Microbead kits

CD34⁺ cells were isolated from fresh umbilical cord blood after written consent. Isolation of CD34⁺ cells and the study were specifically approved by the Ethic Committee of RWTH Aachen University (Permit Number: EK187/08). For antagomiR experiments we used CD133⁺ cells, which were isolated from cord blood from DKMS Nabelschnurblutbank (Dresden, Germany). Isolation of CD133⁺ cells from fresh cord blood (DKMS Nabelschnurblutbank, Dresden, Germany) and the study were specifically approved by the local Ethics Committee of the Ärztekammer Nordrhein (Permit Number: EK103/2011). In brief, mononuclear cells were separated by density gradient centrifugation and CD34⁺ or CD133⁺ cells were enriched using MicroBead Kits according to the manufacturer’s instructions (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) [27 (link)]. For co-culture conditions, MSCs were isolated from the caput femoris after the patient’s written consent and cultivated as described before [4 (link),28 ]. Isolation of MSCs from bone marrow and the study were specifically approved by the Ethic Committee of RWTH Aachen University (Permit Number: EK128/09). MSCs were seeded as feeder cells between passages 3 to 6 (10 to 15 population doublings).

Briefly, lungs were minced and enzymatically-digested using previously described methods^{48 (link)} to generate a single cell suspension. Cells were then isolated by surface marker expression using Miltenyi microbead kits and LS column separation. 15-PGDH enzymatic activity was measured in lysed cell fractions, or in homogenized whole organs, as previously reported^{26 (link)}. Activity was then normalized to input protein and were tabulated graphically with error bars corresponding to standard error of the means and compared using 2-tailed t-tests.

Brain dissociation from 2a^fl/fl and 2aOKO mouse brain was prepared as indicated above. Immunomagnetic cell sorting from single-cell suspensions for OPCs and iOLs was performed on LS columns (Miltenyi Biotec, 130-042-401) using microbead kits from Miltenyi Biotec (for OPCs: CD140a [PDGFRα] MicroBead Kit, mouse, 130-101-502; and for O4: anti-O4 microbeads, human, mouse, rat, 130-096-670) based on the manufacturer’s protocols. Total RNA was extracted using a QIAGEN RNeasy Mini kit, and 900 ng per sample was used for library preparation using NEBNext Ultra TEM RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions and sequenced on an Illumina HiSeq2000. Partek Flow (version 10) was used for analysis, and paired-end sequenced reads were aligned to mm39 genome using STAR alignment 2.7.8a (51 (link)) and annotated using Ref-Seq. Features were filtered using recommended parameters and median ratio normalized. Differential gene expression analysis was performed using DESeq2 (52 (link)) and genes with fold change of at least 1.25 and P < 0.05 were considered significant.