Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors.

International audienceThe cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells

List of fold-change values of selected genes with general or subtype-specific expression in mouse cardiomyocytes.

<p>Positive fold-change values indicate a higher abundance in ITGA6^high as compared to ITGA6^low-sorted cells, negative values demonstrate a higher abundance in ITGA6^low-sorted cells in comparison to ITGA6^high. Differential gene expression was assumed for fold-change values ≥ 3.0 or ≤ -3.0.</p><p>List of fold-change values of selected genes with general or subtype-specific expression in mouse cardiomyocytes.</p

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

<div><p>Rationale</p><p>Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers.</p><p>Methods and Results</p><p>In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis.</p><p>Conclusion</p><p>Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.</p></div

Gene expression analysis of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.

<p><b>(A)</b> Normalized signal intensities of CM-specific marker genes: general CM-specific <i>Tnnt2</i> and <i>Nkx2-5</i>, ventricle-specific <i>Hey2</i> and <i>Irx4</i>, atrium-specific <i>Nr2f2</i> and <i>Fgf12</i>. Data are expressed as mean ± SD, n = 4. Statistical analysis: ANOVA, Benjamini-Hochberg correction for multiple testing p ≤ 0.05, Tukey post-hoc test *** p ≤ 0.001, ns = not significant. <b>(B)</b> Heat-map shows median-centered log2-transformed signal intensities of selected genes. The color code indicates expression relative to the gene-wise median of all samples. Abbreviation: EL = E15.5 ERBB-2⁺/ITGA6^low, EH = E15.5 ERBB-2⁺/ITGA6^high, PL = P2 ITGA6^low, PH = P2 ITGA6^high.</p

Differential expression of ITGA5 and ITGA6 on atrial and ventricular cardiomyocytes.

<p><b>(A)</b> E13.5 whole hearts and mechanically separated atrial and ventricular tissue were co-labeled for ITGA6 or ITGA5 and α-actinin. Histograms, ITGA6 or ITGA5 expression gated on α-actinin+ cells. <b>(B)</b> E13.5 whole-heart preparations co-stained with antibodies against ITGA6 or ITGA5 and MLC-2a or MLC-2v (labeled with AlexaFluor^® 488 goat anti-mouse IgG). Analysis gates set according to the secondary antibody control. Rectangles indicate ITGA6 low (green) and high (red) expressing myocytes. <b>(C)</b> Co-labeling of E11.5 –P2 mouse hearts for ITGA6 and α-actinin.</p

Functional subtype characterization of sorted cells confirms selective enrichment of atrial and ventricular cardiomyocytes.

<p><b>(A)</b> Top graph, typical ventricular-like action potential (AP) of a cell from the EL group. Bottom graph, typical atrial-like AP from a CMs of the EH group. <b>(B)</b> Distribution of the cells in the two sorted groups. <b>(C)</b> Statistical analysis of AP parameters: left, action potential duration at 90% of repolarization (ADP90); mid, maximum rate of rise of the AP (max dV/dt); right, maximum diastolic polarization (MDP). Data are expressed as mean ± SEM. *** p ≤ 0.001 EL vs. EH. <b>(D)</b> Representative voltage ramps recordings from an E15.5 ERBB-2⁺/ITGA6^low CM (left) and an E15.5 ERBB-2⁺/ITGA6^high CM (right) show functional expression of inward and outward current components. Abbreviation: EL = E15.5 ERBB-2⁺/ITGA6^low, EH = E15.5 ERBB-2⁺/ITGA6^high, PL = P2 ITGA6^low, PH = P2 ITGA6^high.</p