Comparison of Differences in Cell Migration during the Osteogenic and Adipogenic Differentiation of the Bone Marrow-Derived Stem Cells
Article information
Abstract
Background
The bone marrow niche comprises diverse cellular populations, including multipotent bone marrow-derived stem cells (BMSCs). Understanding the biology underlying the differentiation of BMSCs into osteogenic and adipogenic commitment in preserving bone health is key due to their inverse correlation. Biological processes such as cellular migration also serve as a crucial player during this differentiation and eventually contribute to various skeletal pathologies such as fractures, osteoporosis, and osteoarthritis. This is also crucial in developing various regenerative therapies involving BMSCs.
Methods
To explore the differential migration of BMSCs, cells were initially directed into osteogenic or adipogenic commitment as confirmed by the mineralized matrix and lipid droplet formation for osteogenic and adipogenic commitment, respectively. The differential level of cellular migration was then assessed using the scratch wound healing assay, cell adhesion assay, and transwell migration assay.
Results
The cellular differentiation was confirmed by the differential expression patterns of key markers, as determined by quantitative real-time reverse transcription-polymerase chain reaction and immunoblotting study. Moreover, the migration data indicates that BMSCs undergoing osteogenic commitment tend to migrate more compared to adipogenic cells, which is possibly attributed to the differential expression of integrins such as Itgα1, and Itgα5. The putative role of the Sdf1/Cxcr4 axis in this account was further established by utilizing a selective inhibitor of Cxcr4.
Conclusions
This study sheds light on the differential migratory property of the BMSCs directed towards a specific lineage. It also highlights the need for a comprehensive understanding of the intricate biological interplay governing this peculiar cellular behaviour.
INTRODUCTION
The bone is an intricately functional and metabolically active endocrine organ, [1,2] plays a crucial role in calcium homeostasis, and hematopoiesis, and provides structural support and protection of vital organs.[3] The highly heterogeneous bone marrow cavity consists of multipotent hematopoietic and musculoskeletal progenitor cells, as well as their terminally differentiated descendants.[4] Bone marrow-derived mesenchymal stem cells (BMSCs) following osteogenic and adipogenic lineages have gained significant attention ever since because of their mutual involvement in various bone metabolic disorders.[5–7] With aging and in pathological conditions such as osteoporosis progress, BMSCs tend to differentiate more toward adipogenic lineages and less toward osteogenic lineages. [8,9] This results in an accelerated decline in bone formation, the occurrence of bone marrow adiposity, and increased fracture risk.
Differentiation of BMSCs occurs in two stages: lineage commitment, in which BMSCs commit to become lineage-specific progenitors, and maturation, where progenitors differentiate into specific cell types.[6] The regulation of osteogenesis and adipogenesis is believed to be influenced by specific signaling pathways, cytokines, and transcription factors, including Runt-related transcription factor 2 (Runx2) and peroxisome proliferator-activated receptor γ (PPAR-γ). [10,11] Moreover, research has identified other crucial signaling pathways, such as transforming growth factor-β/bone morphogenic protein (BMP) signaling, [12] Wnt signaling, [13] Hedgehogs, [14,15] Notch, [16] and fibroblast growth factors (FGFs), [17,18] are also involved in controlling the lineage commitment of BMSCs, highlighting cell proliferation and differentiation.
Interestingly, the osteogenic and adipogenic cells derived from BMSCs showed a significant degree of versatility, for instance, osteoblastic cells can develop into adipocytes, [19] while bone marrow adipocytes are also known to differentiate into osteogenic cells.[20,21] The orchestrated regulations of both cell types have demonstrated highly significant implications in the aetiology of numerous bone metabolic disorders including osteoporosis. Furthermore, besides cellular differentiation, other fundamental cellular processes such as cellular migration of BMSCs are also known to play a crucial role in the pathophysiology of such diseases. Therefore, delivering a comprehensive perspective of this cellular process is essential in understanding the intricate biology governing this fundamental process and knowing other potential variables associated with the pathogenesis.
