusters 3 and 4 were comprised of HAECs from all 3 conditions and appeared to represent partial and complete endMT, respectively. Cluster 3 and 4 were characterized by greater expression of mesenchymal markers (COL1A1, COL1A2, COL3A1, TAGLN, VIM, and CDH2), while cluster 4 was additionally characterized by a reduction in EC markers (ERG, CDH5, PECAM1). When in vitro HAEC and ex vivo plaque data were integrated, cells from in vitro cluster 4 clustered with vascular smooth muscle cells from human atherosclerotic plaques. Taken together, these results reveal that HAECs are heterogeneous both in vitro and ex vivo atherosclerotic conditions. Since endMT occurs spontaneously at some minor frequency in vitro (~5%) with expression signatures observed in vivo, these data establish a clinically relevant in vitro model to understand the molecular processes driving endMT.

Principal protocols for the processing of cultured meat.

The purpose of this study was to establish a basic principal procedure for the processing of cultured meat. The first stage involved isolating satellite cells from the desired muscle of an animal using enzymatic digestion (i.e., by using proteases, collagenases, and pronases). The second stage involved culturing the isolated muscle satellite cells in a growth medium containing fetal bovine serum and penicillin/streptomycin with growth factors for an optimal period of time. The second stage involved a basic method for the isolated muscle cells to proliferate while sub-culturing to further induce differentiation in gelatin-coated culture dishes with the general culture medium. The third stage involved the induction of differentiation of muscle satellite cells or formation of myotubes using myogenic medium. Lastly, the fourth stage involved the identification of cell differentiation or myotube formation (myogenesis) using fluorescent dyes. Moreover, the principle of these protocols can be applied to perform primary culture of animal cells. This study will assist beginners with the technical aspects of culturing meat (isolation, cultivation, and differentiation of muscle satellite cells as well as identification of myotube formation for myogenesis).

Different morphologies of human embryonic kidney 293T cells in various types of culture dishes.

Human embryonic kidney 293T (HEK293T) cells are used in various biological experiments and researches. In this study, we investigated the effect of cell culture environments on morphological and functional properties of HEK293T cells. We used several kinds of dishes made of polystyrene or glass for cell culture, including three types of polystyrene dishes provided from different manufacturers for suspension and adherent cell culture. In addition, we also investigated the effect of culturing on gelatin-coated surfaces on the cell morphology. We found that HEK293T cells aggregated and formed into three-dimensional (3-D) multicellular spheroids (MCS) when non-coated polystyrene dishes were used for suspension culture. In particular, the non-coated polystyrene dish from Sumitomo bakelite is the most remarkable characteristic for 3-D MCS among the polystyrene dishes. On the other hand, HEK293T cells hardly aggregated and formed 3-D MCS on gelatin-coated polystyrene dishes for suspension culture. HEK293T cells adhered on the non- or gelatin-coated polystyrene dish for adherent culture, but they did not form 3-D MCS. HEK293T cells also adhered to non- or gelatin-coated glass dishes and did not form 3-D MCS in serum-free medium. These results suggest that HEK293T cells cultured on non-coated polystyrene dish may be useful for the tool to analyze the characteristics of 3D-MCS.

The nature of the arena surface affects the outcome of host-finding behavior bioassays in Varroa destructor (Anderson & Trueman).

Varroa destructor, an acarian parasite of the Western honey bee Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. The parasite lifecycle occurs in close synchrony with its host development. The females have to discriminate between different developmental stages of the host and trigger an appropriate behavioral response. Many studies have focused on these behavioral aspects, whether it is the choice of a precise host stage or the reproduction of female mites. Behavioral tests often require laboratory settings that are very different from the mite's environment. Our first experiment was designed to study the impact of the surface of test arena on the mite behavior. We found that plastic from Petri dishes commonly used as test arenas disturbs the female mites and can cause death. We searched for a substrate that does not harm mites and found that gelatin-coated plastic Petri dishes responded to these expectations. We then investigated the host choice behavior of phoretic mites confronted with larval stages of the bee on gelatin-coated arenas to watch if the well-documented orientation towards 5th instar larva was observable in our conditions. Pupal stages were included in the host choice experiments, initially to act as neutral stimuli. As white-eyed pupae were revealed attractive to the mite, several pupal stages were then included in a series of host choice bioassays. These additional experiments tend to show that the positive response to the white-eyed pupa stage depends on cues only delivered by living pupae. Further investigation on the nature and impact of these cues are needed as they could shed light on key signals involved in the parasite lifecycle.

