Cells In Serum-free Conditions Research Articles

Abstract 2606: Targeting mesothelin on tumor cells with a novel, completely human fusion construct delivering the serine protease granzyme B

Abstract Mesothelin is a cell-surface antigen identified as an excellent cancer-associated target upregulated on numerous tumors, including pancreatic and ovarian cancers, as well as mesotheliomas and lung cancers. Granzyme B is a highly cytotoxic serine protease nominally delivered by immune effector cells to target cells, resulting in induction of cell death pathways that are both caspase-dependent and -independent. We developed a fusion protein (GrB-Fc-SD1) composed of GrB and containing SD1, a novel human single-domain antibody that uniquely targets Region III of the mesothelin glycoprotein. The SD1 and GrB domains are tethered by a human IgG heavy chain fragment which enables dimerization. The fusion protein was transiently expressed in HEK-293E cells in serum-free conditions with a final yield of 5 mg/L. Western blot analysis confirmed the expression of this construct as a homodimer. The GrB enzymatic activity in GrB-Fc-SD1 was similar to that of commercially available human granzyme B. ELISA studies confirmed the specific recognition of the SD1 antibody to mesothelin extra-cellular domain. Biacore analysis confirmed binding of GrB-Fc-SD1 with a dissociation constant between 10−7 and 10−8 M. In vitro cytotoxicity against a limited panel of lung, pancreatic and ovarian cancer cell lines expressing surface mesothelin indicate cytotoxicity in the nanomolar range, while cytotoxicity against mesothelin-negative cells was at micromolar levels. Ex vivo serum stability studies showed excellent stability of GrB-Fc-SD1 at both 4°C and -20°C over ninety days and a half-life of approximately three days at elevated temperatures (37°C). In vivo efficacy studies against Capan-2 xenografts in nude mice using both weekly x 5 and QOD x 5 doses of GrB-Fc-SD1 (130 mg/mg total dose, delivered IP) indicated highly significant efficacy in both dose regimens, with p < 0.001 compared to vehicle-treated mice. Mouse body weights were not impacted at the efficacy doses used in this study, indicating that this dose was non-toxic. Histopathology studies revealed a significant decrease in the percentage of Ki-67+ nuclei in treated tumors compared to controls. These data warrant continued investigation of the protein as a therapeutic candidate. Research supported by H2Biologics. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Mihai G. Iurascu, Louis R. DePalatis, Claire Thuning-Roberson, Michael G. Rosenblum. Targeting mesothelin on tumor cells with a novel, completely human fusion construct delivering the serine protease granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2606.

Pharmacologic inhibition of astrocyte ApoE release

AbstractBackgroundThe ApoE4 allele is the strongest known genetic risk factor for late onset Alzheimer’s disease. Despite this knowledge, therapeutic strategies seeking to modulate brain ApoE homeostasis remain largely unexplored. Several lines of evidence demonstrate that astrocytes function as the primary producers of ApoE within the brain. In this study, we examine how targeted disruption of astrocyte lipid metabolism and protein prenylation effect the secretion of ApoE from primary astrocyte cultures.MethodPrimary cell cultures of mouse astrocytes were grown from 1‐3 day old pups. Drugs were applied to cells in serum free conditions over a range of timepoints and doses. Astrocyte conditioned media was collected from cultures and ApoE and other proteins of interest were measured by western blot.ResultWe find that inhibiting HMG‐CoA reductase, a rate‐limiting enzyme in the cholesterol synthesis pathway, with simvastatin reduces astrocyte ApoE levels in conditioned media in a dose dependent manner, while secretion of other proteins remain preserved. Inhibition of downstream cholesterol synthesis through blockade of the enzyme FDFT1 with zaragozic acid, however, has no effect on total ApoE secretion. Mice with genetic disruption of brain cholesterol metabolism also have similar levels of ApoE in CSF samples compared to control. These data suggest the effect of HMGCR inhibition on ApoE is not mediated through cholesterol. It is known that HMGCR activity is also required for maintenance of cellular protein prenylation. We tested the prenylation inhibitor FGTI‐2734 and found that it is sufficient to block astrocyte ApoE secretion, suggesting that HMGCR inhibitors may exert their effect on ApoE through this mechanism. Recent work suggests that inflammatory astrocytes exert toxic effects via neurotoxic lipids carried by ApoE and another carrier protein, CLU. We find that inhibiting HMGCR in astrocytes does not prevent cytokine‐induced conversion to an inflammatory phenotype, but remains able to block ApoE releaseConclusionWe conclude that targeted inhibition of prenylation enzymes may provide a therapeutic strategy to modulate ApoE levels in the brain.

