Cultured Cell Populations Research Articles

Development of Photoactive g-C3N4/Poly(vinyl alcohol) Composite Hydrogel Films with Antimicrobial and Antibiofilm Activity

Free-standing, composite hydrogels containing the visible-light responsive metal-free semiconductor graphitic carbon nitride (g-C3N4) as an integral component have been fabricated by direct casting techniques. At 0.67% g-C3N4 loading, intermolecular interactions between the semiconductor particles and the PVA polymer chains enhance both the mechanical and photophysical properties of the resulting hydrogels. In contrast, much higher g-C3N4 loadings of 3.3 or 6.7% g-C3N4 resulted in growth of the average semiconductor particle size and reduction in interactions between the incorporated photocatalyst and the PVA chains. The increased dimensions of the g-C3N4 semiconductor particles had the effect of compromising the mechanical properties of the composite system and reducing the lifetime of photogenerated charge carriers. However, the close proximity of g-C3N4 particles that is realized at increased semiconductor loading densities improves the absorption cross section of the material, resulting in an overall improvement in the photocatalytic activity of the material. Application of visible radiation caused all of the composite hydrogels to generate hydrogen peroxide (H2O2) at catalytic rates of 0.9-2.5 μM/min, while H2O2 decomposition rates remained similar across the different preparations. In studies to examine antimicrobial performance, irradiation of 6.7% g-C3N4/PVA hydrogel samples with visible radiation (400 ≤ λ ≤ 800 nm) generated sufficient H2O2 to significantly reduce both the viable planktonic cell population and biofilm formation in cultures of Pseudomonas aeruginosa.

Transcompp: understanding phenotypic plasticity by estimating Markov transition rates for cell state transitions.

Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. Freely available for download at http://github.com/nsuhasj/Transcompp. Supplementary data are available at Bioinformatics online.

Phenotypic and microRNA characterization of the neglected CD24+ cell population in MCF-7 breast cancer 3-dimensional spheroid culture.

In vitro 3-dimensional (3D) spheroid culture has been widely used as model to enrich CD44CD24 cancer stem cells (CSC) with high aldehyde dehydrogenase 1 (ALDH1) activity. Although CD24 subpopulation was known to be present in 3D spheroids and may influence cancer drug therapies, its characteristics and CSC properties were not well defined. In this study, CD24 population from the Michigan Cancer Foundation-7 (MCF-7) spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test, as well as microRNA expression profiling. Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in nonadherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f, and Nanog was dim in MCF-7 CD24 cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared with that of the parental cells. Moreover, microRNA profiling has shown that the absence of CSC properties was consistent with the downregulation of major CSCs-related pathways including Hedgehog, wingless-related integration site (Wnt), and microtubule associated protein kinase (MAPK) signaling pathways. However, the upregulated pathways such as adherens junctions, focal adhesion, and tight junction suggest that CD24 cells were probably at an epithelial-like state of cell transition. In conclusion, neglected CD24 cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analyzed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.

Culture on Tissue-Specific Coatings Derived from α-Amylase-Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose-Derived Stromal Cells.

While extracellular matrix (ECM)-derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose-derived stem/stromal cells (ASCs) on a range of ECM-derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α-amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α-amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue-like ultrastructure that is lost in the pepsin-digested thin films. ASCs cultured on the α-amylase-digested ECM have a more spindle-shaped morphology, and proliferation is significantly enhanced on the α-amylase-digested DAT coatings. Further, the α-amylase-digested DAT provides a more pro-adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α-amylase digestion as a new approach for generating bioactive ECM-derived coatings, and demonstrates tissue-specific bioactivity using adipose-derived ECM to enhance ASC proliferation and adipogenic differentiation.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. P60 may potentiate CIK cell cytotoxicity against tumor cells.

DNA Damage and Perturbed Topoisomerase IIα as a Target of 1,4-Benzoquinone Toxicity in Murine Fetal Liver Cells.

Benzene is a ubiquitous environmental pollutant. Recent studies have shown a link between the development of childhood leukemias and maternal benzene exposure, suggesting that these leukemias may be initiated in utero. Benzene crosses the placental barrier however the mechanisms behind in utero benzene toxicity have not been well elucidated. This study is the first to show that the benzene metabolite, benzoquinone (BQ), perturbs fetal topoisomerase IIα (Topo IIα), an enzyme essential for DNA repair. Using cultured murine CD-1 fetal liver cells, this study shows that Topo IIα activity decreases following 24 h of exposure to BQ (12.5 and 15.625 µM), with 12.5 µM confirmed to disrupt the c-kit+ Lin- Sca-1- Il7rα- population of cells in culture. Pretreatment with the antioxidant N-acetylcysteine did not prevent the inhibition of Topo IIα by BQ. An increase in Topo IIα-DNA covalent adducts was detected following 24-h exposure to BQ (12.5 and 50 µM). Interestingly, BQ (12.5 µM) exposure did not significantly increase levels of 4-hydroxynonenal (4-HNE), a marker of oxidative stress after 24 h. However, increased levels of the double-stranded DNA break marker γH2AX were detected following 24 h of BQ exposure, confirming that Topo IIα-induced breaks are increased in BQ-treated cells. This study shows that fetal Topo IIα is perturbed by BQ and suggests that this protein is a target of benzene and may be implicated with in utero benzene toxicity.

