Passages In Culture Research Articles

RNA sequence analysis of dermal papilla cells’ regeneration in 3D culture

It is well known that dermal papilla cells (DPCs) are crucial for hair follicle growth and regeneration. However, dermal papilla cells in 2D culture could lose their ability of regeneration after several passage intervals. As opposed to DPCs in 2D culture, the DPCs in 3D culture could passage extensively. However, the molecular mechanisms of DPCs’ regeneration in 3D culture remain unclear. Accordingly, gene sequencing is recommended for the investigation of hair regeneration between 2D and 3D culture, the three groups were established including DPCs in passage 2 in 2D culture, DPCs in passage 8 in 2D culture and DPCs in passage 8 in 3D culture. The differentially expressed genes (DEGs) were identified using the Venn diagram of these three groups, which included 1642 known and 359 novel genes, respectively. A total of 1642 known genes were used for Gene Ontology (GO), Kyoto Gene, Genomic Encyclopedia (KEGG) pathway enrichment and protein‐protein interaction (PPI) analyses, respectively. The functions and pathways of DEGs were enriched in biological regulation, signal transduction and immune system, etc. The key module and the top 10 hub genes (IL1B, CXCL12, HGF, EGFR, APP, CCL2, PTGS2, MMP9, NGF and SPP1) were also identified using the Cytoscape application. Furthermore, the qRT‐PCR results of the three groups validated that the hub genes were crucial for hair growth. In conclusion, the ten identified hub genes and related pathways in the current study can be used to understand the molecular mechanism of hair growth, and those provided a possibility for hair regeneration.

Borrelia miyamotoi strain LB-2001 retains plasmids and infectious phenotype throughout continuous culture passages as evaluated by multiplex PCR

Borrelia miyamotoi is a tick-borne spirochete of the relapsing fever borrelia group and an emerging pathogen of public health significance. The genomes of relapsing fever borreliae and Lyme disease borreliae consist of multiple linear and circular plasmids in addition to the chromosome. Previous work with B. burgdorferi sensu lato found diminished infectivity upon continuous in vitro culture passage that was attributable to plasmid loss. The effect of long-term culture passage on B. miyamotoi is not known. We generated a series of plasmid-specific primer sets and developed a multiplex PCR assay to detect the 14 known plasmids of B. miyamotoi North American strains LB-2001 and CT13−2396. We assessed the plasmid content of B. miyamotoi LB-2001 over 64 culture passages spanning 15 months and determined that strain LB-2001 retained all plasmids upon prolonged in vitro cultivation and remained infectious in mice. We also found that strain LB-2001 lacks plasmid lp20−1 which is present in strain CT13−2396. These results suggest that B. miyamotoi remains genetically stable when cultured and passaged in vitro.

Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

Hydro-climatic variability during last five thousand years and its impact on human colonization and cultural transition in Ladakh sector, India

The Ladakh Range in the Trans Himalaya houses sub-routes of the Silk route through its passes (La in local language) which may have been the main commercial and cultural passage to connect the central Asia and Tibetan region with the rest of India. A ca 6400-220 cal yr B.P. hydroclimatic record of two lakes viz Tsoltak lake and Yaya Tso near the Chang La and Hor La passes of Ladakh Range is presented here. The overall record (mineral magnetic analysis and microbiota) from the Ladakh range shows wetter conditions ca 6400 cal yr BP consistently declining till ~5000 cal yr BP and moderately wet and stable till ~4300 cal yr BP, followed by an arid cold phase (~4300-4000 cal yr BP). This precedes two moderately wet phases (ca 4300-3500 cal yr BP and ca 1260-220 cal yr BP) and the peak arid conditions between 3500 and 2860 cal yr B P and 2230-855 cal yr BP, which may have affected the trade activities and had an adverse affect of cultural transitions during these periods across the Ladakh Range on the to and fro movements from this northern sub-route branch. The biotic assemblage is rich in Non Pollen Palynomorphs (76%) with minor amounts of pollens (24%). An improvement in the conditions since 855 cal yr BP and thereafter from 340 to 220 cal yr BP records the onset of the cold arid conditions again in the Ladakh Range. Presently due to contemporary deglaciation the Ladakh Range is becoming ice free and a number of lakes surrounded by herbaceous meadows are seen in the area due to glacial melt, likely to encourage the human settlements to soon occupy the higher reaches that will negatively affect the natural lake productivity.

Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus.

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

Insights from the comparison of genomic variants from two influenza B viruses grown in the presence of human antibodies in cell culture.

