Effects Of WNT Signaling Pathway On The HMSCs Related Type Ⅱ Osteoporosis

PartⅠThe in Vitro Culture and Identification of Human Bone Marrow Stromal Cells【Objective】To establish the culturing system of hMSCs according to the former established protocols of our lab,and provide cell source for following experiments.【Methods】Bone marrow samples were obtained with IRB approval from femoral tissue discarded during hip replacement surgery.Low-density mononuclear cells were isolated by density centrifugation on Ficoll/Histopaque 1077.Adherent hMSCs were isolated,hMSCs were cuLtured and passaged inα-MEM supplemented with 10%FBS.The cells were induced with adipocytogenic,osteogenic and chondrocyogenic medium separately.The analysis of ALP activity and Alizarin Red S staining were conducted for identification of osteogenesis,Oil Red-O for adipocytogenesis,and Alcian blue for chondrocytogenesis.【ResuLts】The primary cells attached after 24 hours cuLture.They exhibited the shape like spindle or shuttle,the clones of hMSCs formed after 48-72 hours cuLture.The cells proliferated rapidly after 5-7 days cuLture.The cells were almost confluence after 12-14 days cuLture with the appearance like fibroblasts. Some cells overlapped with others.The cells could be passaged after cuLturing for 5-10 days,and they can be passaged for 4-10 times.The ALP activity and Alizarin Red S positive area can be detected after the cells were cuLtured in osteogenic medium.The cells couLd differentiate to Oil Red-O positive cells after adipocytogenic cuLture.There were cells which were positive for Alcian blue after chondrocytogenic induction.【Conclusion】hMSCs can be isolated from bone marrow,and can be expanded after being cuLtured.The cells can differentiate into osteoblasts,chondrocytes, and adipocytes. PartⅡEffects of Age and Gender on WNT Gene Expression in Human Bone Marrow Stromal Cells【Objective】WNT signaling pathways play important roles in the behavior of human bone marrow stromal calls.Although WNT expression has been examined in human bone marrow stromal cells(hMSCs) with limited numbers of subjects or from commercial sources,there are conflicting resuLts on WNT gene expression in hMSCs.Furthermore,the effects of age and gender on WNT expression in hMSCs are largely unknown.【Methods】Bone marrow samples were obtained with IRB approval from femoral tissue discarded during hip replacement surgery.A total of 19 subjects,12 women and 7 men,age ranging from 36 years old to 85 years old,were included in this study. Of them,9 subjects were classified as young(≤50-years,mean 42.0±4.6 years) and 10 were classified as old(≥55-years,mean 69.3±9.5 years).There were 12 samples from women(37 to 85-years,mean 58.6±16.0 years) and 7 from men (36 to 82-years,mean 52.6±16.0 years).hMSCs were cuLtured in 100mm tissue cuLture dishes and Passage 2 or 4 cells were used.In each experiment,standardized conditions were used for all samples;cells were harvested and stored in Trizol reagent at -80℃for analysis at the same time to avoid technical differences between assays. WNT gene expression levels were measured by semi-quantitative RT-PCR.【ResuLts】Twelve of the 19 WNT genes,WNT2,3,4,5A,5B,6,7B,10B,11,13, 14,and16B were expressed in hMSC.WNT7B and 14 were expressed significantly higher in the younggroup.WNT2 and WNT13 showed a trend of higher expression in young group.WNT7B,13,and 14 were inversely correlated with age.Further analysis for gender-specific difference indicated that WNT16 was expressed significantly higher in men than in women.WNT11 showed a trend of higher expression in hMSCs from women.For the hMSCs from women,WNT13 was inversely correlated with age and WNT4 was positively correlated with age.For the hMSCs from men,WNT7B and WNT14 were inversely correlated with age.【Conclusion】These data indicated that most of the age-related WNT genes belong to the canonical WNT signaling pathway.Further,there are gender-specific differences in the expression of WNT4,7B,13,14,and 16 in hMSCs.Age and gender account for many of the sample-to-sample variations in WNT gene expression in human marrow stromal cells PartⅢEffects of WNT Signaling on Adipocytogenesis,osteogenesis,and chondrogenesis in Human Bone Marrow Stromal Cells【Objective】From the body of information available about mechanisms of adipocyte differentiation with murine preadipocytes,it is known that 1) Wnt10b inhibits adipogenesis and stimuLates osteoblastogenesis,2) Wnt4 and Wnt5a stimuLate adipocytogenesis and 3) Wnt3a can enhance the routine C3H10T1/2 cell differentiation into chondrocytes induced by bmp2.Because little is known about WNTs and human adipocytogenesis,osteogenesis,and chondrogenesis,we tested the hypothesis that WNT signaling reguLates adipocytogenesis,osteogenesis,and chondrogenesis of human bone marrow stromal cells(hMSCs);we assessed canonical and non-canonical WNT and effects of a small molecuLe stimuLator ofβ-catenin (SB-216763) during adipocytogenesis,osteogenesis,and chondrogenesis.【Methods】Bone marrow samples were obtained with IRB approval as discarded femoral tissue from Orthopedic department,Brigham and Women's Hospital.Low-density mononuclear cells were isolated by density centrifugation on Ficoll/Histopaque 1077.Upon near-confluence of hMSCs,medium was changed toα-MEM,with adipocytogenic,osteogenic,or chondrogenic supplements.The effect of adipocytogenic medium on the expression(RT-PCR) of adipocyte maker genes and WNT genes was performed at intervals to 10 days.The effects of SB-216763 on expression ofβ-catenin were tested by Western Immunoblotting.Eighteen days after treatment,adipocytes treated with and without SB-216763 were counted in cells positive for staining with 0.3%Oil Red-O.The effects of WNT signaling on osteogenesis and chondrogenesis of hMSCs were assayed by RT-PCR,ALP activity analysis,and Alcian blue staining.