Development of Bifunctional Three-Dimensional Cysts from Chemically Induced Liver Progenitors.

Chemically induced liver progenitors (CLiPs) have promising applications in liver regenerative medicine. Three-dimensional (3D) structures generated from liver progenitor cells possess wide applications in cell transplantation, disease model, and drug testing. Here, we report on the spontaneous formation of 3D cystic structures comprising maturing rat CLiPs on gelatin-coated dishes. Our 3D cysts contained Alb+/+CK19+/− and Ck19+/+Alb+/− cells. These cell types gradually diverged into specialized mature cells, as demonstrated by the expression of mature biliary markers (Cftr, Ae2, and Aqp1) and hepatic markers (Alb and Mrp2). The 3D cysts also expressed functional multidrug resistance protein 1 (Mdr1), as indicated by epithelial efflux of rhodamine. Furthermore, we observed bile canaliculi functions between hepatocytes and cholyl-lysyl-fluorescein extrusions, indicating that the functional characteristics of 3D cysts and active bile salt export pump (Bsep) transporters were intact. Thus, our study revealed a natural characteristic of rat CLiPs to spontaneously form 3D cystic structures accompanied with cell maturation in vitro, offering a platform for studies of liver development and drug screening.

Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels

BackgroundExtracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well. AimThis study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs). MethodsMTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. ResultsMPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1β, TNFα and prostaglandin E2 (PGE2) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB. ConclusionCol I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels.

Gold Nanoparticle-Functionalized Reverse Thermal Gel for Tissue Engineering Applications.

Utilizing polymers in cardiac tissue engineering holds promise for restoring function to the heart following myocardial infarction, which is associated with grave morbidity and mortality. To properly mimic native cardiac tissue, materials must not only support cardiac cell growth but also have inherent conductive properties. Here, we present an injectable reverse thermal gel (RTG)-based cardiac cell scaffold system that is both biocompatible and conductive. Following the synthesis of a highly functionalizable, biomimetic RTG backbone, gold nanoparticles (AuNPs) were chemically conjugated to the backbone to enhance the system's conductivity. The resulting RTG-AuNP hydrogel supported targeted survival of neonatal rat ventricular myocytes (NRVMs) for up to 21 days when cocultured with cardiac fibroblasts, leading to an increase in connexin 43 (Cx43) relative to control cultures (NRVMs cultured on traditional gelatin-coated dishes and RTG hydrogel without AuNPs). This biomimetic and conductive RTG-AuNP hydrogel holds promise for future cardiac tissue engineering applications.

Reactive oxygen species are responsible for the cell aggregation and production of pro-inflammatory mediators in phorbol ester (PMA)-treated U937 cells on gelatin-coated dishes through upregulation of autophagy

ABSTRACTPurpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.

Gelatin promotes cell aggregation and pro-inflammatory cytokine production in PMA-stimulated U937 cells by augmenting endocytosis-autophagy pathway.

Gelatin, denatured collagen, temporarily exists in tissues and may well be pathophysiologically involved in tissue remodeling, inflammation or tissue damage. The present study is aimed to investigate possible biological roles of gelatin by examining its effects on monocyte-like histiocytic lymphoma cell line U937. Once stimulated by phorbol 12-myristate 13-acetate (PMA), U937 cells differentiate into macrophage-like cells, changing from non-adherent to adherent cells with extended pseudopodia. Here we pre-treated the cell dishes with gelatin solution for cell culture. Interestingly, we found that PMA-stimulated U937 cells formed multicellular aggregates on gelatin-coated dishes, accompanying NF-κB-mediated production of pro-inflammatory cytokines, whereas cell aggregation was not detected on non-coated dishes. Moreover, differentiated U937 cells on gelatin-coated dishes showed increased autophagy level and endocytosis. Surprisingly, formation of multicellular aggregates and pro-inflammatory cytokine production were both attenuated by either down-regulation of autophagy with inhibitors, such as 3-methyladenine (3MA) or chloroquine (CQ), or repression of endocytosis with siRNA targeting Endo180. Moreover, autophagy was inhibited by si-Endo180, and endocytosis was suppressed by 3MA, suggesting a positive feedback loop between autophagy and endocytosis. The results revealed that gelatin-coating induced differentiated U937 cells to form cell aggregates and promote NF-κB-mediated pro-inflammatory cytokine production at least partially through an endocytosis-autophagy pathway.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells

