Multi-potential Adult mesenchymal stem cells (MSCs) was induced to differentiate into osteoblast and adipocytes by addition of osteogenic and adipogenic differentiation media respectively. Endogenous biologically active Bone morphogenic proteins (BMPs) and its receptors regulate the lineage differentiation and commitment of MSCs. The potential role of BMPs in adipogenic and osteogenic differentiation is been investigated by the use of BMP inhibitor dorsomophin (1Âµl/well) which is capable of blocking the BMP type 1 receptor. The osteogenic differentiation is remarkably affected with reduced calcium mineralisation when culture is provided with inhibitor. The adipocytes were produced in the culture more or less with same consistency irrespective of the presence or absence of inhibitor. The dormorphin becomes toxic to MSCs undergoing osteogenic differentiation whereas it had less effect on adipogenic differentiation comparatively suggesting its role to be more critical for osteogenic differentiation.
Adult Mesenchymal stem cells (MSCs) are multipotent cells commonly found within adult bone marrow . Characteristics of this type of adult stem cell population have been described previously that they replicate as undifferentiated cells and underlies the potential to differentiate into lineages of mesenchymal tissues such as bone, cartilage, fat, tendon, muscle and marrow stroma . Differentiation of MSCs into neural lineage has also been reported . MSCs derived from bone marrow can be established as monolayer in vitro and can be differentiated into adipogenic and osteocgenic lineages. Therefore, the aim was to obtain these two lineages and to identify its characteristic by staining procedure. Usually within 14-28 days the process of apidogenesis and osteogenesis are completed in vitro .
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MSCs in vitro spontaneously start to differentiation into osteoblast. However, the effective differentiation is attained by addition of differentiation supplements in the culture. So far, effect of ascorbic acid phosphate , beta-Glycerophosphate  and dexamethasone  for osteogenic differentiation and effect of insulin , rosiglitazone , dexamethasone  and IBMX  on adipogenic differentiation have been previously discussed. The differentiation process in this experiment was compared with their controls where only MSC medium was provided.
Bone morphogenic protein (BMPs) which belongs to family of transforming growth factor (TGF-Î²) are to be involved in regulation of commitment and terminal differentiation of MSC-derived lineages . Biologically active BMP-2, BMP-4 and BMP-6 in addition to all BMP-activated receptors are involved in adipogenic and osteogenic differentiation in vitro and in vivo where these BMPs-induced osteoenginc or adipogenic lineage are mutually exclusive . Among this family of BMPs, BMP-2 is found to be essential for osteogenic differentiation where it is responsible for induction of alkaline phosphatase activity and dose-dependent manner for calcium mineralisation . On the other hand, some have reported the BMP-2 alone cannot initiate adipogenic differentiation unless supplemented with some other inducer . Hence, we investigated the effect of BMPs on these two lineages for their abilities to regulate multilineage specific differentiation of MSCs by addition of BMP inhibitor dorsomorphin (also known as compound C) which selectively inhibits the BMP type 1 receptors ALK2, ALK3 and ALK6 to block BMP-mediated phosphorylation .
MATERIALS AND METHODS
The MSC medium containing DMEM supplemented with 10%FCS, 1% L-glutamine, 1% NEAA and 1% antibiotics was prepared.
MSC was extracted from culture flask T25 (passage 33) by trypsin digestion method. 1ml of this solution (150Âµl cells +8ml MSC media) was seeded in each of 8-wells plate and kept incubated at 37â°C, 5%COâ‚‚ for 1 week.
The differentiation media was prepared both for osteogenic (OS) and adipogenic (AD) where 10ml MSC medium was supplemented with ascorbic acid phosphate (50ÂµM), beta-Glycerophosphate (10mM) and dexamethasone (0.1ÂµM) for OS medium whereas for AD medium 10ml MSC medium was supplemented with insulin (10Âµg/ml), rosiglitazone (1ÂµM), dexamethasone (1ÂµM) and IBMX (100ÂµM). The differentiation was initiated after 1 week by addition of 1ml /well differentiation media for their respective plate whereas MSC medium alone was added in control wells. 1Âµl/well of BMP inhibitor, dorsomorphin was added on one control and one test. On regular intervals fresh medium was added.
Fixing of AD plate was carried out after 2 weeks by addition of 1ml ice-cold PFA and subsequent resting for 15 mins followed by removal of PFA in separate tube and addition was 1ml PBS.
The staining of both the plates was carried out after 3 weeks. MSC-derived osteoblast was stained by 1% alizarin-red solution whereas MSC-derived adipocyte was stained with oil red O dye (procedure as given in Mod. 3 practical by V. Sottile).
