Long-term cultivation of MSCs alters their mitochondrial function.

<p>(<b>A</b>): Fluorescence microscopy was used to investigate the morphology of the mitochondrial network within long-term cultivated and primary aMSCs and yMSCs. Upon <i>in vitro</i> aging mitochondrial network appeared to be altered. Images show immunofluorescence of mitochondria and the actin cytoskeleton stained with a specific antibody recognizing cytochrome C and Alexa 594-conjugated phalloidin, respectively. Nuclei were counterstained with DAPI. (<b>B</b>): During <i>in vitro</i> aging the relative mitochondrial area per cell area increases in aMSCs and yMSCs of passage P30 and P100 compared to P2. The mitochondrial network and the cellular area were quantified after staining with MitoTracker™ Red and phalloidin, respectively. Diagram values represent ratio of the mitochondria network area relative to the cell area. (<b>C</b>): The total antioxidant capacity decreases with increasing passage number. Moreover, yMSCs of P2 and P100 exhibited significant higher antioxidant activities than aMSCs of the same passage. The Trolox® equivalent antioxidant assay kit was used to determine the total antioxidant capacity of whole MSC lysates and quantified against a Trolox® standard row. (<b>D</b>): Intracellular ATP levels decline significantly in long-term cultivated aMSCs and yMSCs of passage P30 and P100. Cellular ATP was determined using ATPLite™ bioluminescence luciferase-based assay and normalized to total DNA content determined by CyQuant®. (<b>E</b>): Long-term cultivated yMSCs and aMSCs of P30 and P100 displayed higher ROS production than primary MSCs of P2. After treatment with pyocyanin, which increases ROS levels, the observed difference between P2 and P30/P100 remained but the absolute value amplified about 2-fold. Intracellular ROS level were determined using CM-H2-DCFDA and normalized to total DNA content. (<b>F</b>): Measurement of the mitochondrial membrane potential (ΔΨm) revealed a progressive increase during <i>in vitro</i> aging with highest values in aMSCs of P100. Upon treatment with valomycin, an inhibitor of the mitochondrial respiratory chain, ΔΨm declined in aMSCs and yMSCs of all passages. The mitochondrial ΔΨm was determined with the MitoProbe® JC-1. * indicates statistical significance (p<0.05).</p

Long-term cultivation negatively influences the differentiation and migration potential of aMSCs and yMSCs.

<p>(<b>A</b>): In contrast to primary MSCs of passage 2, <i>in vitro</i> aged aMSCs and yMSCs of P30 and P100 show no matrix mineralization. Osteogenic differentiation was initiated with dexamethason and determined by matrix mineralization (Alizarin Red, AR) and normalized to cell number (alamarBlue®, AB). Dashed lines indicate differentiation potential of the negative control cultured in EM. (<b>B</b>): Under stimulation with BMP2, aMSCs and yMSCs of P2 show strong osteogenic differentiation, while again no matrix mineralization was observed in long-term MSC cultures of P30 and P100. (<b>C</b>): Adipogenic differentiation of aMSCs and yMSCs of P30 and P100, induced by adipogenic medium, was diminished by 50% compared to aMSCs and yMSCs of P2. In reference to the negative control maintained in EM (dashed line), aMSCs and yMSCs of P30 and P100 retained a potential for adipogenic differentiation. Differentiation was determined by using Oil red O (OR) staining and normalized to cell number. Diagram shows values normalized to negative control. (<b>D</b>): The number of migrated cells declined with increased <i>in vitro</i> passage. Moreover, aMSCs of each passage demonstrated significantly lower migratory potential compared to yMSCs. Migration rates were measured with a modified Boyden chamber assay. At least five independent experiments were carried out for all assays. Abbreviations: OD, optical density. * indicates statistical significance (p<0.05).</p

Transcriptional profiling of aMSCs and yMSCs at P2, P30 and P100.