Understanding the extent and limitation of viral genome evolution can provide insight about potential drug and vaccine targets. Influenza B Viruses (IBVs) infect humans in a seasonal manner and causes significant morbidity and mortality. IBVs are negative-sense single-stranded RNA viruses with a segmented genome and can be divided into two antigenically distinct lineages. The two lineages have been circulating and further evolving for almost four decades. The immune response to IBV infection can lead to antibodies that target the strain causing the infection. Some antibodies are cross-reactive and are able to bind strains from both lineages but, because of antigenic drift and immunodominance, both lineages continue to evolve and challenge human health. Here we investigate changes in the genomes of an IBVs from each lineage after passage in tissue culture in the presence of human sera containing polyclonal antibodies directed toward antigenically and temporally distinct viruses. Our previous analysis of the fourth segment, which encodes the major surface protein HA, revealed a pattern of change in which signature sequences from one lineage mutated to the signature sequences of the other lineage. Here we analyze genes from the other genomic segments and observe that most of the quasispecies’ heterogeneity occurs at the same loci in each lineage. The nature of the variants at these loci are investigated and possible reasons for this pattern are discussed. This work expands our understanding of the extent and limitations of genomic change in IBV.

Epigenetic Regulation in Mesenchymal Stem Cell Aging and Differentiation and Osteoporosis.

Mesenchymal stem cells (MSCs) are a reliable source for cell-based regenerative medicine owing to their multipotency and biological functions. However, aging-induced systemic homeostasis disorders in vivo and cell culture passaging in vitro induce a functional decline of MSCs, switching MSCs to a senescent status with impaired self-renewal capacity and biased differentiation tendency. MSC functional decline accounts for the pathogenesis of many diseases and, more importantly, limits the large-scale applications of MSCs in regenerative medicine. Growing evidence implies that epigenetic mechanisms are a critical regulator of the differentiation programs for cell fate and are subject to changes during aging. Thus, we here review epigenetic dysregulations that contribute to MSC aging and osteoporosis. Comprehending detailed epigenetic mechanisms could provide us with a novel horizon for dissecting MSC-related pathogenesis and further optimizing MSC-mediated regenerative therapies.

Molecular Characterization of Hemorrhagic Enteritis Virus (HEV) Obtained from Clinical Samples in Western Canada 2017-2018.

Hemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes an acute clinical disease characterized by hemorrhagic gastroenteritis in 4-week-old turkeys and older. Recurrent incidence of secondary infections (e.g., systemic bacterial infections, cellulitis, and elevated mortality), may be associated with the presence of field-type HEV in Canadian turkey farms. We speculate that field-type HEV and vaccine/vaccine-like strains can be differentiated through analysis of the viral genomes, hexon genes, and the specific virulence factors (e.g., ORF1, E3, and fib knob domain). Nine out of sixteen spleens obtained from cases suspected of immunosuppression by HEV were analyzed. The limited data obtained showed that: (1) field-type HEV circulates in many non-vaccinated western Canadian flocks; (2) field-type HEV circulates in vaccinated flocks with increased recurrent bacterial infections; and (3) the existence of novel point mutations in hexon, ORF1, E3, and specially fib knob domains. This is the first publication showing the circulation of wild-type HEV in HEV-vaccinated flocks in Western Canada, and the usefulness of a novel procedure that allows whole genome sequencing of HEV directly from spleens, without passaging in cell culture or passaging in vivo. Further studies focusing more samples are required to confirm our observations and investigate possible vaccination failure.

Abstract IA17: Patient-derived organoid models of bladder cancer

Abstract The molecular mechanisms of bladder tumorigenesis are poorly understood, in part due to the lack of suitable model systems that reflect the biology of human bladder cancer. In our studies, we have used three-dimensional culture approaches to establish a novel biobank of human patient-derived organoids from primary bladder tumors. These organoid lines recapitulate the histopathologic and molecular diversity of non-muscle invasive bladder cancer as well as muscle-invasive bladder cancer, and can be readily converted into xenografts by orthotopic transplantation. These organoid lines can be propagated by serial passaging in culture while maintaining their tumor properties and display mutational profiles that are highly concordant with their parental tumors. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Furthermore, we have shown that these organoid lines are highly amenable for analyses of drug response, which can be validated in vivo using orthotopic xenografts. I will discuss our recent findings using patient-derived tumor organoid lines to investigate bladder cancer biology and drug response, including ongoing analyses of tumor plasticity and the basis for tumor recurrence. Citation Format: Michael M. Shen. Patient-derived organoid models of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA17.