The current communication explores the intricate biology underlying the modulated cellular migration of BMSCs directed towards osteogenic or adipogenic commitments. The study investigates the molecular mechanism underlying this fundamental cellular behavior. For the study, BMSCs isolated from rats were utilized. Isolated cells were then differentiated into osteogenic and adipogenic lineages by culturing cells in the specific differentiating media. To confirm the respective lineage commitments, expression patterns of certain molecular markers involved in the differentiation process were examined at both mRNA as well as in protein levels. Furthermore, cellular proliferation and estimation of mineralized nodule formation and lipid droplet formation upon osteogenic and adipogenic commitments respectively provide new insights into the cellular, biochemical, and molecular identity of osteoblasts and adipocytes derived from mesenchymal stem cells. Moreover, the study also identified the differential migratory profile of differentiated bone marrow derived from mesenchymal stem cells. Molecular investigations further identified the potential role of the Cxcr/Sdf-1 axis during the enhanced migratory potential of the cells directed to osteogenic commitments compared to the adipogenic one. The finding provides an understanding that helps to determine the pathogenic origins of various disorders connected to fat and bone marrow, develop innovative treatments, and enhance the therapeutic use of BMSCs in tissue engineering and regenerative medicine.
METHODS
1. Isolation and culture of BMSCs
BMSCs were isolated from the bone marrow of 3 to 4-week-old female SD rats. All the experimental procedures were performed in accordance with approval obtained from the Institutional Animal Ethics Committee (IAEC; IAEC/2022/56). The animals were housed at a controlled temperature (22–24°C), suitable light (300 Lux) at floor level, and humidity (50%–60%), and maintained a 12/12 hr light/dark cycle as provided in the animal house facility of CSIR-Central Drug Research Institute (CSIR-CDRI), India. All animal experiments were conducted in accordance with the regulations of the Council for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Social Justice and Empowerment, Government of India.
For the isolation of the BMSCs, briefly, femur and tibia were collected aseptically. Bone marrow was then flushed out from both ends of the long bones into culture medium. The flushed marrow was incubated with red blood cell lysis buffer containing 0.15 M ammonium chloride (NH4Cl), 10 mM potassium bicarbonate (KHCO3), and 0.1 mM ethylenediaminetetraacetic acid (EDTA) for 10 min at room temperature. After centrifugation, the collected cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), glutamax (2 mg/mL), and 1% penicillin-streptomycin at 37°C in a humidified chamber with 5% CO2 and 95% air. All biochemical reagents used for cell culture were obtained from Sigma-Aldrich unless otherwise specified. Cells were passaged twice before subsequent experiments.[22,23]
2. Osteogenic and adipogenic differentiation of cultured cells
For the osteogenic differentiation, cells were grown in osteogenic differentiation medium (ODM) containing α-minimum essential medium media supplemented with 10% FBS, 50 μg/mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone.[24]
While for the adipogenic differentiation, cells were grown in adipogenic differentiation medium (ADM) comprised of high glucose DMEM supplemented with 10% FBS, 1 mM Dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 100 mM indomethacin, and 10 μg/mL insulin.[25]
3. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
For the estimation, total RNAs were isolated from the cultured cells using RNAiso Plus (TaKaRa, Tokyo, Japan) following instructions provided by the manufacturer. A total of 500 ng of RNAs were reverse transcribed into complementary DNA (cDNA) using a High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturing instructions. Then qRT-PCR was executed using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) on QuantStudio 3 (Thermo Fisher Scientific, Inc.) as prescribed by the manufacturer. The relative transcript level expression of target genes was estimated using the comparative Ct (2-ΔΔCt) method after considering the expression of the house-keeping genes. Sequences of the primers used in the study are tabulated in Supplementary Table 1.[26]
4. Scratch wound healing assay
For the assay, BMSCs were seeded in 1×106 cells/well in 12-well plates containing complete growth medium. After achieving the appropriate cell confluence, media was then aspirated and incubated with growth media or in osteogenic or ADM. In a similar set of experiments, cells were incubated in the presence of Cxcr4 inhibitor, AMD 3100 (A5602; Sigma-Aldrich) at a dose of 5 μg/mL or in combination with ODM. The dosage of the inhibitor AMD3100 was used as per the available literature.[27,28] A scratch wound was created on the homogenous layer of the cells with the help of a sterile micropipette tip. The floating cells and cellular debris were then removed by washing with phosphate-buffered saline (PBS). The cells that moved from the wound’s edge to the affected area were captured on camera after 0, 6, 12, and 24 hr of incubation.[29]
5. Cell adhesion assay
Cells were initially cultured under the specified treatment conditions. After completion of the incubation, cells were trypsinized and then seeded in collagen-coated plates after carefully counting the cell number using an automated cell counter. Cells were then incubated for 6 hr. After completion of the incubated time frame, culture media was collected, and free-floating cell number was computed utilizing a cell counter.