Serum-free alginate-C2C12 cells microcapsule as a model of alternative animal protein source.

Due to the climate change crisis, and environmental impacts of the traditional meat sector, the production of artificial animal protein based on in vitro cell culture technology is proposed as an alternative. Furthermore, since traditional animal serum-supplemented cultures pose scientific challenges such as batch variation and contamination risks, artificial animal protein cultures are currently in urgent need of not only serum-free cultures, but also microcarrier culture systems for scalability. However, serum-free microcarrier-based culture system for the differentiation of muscle cells is not available to date. Therefore, we established an edible alginate microcapsules culture system for the differentiation of C2C12 cells in serum-free conditions. Furthermore, metabolites related to central carbon metabolism were profiled based on targeted metabolomics using mass spectrometry. The C2C12 cells cultured in alginate microcapsules displayed high viability throughout 7 days and successfully differentiated within 4 days in serum and serum-free cultures except for AIM-V cultures, which was confirmed by CK activity and MHC immunostaining. Lastly, to the best of our knowledge, this is the first report to compare metabolite profiles between monolayer and alginate microcapsule culture systems. Alginate microcapsule culture showed higher levels of intracellular glycolysis and TCA cycle intermediates, lactate, and the contribution of essential amino acids compared to the monolayer culture. We believe our serum-free alginate microcapsule culture system is adaptable to different species of muscle cells and contributes to future food technology as a proof of concept for the scalability of alternative animal protein source production.

Culturing Adipose-Derived Stem Cells Under Serum-Free Conditions.

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave very different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Single-Cell Analysis of Mesenchymal Stem Cells Reveals Their Molecular and Functional Heterogeneity in Myeloma

IntroductionBone marrow (BM) mesenchymal stem cells (MSCs) are rare but heterogeneous cell populations, and both MSCs and their lineages mediate BM niches, hematopoiesis, and myeloma (MM) growth.The aim of the study was to assess variation in gene expression of unexpanded single MSCs generated from the same source, and study the heterogeneous effect on MM cell growth of MSCs lines generated from single MSCs or normal MSCs primed by MM cells. We also studied changes in MSCs during MM progression and identified MSC factors that mediate MM growth.MethodsCells collected from bone pieces following enzymatic digestion were cultured for 2-4 days. The adherent cells were trypsinized, replated to separate MSCs from macrophages and allowed to adhere overnight. Cells were then trypsinized and single MSCs were sorted into 96-well plates. Validation of single cell sorting and viability was assessed microscopically by staining for CFSE.Single MSCs were generated from two normal samples and two MM patients. Sorted single cells were subjected to qRT-PCR using primers of 34 genes implicated in MSC biology (e.g. CD73), cell cycle (e.g. KI67, CDKN2A), differentiation (e.g. RUNX2, PPARG, CYR61), transcription (e.g. FOXC1), and specific MSC genes we identified to be top overexpressed (e.g. POSTN) or underexpressed (e.g. IGFBP2, LEPR) in bone biopsy samples from high-risk MM patients. Also included were CD45 and CD34 to exclude hematopoietic cells, and GAPDH and ACTB as housekeeping genes. After excluding CD45+ cells and cells with low expression of housekeeping genes a total of 111 and 104 single MSCs from normal and MM subjects, respectively, were analyzed. Single MSCs expressed typical MSC markers such as CD73, CD90, FN1 and FAP.For generating MSC lines from the same source, single MSCs from each source were subjected to long-term culture supplemented with CM from whole BM and MSC cultures.For preparing primed MSCs, normal MSCs generated by standard method were cultured alones (unprimed MSCs) or cocultured with MM cells for 5 days (primed MSCs).The cultured and co-cultured cells were trypsinized, replated for 40 min followed by serial washing that attain >95% purified adherent MSCs. Primed and unprimed MSCs were subjected to gene expression profiling (GEP) and their CM used for proteomics and growth assays.ResultsSingle MSCs from the same source (i.e. normal and MM bones) heterogeneously expressed genes related to cell cycle, differentiation, and extracellular matrix. MM single MSCs had lower expression of markers of proliferation (e.g. KI67, p<0.005) and differentiation (e.g. RUNX2, p<0.03), and higher expression of the senescence marker CDKN2A (p<0.001).To further capture MSC heterogeneity, MSCs lines were generated from single MSCs and tested for their ability to support MM cells. Growth of individual MSCs varied in culture, most had a limited proliferation rate, and few were capable of generating MSC lines. MSC lines generated from the same source (4-10 lines from normal or MM subjects) had variable effects on growth of MM cells in co-cultures, or when their CM used on MM cell grown alone (p<0.0008 between lowest to highest growth stimulating lines).CM from primed MSCs stimulated the growth of primary MM cells compared to CM from unprimed MSCs in short-term culture (n=10, p<0.007) or 10-days co-couture with normal MSCs (n=6, p<0.002).Primed MM cells had reduced expression and secretion of factors related to the IGF1 pathways including IGFBP2, IGFBP3 and IGF2, whereas IGF1 was not impacted. Recombinant IGF1 promoted growth of BM-dependent MM cells in serum-free conditions, an effect that was significantly blocked by recombinant IGFBP2 (p<0.0003).IGFBP2 expression by GEP was lower in biopsy samples from MM patients (n=531) than normal subjects (n=68), lower in samples from high-risk than low-risk patients, and lower in focal lesion than random BM biopsies from the same patients (n=77). Immunohistochemistry showed high IGFBP2 protein expression by BM mesenchymal cells, particularly, small adipocytes, and changes in the proportion of IGFBP2+ cells correlated with the reduced gene expression in MM patients' biopsies, and with disease stage.ConclusionsThese findings suggest that MSCs from normal subjects and MM patients are heterogeneous population of cells, are deregulated as part of MM disease progression, and that production of factors such as IGFBP2 by these cells impacts bioavailability of IGF1. DisclosuresDavies:Bristol-Myers: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Bristol Myers: Consultancy, Honoraria.

