Mesenchymal stem cell isolation and expansion

Bone marrow-derived MSCs were isolated from young (6 weeks) and old (1824 months) C57 black male mice using established techniques42,43 under a protocol approved by the Johns Hopkins University Animal Care and Use Committee. Briefly, immediately following euthanasia, whole bone marrow was flushed out from the bilateral tibias and femurs. After washing by centrifugation at 400g for 10min, cells were plated at 5 106 viable cells per ml. The culture was kept in humidified 5% CO2 incubator at 37C for 72h, when non-adherent cells were removed by changing the media.

All MSC preparations were evaluated using flow cytometry with PE or FITC-conjugated antibodies against murine Sca-1 (1:200; BioLegend 122507), CD31 (1:200; Fisher Scientific BDB554473), CD34 (1:100; eBioscience 14-0341-82), CD44 (1:100; BioLegend 103007), CD45 (1:100; BioLegend 103105), and IgG (1:100; BioLegend 400607) performed on BD LSRII (Becton Dickinson) using DIVA software. At least 10000 events were collected. FlowJo software was used to analyze and create the histograms.

Assessment for osteogenic and adipogenic differentiation was performed using established techniques43. Briefly, to induce osteogenic differentiation, old and young MSCs were seeded into 6-well plates at 1.3 104 cells/well. After 24h the media was replaced with osteogenic differentiation medium containing Iscoves medium supplemented with 100nM dexamethasone, 10mM beta-glycerophosphate, 50 M ascorbic acid, and 1% antibiotic/antimycotic. Cells were maintained in induction media with media changes every 2 days. After 14 days cells fixed in 10% formalin for 15min and calcium deposition was assessed using von Kossa staining. Calcium deposition was then quantified using a colorimetric calcium assay (Calcium CPC Liquicolour Kit StanBio, Boerne, TX) according to the manufacturers instructions. To induce adipogenic differentiation, old and young MSCs were seeded in 6-well plates at 2 105 cells/well. When confluent the media was replaced with adipogenic induction medium containing DMEM-HG, 10% FBS, 5% rabbit serum, 1uM dexamethasone, 10g/mL insulin, 200 M indomethacin, 500 M isobutylmethylxanthine (IBMX), and antibiotic/antimycotic for 3 days followed by exposure to followed by exposure to adipogenic maintenance medium (DMEM-HG, 10% FBS, insulin 10g/ml and P/S) for 3 days. After 3 cycles of induction and maintenance exposure cells were rinsed with PBS and fixed in 10% formalin for 10min. The cells were then stained with Oil Red O to assess for lipid droplets. After imaging Oil Red O extraction was performed using 100% isopropanol. Extract samples were transferred to a 96-well plate and absorbance readings were taken at 490nm to quantify extracted Oil Red O.

Confirmed MSCs were expanded in culture in media prepared by combining 490ml Medium 200 PRF (Gibco Invitrogen, Carlsbad, CA), a standard basal medium intended for culture of large vessel human endothelial cells, with 10ml Low Serum Growth Supplement (LSGS; Gibco Invitrogen). The final preparation contained 2% fetal bovine serum (FBS), 3ng/ml basic fibroblast growth factor (bFGF), 10ng/ml human epidermal growth factor, 10g/ml heparin, and 1g/ml hydrocortisone. Cells were incubated under standard conditions (5% CO2 and 37C). Expanded MSCs at low passage numbers (P2-P5) were used for the experiments. In the event frozen cells were used, they were thawed and grown for one passage prior to use in the experiments.

To prevent cell-cell interaction and assess only paracrine-mediated effects (i.e. those resulting from release of soluble factors), angiogenesis experiments were performed using bioreactor tubes (BT) constructed with CellMax semi-permeable polysulfone membrane tubing (Spectrum Labs, Rancho Dominguez, CA). These allowed the free diffusion of soluble proteins and other molecules released by the cells up to a 500kDa molecular weight cut-off, but not of the cells themselves. To load BTs, MSCs were trypsinized and suspended in Medium 200 PRF without LSGS supplementation (i.e. media devoid of stimulatory growth factors). MSCs were counted using a Scepter automated cell counter (Millipore, Billerica, MA), which had been previously standardized for accuracy. The desired number of MSCs was spun down and resuspended to a total volume of 100 ul that was injected into the BTs using a 0.5mL syringe. To compare paracrine-mediated angiogenesis by old and young MSCs, BTs were loaded with either 105 old or 105 young MSCs. Once cell injection was complete, the tubes were heat-sealed at both ends and the MSC-loaded tubes, fully submerged in media, were grown at standard culture conditions (37C, 5% CO2) for 7 days (Fig. 3a).

ELISA assays were performed to measure paracrine factor (PF) production by the MSCs contained within the BTs grown in culture. Tubes loaded with 2 105 MSCs were submerged in 5mL of alpha-MEM basal medium (Stemcell Technologies, Tukwila, WA) supplemented with 20% FBS (Gibco Invitrogen, Carlsbad, CA) in a 6-well plate. At day 7, conditioned media was collected from each well, spun down for 1min to pellet any debris, and then flash frozen at 80C. Conditioned media samples were assessed for the concentrations of vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF1) and insulin-like growth factor-1 (IGF1) by ELISA (Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturers instructions.

BTs were removed at day 7 and placed in separate wells of a 6-well plate containing human umbilical vein endothelial cells (HUVECs)44. Briefly, 105 HUVECs (Gibco Invitrogen, Carlsbad, CA) suspended in Medium 200PRF were plated per well in Geltrex (Gibco Invitrogen) coated 6-well plates. Negative control wells received a bioreactor loaded with un-supplemented Medium 200PRF only (i.e. no cells). Positive control wells were plated with 105 HUVECs suspended in 1mL of Medium 200PRF supplemented with LSGS, which is known to induce HUVEC tubule formation. After 18h at standard culture conditions (37C, 5% CO2), the wells were imaged to allow quantitative analysis of the resultant HUVEC tubule network. Images were taken in the center of each well and in all four quadrants at pre-determined locations (5 pictures total), at 100x magnification. The total length of the tubule networks captured in the images of each well was measured using ImageJ software. To allow for comparisons between experiments, the total length of the tubule network in each well was normalized to the average length of the tubule network in the negative control wells, and reported as a normalized ratio.

To assess the effect of young MSC-generated PFs on PF-mediated angiogenesis by old MSCs, BTs were prepared as described above containing either 105 young or 105 old MSCs. Two BTs were placed together in a 6-well plate in 5mL MSC media and incubated for 7 days at standard culture conditions (Fig. 3b) using a BT containing old MSCs paired with either a separate BT with other old MSCs (control) or a separate BT with young MSCs. After 7 days the tubes were removed, washed with un-supplemented Medium 200 PRF, and then used separately in the HUVEC assay as described above. After the HUVEC assay was complete (18h) the BTs were placed in separate wells of 6-well plates and grown in culture for 7 additional days with collection of conditioned media for PF release.

Replicates of 105 old MSCs were cultured separately, or in co-culture with young MSCs, for 7 days using a 0.4m Transwell system in 6-well plates (Corning), which allow transfer of soluble paracrine factors released by the cells, but not of the cells themselves. Following RNA purification, library preparation, amplification, and Illumina sequencing, the open source Galaxy pipeline was used for data processing and analyses. After alignment of raw sequencing reads to the UCSC mm10 genome using HISAT2, transcript assembly, alignment quantification, count normalization, and differential expression analyses were conducted with StringTie, featureCounts, DESeq2, and Genesis. Quantitative PCR (KAPA SYBR FAST One-Step qRT-PCR, Wilmington, MA) was used to validate 24 transcripts identified by RNA sequencing. Target genes were selected based on their presence in significantly regulated pathways and quantified relative to 18S ribosomal RNA using the 2Ct method45.

To validate the results of the RNA sequencing and RT-PCR results, a functional autophagy assay was performed to assess relative autophagy between old, young, and rejuvenated old MSCs. Old, young and rejuvenated cells were cultured (or co-cultured, in the case of rejuvenated cells) for 7 days in 6-well plates (105 cells per well). On Day 8, cells were trypsinzed, counted and 104 cells were transferred to each well of a 96-well black plate with clear bottom and incubated for 6h. The Autophagy Assay Kit (Sigma Aldrich, St. Louis, MO) measures autophagy using a proprietary fluorescent autophagosome marker in a microplate reader (ex=360; em=520nm). Three separate experiments were performed in triplicate each for each condition. To account for possible differences introduced by counting cells, results for each cell type were normalized based on absorbance (450nm) of a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. Rockville, MD).

Data are reported as mean standard error of the mean (SEM) unless otherwise indicated. Comparisons between groups for the HUVEC experiments were performed using the permutation test. For the PF ELISA data, groups were compared using the MannWhitney test. The autophagy assay and rt-PCR results were assessed using two-tailed t tests. For these experiments a p-value < 0.05 was deemed significant. In the RNA sequencing differential expression analysis, a false discovery rate (FDR) of <0.05 was considered significant.

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Paracrine-mediated rejuvenation of aged mesenchymal stem cells is associated with downregulation of the autophagy-lysosomal pathway | npj Aging -...