Table 3. Functional clustering of differentially expressed genes of hMSC-OP, hMSC-old and hMSC-senescent when compared to hMSC-C.parathyroid hormone 1 receptor integrin-binding sialoprotein inhibin, alpha insulin-like growth factor binding protein 2 insulin-like growth factor 2 vascular endothelial growth factor B vascular endothelial growth factor A forkhead box C2 (MFH-1, mesenchyme forkhead 1) collagen, type I, alpha 1 runt-related transcription factor 2 ankylosis, progressive homolog SMAD family member 3 secreted phosphoprotein 1 ephrin-B2 alkaline phosphatase, liver/bone/kidney cytochrome P450, family 2, subfamily R, polypeptide 1 forkhead box C1 interleukin 6 signal transducer (Oncostatin M receptor) platelet-derived growth factor alpha polypeptide vitamin D receptor fibroblast growth factor receptor 2 bone morphogenetic protein 6 receptor tyrosine kinase-like orphan receptor 1 ankyrin repeat domain 6transforming growth factor, beta 1 mab-21-like 2 follistatin follistatin-like 3 kringle containing transmembrane protein 1 sclerostin fibroblast growth factor receptor 1 insulin-like growth factor binding protein 5 insulin-like growth factor binding protein 4 epidermal growth factor receptor gremlin 2, cysteine knot superfamily, homolog noggin catenin, beta 1 secreted frizzled-related protein 4 wingless-type MMTV integration site family, member 2 wingless-type MMTV integration site family, member 3Gene name insulin-like growth factor 2 tumor necrosis factor superfamily, member 11 secreted phosphoprotein 1 interleukin 7 thrombospondin 1 interleukin 1, alpha tumor necrosis factor superfamily, member 10 transforming growth factor, beta 2 vascular endothelial growth factor A vascular endothelial growth factor B transforming growth factor, beta 1 runt-related transcription factor 2hyaluronan-mediated motility receptor helicase, lymphoid-specific pleiotrophin superoxide dismutase 2, mitochondrial cyclin B2 cell division cycle 2, G1 to S and G2 to M cyclin A2 cyclin E2 cyclin F cyclin D1 cyclin D2 cell division cycle 25 homolog A cell division cycle 25 homolog B cell division cycle 25 homolog C cyclin-dependent kinase 2pregnancy specific beta-1-glycoprotein 1 pregnancy specific beta-1-glycoprotein 2 pregnancy specific beta-1-glycoprotein 3 pregnancy specific beta-1-glycoprotein 4 pregnancy specific beta-1-glycoprotein 6 pregnancy specific beta-1-glycoprotein 7 Rho GTPase activating protein 29 cyclin-dependent kinase inhibitor 2A cyclin-dependent kinase inhibitor 1Apolymerase (DNA directed), delta 1, catalytic subunit 125kDa polymerase (DNA directed), epsilon 2 (p59 subunit) polymerase (DNA directed), theta polymerase (DNA directed), eta polymerase (DNA directed) kappa Gene name MRE11 meiotic recombination 11 homolog A poly (ADP-ribose) polymerase family, member 3 RAD50 homolog RAD51 homolog RAD51 associated protein 1 topoisomerase (DNA) II alpha 170 kDa exonuclease 1 CHK1 checkpoint homolog high-mobility group box 2arrows pointing downward = at least 2fold reduced expression in comparison to hMSC-C; arrows pointing upward = at least 2fold enhanced expression in comparison to hMSC-C; W = gene associated with WNT signaling; B = gene associated with BMP signaling; * = probesets that refer to the gene are not identical in the indicated comparisons.
Furthermore, we detected indications for osteoporotic stem cells actively enhancing osteoclastogenesis and therefore bone resorption. Besides the enhanced expression of genes coding for osteoclast stimulating ligands, e.g. VEGF, TGFB and CSF1 [4,46,47], we also detected the osteoporosis-induced expression of Parathyroid hormone receptor PTH1R. Activation of PTH1R triggers osteoblast maturation and induces RANKL expression which leads to osteoclast precursor differentiation and activation [26]. The enhanced expression of osteoclastogenesis promoting factors has already been described in fragility fractured bone [48] and is in general consistent with the enhanced bone resorption described for osteoporosis [2]. Because high age is one of the main risk factors for developing osteoporosis, we tried to dissect effects of aging from effects of primary osteoporosis by using hMSC from middle-aged donors as control cells (hMSC-C) for comparisons with hMSC-OP and hMSC-old, respectively, of elderly individuals (Figure 1C and 1D, Table S2). Surprisingly, the patterns of the differential gene expression in aged and osteoporotic hMSC differed widely. Only a few gene products with identical expression profiles in hMSC-old and hMSC-OP were observed and we therefore conclude that osteoporosis-associated changes are very distinct and independent of effects of clock-driven aging. We hypothesize that donors of advanced age who suffered from osteoarthritis but not from osteoporosis, aged in a healthier way than osteoporotic patients, or vice versa that osteoporosis is a distinct syndrome of premature aging. One hypothetical reason for aging is the loss of tissue regeneration due to replicative senescence of stem cells, which accumulates over time and ends in organ failure and death of the organism [33]. Due to the fact that donors of hMSC-OP were of advanced age we analyzed whether these cells exhibited signs of replicative senescence by comparing them to the gene expression pattern of long term-cultivated, senescent hMSC. Thereby we detected a small overlap of genes differentially expressed in hMSC-OP and hMSC-senescent when compared to the identical control group hMSC-C (Figure 1C and D). Despite the distinct gene expression pattern, we found some markers for replicative senescence in osteoporotic hMSC-OP, like the reduced expression of Hyaluronan receptor HMMR, which was described as inversely regulated to tumor suppressor P53 [49], and the induction of CDKN1A, which codes for P21, another inhibitor of cyclindependent kinases (Table 3) [50]. In contrast, analyses of nonosteoporotic hMSC-old of the age-matched donor group revealed no expression of markers for senescence and highlighted even more the differences between aging with and without primary osteoporosis. Our findings suggest that osteoporotic stem cells exhibit deficiencies in proliferation and might already be prone to a pre-senescent state. So far, reduction in proliferative activity in osteoporotic cells has only been described for osteoblasts [51,52]. For confirmation, more detailed investigations of hMSC-OP on protein level and by proliferation or senescence studies are needed.
