Stem Cell Culture Conditions Research Articles

Increased Expression of Oct-4 and Nanog Genes Following Nontoxic Ciprofloxacin Treatment in Adipose-Derived Stem Cells

Background: Recently, because of the demand for cell therapy, stem cells researches have been increased. Improvement in stem cell proliferation promotes the use of these cells in cellular treatments. Objectives: The aim of this study was to improve the culture conditions of adipose-derived stem cells (ADSCs) by treatment of these cells with a nontoxic concentration of ciprofloxacin. Methods: ADSCs were prepared from the human and animal cell bank of Iranian Biological Resource Center. The microbial contamination of cells including bacteria, fungi, and yeast had been tested. Adipogenic and osteogenic differentiation potential of ADSCs was demonstrated and the expression of surface markers including CD45, CD29, CD90, CD34, and CD105 was confirmed by flow cytometry. In the presence of ciprofloxacin at different concentrations, the viability of the cells was measured by MTT assay and the expression of the Oct-4, Nanog and GAPDH were examined by real-time PCR in a nontoxic concentration of ciprofloxacin-treated cells compared to control group. Results: The results showed that 1.5, 3, 6, and 12 mg/mL concentrations of ciprofloxacin have toxic effects on ADSCs (P < 0.0001) while in 0.5 - 150 μg/mL concentrations, there are no toxic effects on the cells. Furthermore, Oct-4 and Nanog expression in the nontoxic concentration of ciprofloxacin-treated cells was increased compared to the control group, which indicates the positive effect of ciprofloxacin on the growth of these cells. Conclusions: The present study shows that due to increased expression of Oct-4 and Nanog in 10 μg/mL ciprofloxacin-treated cells, the use of ciprofloxacin during the ADSCs culture period could provide suitable conditions for maintaining stem cell properties in these cells.

P11.59 Integrin a5 heterogeneous expression in glioblastoma is related to glioma stem cell subpopulations

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive primary brain tumor. Treatment failure and recurrence are explained in part by tumoral heterogeneity. Our previous results showed that the integrin α5β1 is implicated in GBM aggressiveness and represents a relevant therapeutic target. Recently, we observed intra- and inter-tumor heterogeneity of integrin α5β1 expression. Heterogeneity may be linked to different glioma stem cell populations. MATERIAL AND METHODS Ten glioma stem cell lines were grown as neurospheres in stem cell medium and their differentiation was induced by serum and/or ATRA. Two cell lines (NCH421k and NCH644) were selected and were modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Polyclonal lines and individual clones were analyzed phenotypically in vitro, before and after differentiation, and in vivo in orthotopic xenografts of 2x104 cells in nude mice. TCGA datasets were used to validate the heterogeneous expression of α5 integrin in GBM. RESULTS TCGA data validate that α5 integrin mRNA was only over-expressed in the mesenchymal subclass of GBM. Our results show that α5 integrin protein is not expressed in stem cell culture conditions. However, α5 integrin expression is induced after differentiation in only half of the cell lines supporting the notion of tumoral heterogeneity of glioma stem cells. Interestingly, single cell-derived clone evaluation showed that intra-tumoral stem cell heterogeneity also exists at the level of α5 protein expression. When glioma stem cells are programmed or transduced to express α5 integrin, differentiated cells became more aggressive. Notably, they acquired a fibronectin-dependent motility and a proliferative phenotype. Interestingly, integrin α5 remained expressed in secondary stem cells obtained after dedifferentiation. The in vivo assays suggested that glioma stem cells, programmed to express the integrin, were prone to form larger tumors. CONCLUSION Our data support the hypothesis that some glioma stem cells are programmed to express the α5 integrin subunit in their differentiated progeny to form a more aggressive tumor. They add new evidences that both cell populations may be considered for new therapeutic strategies against GBM.