Expansion and characterization of bone marrow derived human mesenchymal stromal cells in serum-free conditions

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

Immunomodulatory effects of rhesus monkey bone marrow-derived mesenchymal stem cells in serum-free conditions

Mesenchymal stem cells (MSCs) have generated tremendous interest for treating various diseases due to their self-renewal and differentiation capacities. Many studies have demonstrated the immunoregulatory capability of MSCs; however, most of these studies were conducted with fetal bovine serum (FBS), which has an uncertain composition. In this study, we established a serum-free, xeno-free, completely chemically defined medium for the proliferation and expansion of rhesus monkey bone marrow (BM)-derived MSCs (rBMSCs) in vitro. The growth kinetics, characteristics, immunophenotype, and immunosuppressive abilities of rBMSCs grown in serum-free media (SFM) were evaluated and compared with those of cells grown in serum-containing media (SCM). Moreover, we employed RNA sequencing to evaluate the expression pattern of genes related to immune responses in both culture conditions. Compared to cells grown in SCM, rBMSCs grown in SFM exhibited better biological characteristics regarding cell proliferation and immunosuppressive abilities. Cells from both media types exhibited similar immunophenotypic expression patterns for CD29, CD34, CD45, HLA-DR, CD73, CD90, and CD105. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and Gene Set Enrichment Analysis (GSEA) revealed that CXCL8 was downregulated by 4.1 fold in SFM-cultured rBMSCs compared with those in SCM. Furthermore, the mixed lymphocyte culture revealed that the proliferation activity and the expression levels of inflammatory factors of peripheral blood mononuclear cells (PBMCs) were significantly decreased after the addition of the CXCL8 neutralizing antibody, which was related to the elevated immunosuppressive abilities of SFM-suspended rBMSCs. These results suggest a possible cell culture method as well as immunoregulatory mechanisms for clinical cell therapies requiring nonanimal-derived components.

Growing Adipose-Derived Stem Cells Under Serum-Free Conditions.

Growing adipose-derived stem cells (ADSC) in serum-free conditions is important as it represents a way of expanding multipotent cells in a clinical grade medium. Most cultured ADSC are expanded and tested in serum-containing media, which can pose significant health risks if these cells were used in clinical applications. Moreover, cells grown in serum-free conditions behave significantly different than those cultured in serum-containing media. Here, we present a technique to culture adipose-derived stem cells in serum-free conditions. The methods described in this chapter were optimized for ovine ADSC. The appropriate optimization should be done for other cell lines.

Changes in Optical Properties of Plasmonic Nanoparticles in Cellular Environments are Modulated by Nanoparticle PEGylation and Serum Conditions.