<p>(<b>A</b>): The absolute number of genes detected after thresholding diminished during advanced <i>in vitro</i> culture independent from donor age (second column). The correlation coefficient (r²) was significantly reduced between aMSCs and yMSCs of P30 and P100 compared to P2. Only minor differences in gene expression were detected between aMSCs and yMSCs of each passage. (<b>B</b>): Functional annotation clustering of genes exclusively expressed either in primary MSC of P2 or <i>in vitro</i> aged MSCs of P30 and P100 revealed 431 and 124 differentially regulated genes, respectively. At P2 genes were mainly associated with chemokine signaling, apoptosis, cell migration, and calcium homeostasis. Whereas at P30 and P100 exclusively expressed genes are involved in Notch signaling, cell cycle progression and receptor signaling. (<b>C</b>): Analysis of pathways down-regulated after long-term <i>in vitro</i> culture revealed involvement of mitochondria, focal adhesions, cytoskeleton organization, TGF-β/BMP, WNT, and PPARγ signaling. Pathways up-regulated upon long-term <i>in vitro</i> culture were associated with cell cycle progression, DNA replication, p53, MAPK, and insulin signaling. (<b>D</b>): Differential statistical analysis summarizes all pathways and genes significantly up- and down-regulated during <i>in vitro</i> culture. The most numerous genes down-regulated during <i>in vitro</i> aging of aMSCs and yMSCs were associated with focal adhesions, actin cytoskeleton organization and mitochondrial function.</p

Generation and characterization of <i>in vitro</i> aged MSCs.

<p>(<b>A</b>): Cumulative population doublings of aMSCs and yMSCs during the first 80 days of culture are shown (n = 5). (<b>B</b>): Long-term cultivation has no influence on short-term proliferation rate of aMSCs and yMSCs of passage 30 and 100. Proliferation assay was performed using CyQuant®. (<b>C</b>): Graphs illustrate quantified signal intensities of p21^WAF1/CIP1 and p16^INK4A relative to GAPDH. (<b>D</b>): Representative Western blots showing increased p21^WAF1/CIP1 and p16^INK4A expression during <i>in vitro</i> aging. GAPDH served as endogenous control. (<b>E</b>): In anchorage-independent growth assays <i>in vitro</i> aged MSCs_P100 did not form colonies, while the breast carcinoma cell line MDA-MB-231, which served as positive control, produced numerous colonies (n = 3). Abbreviations: aMSCs, mesenchymal stromal cells from aged donors; yMSCs, mesenchymal stromal cells from young donors; P: passage. * indicates statistical significance (p<0.05).</p

Functional Comparison of Chronological and <em>In Vitro</em> Aging: Differential Role of the Cytoskeleton and Mitochondria in Mesenchymal Stromal Cells

<div><p>Mesenchymal stromal cells (MSCs) are of high relevance for the regeneration of mesenchymal tissues such as bone and cartilage. The promising role of MSCs in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age, their limited availability in human tissues and the need of <em>in vitro</em> expansion prior to treatment. We therefore aimed to determine to which degree <em>in vitro</em> aging and chronological aging may be similar processes or if <em>in vitro</em> culture-related changes at the cellular and molecular level are at least altered as a function of donor age. For that purpose we established MSCs cultures from young (yMSCs) and aged (aMSCs) rats that were cultured for more than 100 passages. These long-term MSCs cultures were non-tumorigenic and exhibited similar surface marker patterns as primary MSCs of passage 2. During <em>in vitro</em> expansion, but not during chronological aging, MSCs progressively lose their progenitor characteristics, e.g., complete loss of osteogenic differentiation potential, diminished adipogenic differentiation, altered cell morphology and increased susceptibility towards senescence. Transcriptome analysis revealed that long-term <em>in vitro</em> MSCs cultivation leads to down-regulation of genes involved in cell differentiation, focal adhesion organization, cytoskeleton turnover and mitochondria function. Accordingly, functional analysis demonstrated altered mitochondrial morphology, decreased antioxidant capacities and elevated ROS levels in long-term cultivated yMSCs as well as aMSCs. Notably, only the MSC migration potential and their antioxidative capacity were altered by <em>in vitro</em> as well as chronological aging. Based on specific differences observed between the impact of chronological and <em>in vitro</em> MSC aging we conclude that both are distinct processes.</p> </div

Long-term <i>in vitro</i> culture alters MSC morphology independent from the donor age.