The herbal extract deriving from aerial parts of Scutellaria baicalensis shows anti-inflammation and anti-hypoxia responses in cultured fin cells from rabbit fish

A new cell line derived from dorsal fin of rabbit fish Siganus fuscescens was developed and characterized. The cell line was isolated from the dorsal fin, named as rabbit fish fin (RFF) cell line, and which was sub-cultured for 50 cycles since the development. This cell line was tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF cells, amplification of 18S rRNA from genomic DNA and immunostaining of cellular cytokeratin confirmed the proper identity of S. fuscescens fish. After 30th passage of cultures, the cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). The water extract of aerial part of Scutellaria baicalensis (SBA) has been shown in rabbit fish to prevent inflammation. Here, we extended this notion of testing the efficacy of SBA extract in the developed cultured RFF cells. Application of SBA extract inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1β, IL-6, as well as the signaling of NF-κB. The application of CoCl2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, applied SBA extract in the cultures prevented the hypoxia-induced signaling. Our results show the established RFF cell line may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. Additionally, we are supporting the usage of SBA herbal extract in fish aquaculture, which possesses efficacy against inflammation and hypoxia.

Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.

Renal Progenitor Cells Have Higher Genetic Stability and Lower Oxidative Stress than Mesenchymal Stem Cells during In Vitro Expansion.

In vitro senescence of multipotent cells has been commonly associated with DNA damage induced by oxidative stress. These changes may vary according to the sources of production and the studied lineages, which raises questions about the effect of growing time on genetic stability. This study is aimed at evaluating the evolution of genetic stability, viability, and oxidative stress of bone marrow mesenchymal stem cells (MSCBMsu) and renal progenitor cells of the renal cortex (RPCsu) of swine (Sus scrofa domesticus) in culture passages. P2, P5, and P9 were used for MSCBMsu and P1, P2, and P3 for RPCsu obtained by thawing. The experimental groups were submitted to MTT, apoptosis and necrosis assays, comet test, and reactive substance measurements of thiobarbituric acid (TBARS), nitrite, reduced glutathione (GSH), and catalase. The MTT test curve showed a mean viability of 1.14 ± 0.62 and 1.12 ± 0.54, respectively, for MSCBMsu and RPCsu. The percentages of MSCBMsu and RPCsu were presented, respectively, for apoptosis, an irregular and descending behavior, and necrosis, ascending and irregular. The DNA damage index showed higher intensity among the MSCBMsu in the P5 and P9 passages (p < 0.05). In the TBARS evaluation, there was variation among the lines of RPCsu and MSCBMsu, presenting the last most significant variations (p < 0.05). In the nitrite values, we identified only among the lines, in the passages P1 and P2, with the highest averages displayed by the MSCBMsu lineage (p < 0.05). The measurement of antioxidant system activity showed high standards, identifying differences only for GSH values, in the RPCsu lineage, in P3 (p < 0.05). This study suggests that the maintenance of cell culture in the long term induces lower regulation of oxidative stress, and RPCsu presents higher genetic stability and lower oxidative stress than MSCBMsu during in vitro expansion.

Conjunctival reconstruction via enrichment of human conjunctival epithelial stem cells by p75 through the NGF-p75-SALL2 signaling axis.

Severe conjunctival diseases can cause significant conjunctival scarring, which seriously limits eye movement and affects patients' vision. Conjunctival reconstruction remains challenging due to the lack of efficient methods for stem cells enrichment. This study indicated that p75 positive conjunctival epithelial cells (CjECs) were mainly located in the basal layer of human conjunctival epithelium and showed an immature differentiation state in vivo. The p75 strongly positive (p75++) CjECs enriched by immuno‐magnetic beads exhibited high expression of stem cell markers and low expression of differentiated keratins. During continuous cell passage cultivation, p75++ CjECs showed the strongest proliferation potential and were able to reconstruct the conjunctiva in vivo with the most complete structure and function. Exogenous addition of NGF promoted the differentiation of CjECs by increasing nuclear localization of SALL2 in p75++ CjECs while proNGF played an opposite role. Altogether, p75++ CjECs present stem cell characteristics and exhibit the strongest proliferation potential so can be used as seed cells for conjunctival reconstruction, and NGF‐p75‐SALL2 signaling pathway was involved in regulating the differentiation of CjECs.

Genomic Sequence of the New Attenuated Vaccine Strain Neethling-RIBSP of the Lumpy Skin Disease Virus.

We report here the draft genome sequence of the new attenuated strain Neethling-RIBSP of the lumpy skin disease virus, obtained by sequential and alternating passages in cell culture and developing chicken embryos. Genome sequencing allowed the identification of differentiation markers of the new strain.