When plasmonic nanoparticles (NPs) are internalized by cells and agglomerate within intracellular vesicles, their optical spectra can shift and broaden as a result of plasmonic coupling of NPs in close proximity to one another. For such optical changes to be accounted for in the design of plasmonic NPs for light-based biomedical applications, quantitative design relationships between designable factors and spectral shifts need to be established. Here we begin building such a framework by investigating how functionalization of gold NPs (AuNPs) with biocompatible poly(ethylene) glycol (PEG), and the serum conditions in which the NPs are introduced to cells impact the optical changes exhibited by NPs in a cellular context. Utilizing darkfield hyperspectral imaging, we find that PEGylation decreases the spectral shifting and spectral broadening experienced by 100 nm AuNPs following uptake by Sk-Br-3 cells, but up to a 33 ± 12 nm shift in the spectral peak wavelength can still occur. The serum protein-containing biological medium also modulates the spectral changes experienced by cell-exposed NPs through the formation of a protein corona on the surface of NPs that mediates NP interactions with cells: PEGylated AuNPs exposed to cells in serum-free conditions experience greater spectral shifts than in serum-containing environments. Moreover, increased concentrations of serum (10, 25, or 50 %) result in the formation of smaller intracellular NP clusters and correspondingly reduced spectral shifts after 5 and 10 h NP-cell exposure. However, after 24 h, NP cluster size and spectral shifts are comparable and become independent of serum concentration. By elucidating the impact of PEGylation and serum concentration on the spectral changes experienced by plasmonic NPs in cells, this study provides a foundation for the optical engineering of plasmonic NPs for use in biomedical environments.Electronic supplementary materialThe online version of this article (doi:10.1186/s11671-016-1524-4) contains supplementary material, which is available to authorized users.

Chlorpyrifos promotes colorectal adenocarcinoma H508 cell growth through the activation of EGFR/ERK1/2 signaling pathway but not cholinergic pathway.

Aside from the effects on neuronal cholinergic system, epidemiological studies suggest an association between chlorpyrifos (CPF) exposure and cancer risk. This in vitro study examined the effects of CPF and its toxic metabolite, chlorpyrifos oxon (CPF-O), on the growth of human colorectal adenocarcinoma H508, colorectal adenocarcinoma HT-29, normal colon epithelial CCD841, liver hepatocellular carcinoma HepG2, and normal liver hepatocyte THLE-3 cells. The results showed that CPF (5-100 μM) concentration-dependently increased viability of H508 and CCD841 cells in serum-free conditions. This increasing trend was not found in HT-29, HepG2 and THLE-3 cells. In contrast, CPF-O (50-100 μM) reduced the viability of all cell lines. Cell cycle analysis showed the induction of cells in the S phase, and EdU incorporation assay revealed the induction of DNA synthesis in CPF-treated H508 cells indicating that CPF promotes cell cycle progression. Despite the observation of acetylcholinesterase activity inhibition and reactive oxygen species (ROS) generation, atropine (a non-selective muscarinic acetylcholine receptor antagonist) and N-acetylcysteine (a potent antioxidant) failed to inhibit the growth-promoting effect of CPF. CPF increased the phosphorylation of epidermal growth factor receptor (EGFR) and its downstream effector, extracellular signal regulated kinase (ERK1/2), in H508 cells. AG-1478 (a specific EGFR tyrosine kinase inhibitor) and U0126 (a specific MEK inhibitor) completely mitigated the growth promoting effect of CPF. Altogether, these results suggest that EGFR/ERK1/2 signaling pathway but not cholinergic pathway involves in CPF-induced colorectal adenocarcinoma H508 cell growth.

Low-glucose enhances keratocyte-characteristic phenotype from corneal stromal cells in serum-free conditions.

The avascular cornea is a uniquely-isolated organ, with its stroma constituting a nutrient-poor environment. Consequently, the availability of metabolites such as glucose to corneal stromal cells is considerably reduced compared with other tissues, or indeed with media commonly used to culture these cells in vitro. However, the role of glucose in the behaviour of human corneal keratocytes has been overlooked. As such, we sought to investigate the effects of low-glucose formulations on the phenotype of human corneal stromal cells. Cells cultured in low-glucose were able to survive for extended periods when compared to high-glucose, serum-free conditions. Furthermore, low-glucose enhanced their reversal to a keratocyte-characteristic phenotype. Specifically, cells within low-glucose medium assumed dendritic morphologies, with bean-shaped condensed nuclei, absence of alpha-smooth muscle actin or stress fibres, and a corresponding reduction in migratory and contractile activities when compared with high-glucose, serum-free conditions. Moreover, cells within low-glucose uniquely recovered the ability to express a robust keratocyte-characteristic marker, CD34, while still expressing elevated levels of other representative phenotypic markers such as keratocan, lumican, ALDH1A1, and ALDH3A1. These results indicate that low-glucose enhances keratocyte-characteristic phenotype above and beyond established media formulations and thus has important implications for corneal biology in health and disease.