Induced pluripotent stem cell generation from bovine somatic cells indicates unmet needs for pluripotency sustenance

Mechanisms that direct reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs), albeit incomplete in understanding, are highly conserved across all mammalian species studied. Equally, proof of principle that iPSCs can be derived from domestic cattle has been reported in several publications. In our efforts to derive and study bovine iPSCs, we encountered inadequacy of methods to generate, sustain, and characterize these cells. Our results suggest that iPSC protocols optimized for mouse and human somatic cells do not effectively translate to bovine somatic cells, which show some refractoriness to reprogramming that also affects sustenance. Moreover, methods that enhance reprogramming efficiency in mouse and human cells had no effect on improving bovine cell reprogramming. Although use of retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG appeared to produce consistent iPSC-like cells from both fibroblasts and cells from the Wharton's jelly, these colonies could not be sustained. Use of bovine genes could successfully reprogram bothmouse and human cells. These findings indicated either incomplete reprogramming and/or discordant/inadequate culture conditions for bovine pluripotent stem cells. Therefore, additionalstudies that advance core knowledge of bovine pluripotency are necessary before any anticipated iPSC-driven bovine technologies can be realized.

Slc25a36 modulates pluripotency of mouse embryonic stem cells by regulating mitochondrial function and glutathione level.

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a36-deficient colonies under trophoblast stem cell culture conditions. As a result of Slc25a36 deficiency, mtDNA of knockdown cells declined, leading to impaired mitochondria with swollen morphology, decreased mitochondrial membrane potential, and low numbers. The key transcription regulators of mitochondrial biogenesis also decreased. These results indicate that mitochondrial dysfunction leads to an inability to support the pluripotency maintenance. Moreover, down-regulated glutathione metabolism and up-regulated focal adhesion reinforced and stabilized the process of differentiation by separately enhancing OCT4 degradation and promoting cell spread. This study improves the understanding of the function of Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

Human mid-trimester amniotic fluid (stem) cells lack expression of the pluripotency marker OCT4A

Expression of OCT4A is one of the hallmarks of pluripotency, defined as a stem cell’s ability to differentiate into all the lineages of the three germ layers. Despite being defined as non-tumorigenic cells with high translational potential, human mid-trimester amniotic fluid stem cells (hAFSCs) are often described as sharing features with embryonic stem cells, including the expression of OCT4A, which could hinder their clinical potential. To clarify the OCT4A status of hAFSCs, we first undertook a systematic review of the literature. We then performed extensive gene and protein expression analyses to discover that neither frozen, nor fresh hAFSCs cultivated in multipotent stem cell culture conditions expressed OCT4A, and that the OCT4A positive results from the literature are likely to be attributed to the expression of pseudogenes or other OCT4 variants. To address this issue, we provide a robust protocol for the assessment of OCT4A in other stem cells.

Stem cell culture conditions and stability: a joint workshop of the PluriMes Consortium and Pluripotent Stem Cell Platform.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, bio-manufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells.Lay abstractNovel therapies derived from different kinds of precursor cells and stem cells are increasingly moving to clinical trials to restore tissue function in patients who have suffered injury or disease. The manufacture of these new therapies is unusually complex, which means that the manufacturing processes require great attention to assure they are safe and effective. This paper describes a conversation amongst experts in the field who are exploring therapeutic applications of two different kinds of stem cells (pluripotent stem cells and tissue-derived stem/precursor cells). It considers critical issues in developing the manufacturing process for each of these quite different cells types.

182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

PDTM-09. DIFFUSE INTRINSIC PONTINE GLIOMA AND PEDIATRIC GLIOBLASTOMA DERIVED-EXOSOMES HAVE SPECIFIC ONCOGENIC SIGNATURES

Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/106 cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.

Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth.

Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.

Future Perspectives on the Role of Stem Cells and Extracellular Vesicles in Vascular Tissue Regeneration