In vivo cultivation of tuna blood fluke Cardicola orientalis in terebellid intermediate hosts

Some fish blood flukes of the genus Cardicola (Aporocotylidae) are considered important pathogens of farmed/ranched tuna, Thunnus spp. Infections with Cardicola spp. might obstruct the blood flow in the gills via massive accumulations of eggs and often lead to mass mortalities in captive tuna. At present, oral administration of an anthelminthic drug, praziquantel is the most effective treatment, but the tuna farming industries are seeking non-drug control measures. Development of prophylactic and holistic measures have been difficult, owing to a lack of basic knowledge about these parasites. Unlike other trematodes which use molluscs, blood flukes of marine actinopterygian fish use terebellid polychaetes as intermediate hosts. However, information about the development of Cardicola spp. within intermediate hosts is very limited. Recent success in Cardicola opisthorchis sporocyst transplantation into the host polychaete has opened possibilities for the cultivation of Cardicola in the laboratory. Here, we conducted several transplantation trials with another tuna blood fluke, Cardicol orientalis, into its natural and surrogate polychaete hosts. Cardicola orientalis sporocysts were injected into a total of 195 Nicolea gracilibranchis, the natural host, and clear sporocyst development and reproduction was observed in 32 recipients (overall success rate 16.4%). The production of daughter sporocysts in the transplanted polychaete occurred within 14 days post injection, and one sporocystogenous cycle took approximately 4 weeks. Serial passage culture via transplantation of in vivo-cultured sporocysts was also achieved, but with limited sporocyst reproduction. In addition, sporocysts were successfully retrieved from six and one individuals of the surrogate hosts, Thelepus setosus (n = 10) and Thelepus japonicus (n = 5), respectively. These results indicate that the in vivo cultivation of C. orientalis sporocysts is possible, not only in its natural host but also in other terebellids, although the problems of high mortality and inconsistency in successful transplantation need to be resolved.

Adipose-Derived Mesenchymal Stem Cells and Conditioned Medium Attenuate the Memory Retrieval Impairment During Sepsis in Rats.

In this study, we hypothesized that sepsis induction impairs memory retrieval in rats while transplanted mesenchymal stem cells (MSCs) and MSC-conditioned medium (MSC-CM) application are capable of attenuating those complications. MSCs were obtained from adipose tissue of rats and at the second culture passage; MSCs and MSC-CM were collected. Rats were randomly divided into four experimental groups: sham, CLP, MSC, and MSC-CM. Sepsis was induced by cecal ligation and puncture (CLP) model in the CLP, MSC, and MSC-CM groups. The MSC group received 1 × 106 MSCs/rat (i.p., 2h after CLP surgery); the MSC-CM rats received the conditioned medium (CM) from 1 × 106 MSCs intraperitoneally 2 h after sepsis induction. Novel object recognition test, sepsis score, and blood pressure measurement were performed 24h after the treatments. The right hippocampus was taken for western blot analysis. CLP rats showed a significantly higher sepsis score and systolic blood pressure. They also had a significant increase in the phosphorylated form of CAMKII-α, cleaved caspase 3 and Bax/Bcl2 ratio, and a reduction in c-fos protein in the hippocampus tissue samples compared with the sham group. MSC transplantation and MSC-CM administration significantly decreased the mean sepsis score and prevented sepsis-induced attenuation of blood pressure compared with the CLP rats. Animals in the MSC and MSC-CM groups showed a better memory retrieval, attenuation in phosphorylated form of CAMKII-α, cleaved caspase 3 and Bax/Bcl2 ratio, and an increase in c-fos protein expression compared with the CLP group. It seems that CAMKII and c-fos are inversely involved in regulating memory processes in hippocampus. Phosphorylated form of CaMKII-α overexpression may impair the ability of object recognition. Our findings confirmed that MSC-CM application has more advantages compared with transplanted MSCs and may be offered as a promising therapy for inflammatory diseases such as severe sepsis.

Abstract A44: Functional models of chromosome arm-level aneuploidies in cancer

Abstract Aneuploidy, whole-chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. We previously applied methods that define chromosome arm-level aneuploidy and a global cancer aneuploidy score to 10,522 tumors of 33 types in The Cancer Genome Atlas (TCGA). Aneuploidy level was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Overall aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. In contrast, some individual chromosome arm alterations correlated with higher expression of immune signaling genes, including deletion of chromosome 3p, 8p, and 17p. Cancer subtypes are often characterized by tumor specific patterns of arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although yeast and mammalian models of whole-chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. However, recent advances in genome engineering and targeting of endonucleases allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells, validated in 8 clones by whole-genome sequencing. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was partially the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro. We have also grown these cells in spheroid and organoid culture systems to assess the effects of chromosome 3 arm-level aneuploidies on cytokine production and squamous differentiation and are using these systems to identify how chromosome 3p deletion affects drug sensitivity. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison M. Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm-level aneuploidies in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A44.