Human embryonal carcinoma cells in serum-free conditions as an in vitro model system of neural differentiation.

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.

Culture of intestinal stem cells in serum-free conditions

Culture of intestinal stem cells in serum-free conditions

Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

Neuroprotective Effects of GDNF-expressing Human Amniotic Fluid Cells

Brain injury continues to be one of the leading causes of disability worldwide. Despite decades of research, there is currently no pharmacologically effective treatment for preventing neuronal loss and repairing the brain. As a result, novel therapeutic approaches, such as cell-based therapies, are being actively pursued to repair tissue damage and restore neurological function after injury. In this study, we examined the neuroprotective potential of amniotic fluid (AF) single cell clones, engineered to secrete glial cell derived neurotrophic factor (AF-GDNF), both in vitro and in a surgically induced model of brain injury. Our results show that pre-treatment with GDNF significantly increases cell survival in cultures of AF cells or cortical neurons exposed to hydrogen peroxide. Since improving the efficacy of cell transplantation depends on enhanced graft cell survival, we investigated whether AF-GDNF cells seeded on polyglycolic acid (PGA) scaffolds could enhance graft survival following implantation into the lesion cavity. Encouragingly, the AF-GDNF cells survived longer than control AF cells in serum-free conditions and continued to secrete GDNF both in vitro and following implantation into the injured motor cortex. AF-GDNF implantation in the acute period following injury was sufficient to activate the MAPK/ERK signaling pathway in host neural cells in the peri-lesion area, potentially boosting endogenous neuroprotective pathways. These results were complemented with promising trends in beam walk tasks in AF-GDNF/PGA animals during the 7day timeframe. Further investigation is required to determine whether significant behavioural improvement can be achieved at a longer timeframe.

Long‐term maintenance of HepaRG cells in serum‐free conditions and application in a repeated dose study

Chronic repeated-dose toxicity studies are still carried out on animals and often do not correlate with the effects in human beings mainly due to species-specific differences in biotransformation. The human hepatoma cell line HepaRG has been used for human relevant toxicity assessment. However, HepaRG cells are commonly maintained in serum containing medium which limits their use in 'omics'-based toxicology. In this study, we compared the maintenance of HepaRG cells in standard serum-supplemented and serum-free conditions. Viability and Cytochrome P450 (CYP) activity during long-term cultivation were assessed. Liver-specific albumin and urea production was measured. The extracellular metabolome (amino acids, glucose, lactate and pyruvate) was measured to compare different cultivation conditions using metabolic flux analysis. Although metabolic flux analysis reveals differences in certain parts of the metabolism, e.g. production of urea, the overall metabolism of serum-free and serum-supplemented cultured HepaRG cells is similar. We conclude that HepaRG cells can be maintained in optimized serum-free conditions for 30 days without viability change and with high CYP activity. We also tested the acute (24 h) and long-term repeated-dose (7 doses, every second day) toxicity of valproic acid. We calculated an EC50 value of 1.4 mM after repeated exposure which is close to the cmax value for valproic acid. Maintenance of HepaRG cells in serum-free conditions opens up the opportunity for the use of these cells in human long-term repeated-dose hepatotoxicity studies and for application in systems toxicology.

Pancreatic Differentiation of Human Dental Pulp CD117 + stem cells

Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. CD117⁺ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. Our results show that CD117⁺ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.

Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions

The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use.

Serum‐Free Generation of Multipotent Mesoderm (Kdr+) Progenitor Cells in Mouse Embryonic Stem Cells for Functional Genomics Screening

This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions, and for functional genomics screening using these cells. Kdr-positive cells are able to differentiate into a wide array of mesodermal derivatives, including vascular endothelial cells, cardiomyocytes, hematopoietic progenitors, and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. In addition, the oligonucleotide transfection protocol used to functionally screen siRNA and miRNA libraries is a powerful tool to reveal networks of genes, signaling proteins, and miRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. Technical limitations, troubleshooting, and potential applications of these methods are discussed.

Primary human cervical carcinoma cells require human papillomavirus E6 and E7 expression for ongoing proliferation

Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells.