Bovine Serum-containing Medium Research Articles

A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an "Off-the-Shelf" Bioartificial Liver System.

To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.

Bioinspired Silicification of mRNA-Loaded Polyion Complexes for Macrophage-Targeted mRNA Delivery.

In vitro transcribed messenger RNA (mRNA) delivery to macrophages is a promising therapeutic modality for inflammatory diseases because it can modulate the immunological activity of macrophages. However, efficient macrophage-targeted mRNA delivery remains challenging. Herein, we fabricated silica-coated polyion complexes (PICs), termed SilPICs, via bioinspired silicification for stable encapsulation of mRNA and scavenger receptor (SR)-mediated macrophage targeting. Silica coating was readily performed by simply mixing mRNA-loaded PICs with tetramethyl orthosilicate in aqueous media at 25 °C. The silica shell formation was verified by a slight increase in size (∼18 nm), a conversion of ζ-potential from positive (+22 mV) to negative (-23 mV), the peak appearance derived from silanol groups and siloxane bonds in the IR spectra, and elemental analyses by scanning transmission electron microscopy-energy-dispersive X-ray spectrometry (STEM-EDS). The silica shell efficiently protected the mRNA payload from enzymatic degradation in a fetal bovine serum-containing medium. Meanwhile, the reversibility of the silica shell allowed mRNA release from SilPICs after silica dissolution into silicic acids under diluted conditions. Furthermore, SilPICs elicited 20-fold higher mRNA transfection efficiency in the macrophage cell line RAW264.7 compared to noncoated PICs, presumably due to the facilitated cellular internalization by the silica shell. These enhancements were compromised in the RAW264.7 cells incubated with dextran sulfate and poly(inosinic acid) as inhibitors of SR type A1 and were not observed in cultured CT26 colon cancer cells, which are SR-negative cells. Collectively, SilPIC is a promising mRNA delivery vehicle with both mRNA protectability and macrophage targetability.

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture

BackgroundHuman dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs.MethodsWe selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells).ResultsOur results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2β1 and α11β1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization.ConclusionThe use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

Evaluation of characteristic of human turbinate derived mesenchymal stem cells cultured in the serum free media.

We evaluated the effect of serum-free and xeno-cultivation (SFXFM) on the characterization, proliferation, and differentiation properties of human nasal stem cells (airway tissue; hTMSCs). hTMSCs were isolated from 10 patients, after which patient samples were separated into two groups, an SFXFM group and a control group. The control group was treated with bovine serum-containing medium. FACS analysis revealed that SFXFM-cultured hTMSCs maintained a characteristic mesenchymal stem cell phenotype. hTMSC proliferation was not influenced by SFXFM. In addition, upregulation of IL-8 and GM-CSF and downregulation of RANTES expression were shown in response to SFXFM. Moreover, two-lineage differentiation properties (osteocyte and adipocyte) of hTMSCs were enhanced under SFXFM. Finally, the genetic stability of SFXFM-cultured hTMSCs was demonstrated by normal karyotype results. SFXFM enables good expansion, multipotentiality, and normal genotype maintenance of MSCs. Moreover, this approach serves as a substitute to conventional media for the cultivation of capable MSCs for upcoming medical applications.

Improved Chondrogenic Potential and Proteomic Phenotype of Porcine Chondrocytes Grown in Optimized Culture Conditions.

For successful cartilage tissue engineering, the ability to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation is critical. The ability to accomplish this by using the abundant and easily sampled costal cartilage could provide a practical alternative to the use of articular cartilage and mesenchymal stem cells. Chondrocytes isolated from pig costal cartilage were expanded in either serum-free medium with FGF2 (SFM) or fetal bovine serum-containing medium (SCM), under either high (21%) or low (5%) oxygen conditions. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (p < 0.05). The effect of passage number on the differentiation status of the chondrocytes was analyzed by alkaline phosphatase (AP) staining and real-time PCR for known chondrocyte quality markers. AP staining indicated that chondrocytes grown in SCM had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (p < 0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (p < 0.05) in cells expanded in Low-SFM, indicating reduced de-differentiation. In vitro re-differentiation capacity was assessed after a 6-week induction, and chondrocytes grown in Low-SFM showed similar expression ratios of COL2/COL1 and ACAN/VCAN to native cartilage. Proteomic analysis of in vitro produced cartilage indicated that the Low-SFM condition most closely matched the proteomic profile of native costal and articular cartilage. In conclusion, Low-SFM culture conditions resulted in improved cell growth rates, reduced levels of de-differentiation during expansion, greater ability to re-differentiate into cartilage on induction, and an improved proteomic profile that resembles that of in vivo cartilage.