Dose-dependent spatiotemporal responses of mammalian cells to an alkylating agent.

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances.

Cohesin-Mediated Genome Architecture Does Not Define DNA Replication Timing Domains.

3D genome organization is strongly predictive of DNA replication timing in mammalian cells. This work tested the extent to which loop-based genome architecture acts as a regulatory unit of replication timing by using an auxin-inducible system for acute cohesin ablation. Cohesin ablation in a population of cells in asynchronous culture was shown not to disrupt patterns of replication timing as assayed by replication sequencing (RepliSeq) or BrdU-focus microscopy. Furthermore, cohesin ablation prior to S phase entry in synchronized cells was similarly shown to not impact replication timing patterns. These results suggest that cohesin-mediated genome architecture is not required for the execution of replication timing patterns in S phase, nor for the establishment of replication timing domains in G1.

Determining the function of proteins degraded by Human Cytomegalovirus

Human Cytomegalovirus (HCMV) is associated with significant morbidity and mortality in the immunocompromised, and is a leading cause of congenital infection. Only three drugs are available for treatment, all with significant toxicities. New therapies and a vaccine are urgently required. Susceptibility to viral infection and disease is determined in part by antiviral restriction factors (ARFs) and the viral proteins that have evolved to degrade them. Small-molecule disruption of the interaction between an ARF and a viral antagonist can inhibit viral replication and may be utilised for antiviral therapies. To identify novel restriction factors against HCMV, we previously developed a multiplexed proteomic approach to identify proteins that are actively degraded early during HCMV infection. We reasoned that these would be enriched in known and novel ARFs that the virus must degrade in order to replicate. 35 proteins were shown to be degraded according to stringent statistical criteria, which included the known anti-HCMV restriction factors Sp100 and MORC3, and a novel ARF, HLTF. Here, we present preliminary results from a combination of viral replication assays to identify other novel ARFs. These include a new ‘two-colour’ approach to characterise HCMV restriction, which aims to eliminate variability in cell density by mixing populations of cells in a single cell culture well. Preliminary studies have identified a number of proteins that inhibit virus replication, as well as a novel dependency factor which the virus may degrade in order to attenuate cellular immune signalling or help establish latency.

Heterogeneity of Stem Cells in theHuman Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal, and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

Regulation of Ca2+ signaling by acute hypoxia and acidosis in cardiomyocytes derived from human induced pluripotent stem cells

AimsThe effects of acute (100 s) hypoxia and/or acidosis on Ca2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time. Methods and results1) hiPSC-CMs express two cell populations: rapidly-inactivating ICa myocytes (τi<40 ms, in 4–5 day cultures) and slowly-inactivating ICa (τi ≥ 40 ms, in 6–8 day cultures). 2) Hypoxia suppressed ICa by 10–20% in rapidly- and 40–55% in slowly-inactivating ICa cells. 3) Isoproterenol enhanced ICa in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba2+ was the charge carrier through the calcium channels, implicating Ca2+-dependent inactivation in O2 sensing. 5) Acidosis suppressed ICa by ∼35% and ∼25% in rapidly and slowly inactivating ICa cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ∼25% in global and ∼37% in RyR2 Ca2+-microdomains in either cell type, whereas with hypoxia suppression was ∼20% and ∼25% respectively in global and RyR2-microdomaine in rapidly and ∼35% and ∼45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. ConclusionsVariability in ICa inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca2+-signaling in the two-cell types arises from differential Ca2+-dependent inactivation of the Ca2+-channel caused by proximity of Ca2+-release stores to the Ca2+ channels.