Endothelial cells release ATP in response to fluid shear stress, which activates purinergic (P2) receptor-mediated signaling molecules including endothelial nitric oxide (eNOS), a regulator of vascular tone. While P2 receptor-mediated signaling in the vasculature is well studied, the role of P2Y2 receptors in shear stress-associated endothelial cell alignment, cytoskeletal alterations, and wound repair remains ill defined. To address these aspects, human umbilical vein endothelial cell (HUVEC) monolayers were cultured on gelatin-coated dishes and subjected to a shear stress of 1 Pa. HUVECs exposed to either P2Y2 receptor antagonists or siRNA showed impaired fluid shear stress-induced cell alignment, and actin stress fiber formation as early as 6 h. Similarly, when compared to cells expressing the P2Y2 Arg-Gly-Asp (RGD) wild-type receptors, HUVECs transiently expressing the P2Y2 Arg-Gly-Glu (RGE) mutant receptors showed reduced cell alignment and actin stress fiber formation in response to shear stress as well as to P2Y2 receptor agonists in static cultures. Additionally, we observed reduced shear stress-induced phosphorylation of focal adhesion kinase (Y397), and cofilin-1 (S3) with receptor knockdown as well as in cells expressing the P2Y2 RGE mutant receptors. Consistent with the role of P2Y2 receptors in vasodilation, receptor knockdown and overexpression of P2Y2 RGE mutant receptors reduced shear stress-induced phosphorylation of AKT (S473), and eNOS (S1177). Furthermore, in a scratched wound assay, shear stress-induced cell migration was reduced by both pharmacological inhibition and receptor knockdown. Together, our results suggest a novel role for P2Y2 receptor in shear stress-induced cytoskeletal alterations in HUVECs.

Effects of Activin in Embryoid Bodies Expressing Fibroblast Growth Factor 5.

Nodal/activin signaling is indispensable for embryonic development. We examined what activin does to the embryoid bodies (EBs) produced from mouse embryonic stem cells (mESCs) expressing an epiblast marker. The EBs were produced by culturing mESCs by the hanging drop method for 24 hours. The resulting EBs were transferred onto gelatin-coated dishes and allowed to further differentiate. The 24-hour EBs showed a stronger expression of fibroblast growth factor (FGF)5 and Brachyury (specific to the epiblast) in comparison with mESCs. Treating the transferred EBs with activin A maintained transcript levels of FGF5 and Oct4, while inhibiting definitive endoderm differentiation. The activin A treatment reversed the endoderm differentiation induced by retinoic acid (RA), while the inhibition of nodal/activin signaling promoted RA-induced endoderm differentiation. Inhibition of nodal/activin signaling in EBs, including epiblast-like cells, promotes differentiation into the endoderm, facilitating the transition from the pluripotent state to specification of the endoderm.

Abstract 566: Functional Analysis of Inducible Pluripotent Stem Cell-derived Vascular Endothelial Cells From Plasminogen Activator Inhibitor-1 Deficient Patients

Background: Plasminogen Activator inhibitor-1 (PAI-1) is the principle regulator of PA system and its enhanced expression levels are related to many pathological conditions including thrombosis and malignant tumors. We have identified two distinct PAI-1 deficient patients having apparent phenotypes of severe bleeding and impaired wound healing, both of which are not seen in the PAI-1 deficient mice. With employing inducible Pluripotent Stem (iPS) cells established from the patients, we investigated the intrinsic function of PAI-1 in endothelial cells. Methods: iPS cells, generated from PAI-1 deficient patients and control subject, were differentiated to endothelial cells. Endothelial cells were isolated by magnetic sorting with anti-VEGFR-2 antibody, and their functions and the related gene expressions were analyzed. Results: (1) Endothelial cells derived from PAI-1 deficient iPS cells (PAI-1 iPS-ECs) detached more easily from gelatin-coated dishes. (2) PAI-1 iPS-ECs migrated faster in migration assay. (3) Tube formation activities of PAI-1 iPS-ECs, especially vessel branching, were impaired compared with control iPS-ECs. (4) The expression of Dll4 gene, known to be mainly observed at the region of angiogenic-edge, was reduced in PAI-1 iPS-ECs. Conclusion: PAI-1 appeared to play important roles in the function of endothelial cells and to contribute to angiogenesis.