6. Transwell migration assay
A Transwell device (Millipore, Bedford, MA, USA) with an 8μm pore size polycarbonate filter membrane was used to detect cellular migration. For the estimation, 150 μL of cell suspension (1×106 cells/mL) was added to the upper chamber of the transwell in the differentiating/growth media/media supplemented with Cxcr4 inhibitor AMD 3100 (5 μg/mL). After 12 hr of incubation, cellular migration was tested after staining with crystal violet. Non-migrating cells were removed from the top chamber using cotton-tipped swabs. Migrated cells from the underside of the chamber were then photographed and migration level was quantitatively measured from random fields.
7. Protein extraction and immunoblot assay
Total protein was extracted from the cells after given treatment utilizing lysis buffer (1X RIPA (Millipore) supplemented with 1X protease inhibitor (Thermo Scientific). Immunoblotting was then performed using the extracted protein, Briefly, total protein content was measured using a BCA assay. The 30 μg of protein was separated on SDS polyacrylamide gel of 10% to 15%. Separated Proteins were then transferred to a polyvinylidene difluoride membrane (Millipore) and blocked in 5% BSA in PBS-Tween solution. Membranes were probed with the mentioned primary antibodies; Runx2 1:1,000 (Cat. No. 12556; Cell Signaling Technology, Danvers, MA, USA), Bmp2 1:1,000 (Cat. No. ab284387; Abcam, Cambridge, MA, USA), Col1a1 (Cat. No. A1352; ABclonal Science, Inc., Woburn, MA, USA), ap2 1:1,000 (Cat. No. 15872-1-AP; Proteintech, Chicago, USA), PPAR-γ 1:500 (Cat. No. ab19481; Abcam), C/EBP-α 1:500 (Cat. No. 74404; Abcam), Vcam1 1:2,000 (Cat. No. A19131PM; ABclonal), Cxcr4 1:1,000 (Cat. No. sc-53534; Santa Cruz Biotechnology, Dallas, TX, USA), Sdf-1 1:1,000 (Cat. No. A18225; ABclonal), Itgα5 1:1,000 (Cat. No. A19069; ABclonal), Itgα1 1:1,000 (Cat. No. A16054; ABclonal) or β-actin 1:50,000 (Cat. No. A3854; Sigma-Aldrich) overnight at 4°C. Expression pattern of the tested markers was checked after incubating the membranes in HRP-conjugated appropriate secondary antibody. Immunoblots were visualized in Imager Quant LAS 4010 Chemidoc (GE Healthcare, Little Chalfont, UK) after adding peroxidase substrate (Immobilon; Millipore) following the manufacturer’s instructions. The mean intensity of the immunoblots was quantified using ImageJ software.[30]
8. Statistical examination
All the data were presented as mean±standard error of the mean, for statistical analysis unpaired student t-test or one-way ANOVA was applied with multiple comparisons followed by Tukey’s multiple comparison test of significance with the help of GraphPad Prism (version 5.0; Graph-Pad Software Inc., San Diego, CA, USA).
RESULTS
1. Determination of differential expression level of tested osteogenic markers in the BMSCs undergoing osteogenic differentiation at different phases
To confirm the osteogenic differentiation of the BMSCs cultured in the ODM, the expression pattern of certain key markers involved in this process was checked at different time points of the differentiation as mentioned. The mRNA expression data indicates that the expression of Runx2, the master transcription factor responsible for osteogenic commitments, was found most upregulated at 5 days (P<0.001), a similar increment pattern was also noticed throughout the tested differentiation phases compared to undifferentiated control (Fig. 1A). The elevated expression level of Bmp2 and Col1a1 was observed at the later stage of osteogenic differentiation as compared to the early phases (Fig. 1B, C). Significant elevation in the expression pattern of both was observed after 7 days of differentiation. Another osteoblast-specific marker, Ocn was observed maximally expressed in the BMSCs incubated in ODM for 5 days as compared to other time points (Fig. 1D). While the maximum expression level of both Sparc and Ibsp was found at the 5-day time point as compared to others (Fig. 1E, F). Osteogenic marker sp7 was found elevated after 5 days of ODM incubation to the endpoint (Fig. 1G). Whereas the mRNA expression level of Opn was found elevated in all tested ODM-treated groups as compared to the untreated control (Fig. 1H).