Vascular tissue engineering is an area of regenerative medicine that attempts to create functional replacement tissue for defective segments of the vascular network. One approach to vascular tissue engineering utilizes seeding of biodegradable tubular scaffolds with stem (and/or progenitor) cells wherein the seeded cells initiate scaffold remodeling and prevent thrombosis through paracrine signaling to endogenous cells. Stem cells have received an abundance of attention in recent literature regarding the mechanism of their paracrine therapeutic effect. However, very little of this mechanistic research has been performed under the aegis of vascular tissue engineering. Therefore, the scope of this review includes the current state of TEVGs generated using the incorporation of stem cells in biodegradable scaffolds and potential cell-free directions for TEVGs based on stem cell secreted products. The current generation of stem cell-seeded vascular scaffolds are based on the premise that cells should be obtained from an autologous source. However, the reduced regenerative capacity of stem cells from certain patient groups limits the therapeutic potential of an autologous approach. This limitation prompts the need to investigate allogeneic stem cells or stem cell secreted products as therapeutic bases for TEVGs. The role of stem cell derived products, particularly extracellular vesicles (EVs), in vascular tissue engineering is exciting due to their potential use as a cell-free therapeutic base. EVs offer many benefits as a therapeutic base for functionalizing vascular scaffolds such as cell specific targeting, physiological delivery of cargo to target cells, reduced immunogenicity, and stability under physiological conditions. However, a number of points must be addressed prior to the effective translation of TEVG technologies that incorporate stem cell derived EVs such as standardizing stem cell culture conditions, EV isolation, scaffold functionalization with EVs, and establishing the therapeutic benefit of this combination treatment.

PO-295 Heterogeneity of the alpha5 integrin subunit expression in glioblastoma

IntroductionGlioblastoma (GBM) is the most aggressive primary brain tumour. Treatment failure and recurrence are explained by intratumoral heterogeneity. Our previous results showed that the integrin α5β1, the fibronectin receptor, is implicated in GBM aggressiveness and represents a pertinent therapeutic target. Recently, we observed that its expression was heterogeneous between patient tumours but also between different areas in a given tumour. We hypothesised that this intratumoral heterogeneity may be linked to different glioma initiating cells (GIC).Material and methodsGICs were grown as neurospheres in stem cell medium and their differentiation was induced by serum. We characterised α5β1 expression in 9 GICs cell lines before and after differentiation. Two cell lines were selected and were genetically modified by depletion (CrisprCas9) or transfection of the α5 integrin gene. Different clones were selected expressing or not the integrin. Aggressiveness of polyclonal lines and individual clones was analysed in vitro before and after differentiation (proliferation, migration, evasion from spheroids) and in vivo (orthotopically xenografted cells).Results and discussionsOur results show that α5 integrin is not expressed in stem cell culture conditions. However, α5 expression is induced after differentiation in about half of the cell lines supporting the notion of inter-tumoral heterogeneity of GICs. Interestingly, single cell-derived clone evaluation showed that intra-tumoral GICs heterogeneity also exists. We noticed that when GICs are programmed or forced to express α5 integrin, differentiated cells became more aggressive. Notably, differentiated cells, expressing the integrin, acquired a fibronectin-dependent motility and a proliferative phenotype. The in vivo assays demonstrated that GICs, programmed to express the integrin, were prone to form larger tumours.ConclusionOur data support the hypothesis that some GICs are programmed to express the α5 integrin subunit to form a more aggressive tumour. Further studies will be needed to explore the implication of such heterogeneity in resistance to anti-integrin therapies but also to conventional chemo/radiotherapies.

Abstract 4632: Decitabine for neuroblastoma cure: Target therapy induced ongoing acquisition of RD3 loss and reverts tumor evolution

Abstract Recently, we defined the novel tumor evolution stabilization role of Retinal degeneration protein 3 (RD3), regulating the metastatic state of neuroblastoma (NB) cells in vitro and their metastatic potential in vivo. Our earlier studies in various tumor models recognized the role of therapy resistant residual cells in tumor recurrence and relapse. Herein, we investigated the ongoing acquisition of RD3 transcriptional regulation in radiotherapy surviving NB cells and further identified the potential drug candidates that can reprogram and reinstate RD3 transcription in these cells. Human NB (SH-SY5Y, SK-N-AS) cells exposed to single dose (10, 20, 50cGY, 1, 2, 4Gy) or fractionated (FIR, 2Gy/Day for 5 days) irradiation resulted in significant transcriptional repression of RD3. Drug screening with a panel of drugs that are in current clinical use, those in pipelines or in developmental stages by exposing radioresistant cells identified many potential drug candidates that could effectively restore RD3 transcription in this setting. Evidently, we found RD3 promoter region hypermethylation drives its transcriptional regulation in therapy resistant cells. To that note, FDA approved hypomethylating agent decitabine significantly reinforced RD3 transcription in this setting and further promoted cellular differentiation and completely inhibited the formation of organized tumorosphere under stem-cell culture conditions. Together, these results demonstrate the ongoing acquisition of RD3 loss in therapy resistant cells and further recognized the mechanism of how RD3 is regulated in this setting. More importantly, this results of this study identified a drug candidate, that has been already in clinical use, that could be beneficial in mitigating therapy resistance and better clinical outcomes for the patients presented with high-risk advanced disease. Funding: NIH-COBRE-IP20GM103639-01, OUHSC-COM-Radiation Oncology Research Development Funds Citation Format: Aravindan Natarajan, Dinesh Babu Somasundaram, Sheeja Aravindan, Mohan Natarajan, Terence Herman. Decitabine for neuroblastoma cure: Target therapy induced ongoing acquisition of RD3 loss and reverts tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4632.