Development of Surface-Variable Polymeric Nanoparticles for Drug Delivery to Tumors.

To develop nanoparticle drug carriers that interact with cells specifically in the mildly acidic tumor microenvironment, we produced polymeric nanoparticles modified with amidated TAT peptide via a simple surface modification method. Two types of core poly(lactic-co-glycolic acid) nanoparticles (NL and NP) were prepared with a phospholipid shell as an optional feature and covered with polydopamine that enabled the conjugation of TAT peptide on the surface. Subsequent treatment with acid anhydrides such as cis-aconitic anhydride (CA) and succinic anhydride (SA) converted amines of lysine residues in TAT peptide to β-carboxylic amides, introducing carboxylic groups that undergo pH-dependent protonation and deprotonation. The nanoparticles modified with amidated TAT peptide (NLpT-CA and NPpT-CA) avoided interactions with LS174T colon cancer cells and J774A.1 macrophages at pH 7.4 but restored the ability to interact with LS174T cells at pH 6.5, delivering paclitaxel efficiently to the cells following a brief contact time. In LS174T tumor-bearing nude mice, NPpT-CA showed less accumulation in the lung than NPpT, reflecting the shielding effect of amidation, but tumor accumulation of NPpT and NPpT-CA was equally minimal. Comparison of particle stability and protein corona formation in media containing sera from different species suggests that NPpT-CA has been activated and opsonized in mouse blood to a greater extent than those in bovine serum-containing medium, thus losing the benefits of pH-sensitivity expected from in vitro experiments.

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth

Lytic growth of intracellular Toxoplasma gondii tachyzoite stages over a period of days results in plaques within mononolayers of host cells. Plaque assays are in frequent use to isolate single clones and to investigate invasion, replication and egress over a longer time frame. To allow correlating plaque morphology and/or size with ultrastructural examination of individual parasites we introduce a simple protocol for correlative light and electron microscopy (CLEM) of entire plaques. We also illustrate the advantages of visualizing only the boundaries of plaques by staining for infected cells (‘positive staining’) rather than the traditional staining of the intact cell monolayer, thus outlining the area of lysed cells (‘negative staining’). Tachyzoites expressing β-galactosidase of Escherichia coli are an easy to visualize histochemical marker for this purpose. Quantitative measurements of plaque area with our compiled user-friendly ImageJ macros are compared to commercial software for ease and shown to be more accurate for some applications. Finally, a chemically defined medium is shown to be superior to the fetal bovine serum-containing medium for plaque assays, resulting in larger plaques. The reported additions and changes of the plaque assay procedure offer improved ways to analyze subtle differences in invasion, pathogen growth and egress. Our chemically defined medium will improve standardization of e.g. drug screening assays.

T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway

T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10ng/ml) and androstenedione (3ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100nM, 24h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.

Graphene supports in vitro proliferation and osteogenic differentiation of goat adult mesenchymal stem cells: potential for bone tissue engineering.

Current treatments for bone loss injuries involve autologous and allogenic bone grafts, metal alloys and ceramics. Although these therapies have proved useful, they suffer from inherent challenges, and hence, an adequate bone replacement therapy has not yet been found. We hypothesize that graphene may be a useful nanoscaffold for mesenchymal stem cells and will promote proliferation and differentiation into bone progenitor cells. In this study, we evaluate graphene, a biocompatible inert nanomaterial, for its effect on in vitro growth and differentiation of goat adult mesenchymal stem cells. Cell proliferation and differentiation are compared between polystyrene-coated tissue culture plates and graphene-coated plates. Graphitic materials are cytocompatible and support cell adhesion and proliferation. Importantly, cells seeded on to oxidized graphene films undergo osteogenic differentiation in fetal bovine serum-containing medium without the addition of any glucocorticoid or specific growth factors. These findings support graphene's potential to act as an osteoinducer and a vehicle to deliver mesenchymal stem cells, and suggest that the combination of graphene and goat mesenchymal stem cells provides a promising construct for bone tissue engineering.

