Autophagy in mesenchymal progenitors protects mice against bone marrow failure after severe intermittent stress

Key points Defective autophagy in mesenchymal progenitors associates with BM hypocellularity and mortality after severe stress. A short 4-day pharmacological in vivo treatment of mice to attenuate CDC42 activation protects against cytopenia and improves survival.


Long-term culture of MSPCs
For determination of growth curves, MSPCs were cultured for at least 8 passages.
Growing cells were passaged at approximately 70% confluence and reseeded at a density of 1x10 3 cells/cm 2 . Cumulative population doublings (PD) were calculated from the number of cells seeded and the number of cells harvested at the next passage as described previously 1 .

Differentiation assay
Adipogenic and osteogenic potential of MSPCs was determined using commercially available differentiation media as described by the manufacturer (StemXVivo®; R&D Systems). For staining, cells were rinsed with PBS and fixed in 10% paraformaldehyde (PFA) for 15 min at RT. Adipogenic potential was determined by staining with Oil Red O and visual examination and quantitation using propanol-extracted dye and measurement at 520 nm. Osteogenic potential was determined by Alizarin Red S staining with visual examination and quantitation using destaining solution (10% Cetylpyridiumchloride in 10 mM Sodiumphosphate (pH 7.0))-eluted dye measurement at 562 nm.

Cytomorphology
BM smears were stained with May Grunwald and Giemsa staining.
In some experiments, MSPCs were treated with recombinant murine WNT5A (rWNT5A), a low dose of 100 ng/ml rWNT5A (R&D Systems) was added to MSPCs at 2 consecutive days, 24 hrs apart.

In vitro treatment with stress inducers
To show MSPC responses to different types of stress, compact bone-derived MSPCs (P4) were spotted on chamber slides and treated with poly(I:C) (100µM, Apexbio) for 4 h or with 5-FU (15µM, Ribosepharm) for 16 hrs. For the irradiation experiment, the cells were irradiated with 15 Gy (gamma-irradiation). Cells were fixed and analyzed by IF microscopy as described below.
Staining of cells were analyzed by flow cytometry on a CyAn ADP LxP8.

Analysis of immunofluorescent labeled cells
To determine total protein content, the number of pixels per cell was assessed using ImageJ software (v1.52). Pictures within one experiment were taken under standardized conditions (light intensity, exposure, diaphragm).
To determine stress fiber formation, each field was counted for 1) the total number of For mean fluorescence intensity, the average intensity of pixels per cell was determined.
For foci analysis, the contrasts were visible by black and white processing within the digital images (Affinity Photo, Serif Europe Ltd). After threshold setting, cell structures were selected and counted automatically using ImageJ software.
For diameter analysis, contrasts were visualized using relief filters (Adobe Photoshop, Version 21.1.1). After threshold setting, cell structures were selected and length was determined using ImageJ software.
Colocalization was assessed using ImageJ after picture processing by selection of single cells, background subtraction with the rolling ball algorithm, and threshold adjustment by the autothresholding method "Moment". The ImageJ "Colocalization'' plugin was then used to recover the overlapping pixels of two analysed proteins. The pixel area occupied were then collected by using the "Analyse particles" function. To identify colocalization bias either in nuclear proximity or the cytoplasm, the DAPI signal was used to delimit the nuclear area and the nuclear and cytoplasmic fractions were reported separately.