<p>(<b>A</b>): Cell diameter of aMSCs and yMSCs decreases during the course of long-term cultivation. Diagram shows the cell size distribution of MSCs measured by CASY® TT cell analyzer system at indicated passages after trypsinization. (<b>B</b>): Cellular area of attached aMSCs and yMSCs significantly decreases during <i>in vitro</i> aging. Measurements were performed from fluorescence images of identical exposure conditions. (<b>C</b>): Representative images of phalloidin labeled MSCs highlight reduction of cellular expansion. Additionally, <i>in vitro</i> aged aMSCs and yMSCs exhibited less filopodia, lamellipodia and cell spreading (white arrows). * indicates statistical significance (p<0.05).</p

Gfi1-ko/ko mice kept under nonSPF conditions develop osteopenia.

<p><b>(A)</b> Trabecular bone of vertebrae was measured with microCT. Gfi1-ko/ko mice kept under nonSPF conditions developed severe osteopenia as indicated by 60% BV/TV reduction compared to Gfi1-wt/wt mice. Upon SPF breeding Gfi1-ko/ko mice show a 15% reduction in BV/TV. However, SPF+nonSPF conditions induced intermediate osteopenia characterized by approx. 30% BV/TV reduction. Please note Gfi1-ko/ko mice kept at nonSPF conditions develop cortical bone osteopenia in femur. <b>(B)</b> Representative bone sections stained with von Kossa/ Kernechtrot illustrate trabecular bone in vertebrae of mice kept under nonSPF, SPF and SPF+nonSPF conditions. <b>(C)</b> Quantification of bone tissue by histomorphometry revealed reduced BV/TV values in Gfi1-ko/ko vertebrae under nonSPF and SPF+nonSPF conditions compared to controls. Breeding within the SPF environment did not significantly affect bone mass of Gfi1-ko/ko mice. <b>(D)</b> Histomorphometric quantification of the osteoblast covered bone surface (Ob.S/BS) shows reduced but elevated values upon nonSPF and SPF+nonSPF breeding, respectively. SPF breeding did not affect Ob.S/BS between control and mutant mice. <b>(E)</b> Quantification of the osteoclast covered bone surface (Oc.S/BS) revealed diminished but raised values in Gfi1-ko/ko mice upon nonSPF and SPF+nonSPF breeding compared to their corresponding Gfi1-wt/wt controls, respectively. Gfi1-ko/ko mutants grown under SPF conditions showed elevated Oc.S/BS counts compared to Gfi1-wt/wt mice. For better comparability, Gfi1-wt/wt values were set to 1 and Gfi1-ko/ko values were relatively calculated for each breeding condition. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s009" target="_blank">S3</a> Tables for absolute values. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Housing conditions determine Gfi1-ko/ko mice body mass and survival.

<p><b>(A)</b> Average growth curves from control mice kept under nonSPF conditions indicate evolving development also beyond weaning (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). However, soon after weaning (P19) Gfi1-ko/ko mutants display significantly delayed growth. Development under SPF growth conditions did not affect growth of Gfi1-ko/ko mice (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3). Upon SPF+nonSPF housing Gfi1-ko/ko mice demonstrated relative normal growth compared to controls (Gfi1-wt/wt n = 4, Gfi1-ko/ko n = 3); however, Gfi1-ko/ko mice show significant body mass reduction. All curves show values of male mice. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s007" target="_blank">S1 Table</a> for health monitoring. <b>(B)</b> At SPF and SPF+nonSPF conditions Gfi1-ko/ko mice showed a mortality rate of approx. 9% and 6%, respectively. <b>(C)</b> Final body mass of controls and Gfi1-ko/ko mice was assessed at indicated time points for SPF and SPF+nonSPF conditions. Combined SPF+nonSPF breeding caused a body mass reduction of approx. 25% in male and female Gfi1-ko/ko mice. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Elevated G-CSF and GM-SCF but normal M-CSF levels in Gfi1-ko/ko mice.