Estimation of the expression level of osteogenic markers in bone marrow-derived mesenchymal stem cells. Relative mRNA expression pattern of (A) Runx2, (B) Bmp2, (C) Col1a1, (D) Ocn, (E) Sparc, (F) Ibsp, (G) sp7, and (H) Opn as estimated from quantitative real-time reverse transcription-polymerase chain reaction. (I) Immunoblots indicate the expression pattern of certain key markers such as Runx2, Bmp2, and Col1a1. Statistically significant differences were indicated. a)P<0.05, b)P<0.01, c)P<0.001 compared with undifferentiated control.
The observed data obtained from the qRT-PCR was further validated from the expression pattern of certain key markers such as Runx2, Bmp2, and Col1a1 at the protein level utilizing immunoblot assay. An increased expression pattern of all three tested markers was noted at all the levels of osteogenic differentiation as compared to the undifferentiated control cells (Fig. 1I).
2. Estimation of the differential expression level of different adipogenic markers in the BMSCs differentiated into adipogenic differentiation at different phases
Like the previous estimations, differential expression level of certain adipogenic markers were also checked to deduce the differential level of adipogenic commitment of the BMSCs. Expression patterns of some known markers involved in the process were checked at the mRNA level and then the obtained data were further confirmed via immunoblot assay.
The key markers responsible for adipogenic commitments such as PPARs and C/EBPs were found elevated in the cells incubated in ADM as compared to undifferentiated control. The mRNA expression of Ppar-γ was significantly elevated in the cells incubated for 5, 7, and 9 days (P<0.001) compared to control (Fig. 2A). On the other hand, the expression level of tested C/EBPs such as C/EBP-α (Fig. 2B), C/EBP-β (Fig. 2C), and C/EBP-γ (Fig. 2D) were found significantly enhanced in the cells incubated in ADM for 5 to 14 days. While Lpl was maximally elevated at the 7-day time point as compared to others (Fig. 2E). Adipocyte markers such as ap2 and Adipoq were found significantly elevated in the 5, 7, and 9 days time points as compared to undifferentiated cells (Fig. 2F, G). While Cfd was seen maximally elevated at the later stage of the adipogenic differentiation (Fig. 2H).
Estimation of the expression level of adipogenic markers of bone marrow-derived mesenchymal stem cells. Relative mRNA expression of (A) Ppar-γ, (B) C/EBP-α, (C) C/EBP-β, (D) C/EBP-γ, (E) Lpl, (F) ap2, (G) Adipoq, and (H) Cfd as estimated from quantitative real-time reverse transcription-polymerase chain reaction. (I) Representative immunoblots of Ppar-γ, C/EBP-α, and ap2 indicate the expression pattern of the tested markers at the protein levels. Statistically significant differences were indicated. a)P<0.05, b)P<0.01, c)P<0.001 compared with undifferentiated control.
These findings were further corroborated by data obtained from the immunoblot assay. Increased expression of key adipogenic markers, including PPAR-γ, C/EBP-α, ap2 was observed across various stages of adipogenic differentiation in BMSCs, compared to undifferentiated cells (Fig. 2I).
3. Assessment of the migratory potential of BMSCs differentiating into osteogenic and adipogenic lineages
The changes in migratory potential of BMSCs undergoing osteogenic or adipogenic commitments were also studied. For the estimation of cell migration, scratch assay was utilized. Computed wound closure (%) indicates cells undergoing osteogenic differentiation migrate more to close the wound as compared to control cells, while cells incubated in ADM exhibit delayed migration (Fig. 3A, B). Moreover, even after 24 hr of incubation, no such significant changes in wound closure was observed in the ADM group as compared to the control (Fig. 3A, B).