Abstract A56: Chemically induced alkaline phosphatase on osteosarcoma stem cells derived from a novel osteosarcoma cell line as a potential molecular target

Abstract Osteosarcoma is the sixth most common pediatric tumor in the under 15 age group. Relapse observed after chemotherapy is associated with &amp;lt; 20 % survival. Osteosarcoma stem cells (OSCs) have been implicated in the poor response to chemotherapy and ultimate relapse of osteosarcoma. Thus, to achieve successful osteosarcoma therapy, it may be crucial to target OSCs. To this end, a molecular target that is overexpressed in osteosarcoma cells and OSCs and either not expressed or underexpressed in normal cells is desirable. While the search for such a targeting molecule is ongoing, we endeavored to test the utility of a chemical treatment to induce overexpression of a potential molecular target in osteosarcoma cells and OSCs. We hypothesized that a chemical cocktail could be used to induce overexpression of bone specific alkaline phosphatase (bsALP) in both osteosarcoma cells and OSCs derived from a novel osteosarcoma cell line (CHA59). Towards this goal, an OSC-enriched culture was derived from CHA59 using serum-free culture medium. This was accomplished by changing the cell growth media, as recently reported for other cell lines, from 10% fetal bovine serum-containing medium to serum-free medium containing 15% KnockOut Serum Replacement (serum substitute (SS)). This resulted in a rapid change in cell growth from adherent monolayers (in serum) to floating spheres (in SS). Monolayers, containing osteosarcoma cells and a few OSCs, and OSC-enriched spheres were then treated with a chemical cocktail (0.1 µM dexamethasone, 50 M ascorbic acid, and 10 mM beta-glycerolphosphate) that has been previously reported to induce the expression of bsALP in mesenchymal stem cells. A colon tumor cell line, HT-29, served as the negative control for ALP induction. Nine days post-treatment, ALP expression was monitored by Fast Red staining. Cocktail treated monolayer and OSC-enriched spheres stained intensely for ALP compared with the untreated control and the HT-29 (cocktail treated) negative control. To conclude, the results support our hypothesis that ALP can be overexpressed in both monolayer and OSC-enriched spheres. This opens the possibility of targeting therapeutic drugs, gene therapy vectors, and novel nanotechnology to OSCs along with the osteosarcoma cells using anti-bsALP monoclonal antibodies, which is our future goal. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A56.

Smad3 Differentially Regulates the Induction of Regulatory and Inflammatory T Cell Differentiation

Transforming growth factor beta (TGF-beta) is a crucial cytokine with pleiotropic functions on immune cells. In CD4(+) T cells, TGF-beta is required for induction of both regulatory T and Th17 cells. However, the molecular mechanism underlying this differential T cell fate decision remains unclear. In this study, we have evaluated the role of Smad3 in the development of Th17 and regulatory T cells. Smad3 was found to be dispensable for natural regulatory T cell function. However, induction of Foxp3 expression by TGF-beta in naive T cells was significantly reduced in the absence of this molecule. On the contrary, Smad3 deficiency led to enhanced Th17 differentiation in vitro and in vivo. Moreover, Smad3 was found to interact with retinoid acid receptor-related orphan receptor gammat (RORgammat) and decrease its transcriptional activity. These results demonstrate that Smad3 is differentially involved in the reciprocal regulatory and inflammatory T cell generation.

Comparative Proteomics Approach to Screening of Potential Diagnostic and Therapeutic Targets for Oral Squamous Cell Carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.

Analysis ofPRA1and Its Relationship toCandida albicans- Macrophage Interactions

Phagocytosis of Candida albicans by either primary bone marrow-derived mouse macrophages or RAW 264.7 cells upregulated transcription of PRA1, which encodes a cell wall/membrane-associated antigen previously described as a fibrinogen binding protein. However, a pra1 null mutant was still able to bind fibrinogen, showing that Pra1p is not uniquely required for fibrinogen binding. As well, Pra1 tagged with green fluorescent protein did not colocalize with AlexaFluor 546-labeled human fibrinogen, and while PRA1 expression was inhibited when Candida was grown in fetal bovine serum-containing medium, Candida binding to fibrinogen was activated by these conditions. Therefore, it appears that Pra1p can play at most a minor role in fibrinogen binding to C. albicans. PRA1 gene expression is induced in vitro by alkaline pH, and therefore its activation in phagosomes suggested that phagosome maturation was suppressed by the presence of Candida cells. LysoTracker red-labeled organelles failed to fuse with phagosomes containing live Candida, while phagosomes containing dead Candida underwent a normal phagosome-to-phagolysosome maturation. Immunofluorescence staining with the early/recycling endosomal marker transferrin receptor (CD71) suggested that live Candida may escape macrophage destruction through the inhibition of phagolysosomal maturation.