The Chick Caudolateral Epiblast Acts as a Permissive Niche for Generating Neuromesodermal Progenitor Behaviours

Neuromesodermal progenitors (NMps) are a population of bipotent progenitors that maintain competence to generate both spinal cord and paraxial mesoderm throughout the elongation of the posterior body axis. Recent studies have generated populations of NMp-like cells in culture, which have been shown to differentiate to both neural and mesodermal cell fates when transplanted into either mouse or chick embryos. Here, we aim to compare the potential of mouse embryonic stem (ES) cell-derived progenitor populations to generate NMp behaviour against both undifferentiated and differentiated populations. We define NMp behaviour as the ability of cells to: (i) contribute to a significant proportion of the anterior-posterior body axis, (ii) enter into both posterior neural and somitic compartments, and (iii) retain a proportion of the progenitor population within the posterior growth zone. We compare previously identified ES cell-derived NMp-like populations to undifferentiated mouse ES cells and find that they all display similar potentials to generate NMp behaviour in vivo. To assess whether this competence is lost upon further differentiation, we generated anterior and posterior embryonic cell types through the generation of 3D gastruloids and show that NMp competence is lost within the anterior (Brachyury-negative) portion of the gastruloid. Together this suggests that in vitro-derived NMp-like cells maintain an ability to contribute to multiple germ layers that is also present within pluripotent ES cells, rather than acquiring a neuromesodermal competent state through differentiation.

47P - Characterization of the different cell population in primary culture of breast tumor

47P - Characterization of the different cell population in primary culture of breast tumor

In situ transcriptome characteristics are lost following culture adaptation of adult cardiac stem cells

Regenerative therapeutic approaches for myocardial diseases often involve delivery of stem cells expanded ex vivo. Prior studies indicate that cell culture conditions affect functional and phenotypic characteristics, but relationship(s) of cultured cells derived from freshly isolated populations and the heterogeneity of the cultured population remain poorly defined. Functional and phenotypic characteristics of ex vivo expanded cells will determine outcomes of interventional treatment for disease, necessitating characterization of the impact that ex vivo expansion has upon isolated stem cell populations. Single-cell RNA-Seq profiling (scRNA-Seq) was performed to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses demonstrate that identity marker genes expressed in freshly isolated cells become undetectable in cultured CPCs while low level expression emerges for thousands of other genes. Transcriptional profile of CPCs exhibited greater degree of similarity throughout the cultured population relative to freshly isolated cells. Findings were validated by comparative analyses using scRNA-Seq datasets of various cell types generated by multiple scRNA-Seq technology. Increased transcriptome diversity and decreased population heterogeneity in the cultured cell population may help account for reported outcomes associated with experimental and clinical use of CPCs for treatment of myocardial injury.

Use of fluorescent Fab/Ab complexes and IncuCyte live-cell analysis to dynamically track cell surface markers and cell populations in mixed cultures

Abstract Fluorescently-labeled antibodies are widely used for visualising cellular protein expression/distribution (e.g. immunocytochemistry) and immuno-phenotyping (e.g. flow cytometry). However, their applications are largely confined to end-point or short term (min-h) detection, and the cell processing and labeling steps that are required often perturb the biology of interest. To enable longer-term, fully kinetic applications of cell surface protein markers in living cells, we have developed a novel strategy based on fluorescently-labeled antibody fragments (Fabs) and IncuCyte live-cell analysis. An Fc-targeted anti-mouse Fab fragment conjugated to a green-emitting fluoroprobe (IncuCyte FabFluor-488) was used to tag Abs to surface markers (e.g. CD4, CD20, Her2) via a simple one-step, no-wash protocol. Addition of the corresponding FabFluor-488-Ab complex to living cells (e.g. Jurkats, RAMOS, SKOV-3) produced long lasting (up to 5d), specific and stable cellular fluorescence at relatively low Ab concentrations (1mg ml−1) that did not perturb cell morphology or growth (IncuCyte S3). To illustrate the application of this approach the method was used to (a) quantify upregulation of the checkpoint protein PDL-1 over 48h in IFN-gamma-treated MDA-MB231 and SKOV-3 and tumor cells, (b) identify cellular subsets in PBMCs, and (c) observe proximity and engagement of A549 target cells by CD-8 positive PBMCs in immune-cell killing experimentsThis FabFluor-488/IncuCyte method enables long-term tracking and quantification of cell surface protein expression and the ability to identify cell subsets in living cultures over time. This method should prove powerful in analyses on complex and advanced heterogeneous cell models.

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Ex vivo expansion of human peripheral blood natural killer cells and cytotoxic T lymphocytes from lung cancer patients.