53 Alterations in the epigenetic profile of glioblastoma tumors within hypoxic tumor regions

Glioblastomas are the most frequent and aggressive primary brain tumor in adults and despite recent therapeutic advances, they are resistant to treatment. Increasing malignancy of gliomas correlates with an increase in cellularity and a poorly organized tumor vasculature, leading to insufficient blood supply, hypoxic areas, and ultimately to the formation of necrosis. Hypoxia induces direct or indirect changes in the biology of solid tumor and their microenvironment through the activation of HIF transcription factors, leading to increased aggressiveness and tumor resistance to therapy. Not much is known about the epigenetic alterations induced by hypoxia and how they could alter tumor biology. In the present study, we have utilized PIMO as a specific marker of hypoxia in glioblastoma patients, treated with PIMO preoperatively. We have estimated PIMO positivity in each tumor (5-45%) and determined that it positively correlates with the hypoxia marker CA IX (r=0.57). In addition, 10 surgical PIMO cases were dissociated, immune labeled using PIMO antibody, followed by DNA isolation and methylation profiling. Our analysis of differentially top 4000 differentially methylated probes suggests that PIMO-positive (hypoxic) cells are differentially methylated compared to the PIMO-negative cells and these changes are associated with genes involved in hypoxic cellular response. We will validate these findings in additional glioblastoma cases and assess the mechanism of these epigenetic alterations in vitro in glioma stem cell culture conditions and upon exposure of the cells hypoxic conditions.

Expression of microRNAs associated with apoptosis in neurospheres of glioblastoma cell line subjected to treatment with ionizing radiation and temozolomide.

IntroductionGlioblastomas (GBM) are the most common malignant primary tumors in the brain, and show high mortality rate. Despite the current advances in the therapy, the GBMs are extremely resistant to ionizing radiation and chemotherapy, and the number of relapse is high. Studies bring this eminent tumorigenic potential due to the presence of a subpopulation of neoplastic cells with characteristics of stem cells, called cancer stem cells (CSCs). In gliomas, the isolation of tumor stem cells have been done by antigenic markers and observing the culture conditions of normal neural stem cells in vitro. That is, the floating proliferation of tumor cells when cultured, are analogous to neurospheres derived from normal neural stem cells in defined culture conditions. It is believed that CSCs are responsible for the restoration of the tumor and the low efficiency of the treatment, as these cells demonstrate malignant properties as tumorigenesis, chemoresistance and radioresistance. The practical implication of this finding is that no current therapy is able to suppress or stop the proliferation of these cells. Several microRNAs have been linked to the development and proliferation of glioblastomas, associated with different molecular mechanisms, among them programmed cell death. Studies have shown that these microRNAs in brain tumors exhibit altered expression levels, one of the essential mechanisms for the regulation of tumor stem cells (CSCs).ObjectivesTo evaluate the effect of ionizing radiation and temozolomide, alone or associated, on the expression of miRNAs that act as tumor suppressors (miR‐15 and miR‐16) and as oncogene (miR‐21), and adhered cells in neurospheres in culture of glioblastoma cell line (U343‐MG).Materials and MethodsThe trypan blue was used to examine cell viability before and after treatment, and method of real time PCR to check the expression of microRNAs in time 0h, considered to be immediately after the treatments, and 48h after exposure to treatments.Results and ConclusionsThe presented microRNAs are differentially expressed when comparing the different treatment modalities and time. We also observed differences between adhered cells and neurospheres. The oncogene miR‐21 remained with higher expression on treatment with ionizing radiation in neurospheres compared with the group of adherent cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Physiological Hypoxia Enhances Stemness Preservation, Proliferation, and Bidifferentiation of Induced Hepatic Stem Cells.

