Stemmacs trilineage differentiation kit

Expression of specific markers of the mesoderm, ectoderm and endoderm were analyzed according to the StemMACS Trilineage Differentiation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Five hundred thousand cells were stained with antibodies. For staining of surface markers, the cells were alive, whereas for intracellular staining, the cells were fixed by BD Cytoperm/Cytofix Plus (Becton Dickinson, Franklin Lakes, NJ, USA). The differentiated cells were incubated with fluorochrome-conjugated antibodies (fluorescein allophycocyanin-APC and phycoerythrin-PE): anti-CD144-PE, anti-CD140b-APC (mesodermal markers); anti-PAX6-APC, anti-SOX2-PE (ectodermal markers); and anti-CD184-PE and anti-SOX17-APC (endodermal markers). According to the protocol, the negative controls were the unstained cells. The staining was analyzed using Attune NxT Software v2.2 on the Attune Nxt Flow cytometer (Thermo Fisher Scientific).

Trilineage differentiation was performed as described previously [12 (link),24 (link)] using a StemMACS™ Trilineage Differentiation Kit (#130-115-660; Miltenyi Biotec, Gaithersburg, MD, USA). Differentiated germ layers were confirmed by staining with antibodies: anti-PAX6 antibody for the ectoderm, anti-SM22A antibody for the mesoderm, and anti-FOXA2 antibody for the endoderm.

All iPSC lines were differentiated into Endoderm, Ectoderm and Mesoderm using the StemMACS™ Trilineage Differentiation Kit (Miltenyi Biotech; 130-115-660) according to manufacturer instructions.

To proof pluripotency, iPSCs (CMGANTi003-A passage 16 and CMGANTi004-A passage 15) were differentiated into the three embryonic germ layers (mesoderm, endoderm and ectoderm) using the StemMACS Trilineage Differentiation Kit (Miltenyi Biotec) according to manufacturer’s protocol at 37 °C, 5 % CO₂, 20 % O₂. On day seven, cells were collected for RNA extraction and cDNA synthesis. Expression of the selected germ layer markers (Table 1) was verified using RT-qPCR as described above (Table 2).

Fibroblasts were obtained from skin biopsies of two MSA patients and an age‐matched healthy control (Table S1) at the Department of Neurology, Medical University of Innsbruck with the appropriate consent and ethics approval. Cells were transduced with CytoTune™ 2.0 Sendai reprogramming vectors (A16517, Thermo Fisher Scientific) encoding for the classical Yamanaka’s factors (Klf4, Oct4, Sox2 and c‐Myc). The clearance of the Sendai virus (SeV) vector used for the somatic reprogramming was proven in the iPSCs cultures by quantitative RT‐PCR (qRT‐PCR) and immunocytochemistry and according to manufacturer’s instructions. Karyotyping was performed for each iPSC line by classical G‐banding at the Division of Human Genetics, Medical University of Innsbruck. Cells in metaphase arrest, induced by cocamide solution (0.1 µg/ml) were fixed, and Giemsa staining was performed. Chromosomes were analysed in at least 20 metaphases per cell line.
To confirm the pluripotency of the generated iPSCs, differentiation into the three germ layers (ectoderm, endoderm and mesoderm) was performed using StemMACS™ Trilineage Differentiation Kit (Miltenyi) according to the manufacturer’s protocols. Cells were analysed by immunocytochemistry at day 7 after initiation of the differentiation.