Serum Free Cultured Bone Marrow Mesenchymal Stem Cells as a Platform to Characterize the Effects of Specific Molecules (original) (raw)
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Journal of Applied Oral Science
Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective: Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods: ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA ® software. Results: RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions: In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.
Avicenna journal of medical biochemistry, 2022
Background Abdominal obesity occurs with an increase in both size and number of fat cells (1) and could therefore be the major risk factor for metabolic syndrome, heart diseases, stroke, and diabetes (2). Similar to bone marrow-derived mesenchymal stem cells (BM-MSCs), human adiposederived mesenchymal stem cells (hAD-MSCs) are the preexisting source of new fat cells, which play an important role in maintaining function and the mass of adult adipose tissue (3,4). These multipotent non-hematopoietic stem cells that are capable of differentiating into a variety of cell types could be found in many tissues such as adipose tissue. In addition, they can express cell surface markers such as CD105, CD73, and CD90 (5,6). To study the earliest regulation of adipocyte differentiation, in vitro differentiation of MSCs can be used as a model of adipogenesis (7). During this process, from pre-adipocytes to adipocytes, gene promoter activities are necessary, which are promoted by two important transcription factors including CCAAT/enhancer-binding protein α (C/EBPα) and nuclear receptor peroxisome proliferator-activated receptor γ 2 (PPARγ 2) (8). PPARγ 2 activation modulates the activity of AMPK and glucose uptake through glucose transferase-4 (GLUT4) and stimulates adipogenesis (9).
Experimental Cell Research, 2008
Human Mesenchymal Stem Cells (hMSC), derived mainly from adult bone marrow, are valuable models for the study of processes involved in stem cell self-renewal and differentiation. As the Extracellular signal-Regulated Kinase (ERK) signalling pathway is a major contributor to cellular growth, differentiation and survival, we have studied the functions of this kinase in hMSC activity. Ablation of ERK2 gene expression (but not ERK1) by RNA interference significantly reduced proliferation of hMSC. This reduction was due to a defect in Cyclin D1 expression and subsequent arrest in the G0/G1 phase of the cell cycle.
Biochemical Effects of Retinoid Derivatives on Mesenchymal Stem Cells In Vitro
The studies we performed targeted the effects of all-trans retinol (vitamin A) and some retinoid derivatives (including tretinoin or all-trans retinoic acid, retinyl propionate, 9-cis retinoic acid, 13-cis retinoic acid), as well as of tazarotenic acid on apoptosis of rat mesenchymal stem cells, cultured after isolation. Tazarotenic acid is considered to be relatively selective and a potent agonist for RARβ and RARγ and less for RARα. The same time, tazarotenic acid is not binding to RXRs (retinoid X receptors). The relevant analysis of our experimental results demonstrated that 13-cis retinoic acid was the most potent inducer of apoptosis of cultured mesenchymal stem cells of rat origin when compared to other retinoid derivatives, as follows: 13cis retinoic acid > 9-cis retinoic acid > tazarotenic acid > all-trans retinoic acid > retinyl propionate > retinol (or vitamin A). Very interesting and unexpected were the apoptotic effects of 1 µM tazarotenic acid for 24 hours in our experiments, very close to those induced by all-trans retinoic acid (tretinoin). The apoptosis induced by 13-cis retinoic acid, a principal activator of RARβ and RARγ, and that induced by 9-cis retinoic acid, a major activator of RXRs, suggests different pathways activated by these retinoid derivatives.
Osteogenic Effects of Pigment Epithelium Derived Factor on Mesenchymal Stem Cells
2015
The field of bone tissue engineering has expanded in the recent decade to meet the increasing need to replace bone tissue in skeletal disease, congenital malformation, trauma, and tumours. Stem cell encapsulation has become a promising method in the future of this field. Alginate is a natural polymer that has been used widely for stem cell transplantation due to its biocompatibility. Pigment epithelium-derived factor (PEDF) is known for its anti-cancer properties due to its anti-angiogenic and anti-proliferative properties, particularly against osteosarcoma, a type of primary bone cancer. This study investigated the osteogenic effect of PEDF on mesenchymal stem cells (MSCs) in monolayer cell cultures and encapsulated in alginate beads in vitro and in vivo. Stem cells were isolated from the bone marrow of mouse long bones, and PEDF was used as an osteogenic supplement to differentiate MSCs to osteoblasts in both monolayers and in alginate beads (3D structure). Differentiation to oste...
Journal of Cellular Biochemistry, 1994
The effects of retinoic acid (RA) on the expression of osteoblastic-related cell markers was examined. A marrow stromal osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen a2 (I), osteonectin, osteopontin, biglycan, and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24 hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CDI O/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CDIO/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CDI O/NEP activity at the beginning of the culture period, reaching basal level with time.
Phytomedicine, 2007
In the present study, we investigated the in vitro effect of resveratrol (RSVL), a polyphenolic phytoestrogen, on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10 À8 -10 À5 M) increased cell growth dose-dependently, as measured by [ 3 H]-thymidine incorporation, and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity, calcium deposition into the extracellular matrix, and the expression of osteoblastic markers such as RUNX2/CBFA1, Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10 À6 M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17b-estrodial (10 À8 M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor, PD98059, significantly attenuated RSVL-induced ERK1/2 phosphorylation, consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast, SB203580, a p38 MAPK pathway blocker, blocked RSVL-induced p38 phosphorylation, but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation.
Journal of Biological …, 2000
Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogenactivated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor ␥2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively. Human bone marrow-derived mesenchymal stem cells (hM-SCs) 1 are multipotent, capable of differentiating into at least three lineages (osteogenic, chondrogenic, and adipogenic) when
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2018
Retinoic acid receptor (RAR) signaling regulates bone structure and hematopoiesis through intrinsic and extrinsic mechanisms. This study aimed to establish how early in the osteoblast lineage loss of Rarg disrupts the bone marrow microenvironment. Bone structure was analyzed by microCT in Rarg mice and mice with Rarg conditional deletion in Osterix-Cre-targeted osteoblast progenitors or Prrx1-Cre-targeted mesenchymal stem cells. Rarg tibiae exhibited less trabecular and cortical bone and impaired longitudinal and radial growth. The trabecular bone and longitudinal, but not radial, growth defects were recapitulated in Prrx1:Rarg mice but not Osx1:Rarg mice. While both male and female Prrx1:Rarg mice had low trabecular bone mass, males exhibited increased numbers of trabecular osteoclasts and Prrx1:Rarg females had impaired mineral deposition. Both male and female Prrx1:Rarg growth plates were narrower than controls and their epiphyses contained hypertrophic chondrocyte islands. Flow ...