Induced hepatic stem cells (iHepSCs) have great potential as donors for liver cell therapy due to their self-renewal and bipotential differentiation properties. However, the efficiency of bidifferentiation and repopulation efficiency of iHepSCs is relatively low. Recent evidence shows that physiological hypoxia, a vital factor within stem cell “niche” microenvironment, plays key roles in regulating tissue stem cell biological behaviors including proliferation and differentiation. In this study, we found that physiological hypoxia (10% O2) enhanced the stemness properties and promoted the proliferation ability of iHepSCs by accelerating G1/S transition via p53-p21 signaling pathway. In addition, short-term hypoxia preconditioning improved the efficiency of hepatic differentiation of iHepSCs, and long-term hypoxia promoted cholangiocytic differentiation but inhibited hepatic differentiation of iHepSCs. These results demonstrated the potential effects of hypoxia on stemness preservation, proliferation, and bidifferentiation of iHepSCs and promising perspective to explore appropriate culture conditions for therapeutic stem cells.

Effects of Cell Cycle Regulators on the Cell Cycle Synchronization of Porcine induced Pluripotent Stem Cells.

ABSTRACTUnlike mouse results, cloning efficiency of nuclear transfer from porcine induced pluripotent stem cells (piPSCs) is very low. The present study was performed to investigate the effect of cell cycle inhibitors on the cell cycle synchronization of piPSCs. piPSCs were generated using combination of six human transcriptional factors under stem cell culture condition. To examine the efficiency of cell cycle synchronization, piPSCs were cultured on a matrigel coated plate with stem cell media and they were treated with staurosporine (STA, 20 nM), daidzein (DAI, 100 μM), roscovitine (ROSC, 10 μM), or olomoucine (OLO, 200 μM) for 12 h. Flow Cytometry (FACs) data showed that piPSCs in control were in G1 (37.5±0.2%), S (34.0±0.6%) and G2/M (28.5±0.4%). The proportion of cells at G1 in DAI group was significantly higher than that in control, while STA, ROSC and OLO treatments could not block the cell cycle of piPSCs. Both of viability and apoptosis were affected by STA and ROSC treatment, but there were no significantly differences between control and DAI groups. Real-Time qPCR and FACs results revealed that DAI treatment did not affect the expression of pluripotent gene, Oct4. In case of OLO, it did not affect both of viability and apoptosis, but Oct4 expression was significantly decreased. Our results suggest that DAI could be used for synchronizing piPSCs at G1 stage and has any deleterious effect on survival and pluripotency sustaining of piPSCs.

Optimal culture conditions are critical for efficient expansion of human testicular somatic and germ cells in vitro

To optimize culture conditions for human testicular somatic cells (TSCs) and spermatogonial stem cells. Basic science study. Urology clinic and stem cell research laboratory. Eight human testicular samples. Testicular tissues were processed by mechanical and enzymatic digestion. Cell suspensions were subjected to differential plating (DP) after which floating cells (representing germ cells) were removed and attached cells (representing TSCs) were cultured for 2 passages (P0-P1) in StemPro-34- or DMEM-F12-based medium. Germ cell cultures were established in both media for 12days. TSC cultures: proliferation doubling time (PDT), fluorescence-activated cell sorting for CD90, next-generation sequencing for 89 RNA transcripts, immunocytochemistry for TSC and germ cell markers, and conditioned media analysis; germ cell cultures: number of aggregates. TSCs had significantly prolonged PDT in DMEM-F12 versus StemPro-34 (319.6 ± 275.8h and 110.5 ± 68.3h, respectively). The proportion of CD90-positive cells increased after P1 in StemPro-34 and DMEM-F12 (90.1 ± 10.8% and 76.5 ± 17.4%, respectively) versus after DP (66.3 ± 7%). Samples from both media after P1 clustered closely in the principle components analysis map whereas those after DP did not. After P1 in either medium, CD90-positive cells expressed TSC markers only, and fibroblast growth factor 2 and bone morphogenetic protein 4 were detected in conditioned medium. A higher number of germ cell aggregates formed in DMEM-F12 (59 ± 39 vs. 28 ± 17, respectively). Use of DMEM-F12 reduces TSC proliferation while preserving their unique characteristics, leading to improved germ cell aggregates formation compared with StemPro-34, the standard basal medium used in the majority of previous reports.