Estimation of the migratory potential of bone marrow-derived mesenchymal stem cells (BMSCs) undergoing osteogenic and adipogenic commitments. (A) Microscopic image indicating different levels of cell migration after different time points of osteogenic differentiation medium (ODM) or adipogenic differentiation medium (ADM) incubation as estimated from scratch assay (scale bar 400 μm). (B) Estimated wounded closure (%) computed from microscopic images taken during scratch assay. (C) Estimation of the percentage of seeded cells as obtained from the cell adhesion assay. (D) Computed migratory cells/microscopic field as estimated from transwell migration assay (scale bar 200 μm). (E) Representative microscopic images of crystal violet-stained migrated cells as obtained after incubating BMSCs in ODM or ADM for 24 hr. Statistically significant differences were indicated. a)P<0.05, b)P<0.01, c)P<0.001 compared with undifferentiated control.
Furthermore, to get more intricate details regarding the above inference, a cell adhesion assay was conducted to estimate the level of adhesion of BMSCs following differentiation. Similar to previous observations, BMSCs undergoing osteogenic differentiation tend to adhere more as compared to the undifferentiated BMSCs. However, a compromised cell adherence property was observed in the BMSCs that were directed to adipogenic commitments as compared to the undifferenced cells (Fig. 3C).
Transwell migration assay was also conducted to further confirm the observed modulation in cellular migration during differentiation. Transwell migration of the cells was examined after 12 hr of incubation in either ODM or ADM. Like the previous observations, obtained data from the assay indicate a significantly elevated migration in the ODM incubated cells (P<0.001). And on the contrary, delayed migration was evident in the cells incubated in ADM (Fig. 3D, E).
4. Estimation of the expression of the molecular markers involved in the migration of BMSCs undergoing osteogenic and adipogenic commitment
Next, molecular studies were further performed to check the expression of some markers involved in the cellular migration process at both mRNA as well as protein levels. The mRNA data indicate a significant elevation in the expression level of Itgα1 (Fig. 4A), Itgα5 (Fig. 4B), Sdf-1 (Fig. 4C), and Cxcr4 (Fig. 4D) was seen in the ODM-treated cells as compared to control. Vcam1, on the other hand also showed an increased expression pattern compared to the control. However, cells incubated in ADM exhibit a significant decrease in the expression level compared to the control group (Fig. 4E).
Evaluation of the expression profile of certain markers involved in cell migration of the bone marrow-derived mesenchymal stem cells undergoing osteogenic and adipogenic commitments. Relative mRNA expression of (A) Itgα1, (B) Itgα5, (C) Sdf-1, (D) Cxcr4, and (E) Vcam1. (F) Representative immunoblots of the tested markers and the respective quantitative expression level of the same. Statistically significant differences were indicated. a)P<0.05, b)P<0.01, c)P<0.001 compared with undifferentiated control. ADM, adipogenic differentiation medium; ODM, osteogenic differentiation medium.
The results from mRNA expression analysis were further validated by assessing the protein expression levels of the tested markers. Similar to the previous data, an elevation in the expression of all the tested markers was noticed in the BMSCs directed into osteogenic commitments. A compromised expression profile of all the tested markers was noted in the BMSCs incubated in ADM as compared to control cells (Fig. 4F).
5. ODM modulates the cellular migration of BMSCs via the regulation of Cxcr4 axis
To elucidate the putative molecular mechanism governing the enhanced cellular migration of the BMSCs directed into the osteogenic commitment, a selective inhibitor of Cxcr4, AMD 3100 was employed. As suggested in the literature, Cxcr4 plays the most crucial role in migrating BMSCs cells. Considering this key role of Cxcr4, its selective inhibitor AMD3100 was utilized in our experimental set-up. Data obtained from scratch assay indicates a suppressive cellular migration of the BMSCs when treated with AMD 3100. The inhibitor was also employed in the BMSCs directed to the osteogenic lineages as well as in the undifferentiated cells. In contrast to previous observations, ODM treatment did not enhance cell migration when combined with the Cxcr4 inhibitor AMD 3100 (Fig. 5A, B). The estimated wound closure obtained scratch wound healing assay was further confirmed by the estimation of cell adhesion (Fig. 5C).
