Tuesday, June 25, 2013

Protocol for Flow Cytometric Sorting of Enriched Neuronal Cultures from iPSCs

Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, the authors present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. They have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f-/CD200high surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology: Turaç G, Hindley CJ, Thomas R, Davis JA, Deleidi M, et al. (2013) Combined Flow Cytometric Analysis of Surface and Intracellular Antigens Reveals Surface Molecule Markers of Human Neuropoiesis. PLoS ONE 8(6): e68519. doi:10.1371/journal.pone.0068519

Figure 1. Experimental outline. Schematic illustrating the research strategy of identifying novel surface marker combinations on a target population in neural and other stem cell differentiation systems for which intracellular, standard immunocytochemical markers are well established. Following harvesting, the resulting single cell suspension is subject to surface antigen candidate staining, followed by gentle fixation, permeabilization and subsequent co-staining with known intracellular markers. CD markers co-labeling the target population serve as positive markers, those absent on the target population serve as negative markers. In a separate, subsequent step, a combination of the identified positive and/or negative CD markers enables the flow cytometric enrichment of the viable population of interest from a heterogeneous cell suspension for further study and biomedical applications. doi:10.1371/journal.pone.0068519.g001.
Figure 2. Accurate detection of intracellular antigens with optimized fixation-permeabilization conditions preserving surface antigens. Flow cytometric detection of TUJ1, MAP2 and nestin antigens in BJ fibroblasts and the neural SH-SY5Y cell line (A). TUJ1 and nestin are present in both cell lines, while the mature neuronal marker MAP2 was only detected in SH-SY5Y cells (arrows). Note stable fluorescent levels of the negative population, indicating low background staining using this protocol. Representative experiment of three independent repeats shown. (B) Corresponding validation by immunofluorescence analysis. (C) Quantitation of TUJ1, MAP2 and nestin intracellular antigen detection (n=3). Error bars indicate standard deviation. (D) Response of TUJ1 and MAP2 intracellular antigen expression to 6 DIV of 10 µM retinoic acid (RA) treatment of SH-SY5Y cells. Note disappearance/reduction of subsets negative for these markers (upward shift, green arrows), as well as a shift toward CD184low expression with differentiation (blue arrows). doi:10.1371/journal.pone.0068519.g003.

Please note: In addition to the our Human Mouse Monoclonal Nestin Antibody used in the study, we have an extensive catalog of stem cell solutions. I will continue to post highlights of new applications.

Monday, June 17, 2013

Super-Charging Pluripotent Stem Cells

Adapting Stem Cells to Cellular Stress for Regenerative Medicine and Cell-Based Therapies

This approach for the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells could represent a breakthrough. The authors have identified these cells as "Multilineage Differentiating Stress-Enduring (MUSE) Cells": Heneidi S, Simerman AA, Keller E, Singh P, Li X, et al. (2013) Awakened by Cellular Stress: Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue. PLoS ONE 8(6): e64752. doi:10.1371/journal.pone.0064752.

Highlights: Although adult stem cells have been considered an attractive source for cell therapy, their effectiveness and efficiency is hindered by a frequently low survival rate due to their exposure to a high cellular stress environment upon transplantation. This key limitation is observed when utilizing adult stem cells for regenerative purposes, as typical cell engraftment yields are extremely low (less than 3%). This low survival rate limiting in the use of stem cells for therapies.

The authors have developed methods for isolating MUSE Cells that are preconditioned to survive engraftments. These cells display down regulation of genes involved in cell death and survival, embryonic development, organism survival, cellular assembly and organization, mitosis, DNA replication, recombination and repair.
Figure 1. Isolation and morphologic characterization of Muse-ATs. (A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity. doi:10.1371/journal.pone.0064752.g001.

Muse-AT cell isolation requires a simple yet highly efficient purification technique, Muse-AT cells could provide an ideal source of pluripotent-like stem cells with the potential to have a critical impact on regenerative medicine and cell-based therapy. The capabilities of these cells need to be further validate. I will keep you posted.

Thursday, June 06, 2013

mGluR5 and Fragile-X Syndrome (FXS)

Upregulation of mGluR5 uncovered in postmortem prefrontal cortex of 14 FXS patients

We are proud that our mGluR5 antibody was selected for this important study. The investigators found that mGluR5 binding density and protein expression were increased in the brains of FXS patients or carriers: Talakad G Lohith, Emily K Osterweil, Masahiro Fujita, Kimberly J Jenko, Mark F Bear, Robert B Innis. Is metabotropic glutamate receptor 5 upregulated in prefrontal cortex in fragile X syndrome? Molecular Autism 2013, 4:15 (24 May 2013).

Protocol: Tissue samples were homogenized using a mortar and pestle in fresh ice-cold, 50 mM Tris– HCl buffer (1:10 w/v) completing 3 × 10 passes with cooling on ice between homogenizations. Homogenates were centrifuged at 20,000g and 4°C for 25 minutes,followed by removal of the supernatant. Pellets were then resuspended in fresh ice-cold 50 mM Tris–HCl buffer and centrifuged again at the same settings. Pellets were resuspended in fresh ice-cold 50 mM Tris–HCl buffer at a protein concentration of approximately 1 mg of protein/mL. Aliquots were stored in a freezer at −80°C until further use. Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA), and absorption was read at 595 nm.

Figure: mGluR5 receptor density and expression in prefrontal cortex of FXS individuals and healthy controls. (A) Binding curves from homologous competition binding of 0.48 nM of [3H] MPEPy to membrane preparation from FXS and control subject samples at concentrations of unlabeled MPEPy ranging from 0.1 nM to 1μM. Individual binding curves were obtained from the average of triplicate measurements for e ach unlabeled ligand concentration. Data represent mean ± standard error in the mean from 14 FXS patients or carriers (FX) and 17 healthy controls (HC). At low concentration of unlabeled ligand, specific binding was higher for FXS than control samples. (B) Results of unpaired t-test tocompare the two groups. mGluR5 density tended to be higher (+16%;P = 0.058) in FXS patients than in the control group. Data represent mean ± standard deviation. Solid triangles (▲) in the FX group indicate the location of three FXS carriers; semisolid triang les indicate the location of a carrier with FXTAS. (C) Representative immunoblot for mGluR5. The mGluR5 band intensity was stronger for the FXS than control subject. Total protein stain of the same lanes confirmed equal-protein loading. (D) Average mGluR5: total protein ratio normalized to control subjects. The ratio was high and marginally significant (+32%; P=0.048) for the FXS group compared with controls. Data represent mean ± standard deviation.Solid triangles (▲) in the FX group indicate the location of three FXS carriers; semisolid triangles indicate the location of a carrier with FXTAS.
This study was the first to identify upregulation of mGluR5 density and expression in the prefrontal cortex of FXS patients or carriers compared to an age- and sex-matched control group. This is consistent with several studies in FXS model mice that postulate that the syndromic features of FXS are caused by an upregulated mGluR5 signaling pathway. Although the sample size was relatively small and the results could be secondary to prior medication treatment, these initial findings provide strong rationale for measuring mGluR5 in live patients using PET. Such in-vivo studies could measure mGluR5 in all brain regions; the results could also be correlated with treatment response to mGluR5 negative allosteric modulators.