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The adult MSCs were seeded on 12-well plate on day 1 and differentiation was initiated after 1 week which was observed for over 10 days with twice medium change after every week.
Fig1. Seeding of adult MSCs. The MSCs in figure are shown as monolayer in T25 flask at passage 33(Scale bar: 125Âµm)
The Morphology of adult MSCs appears like fibroblastic, elongated forming a dense network throughout the T25 flask. This cell appears healthy but is not completely homogeneous. There is presence of some hematopoietic cells which appears circular in shape. The Flask was approximately 70% confluent prior to seeding.
After 1 week, the MSCs medium was replaced with adipogenic and osteogenic differentiation medium except for control wells. After initiation of differentiation, the adipogenesis was carried out for 8 days whereas osteogenesis was carried out for 10 days and the cells were fixed on that respective day. Following the fixation, the cells were stained after 1 week.
Fig2. Medium change of osteogenic (A) and adipogenic differentiation (B) plates. The control wells contained only the MSC medium whereas adipogenic and osteogenic differentiation wells contained their respective differentiation media. (-) indicates the absence of the BMP inhibitor and (+) indicates the presence of BMP inhibitor (1Âµl/well). (Scale bar: 250Âµm).
The differentiation process is initiated can be clearly seen the above figure. The control wells show no differentiation whereas wells with BMP inhibitor shows dispersed cells and very little differentiation process.
Fig3. Osteoblast differentiation in absence (A) and presence of BMP inhibitor (B). The control wells contained only the MSC medium whereas osteogenic differentiation wells contained their osteogenic differentiation media. (-) indicates the absence of the BMP inhibitor and (+) indicates the presence of BMP inhibitor (1Âµl/well). The arrow indicates the mineralisation of osteoblast. (Scale bar: 250Âµm).
The figure shows Osteoblast differentiation after 1 week. This differentiation process was continued for 3 more days and then cells where fixed. When the MSCs where cultured with osteogenic differentiation factors i.e. dexamethosone, ascorbic acid and Î²-glycerophosphate, MSCs differentiation into osteogenic lineage producing bone-like nodules with a littler mineralization extracellular matrix containing hydroxyapatite took place. The formations of osteobalst cells are visible which are tightly packed and bluntly linear in shape. However, this mineralization process was much reduced or not occurring in presence of BMP inhibitor. Also, its control (+) shows apoptosis of MSCs as compared to control (-) where the MSCs are still intact and to some extent undifferentiated.
Fig4. Adipocyte differentiated cells in absence (A) and presence of BMP inhibitor (B). The control wells contained only the MSC medium whereas adipogenic differentiation wells contained adipogenic differentiation media. (-) indicates the absence of the BMP inhibitor and (+) indicates the presence of BMP inhibitor (1Âµl/well). The arrow indicates the lipid filled lobular structure. (Scale bar: 250Âµm).
The adipocyte differentiation after 1 week is shown in this figure. When MSCs were cultured with adipogenic supplements i.e. insulin, rosiglitazone, dexamethasone and IBMX it triggered adipocyte differentiation producing lipid filled lobules appearing circular. The control appears to be undifferentiated only till certain extent. The addition of BMP inhibitor also resulted adipocyte differentiation but it appears to be toxic to cells in control (+).
Fig5. Alzarin-Red Staining of MSCs-derived osteoblast in absence (A) and presence of BMP inhibitor (B). The figure shows staining of osteoblast within the wells along with whole plate picture. The control wells (C) contained only the MSC medium whereas osteogenic differentiation wells (OS) contained osteogenic differentiation media. (-) indicates the absence of the BMP inhibitor and (+) indicates the presence of BMP inhibitor (1Âµl/well). (Scale bar: 125Âµm).
After fixation of MSC-derived osteoblast cells, the cells were stained with Alzarin-red which resulted in the dark red colourisation of calcium deposits mineralisation in the extracellular matrix which was clearly visible. However, in BMP inhibitor plates, the staining of mineralised areas was comparatively less. In whole plate picture also, the difference in staining is clearly visibly and so the effect of BMP inhibitor on osteoblast differentiation.
Fig6. Oil Red O Staining of MSCs-derived mature adipocytes in absence (A) and presence of BMP inhibitor (B). The figure shows staining of adipocytes within the wells along with whole plate picture. The control wells (C) contained only the MSC medium whereas adipogenic differentiation wells (AD) contained adipogenic differentiation media. (-) indicates the absence of the BMP inhibitor and (+) indicates the presence of BMP inhibitor (1Âµl/well). (Scale bar: 125Âµm).