Elucidation of the migratory potential of bone marrow-derived mesenchymal stem cells (BMSCs) undergoing osteogenic commitment in the presence of Cxcr4 inhibitor. (A) Microscopic image indicating different levels of cell migration following mentioned treatments as estimated from scratch assay. (B) Estimated wounded closure (%) computed from microscopic images taken during scratch assay. (C) Estimation of the percentage of seeded cells as obtained from the cell adhesion assay. (D) Computed migratory cells/microscopic field as estimated from transwell migration assay. (E) Representative microscopic images of crystal violet-stained migrated BMSCs of the respective groups. Statistically significant differences were indicated. a)P<0.05, b)P<0.01, c)P<0.001 compared with undifferentiated control. ODM, osteogenic differentiation medium.
To further confirm the above inferences, a transwell migration assay was employed with the same experimental setup. The data obtained from the assay showed a similar pattern as noted previously. Cell migration was impaired in BMSCs treated with AMD 3100 compared to untreated control cells. Moreover, no such positive modulation was noted even after the exposure of ODM in the BMSCs, which may be attributed to the inhibition of Cxcr4 by the induction of AMD 3100 (Fig. 5D, E).
DISCUSSION
The complexity of skeletal tissue can be exemplified by the diverse cellular populations in the bone marrow niche. To meet the structural and metabolic requirements of any particular changes in physiology, heterogenic precursor cellular populations differentiate into particular cellular types. For instance, the osteogenic commitment of BMSCs is considered crucial during embryogenesis, skeletal development, and bone remodeling.
The reciprocal relationship between the differentiation of BMSCs towards osteogenic and adipogenic lineages has long been considered as one of the most exciting and complex highlights of stem cell biology. This is dependent on several variables, including highly controlled signaling pathways, hormonal influences, epigenetic regulators, and the function of certain transcription factors. It is well believed that the commitments of BMSCs towards osteogenic and adipogenic commitments are governed by multiple transcription factors, among which Runx2 and PPAR-γ, are known to play key roles in directing BMSCs towards osteogenic and adipogenic commitments.[31,32] All this accounts for creating a very complex molecular network, and understanding the relevant molecular signals at different stages of cellular differentiation provides their fine role in regulating the process.
Significant progress has been made in understanding the complex cellularity and functionality of the BMSCs over the past decades, nevertheless, a few aspects are still in their infancy. Cell migration is one such key area that needs to be explored. The migratory property of the BMSCs makes it a unique cell depot. The in-depth understanding of the intricate biology governing this fundamental yet complex biological process is still an unmet need. This study aims to explore the modulation in cell migration as influenced by the differential lineage allocations of the BMSCs.
BMSCs are well defined by the presence of certain surface markers such as CD29 and CD105 and the absence of CD34 and CD45.[33–35] After characterizing these noted markers, the differential migratory behaviour of the cells was studied using cultured BMSCs through a series of different experimental settings.
To elucidate changes occurring during the osteogenic and adipogenic differentiation of BMSCs, cellular proliferation was initially assessed. Results suggest that cells that underwent osteogenic commitment tend to proliferate more, while during adipogenic commitment, no notable change in cellular proliferation was observed.