【ResuLts】Analysis of adipocyte marker genes indicated that the expression level of PPARγ2 and LPL was detectable after 1 day in adipocytogenic medium and increased thereafter.The expression of Adipsin also increased with time.Upon adipocytogenic differentiation of hMSCs(day 1),the expression of canonical WNT genes(2,10B,13,and 14) decreased,whereas non-canonical WNT genes (4 and 11),but not WNT5A increased.WNT11 was the only one to change prior to upreguLation of adipocyte signature genes.SB-216763(5μM),which increasedβ-catenin levels inhibited adipocytogenesis by blocking induction of PPARγ2,LPL,and Adipsin.Consistent with the molecuLar effects,SB-216763 inhibited generation of Oil Red-O adipocytes with duration- and dosage-dependence. Activation of canonical WNT signaling pathway in hMSCs by SB-216763 inhibited the expression of osteoblasts mark gene ALP,further confirmed by the ALP activity analysis.The chondrocytes mark gene,RUNX2 and COLⅡexpression were enhanced,accompanied by the collagenⅡproductivity in vitro confirmed by Alcian blue staining.【Conclusion】These study indicate that for human adipocytogenesis,non-canonical WNT11 and 4 may be major enhancers and that activation of canonical WNT signaling pathway prevents hMSCs from differentiating into adipocytes and osteoblasts,and stimuLates hMSCs differentiation into chondrocytes.Thus,there may be fundamental species differences in WNT signaling during adipocytogenesis and osteogenesis.PartⅣEffects of Age and Gender on Adipocyte Differentiation of Human Bone Marrow Stromal Cells【Objective】Our previous studies indicated an effect of age on many properties of hMSCs,including the decreased ability to differentiation into osteoblasts.In order to further identify the mechanism of the enhanced volume of fat tissue in the bone marrow of typeⅡosteoporosis patients,we surveyed the effects of age and gender on the adipocytogenesis of hMSCs,which were from different ages, both women and men.【Methods】Bone marrow samples were obtained with IRB approval as discarded femoral tissue from Orthopedic department,Brigham and Women's Hospital.A total of 15 subjects,8 women and 7 men,age ranging from 17 years old to 90 years old, were included in this study.Of them,5 subjects were classified as young and 10 were classified as old.The Oil Red-O positive cells were counted at after the hMSCs were cultured in adipocytogenic medium for 18 days.For the adipocyto marker gene and osteoblast marker gene expression experiment,a total of 19 subjects,12 women and 7 men,age ranging from 36 years old to 85 years old,were included in this study.Of them,9 subjects were classified as young and 10 were classified as old.There were 12 samples from women and 7 from men.RT-PCR was conducted to detect the gene expression.【ResuLts】Under the adipocytogenic induction,the number of adipocytes from old hMSCs was 2.35 folder as the ones from young group,but it was not statistically significant.The correlation analysis of the number of adipocytes form different subjects with age showed there was no significant correlation between them (Spearman,r=0.01899,P=0.95).There is no significant difference of the adipocytes number induced from hMSCs between women and men.The adipocyte marker genes,PPARγ2,LPL,and Adipsin were expressed higher in the hMSCs from old group than the young group,but there is no significant difference.For the osteoblast gene expression,the expression of RUNX2 was lower;OC and ALP were higher in hMSCs from old group as compared to young group.LPL expression showed a trend of positive correlation with age,while PPARγ2,Adipsin,RUNX2,OC, and ALP did not show significant correlations with age.【Conclusion】The in vitro adipocytogenic differentiation ability of hMSCs is not related to age and gender,there is difference of the adipocytogenic ability of hMSCs between in vivo and in vitro. Reflections and ConclusionsWe draw the following the theoretical considerations based on our resuLts:1.The cell isolation and cuLture system of our lab is stable and reliable,sufficient cells can be acquired for the following experiments.2.TweIve of 19 known WNT genes expressed in hMSCs,and there are differences of gene expression between young and old subjects,and between both genders.3.The non-canonical WNTs,WNT11 and WNT4 may be the enhancers of hMSCs differentiation into adipocytes.Activation of WNT/β-catenin signaling pathway can inhibit the adipocytogenic and osteogenic differentiation and enhance chondrogenic differentiation of hMSCs.The novelty of this study is threefold:1.These studies show variances in constitutive expression of many WNT genes in 19 samples of freshly isolated human bone marrow stromal cells from young and old subjects,both women and men.Further,they indicate effects of age and gender on expression of some WNT genes.2.Tested the effects of WNT signaling pathway during adipocytogenesis, osteogenesis,and chondrocytogenesis of hMSCs,and found there may be fundamental species differences in WNT signaling during adipocytogenesis and osteogenesis.3.Assessed the effects of age and gender on the adipocytogenic differentiation of hMSCs from young and old subjects both in women and men.