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After fixation of MSC-derived mature adipocyte cells, the cells were stained with Oil-Red O dye which resulted in the faint red colourisation of lipid lobules formed was clearly visible irrespective of presence or absence of BMP inhibitor in plates. The staining of adipocytes in presence and absence of BMP inhibitor was comparatively more or less the same. In whole plate picture also, the difference in staining is not obvious.
Multi-potent Adult MSCs can be induced to differentiate into adipogenic and osteogenic lineages in vitro . In developing mammal, there exists balance between the formation of osteogenic lineage and adipogenic lineages in bone marrow under normal condition . However, this balance is disturbed in conditions like osteoporosis and osteopenia . This shows that there underlie regulation of several signalling mechanism which are responsible for formation these two lineage eventually from the same cell type. However, it is difficult to recapitulate the same in vitro.
The process of osteoegenesis usually takes approximately 21-28 days whereas the process of adipogenesis take places approximately 14 days in vitro . The differentiation protocol in this experiment was employed only for 7 days and 10 days for adipocyte and osteoblast differentiation respectively. Therefore, it cannot be said that they formed MSCs-derived fully mature adipocytes or osteoblast with confirmation. However, most literatures have shown that the differentiation process becomes prominent over a week [4,17,18].
The differentiated tightly packed linear shaped osteoblast with prominent calcium mineralisation on the matrix stained with alizarin red becomes visible (Fig3. & Fig5.) when cultured with osteogenic differentiation media including dexamethasone, ascorbic acid phosphate and Î²-glycerophosphate. Also, the MSCs-derived adipocytes characterised by the presence of lipid filled circular lobules stained with oil-red O dye is obtained (Fig4. & Fig6.) when cultured with adipogenic differentiation media including insulin, dexamethasone. Rosiglitazone and IBMX. Thus, osteogenic and adipogenic differentiation was successfully derived from MSCs when there was availability of endogenous BMPs.
The morphology of the MSCs when first seeded (Fig1.) changes throughout in the control wells (Fig2, Fig3.A (i) & Fig4.A (i)) as the differentiation progresses. Ideally the MSCs should have remained intact and of same morphology in the controls wells but they lose their undifferentiated state to some extent. Hence, as the days goes by the MSCs looses it self-renewal capacity. Also, MSCs in vitro spontaneously differentiate into the osteogenic lineage if kept unattended even without availability of osteogenic supplements . Hence, the some osteogenic type and less adipogenic type cell morphology are visible in the control wells with absence of inhibitor.
BMPs have been known for varied effects on the fate of MSCs which depend on the concentration and type of BMP, the type of precursor cells and the presence of regulators . The MSCs weren't seeded with known seeding density. Hence, this experiment cannot provide any quantitative justification. Among the BMPs, BMP-2 has been reported to be necessary for osteogenic differentiation whereas its role in adipogenesis is contradictory. It was shown to be strong inducer in murine 3T3-L1 preadipocytes only if given with thiazolidinedione . Also, low BMP-2 concentration favours adipogenises of multipotent C3H10T1/2 cells whereas favours osteogenises at high concentration . Some explained that BMP-2 has prominent effect only in the early stages of adipogensis using 3T3-F442A pre-adipocytes [15, 20]. Therefore, its effect on adipogenesis is not well understood but it can be deduce from literature that BMP-2 alone has very little effect on adipogenesis. Hence, we observed its effect on differentiation of MSCs into osteoblast and adipocyte by blocking the endogenous BMP with the help of dormorphin (1Âµl/well).
The osteogenic differentiation was drastically reduced with very little calcium mineralisation when the BMP receptors are blocked by the dorsomorphin (Fig5B.) Park, K.W. et al.  also demonstrated osteogenesis gene expression and alkaline phosphatase (ALP) activity significantly reduced by blocking BMP by dorsomorphin. Spontaneous mineralisation was also aborted. Apart from unable to differentiate into osteogenic lineage, the control MSCs was undergoing cell death (Fig5. and Fig6.). Inhibiting the access to endogeous BMPs to the cells led to toxic condition for MSCs. Nam, M. et al.  reported compound C significantly inhibited adipogenic differentiation of 3T3-L1 cells in intial stage of differentiation does-dependently. However, in our experiment reduced adipocyte differentiation was obtained by subsequent treatment with BMP-inhibitor (Fig6B.) but its effect was not as remarkable as it was on osteogenic differentiation. Since, BMP is known to play less important role in adipogenesis, its subsequent inhibition by dorsomorphin also didn't result in significant change.
Hence, it can be said that especially BMP is more critically involved in osteogenic differentiation rather than adipogenesis as BMP inhibition resulted in significant reduced osteogenesis throughout. More, studies need to be done to investigate exact role of endogenouse BMP in adipogensis. The analysis of gene expression and the test for ALP activity could give more semi-quantitative information valid for comparison.