To understand the changes underlying the osteogenic and adipogenic differentiation of BMSCs, studies were conducted in C3H10T1/2 cells in addition to primary BMSCs isolated from rats. The mouse-derived C3H10T1/2 cells are well known for the ability to differentiate into both osteogenesis[36,37] and adipogenesis.[38]
The transcription factors Runx2 and PPAR-γ, which have been proven to be crucial molecular regulators of osteogenic or adipogenic differentiation of BMSCs respectively. [39,40] It is also believed that the choice between osteoblastogenesis and adipogenesis of the mesenchymal progenitors depends on the relative expression and activity of Runx2 and PPAR-γ. However, their dynamics in expression level have remained elusive. In the current study, the dynamic expression of these two markers along with other key markers, involved in osteogenic and adipogenic differentiation, were checked at varied time points of the respective differentiation of BMSCs. Increased expression of Runx2 was seen in the early phases of osteogenic commitment of both BMSCs and C3H10T1/2 cells. The increased expression may further enhance other osteogenic markers, including Col1a1, Ocn, and Sparc. Heightened expression of osteogenic markers ultimately contributes to enhanced osteogenic differentiation as seen by increased mineralized nodule formation that mirrors the morphology, ultrastructure, and biochemistry of woven or embryonic bone.[41] However, some variations in the dynamics of transcriptional expression of the tested markers were noted between the two cell types, which may be attributed to different origins and passage. For instance, in BMSCs, Bmp2, and Col1a1 were found elevated at the mid or late phases of osteogenic differentiation, while a similar level of expression was seen in the early and mid-phases of osteogenic differentiation in C3H10T1/2 cells. The expression levels of Sparc, sp7, and Opn were found elevated mostly in the later stage of the differentiation. Similarly, the expression level of Ppar-γ, C/EBP-α, C/EBP-β, and C/EBP-γ was found elevated in all the differentiated groups. This was evidenced in both BMSCs and C3H10T1/2 cells. Other tested markers such as Lpl, ap2, and Cfd reached their maximum expression level in the mid-phase of adipogenic differentiation. All this enhanced expression further contributes to the adipogenic commitment of the cells, as confirmed by the enhanced lipid droplet formation in tandem with the differentiation. [42,43]
Since, understanding cellular migration is very crucial for regenerative medicine and treating other bone diseases such as osteoporosis, osteoarthritis, fracture, and periodontitis, [44,45] the study aims to explore the differential migration pattern of the BMSCs directed to either osteogenic or adipogenic lineage. This study was conducted after confirming the successful induction of the respective differentiation through a series of obtained data. Further experimental evidence obtained from migration-related studies suggests BMSCs that are undergoing osteogenic commitments have a higher tendency to migrate, while delayed migration was noticed during adipogenic commitments. This may be attributed to the elevation of various integrins known to be involved in the process of cell migrations such as Itgα1, Itgα5, and Vcam1.
Besides this, the Sdf-1/Cxcr4 axis is known as a well-established molecular regulator in modulating the migration and homing of BMSCs.[46] To confirm the key involvement of this axis underlying the enhanced migration in the BMSCs committing to the osteogenic commitments, a selective inhibitor of Cxcr4, AMD 3100 was employed.[47] AMD 3100 inhibits cellular migration by interfering with the interaction between Cxcr4 and its natural ligand Sdf-1.[48] A series of test results utilizing AMD 3100, indicated a positive contribution of this molecular axis in regulating the elevated migration of the BMSCs underwent osteogenic lineages. In addition to cellular migration, cellular adhesion was also found to be modulated in a similar manner. However, cell adhesion and migration are two fundamental biological processes, but they are well connected and known to influence each other’s. Further studies confirmed that the migratory behaviour of C3H10 T1/2 cells follows a similar pattern to that of BMSCs. This similarity may be attributed to the resemblance in the molecular and biochemical profiles of the cells with BMSCs at various stages of differentiation.
The identified molecular markers, which were found modulated following the induction to differentiate are also known to articulate other cellular processes including proliferation, differentiation, and adhesion, all of which are believed to be crucial in implementing BMSCs as regenerative medicine.[49] The experimental evidence presented in the study can further contribute to gaining knowledge regarding the complicated biology that governs the differentiation of BMSCs and understanding the fundamental cellular processes such as migration in more detail. All these understandings can be further utilized in redesigning BMSCs in the field of regenerative medicine.
Notes
Acknowledgments
The authors are thankful to the Director, CSIR-CDRI, for providing necessary research facilities. Instrumentation facilities from SAIF, CSIR-CDRI, are gratefully acknowledged for the FACS microscopy. The author also acknowledges Aprajita from Patna University for helping in data curation. The manuscript bears CSIR-CDRI communication number 11002.
Funding
A.S. is thankful to the Council of Scientific & Industrial Research, New Delhi; S.V., A.R., and B.M. are grateful to the University Grants Commission for the financial assistance in the form of fellowships.
Ethics approval and consent to participate
All studies were conducted in accordance with the guidelines approved by the Institutional Animal Ethics Committee (IAEC) of CSIR-Central Drug Research Institute.
Conflicts of interest
No potential conflict of interest relevant to this article was reported.