Dominik Eckardt

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

The 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.

Multimodal Imaging for In Vivo Evaluation of Induced Pluripotent Stem Cells in a Murine Model of Heart Failure

Myocardial stem cell therapy in heart failure is strongly dependent on successful cellular transfer, engraftment, and survival. Moreover, massive cell loss directly after intramyocardial injection is commonly observed, generating the need for efficient longitudinal monitoring of transplanted cells in order to develop more efficient transplantation techniques. Therefore, the aim of the present study was to assess viability and cardiac retention of induced pluripotent stem cells after intramyocardial delivery using in vivo bioluminescence analysis (BLI) and magnetic resonance imaging (MRI). Murine induced pluripotent stem cells (iPSCs) were transfected for luciferase reporter gene expression and labeled intracellularly with supraparamagnetic iron oxide particles. Consequently, 5 × 105 cells were transplanted intramyocardially following left anterior descending coronary artery ligation in mice. Cardiac iPSCs were detected using BLI and serial T2* sequences by MRI in a 14-day follow-up. Additionally, infarct extension and left ventricular (LV) function were assessed by MRI. Controls received the same surgical procedure without cell injection. MRI sequences showed a strong MRI signal of labeled iPSCs correlating with myocardial late enhancement, demonstrating engraftment in the infarcted area. Mean iPSC volumes were 4.2 ± 0.4 mm3 at Day 0; 3.1 ± 0.4 mm3 at Day 7; and 5.1 ± 0.8 mm3 after 2 weeks. Thoracic BLI radiance decreased directly after injection from 1.0 × 106  ± 4.2 × 104 (p/s/cm2 /sr) to 1.0 × 105  ± 4.9 × 103 (p/s/cm2 /sr) on Day 1. Afterward, BLI radiance increased to 1.1 × 106  ± 4.2 × 104 (p/s/cm2 /sr) 2 weeks after injection. Cardiac graft localization was confirmed by ex vivo BLI analysis and histology. Left ventricular ejection fraction was higher in the iPSC group (30.9 ± 0.9%) compared to infarct controls (24.0 ± 2.1%; P < 0.05). The combination of MRI and BLI assesses stem cell fate in vivo, enabling cardiac graft localization with evaluation of LV function in myocardial infarction.

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

Rationale 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. Methods and Results 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. Conclusion 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.

Abstract 174: Isolation of primary human tumor cells significantly reduces bias in molecular analysis and improves culture of target cells

Solid tumors are infiltrated by cells of non-tumor origin, including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The amount and composition of infiltrating cells is highly variable and patient dependent, which makes analyses of primary tumor samples difficult. The contaminating cells lead to hybridization of non-tumor cell derived mRNA molecules to probes on microarrays and a significant reduction of sensitivity caused by measurement of irrelevant signals during next-generation sequencing or proteome analysis can be expected. In addition, the culture of human tumor cells is frequently hampered by fibroblasts overgrowing the target cells, which bias assays such as drug sensitivity tests. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from primary tissue. This procedure is based on the comprehensive depletion of cells of non-tumor origin by combining automated tissue dissociation and magnetic cell sorting (MACS). A negative selection strategy enables the isolation of the tumor cell population without knowledge of surface protein expression on these cells. Even tissues that initially contain low numbers of tumor cells ( samples. The reduction of non-tumor cell derived DNA, therefore, led to the detection of a higher number of tumor specific SNPs and INDELS, and consequently in a higher confidence in the results, in particular for mutations only present in a subset of tumor cells. Taken together, removal of non-tumor cells strongly improves their subsequent culture and molecular analysis of primary human tumor tissue. Citation Format: Lena Willnow, Stefan Tomiuk, Jutta Kollet, Stefan Wild, Silvia Ruberg, Claudius Fridrich, Peter Mallmann, Frauke Alves, Philipp Strobel, Dominik Eckardt, Andreas Bosio, Olaf Hardt. Isolation of primary human tumor cells significantly reduces bias in molecular analysis and improves culture of target cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 174.

FAS-based cell depletion facilitates the selective isolation of mouse induced pluripotent stem cells.

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues.

Wachstumsfaktoren für die Differenzierung pluripotenter Stammzellen

Due to their potential to differentiate into virtually any cell-type, pluri — potent stem cells (PSCs) are a highly valuable tool for both academia and industry. Most in vitro differentiation protocols mimic embryonic development by activation or inhibition of crucial signalling pathways. Growth factors play a central role in the manipulation of these pathways. In light of therapeutic applications, GMP manufacturing and tools for automation, scale-up and standardized processing become essential.