A porous PCL scaffold promotes the human chondrocytes redifferentiation and hyaline‐specific extracellular matrix protein synthesis

The redifferentiation, proliferation, and hyaline-specific extracellular matrix (ECM) protein synthesis of chondrocytes cultured in a polycaprolactone (PCL) scaffold were analyzed. Gene expression of the type II collagen and aggrecan was assessed by real-time PCR in cells from PCL scaffolds, monolayer, and pellet cultures. The proliferative activity was assessed using Ki-67 immunodetection, and the chondrocytic differentiation was evaluated using S-100 immunodetection. The synthesis and deposition into scaffold pores of type II collagen and glycosaminoglycan were analyzed by immunohistochemistry and Alcian blue staining, respectively. All parameters were assessed throughout 28 days of cultures maintained in either fetal bovine serum-containing medium (FCM) or Insulin-Transferrin-Selenium-containing medium (ICM). Expression of the type II collagen gene was lower in FCM cultures than in ICM cultures for all culture systems (p < 0.05). Moreover, PCL scaffolds cultured in ICM were able to induce collagen gene expression more efficiently than pellet and monolayer cultures. Aggrecan gene expression did not vary significantly between mediums and three-dimensional system cultures, but in ICM cultures, the monolayer cultures had significantly higher levels of aggrecan gene expression than did either the PCL or pellet cultures. Chondrocytes cultured in PCL scaffolds or pellets with FCM did not proliferate to a great extent but did maintain their differentiated phenotype for 28 days. Levels of cartilage ECM protein synthesis and deposition into the scaffold pores were similar among PCL and pellet cultures grown in FCM and in ICM. In conclusion, chondrocytes seeded into PCL scaffolds, cultured in ICM, efficiently maintained their differentiated phenotype and were able to synthesize cartilage-specific ECM proteins.

Enhancement of Insulin Transport Across Primary Rat Alveolar Epithelial Cell Monolayers by Endogenous Cellular Factor(s)

To characterize factor(s) contained in apical medium of primary cultured rat alveolar epithelial type II cell-like monolayers (RAECM-II) that enhance insulin absorption across alveolar epithelial cells. Primary rat alveolar epithelial cell monolayers cultured on Transwells in the presence and absence of 10 ng/ml keratinocyte growth factor for 6 days were dosed from the apical compartment with radiolabeled insulin in: newborn bovine serum-containing medium (SM), conditioned medium from apical compartment of rat alveolar epithelial type I cell-like monolayers (RAECM-I) (CMI), or conditioned medium from apical compartment of RAECM-II (CMII). At the end of 2 h incubation, basolateral medium was collected and amounts of transported radiolabeled insulin were determined using a gamma counter. In order to determine the molecular size range of the enhancing factor(s), CMII was centrifuged in 50 kDa molecular weight cut-off Centricon tubes, and both retentate and filtrate were used as separate dosing solutions. Heat denaturation and ammonium sulphate precipitation were used to determine if the involved factor(s) represent proteins or other smaller soluble factors. Transalveolar transport rates of a paracellular marker, (14)C-mannitol, and fluid-phase marker, horseradish peroxidase, were determined in the presence and absence of the factors. Effects of temperature (4, 16 and 37 degrees C) on radiolabeled insulin fluxes were also measured. Conditioned medium obtained from the apical compartment of RAECM-II, CMII, increased transport of insulin across the monolayers when compared to SM or CMI. The enhancing effect of CMII was retained in the precipitate following ammonium sulfate treatment and in the retentate after Centricon filtration. The enhancing effect of CMII was significantly decreased when heated at 80 degrees C for 15 min. CMII did not affect the transport of (14)C-mannitol or HRP, while its effect on insulin transport was decreased by 87% when temperature was lowered to 4 degrees C from 37 degrees C. Conditioned medium from type II cell-like monolayer cultures appears to contain protein factor(s) which seem to be involved in facilitating active transcellular transport of insulin across primary cultured RAECM-II.

Culture conditions for human embryonic stem cells

Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.

Galectin‐1 supports survival of naive T cells without promoting cell proliferation

Naive T cells do not proliferate but remain alive in vivo. In contrast, naive T cells rapidly die in an in vitro culture, suggesting that some factors that are present at the sites of naive T cell circulation in vivo but missing in the bovine serum-containing culture medium, are necessary for their survival. The present study was designed to search for such factors. By functional screening of the cDNA library from murine lymph node-derived stromal cells (LNS) that effectively support the survival of naive T cells, we found that nascent polypeptide-associated complex (alpha-NAC) promoted T cell survival. A conditioned medium derived from culture supernatant of Cos7 cells transfected with alpha-NAC gene supported T cell survival, indicating that alpha-NAC induced production of soluble factor(s) that were secreted into the medium. By examining the products that were cloned from a functional screening of the cDNA library from alpha-NAC-transfected NIH3T3 cells but were not detected in that from control vector-transfected cells, galectin-1 was found as a soluble factor in the conditioned medium of the LNS. Our study demonstrates the novel role of galectin-1 as a soluble factor that functions to maintain naive T cell survival without inducing cell proliferation.