Lung cancer is the most common type of cancer with the highest cancer-associated mortality rates worldwide, as well as in Vietnam. Numerous studies have demonstrated that higher numbers and higher rate of activity of infiltrating natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) in the tumor are closely correlated with positive prognosis, tumor size decrease and longer survival of lung cancer patients. In the present study, the effectiveness of BINKIT® kit in the ex vivo expansion of NK cells and CTLs in the peripheral blood of 7 patients aged between 30 and 84 years with metastatic lung cancer was evaluated. After 21 days of culture, the average number of CTLs (CD3+CD8+) increased by 742.3-fold in the CTL culture, accounting for 72.2% of the cultured cell population, and the mean cell viability was 95.7%. In the NK cell culture, the average number of NK cells (CD3−CD56+) increased by 637.5-fold, accounting for 84.3% of the cultured cell population, with an average viability of 94.7%. The percentage of active NK cells (CD3−CD56+ bright) was 82.1%, which increased by 408.9-fold. Notably, a close correlation was identified between the numbers of cytokine-induced killer (CD3+CD56+) and NK (CD3−CD56+) cells in the NK cell culture (P<0.05). In the two culture conditions (namely NK cell and CTL cultures), no clear correlation was identified between the rate of initial immune cells in the peripheral blood and the corresponding number following ex vivo expansion (P>0.05). These results revealed that the method of expansion and activation of NK cells and CTLs from peripheral blood was successfully applied using BINKIT, and reached the requirements for clinical applications in cancer treatment in Vietnam.

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

The mammalian hair follicle arises during embryonic development from coordinated interactions between the epidermis and dermis. It is currently unclear how to recapitulate hair follicle induction in pluripotent stem cell cultures for use in basic research studies or invitro drug testing. To date, generation of hair follicles invitro has only been possible using primary cells isolated from embryonic skin, cultured alone orin a co-culture with stem cell-derived cells, combined with invivo transplantation. Here, we describe the derivation of skin organoids, constituting epidermal and dermal layers, from a homogeneous population of mouse pluripotent stem cells in a 3D culture. We show that skin organoids spontaneously produce de novo hair follicles in a process that mimics normal embryonic hair folliculogenesis. This invitro model of skin development will be useful for studying mechanisms of hair follicle induction, evaluating hair growth or inhibitory drugs, and modeling skin diseases.

Immunophenotyping of Live Human Pluripotent Stem Cells by Flow Cytometry.

Human pluripotent stem cells (hPSCs) have great potential for use in regenerative medicine and cell replacement therapies; however, prior to clinical application, cultured cell populations need to be screened to ensure the quality of the culture, as well as the capacity of these pluripotent cells to differentiate into desired cell types. Flow cytometry, utilizing antibodies recognizing targets restricted to the hPSC surfaceome, offers an invaluable tool for high-throughput validation of hPSC lines. Here we describe the immunophenotyping of live human embryonic stem cell (hESC, H9) and human induced pluripotent stem cell (hiPSC, KB3) lines by flow cytometry using a panel of antibodies identified as either stem cell reference markers (CD90, EpCam) or reported as being prevalent or restricted (c-Kit, HPI-1, Integrin α6, Semaphorin-6A) to these cells. The protocols described here with hPSCs are also applicable to differentiated hPSC progeny and should be instrumental in the immunophenotyping and isolation of well-defined homogeneous cell populations useful in regenerative medicine.

A Novel Soft Sensor Approach for Estimating Individual Biomass in Mixed Cultures with Applications to Agricultural Waste and Wastewater Treatment

Mixed cultures are prevalent in nature, which are biological systems with more than one type of organism sharing or competing for available nutrients. Compared to single cultures, mixed culture present many advantages. For example, when organisms in mixed cultures work symbiotically, the cells’ metabolic pathways complement each other and can result in efficient utilization of substrates and increased product yield. Other advantages include the use of mixed or cheaper substrates, reduction in sterilization and increased robustness to environmental changes. The application of mixed cultures in biotechnology has expanded rapidly in the last a few years, with applications ranging from wastewater treatment, biogas production, soil remediation, to chemical production such as solvent, antibiotics, acids and other valued commodities. At the same time, many challenges remain for effective mixed culture applications. One of them is how to efficiently and accurately monitor the individual cell populations in a mixed culture. The current approaches on individual cell mass quantification are suitable for off-line, infrequent characterization of mixed cultures. In this work, we propose a fast and accurate ‘soft sensor’ approach for estimating individual cell concentrations in mixed cultures. The proposed approach utilizes absorption spectrum of a mixed culture sample measured by a spectrophotometer over a range of wavelengths. A multivariate linear regression method, partial least squares or PLS, is applied to correlate individual cell concentrations to the spectrum. The proposed soft sensor is demonstrated using a methanotroph microalgae coculture. Recent findings published in Nature and Nature Geoscience suggest that the coupling of methane oxidation and oxygenic photosynthesis are prevalent in nature and reduce CH4 emissions and reuse CO2. These findings suggest that the coculture of methanotrophs and microalgae represents not only a feasible, but also a highly promising strategy for agriculture waste and wastewater treatment: the biogas and wastewater produced from anaerobic digestion of agriculture waste can be converted by the coculture of methanotroph and microalgae to cell biomass for animal food or biodiesel production, which not only significantly reduces the greenhouse gases emission, but also captures the valuable nutrients contained in digester effluent. Several case studies are presented to demonstrate the effectiveness and robustness of the soft sensor approach.