Stem Cell Organoids in Primary Cultures of Human Non-Malignant and Malignant Colon

Aims: A sub-population of cells named cancer stem cells (CSCs) that initiate and promote tumour growth have been demonstrated to exist in several malignancies including colon carcinoma. The objective of our pilot study was to isolate CD133+CD26+CD44+ CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. Parallel cultures of non-cancer controls from colon normal lining and nonadenomatous polyps were set up. Methods: Magnetic activated cell sorting was used to isolate CD133+CD26+CD44+ cell populations followed by primary cell culturing under stem cell culture conditions. Number, cells/organoid and daughter generations of organoids were calculated using phase contrast microscope. Trypan blue exclusion method was used to test the viability of the cells. Results: Both colon tumour and colon non-adenomatous polyp formed floating organoids in suspension; however non-adenomatous polyp cultures did not show self-renewal properties for more than 1 passage. Normal colon singlecell suspension did not create organoids. Metastatic colon tumours rapidly produce cancer cell organoids in less than 24 hours in larger numbers compared to non-metastatic colon tumours (1-3 weeks). Metastatic colon tumour organoids have the ability for proliferation for upto five daughter generations in primary culture compared to three generations for those grown from non-metastatic tumours. Conclusions: This in vitro CSC organoid model will help study colon cancer biology, in particular providing a valuable source of primary cell-derived tissue for studying personalized molecular profiling using ‘omics strategies to direct therapeutic intervention.

Survival, proliferation, and migration of human meningioma stem-like cells in a nanopeptide scaffold.

Objective(s):In order to grow cells in a three-dimensional (3D) microenvironment, self-assembling peptides, such as PuraMatrix, have emerged with potential to mimic the extracellular matrix. The aim of the present study was to investigate the influence of the self-assembling peptide on the morphology, survival, proliferation rate, migration potential, and differentiation of human meningioma stem-like cells (hMgSCs).Materials and Methods:The efficacy of a novel method for placing hMgSCs in PuraMatrix (the injection approach) was compared to the encapsulation and surface plating methods. In addition, we designed a new method for measurement of migration distance in 3D cultivation of hMgSCs in PuraMatrix.Results:Our results revealed that hMgSCs have the ability to form spheres in stem cell culture condition. These meningioma cells expressed GFAP, CD133, vimentin, and nestin. Using the injection method, a higher proliferation rate of the hMgSCs was observed after seven days of culture. Furthermore, the novel migration assay was able to measure the migration of a single cell alone in 3D environment.Conclusion:The results indicate the injection method as an efficient technique for culturing hMgSCs in PuraMatrix. Furthermore, the novel migration assay enables us to evaluate the migration of hMgSCs.

Degradation of alkanethiol self-assembled monolayers in mesenchymal stem cell culture.

Alkanethiol self-assembled monolayers (SAMs) have been used extensively in studying the role of surface functionality and geometry on stem cell differentiation; however, the effects of stem cell culture conditions on SAM stability over time are not well understood. In this work, we examined the physical and chemical changes occurring on gold (Au)-SAM surfaces over time as a function of Au thickness. Within a narrow range of thicknesses (4, 8, and 10 nm), we observed significant differences in temporal SAM stability for a commonly utilized, hydrophilic, protein and cell repulsive oligo(ethylene) glycol alkanethiol (HS-(CH2 )11 (O(CH2 )2 )6 OH) SAM and the hydrophobic, protein adhesive hexadecanethiol (SH-(CH2 )15 CH3 ) SAM. Within both acellular and stem cell culture conditions, 8 nm Au resulted in the most stable SAMs (∼7 days). The 4 and 10 nm Au SAMs exhibited loss in stability following 5 days at varying degradation rates, showing 4 nm Au to be the least stable. Migration of human mesenchymal stem cells seeded on SAM surfaces showed that SAM degradation rates in acellular conditions were directly correlated with the cellular migration behavior. Findings of this study can be used to develop SAM surfaces with controlled degradation rates for applications in stem cell engineering and regenerative medicine. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 464-474, 2017.