Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry. RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARγ, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways

1,25-Dihydroxyvitamin D3 Treatment Delays Cellular Aging in Human Mesenchymal Stem Cells while Maintaining Their Multipotent Capacity

<div><p>1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in <em>fibroblast growth factor-23</em> (<em>Fgf23</em>) and <em>klotho</em> deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and <em>P16</em> expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.</p> </div

Morphological characteristics of 1,25D3 cultured hMSC and ß-galactosidase quantification.

<p><b>A</b> Morphology of hMSC in P1 (upper row) and P3 (lower row) treated with 1,25D3 over three passages compared to control cells. 1,25D3 incubated hMSC lost their typical fibroblast-like features and showed a broadened and extended cellular morphology. Scale bar = 50 µm. <b>B</b> The induction of ß-galactosidase was significantly reduced in 1,25D3 cultured cells compared to control hMSC. Cells from three different donors were used and were cultured up to three passages. The results are shown as mean+SEM of three independent experiments, each normalized to its control. Each time five pictures of ß-galactosidase staining were analyzed using the AutMess tool of AxioVision Rel. 4.6 software. (***, p<0.001, student's t-test).</p

Primers used for RT-PCR experiments.

<p>Primer sequences, fragment lengths and PCR conditions including number of cycles, annealing temperature and magnesium supplementation.</p

Trilineage differentiation capacity of 1,25D3 pretreated hMSC.

<p><b>A</b> Chondrogenic, adipogenic and osteogenic differentiation of hMSC after 1,25D3 pretreatment over three passages. Alcian Blue staining of chondrogenic differentiated hMSC (upper panel), Oil Red O staining for intracellular lipid vesicles during adipogenic differentiation (middle panel) and Alizarin Red S staining of mineralized extracellular matrix after osteogenic differentiation (lower panel) are shown (n.d. = not differentiated; C = differentiated control; +1,25D3 = differentiated, cells pretreated for three passages with 1,25D3). The figure is representative for three independent experiments. The bar represents 100 µm and 10 µm, respectively as indicated. <b>B</b> Quantification of chondrogenic (black bar), adipogenic (dark gray bar) and osteogenic differentiation (light gray bar) after three passages 1,25D3 pretreatment in comparison to untreated cells. For each differentiation lineage the Fold Change was calculated by comparing the induction of differentiation of 1,25D3 pretreated cells with control cells. The results are shown as means of three independent experiments+SEM using three different preparations of hMSC. Each time 5 to 9 pictures of each differentiation experiment were analyzed using the AutMess tool of the software of AxioVision Rel. 4.6.</p

Effects of 1,25D3 treatment on quiescence- and senescence-associated genes.

<p><b>A</b> Expression of quiescence markers and densitometric quantification of semiquantitative RT-PCR results of hMSC (P3) treated with or without 1,25D3. Cells from five different donors were used. For each donor the Fold Change was calculated by comparing the expression of 1,25D3 treated cells with control cells. The results are shown as mean+SEM. The gene expression levels of <i>FOXO1a</i>, <i>FOXO3a</i>, <i>FOXO4</i>, <i>NANOG</i>, <i>TxNIP</i>, <i>TP53</i> were induced by 1,25D3 treatment over 3 passages. (FOXO1a: forkhead box 1a, FOXO3a: forkhead box 3a, FOXO4: forkhead box 4, NANOG: Nanog homeobox, TxNIP: thioredoxin interacting protein, TP53: tumor protein p53). <b>B</b> Expression of senescence markers and densitometric quantification of semiquantitative RT-PCR results of hMSC treated with or without 1,25D3 over at least four passages. 1,25D3 treatment reduced <i>P16</i> expression (p<0.05) and induced <i>P15</i> expression in cells at P4 compared to control cells. <i>P27</i> and <i>P21</i> gene expression in P4 showed no (<i>P27</i>) or only very weak (<i>P21)</i> induction by 1,25D3 treatment. Densitometric quantification of the semi-quantitative PCR results revealed downregulation of <i>PSG1</i> and almost no change in the expression of <i>PSG5</i> at P4 in 1,25D3 stimulated hMSC. The Relative Fold change represents the factor of different gene expression levels in 1,25D3 treated hMSC versus control cells. Expression profile of senescence-associated genes was determined in P4. Cells from three different donors were used. For each donor the Fold Change was calculated by comparing the expression of 1,25D3 treated cells with control cells. The results are shown as mean+SEM. (*, p<0.05, student's t-test).</p

1,25D3 response in hMSC.

<p><b>A/B</b> 1,25D3 stimulation of hMSC resulted in a strong increase of <i>CYP24A1</i> expression in all three donors. Control cells showed no <i>CYP24A1</i> expression, therefore densitometric quantification was not possible (A). The induction of <i>OC</i> expression is considerable and significant (**, p<0.01, student's t-test), the <i>OPN</i> expression is less intense. Densitometric quantification of <i>OC</i> and <i>OPN</i> expression was performed. For each donor the Fold Change was calculated by comparing the expression of 1,25D3 treated cells with control cells. The results were obtained from three independent donors and are shown as mean+SEM (B). <b>C</b> Immunostaining of VDR in hMSC after stimulation with 1,25D3 for 24 h. Stimulated cells show an increased localization of VDR in the nucleus, whereas the staining of the control cells is more diffuse (upper panel). Counterstaining with DAPI displays the localization of the nucleus (lower panel). One representative experiment is shown. Experiments were performed three times.</p

1,25D3 effects on proliferation, apoptosis and cumulative population doublings.

<p><b>A/B</b> Measurement of proliferation capacity and apoptosis induction of hMSC from three different donors after stimulation with 1,25D3 for 24, 48 and 72 h. The proliferation rate was decreased time-dependently after 1,25D3 treatment (gray bars) compared to control cells (black bars), with significant effects after 48 h and 72 h (A). 1,25D3 stimulation showed the strongest reduction of apoptosis rates after 72 h 1,25D3 treatment (gray bars) compared to control cells (black bars, B). The results are shown as mean+SEM of three independent experiments, each normalized to its control and performed in triplicates. Cells from three different donors were used. (*, p<0.05; **, p<0.01;***, p<0.001, student's t-test). <b>C</b> The growth rates of cells were determined by population doublings at each subcultivation. Cumulative population doubling (CPD) was first determined for P2. Population doublings were observed for up to six cell passages. Persistent 1,25D3 supplementation resulted in a reduction of population doublings (gray bar), but the difference was not statistically significant. Independent experiments were performed using hMSC from several human donors. Control: P1, P2, n = 6; P3, P4, n = 5; P5, n = 3; P6, n = 2. 1,25D3: P1, P2, n = 6; P3, P4, n = 4; P5, P6, n = 1. <b>D</b> A representative example of CPDs of cells from one donor. 1,25D3 cultured cells exhibited lower CPDs compared to control hMSC (**, p<0.01, student's t-test). <b>E</b> The time required to reach subconfluence was determined. 1,25D3 treated cells (gray bars) needed more days until they reached subconfluence compared to control hMSC (black bars) (**, p<0.01, student's t-test). Independent experiments were performed using hMSC from several human donors. Control: P1, P2, P3, P4, n = 6; P5, n = 5; P6, n = 3. 1,25D3: P1, P2, P3, n = 6; P4, n = 5; P5, n = 4; P6, n = 1.</p