Int J Stem Cells.  2020 Mar;13(1):65-79. 10.15283/ijsc19069.

Molecular Characterization of Embryonic Stem Cell-Derived Cardiac Neural Crest-Like Cells Revealed a Spatiotemporal Expression of an Mlc-3 Isoform

• Schmitteckert S
• Ziegler C
• Rappold GA
• Niesler B
• Rolletschek A

• KMID: 2500567
• DOI: http://doi.org/10.15283/ijsc19069

Abstract

Background and Objectives
Pluripotent embryonic stem (ES) cells represent a perfect model system for the investigation of early developmental processes. Besides their differentiation into derivatives of the three primary germ layers, they can also be differentiated into derivatives of the ‘fourth’ germ layer, the neural crest (NC). Due to its multipotency, extensive migration and outstanding capacity to generate a remarkable number of different cell types, the NC plays a key role in early developmental processes. Cardiac neural crest (CNC) cells are a subpopulation of the NC, which are of crucial importance for precise cardiovascular and pharyngeal glands’ development. CNC-associated malformations are rare, but always severe and life-threatening. Appropriate cell models could help to unravel underlying pathomechanisms and to develop new therapeutic options for relevant heart malformations.
Methods
Murine ES cells were differentiated according to a mesodermal-lineage promoting protocol. Expression profiles of ES cell-derived progeny at various differentiation stages were investigated on transcript and protein level.
Results
Comparative expression profiling of murine ES cell multilineage progeny versus undifferentiated ES cells confirmed differentiation into known cell derivatives of the three primary germ layers and provided evidence that ES cells have the capacity to differentiate into NC/CNC-like cells. Applying the NC/CNC cell-specific marker, 4E9R, an unambiguous identification of ES cell-derived NC/CNC-like cells was achieved.
Conclusions
Our findings will facilitate the establishment of an ES cell-derived CNC cell model for the investigation of molecular pathways during cardiac development in health and disease.

Keyword

Embryonic stem cells; Mice; Neural crest; Cell differentiation

Figure

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  Fig. 1 Comparative expression analyses of undifferentiated ES cells and ES cell-derived progeny using selected CNC-associated markers. (A∼E) Investigated genes were arranged according to their putative role in the CNC (adapted from (30)). Undifferentiated ES cells (0 days; d) are shown in black and embryoid body formation stages are indicated in dark grey (2d, 5d). Differentiation stages 5＋2d-5＋35d are shown in light grey. Column bar graphs are presenting the mean values of ten independent experiments with corresponding standard deviations.

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  Fig. 2 Immunofluorescence analyses of cardiac and non-cardiac ES cell-derived progeny. (A∼E, G∼J) Representative immunofluorescence analyses of isolated beating cardiac clusters using CNC-associated (all in red) and the cardiomyocyte-specific markers α-Actinin2 and Troponin T (both in green) (A, B, D, E, G∼J: 5＋7＋1d; C: 5＋4＋1d). (F) Staining of non-isolated ES cell-derived cardiac progeny at stage 5＋9d using α-Actinin2 and ET-AR (endothelin-1). (K∼M) Immunofluorescence analyses of non-cardiac ES cell-derived progeny using CNC-associated markers (all in red) and endothelial cell-(CD144/VE-Cadherin), neuronal cell-(Tubb3) and skeletal muscle cell-specific (α-Actinin2) markers (K: 5＋4d; L, M: 5＋15d). White arrows indicate co-expression. Nuclei were counterstained with DAPI in blue. Bars: 50 μm (D, L); 20 μm (B, C, F∼K, M); 10 μm (A, E).