A Pool of Eight Virally Vectored African Swine Fever Antigens Protect Pigs Against Fatal Disease.

Classical approaches to African swine fever virus (ASFV) vaccine development have not been successful; inactivated virus does not provide protection and use of live attenuated viruses generated by passage in tissue culture had a poor safety profile. Current African swine fever (ASF) vaccine research focuses on the development of modified live viruses by targeted gene deletion or subunit vaccines. The latter approach would be differentiation of vaccinated from infected animals (DIVA)-compliant, but information on which viral proteins to include in a subunit vaccine is lacking. Our previous work used DNA-prime/vaccinia-virus boost to screen 40 ASFV genes for immunogenicity, however this immunization regime did not protect animals after challenge. Here we describe the induction of both antigen and ASFV-specific antibody and cellular immune responses by different viral-vectored pools of antigens selected based on their immunogenicity in pigs. Immunization with one of these pools, comprising eight viral-vectored ASFV genes, protected 100% of pigs from fatal disease after challenge with a normally lethal dose of virulent ASFV. This data provide the basis for the further development of a subunit vaccine against this devastating disease.

3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

Human mesenchymal stem cells, such as human adipose-derived stem cells (hASCs), are typically cultured on a two-dimensional (2D) monolayer material surface, on which 2D culturing methods are easily performed and time-saving. However, hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2D monolayer culturing method compared to hASCs cultured with a three-dimensional (3D) culturing method, such as suspension culture. In this study, we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2D and 3D culturing processes. The hASCs cultivated with a 3D culturing process after 2D culture showed at least 2-fold enhanced pluripotency (Sox2, Nanog, and OCT4) compared with that of hASCs cultured with the 2D culture process alone. Furthermore, hASCs obtained from the 3D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2D culture process when hASCs were induced to differentiate. However, their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3D culture to 2D culture and vice versa, which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2D and 3D culture. The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2D and 3D culturing processes. Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs, which may reduce the effects of the aging process in hASCs. We discuss whether the environment of stem cell culture (i.e., 2D or 3D cultivation) can affect stem cell fate in terms of pluripotency and differentiation reversibility.

Successful scale up expansion of Wharton's jelly mesenchymal stromal cells in different commercial xeno-free and serum-free media

Background & Aim Cell-based therapies are becoming widely accepted in regenerative medicine and their demand and potential is increasing. Human multipotent Mesenchymal Stromal Cells (MSC) derived from the Wharton's jelly (WJ) of the umbilical cords can be obtained from samples typically discarded from public cord blood banking programmes. However, current cell culture bioprocesses for the derivation, scale up expansion, and GMP-compliant product manufacturing for early phase clinical trials use serum and xenogeneic proteins in medium formulation. Therefore, the use of xeno-free and serum-free media (XSFM) formulations is required to ensure more homogenous cell populations. The aim of this study was to compare four commercially available XSFM as an alternative to human serum-containing media in WJ-MSC culture. Methods, Results & Conclusion Cryopreserved MSC derived from WJ in serum-conditions were cultured in four commercial XSFM, namely PRIME-XV MSC Expansion XSFM, MSC Nutristem® XF, MesenCult™-ACF Plus Medium, and MSC-Brew GMP Medium and were further compared with conventional expansion medium Dulbecco's modified Eagle's medium (DMEM) containing 2 mM glutamine and supplemented with 10% of human serum B (hSerB). Cells were successfully expanded in culture in all XSFM conditions. Cumulative population doublings and harvest cell densities were higher in early passages for 3 out of 4 XSFM than serum-containing expansion medium. WJ-MSC proliferation was higher than 1 × 105 cells/cm2 in early culture (passage 2) while it decreased under 1 × 105 cells/cm2 in late culture (passage 8) in XSFM conditions. Spindle shaped morphology was kept upon passages in XSFM conditions. In all cases, cells were highly positive for CD73, CD90 and CD105 and negative for CD31, CD45 and HLA-DR expression in passage 1 and 6 and multilineage capacity to osteogenic, adipogenic and chondrogenic lineages was confirmed after in vitro induction. These data demonstrate that WJ-MSC can be successfully cultured and expanded in commercially available xeno-free and serum-free media and retain all necessary characteristics attributed to MSC for potential therapeutic use.