Collagen Membranes Adsorb the Transforming Growth Factor-β Receptor I Kinase-Dependent Activity of Enamel Matrix Derivative.

Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)-β signaling in oral fibroblasts. Three commercially available collagen products were exposed to EMD or recombinant TGF-β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF-β target genes interleukin (IL)-11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen. EMD or TGF-β1 provoked a significant increase of IL-11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF-β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin-coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL-11 and PRG4. These in vitro findings suggest that collagen products adsorb a TGF-β receptor I kinase-dependent activity of EMD and make it available for potential target cells.

Gelatin promotes murine fibrosarcoma L929 cell detachment and protects the cells from TNFα-induced cytotoxicity

ABSTRACTPurpose: Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammation Materials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)–induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined. Results: L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy. Conclusion: This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions.

A strategy for culturing human pluripotent stem cells for translational research

The tremendous potential of human pluripotent stem cells (hPSCs) in regenerative medicine has garnered the focus of the scientific community worldwide. However, many limitations need to be addressed before we can use the knowledge for clinical applications. We have had a longstanding interest in establishing a safe, clinical-grade environment for culturing hPSCs. We observed that human placenta-derived (chorionic villi) cells could support and maintain hPSC characteristics without the exogenous supplementation of growth factors. We developed a conditioned medium from human placenta-derived feeder cells. This medium could support hPSC growth on a 0.1% gelatin-coated dish that is commonly used for culturing human cells. Thereafter, we identified candidates affecting the pluripotency of hPSCs through CXCR2 ligands in the absence of bFGF. Our previous study was the first to describe the use of a unique feeder-free humanized culture system to support hPSCs on a gelatin substratum regardless of the presence bFGF. Clearly, the human placenta can be used to not only circumvent many limitations but also is an excellent material for translational research.

Abstract P119: Human Podoplanin+/vegfr-3+/lyve-1+ Monocytes are Lymphatic Endothelial Precursors

Lymphatic vessels are involved in the development of various inflammatory disorders, and lymphatic vessel regeneration has been increasingly investigated to develop therapies for lymphatic diseases. Here we report that Podoplanin+/VEGFR-3+/LYVE-1+ is a valid marker for human lymphatic endothelial precursors and the triple-positive cells can be used in lymphatic regeneration. During 5-day culture on an ultra-low attachment surface dish, human peripheral blood mononuclear cells (PBMCs) underwent exponential growth, aggregating into a sphere-like structure and expressing several lymphatic endothelial cell (LEC) markers and lymphangiogenic transcription factors. When dissociated from the aggregate and cultured on a gelatin-coated dish, the cells were attached to the surface. The attached cells were triple positive for LEC markers e.g. Podoplanin, LYVE-1, VEGFR-3. Furthermore, seeded in Matrigel with LECs, the 5-day aggregate-derived cells were incorporated into lymphatic endothelial network. The 5-day aggregates were largely positive for CD14+, a monocyte marker. The CD14+ population was sorted into Podoplanin-positive and negative group for further characterization. Notably, CD14+/Podoplanin+ cells showed increased expression of lymphangiogenic molecules (e.g. VEGFR-3, LYVE-1) both at the genetic and protein levels. Also, CD14+/Podoplanin+ cells secreted higher levels of lymphangiogenic cytokines (VEGF, HGF, PDGF-BB). ELISA results showed that CD14+/Podoplanin+ cells produced more lymphangiogenic cytokines than CD14+/Podoplanin- cells. Local injection of monocyte aggregates significantly increased lymphatic neovascularization and facilitated healing of the skin wound model of nude mice, with CD14+/Podoplanin+ group showing the most dramatic result. Our data suggests that Podoplanin-positive monocytes can be transdifferentiated into lymphatic endothelial precursor cells, and cells with triple positivity for Podoplanin, VEGFR-3, and LYVE-1 can be a promising cell source for therapy against human lymphatic vessel diseases.

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.