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  Fig. 3 Western blot analyses of primary CNC cells and ES cell specimen using the 4E9R antibody. Comparative immunoblot analysis of CNC cells (1) as well as ES cell-derived specimens at various differentiation stages: 2) undifferentiated ES cells, 3) 5d EBs; 4) 5＋2d; 5) 5＋4d; 6) 5＋7d; 7) 5＋9d; 8) 5＋11d; 9) 5＋14d; 10) 5＋18d; 11) 5＋21d; 12) was performed using 4E9R antibody. For protein integrity the reference protein Gapdh was used as loading control. E: embryonic day, undiff.: undifferentiated, d: day, EB: embryoid body.

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  Fig. 4 Immunofluorescence analyses of multilineage ES cell-derivatives using the NC/CNC-specific marker 4E9R. (A) Immunocytochemistry of ES cell-derived non-CNC-related skeletal muscle cells applying 4E9R (green) and the skeletal muscle cell-specific antibody α-Actinin2 (red). (B∼D): Representative immunofluorescence staining of ES cell-derived CNC progeny using 4E9R (in green) and lineage-specific markers as α-Actin2 for smooth muscle cells (B) as well as Nestin (C) and Tubb3 (D) for neuronal cells (all in red). (E∼J) Immunofluorescence analysis of ES cell-derived progeny using 4E9R (in green) and CNC-associated markers such as Mef2c (E), Cx43 (F), Hand2 (G, J), Pax3 (H) and Lbx1 (I) (all in red). White arrows indicate co-expression. Nuclei were counterstained using DAPI (blue). Bars: 50 μm (A∼E, G∼J); 10 μm (F).

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  Fig. 5 Western blot and IP analyses of various heart specimen. (A) Comparative Western blot analysis of NC cells, embryonic, postnatal and adult heart tissue immunostained against 4E9R and Gapdh: 1) CNC cells E10.5; 2∼9) embryonic heart tissue (E10.5∼E17.5); 10) postnatal heart P8; 11) postnatal heart P15; 12) adult heart. For protein integrity the reference protein Gapdh was used as loading control. E: embryonic day, P: postnatal day, d: day. (B) Coomassie Blue stained SDS-PAGE resulting from immunoprecipitation with 4E9R antibody. The IP was performed with protein lysates of several tissue and ES cell-derived specimen at various differentiation stages. Numbers 1∼17 indicate the gel bands analyzed by nLC-ESI-MSMS. All investigated samples showed a triplet band pattern between 15 and 17 kDa except the adult heart tissue, where the lowest band is missing (indicated by the red arrow). (B’) Magnified section showing a selection of analyzed samples including adult heart tissue. Identified proteins and corresponding accession numbers are listed in Supplementary Tables S5 and S6.

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  Fig. 6 Comparative expression analyses elucidating the respective immunoreactivity of Mlc-3 and 4E9R. (A) Western blot analysis of primary murine heart tissues immunostained with Mlc-3 (13 kDa, predicted: 17 kDa) and 4E9R (15 kDa). 1∼8 embryonic heart tissues (1: E10.5; 2: E11.5; 3: E12.5; 4: E13.5; 5: E14.5; 6: E15.5, 7: E16.5; 8: E17.5); 9) postnatal heart tissue P8; 10) postnatal heart tissue P15; 11) adult heart tissue. (B) Immunoblot of undifferentiated (undiff.) and differentiated ES cell-derived specimens. 1) undiff. ES cells; 2) 5d EBs; 3) 5＋2d; 4) 5＋4d; 5) 5＋7d; 6) 5＋9d; 7) 5＋14d; 8) 5＋25d. As a loading control, the reference protein Gapdh (40 kDa) was used. (C∼E) Immunofluorescence analyses comparing the staining patterns of Mlc-3 (red) and 4E9R (green) in non-cardiac (C; 5＋5d), neuronal (D; 5＋7d) and cardiac (E, E’; 5＋7d) ES cell-derived progeny. The white box indicates the magnified area (E), whereas the white arrow shows the co-labeling of Mlc-3 and 4E9R (E’). Nuclei were counterstained using DAPI (blue). Bars: 50 μm (C), 20 μm (D, E).

Reference

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