<p>The plasma levels of M-CSF, G-CSF, and GM-CSF were assessed by ELISA in mice kept at nonSPF, SPF, and SPF+nonSPF conditions. <b>(A)</b> M-CSF levels of Gfi1-ko/ko mutants were unaffected compared to controls upon all breeding conditions. <b>(B)</b> G-CSF is massively elevated in Gfi1-ko/ko mice compared to controls housed at nonSPF, SPF, and SPF+nonSPF conditions. <b>(C)</b> At all conditions Gfi1-ko/ko mice demonstrate significantly elevated GM-CSF levels. GM-CSF levels were measured by Q-Plex ELISA assay. The GM-CSF was below the limit of detection (b.l.d.) in Gfi1-wt/wt under all breeding conditions. Error bars represent SD. <b>(D)</b> The plasma levels of inflammatory cytokines were assessed by Q-Plex assay in control and Gfi1-ko/ko mice kept at nonSPF, SPF, and SPF+nonSPF conditions. Inflammatory cytokines such as Il-1β, Il-6, IFN-gamma, and TNF-alpha are significantly elevated in Gfi1-ko/ko mice kept at SPF+nonSPF conditions compared to their controls. Highest levels of Il-1β, Il-6, IFN-gamma, and TNF-alpha are present in Gfi1-ko/ko mice exclusively kept at nonSPF conditions. Due to sample limitations we measured for nonSPF mice serum pools (n = 2 with n = 4/pool) that were not statistically evaluated. Please note that lowest cytokine concentrations in Gfi1-ko/ko mice are present upon SPF conditions. In control mice IFN-gamma was below the limit of detection independent from the breeding condition. Full data set of analyzed cytokines is available as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198510#pone.0198510.s011" target="_blank">S5 Table</a>. Error bars represent SEM. Statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01.</p

Diminished osteoblast and osteoclast activity in nonSPF Gfi1-ko/ko mice.

<p><b>(A)</b> The Ob. and Oc. activity in Gfi1-ko/ko and Gfi1-wt/wt mice kept at nonSPF conditions was determined by measuring the plasma markers receptor activator of NF-kB ligand (Rankl), osteoprogerin (Opg), cross-linked carboxy-terminal telopeptide of type I collagen (CTX-I), and osteocalcin. Elevated Opg and diminished Rankl levels in Gfi1-ko/ko mutants suggest reduced osteoclast activity. Diminished bone resorption and formation in Gfi1-ko/ko mice is indicated by lowered CTX-I and osteocalcin levels, respectively. Error bars represent SD and statistical significance was calculated with t-test, * p ≤ 0.05 and ** p ≤ 0.01. <b>(B)</b> Basal expression of Gfi1 mRNA in different tissues shows highest values in spleen, bone marrow, and cortical bone. Non-haematopoietic tissues such as liver or cartilage and <i>in vitro</i> differentiated Oc. as well as Ob. demonstrated very low Gfi1 mRNA expression (n = 3 with 3 technical replicates/sample). Gfi1 expression was assessed with quantitative PCR (qPCR) and Gapdh was used as endogenous control. <b>(C)</b> All markers of Ob. proliferation and differentiation are diminished in Gfi1-ko/ko mutant lysates. Please note the low levels of osteoblast markers Ocn, Runx2, and Osx but normal expression of the osteocyte marker Dmp1. Values are shown as ratio of Gfi1-ko/ko vs. Gfi1-wt/wt mRNA expression (n = 3 with 3 technical replicates/sample). Normal expression is indicated with the dotted line at 1. Relative expression of bone marker genes was assessed by qPCR in full RNA preparations of cortical bone. Gapdh was used as endogenous control. <b>(D)</b> Immunohistology of the Ob. marker osteocalcin (Bglap) demonstrates abundant signals (red) in controls at the bone marrow to bone junction but low levels in Gfi1-ko/ko mutants. Please note abundant osteocalcin signals also in bone marrow cells. Immunostaining was performed on film supported cryosections. Counterstaining occurred with DAPI visualizing cell nuclei.</p