CD49f and CD146: A Possible Crosstalk Modulates Adipogenic Differentiation Potential of Mesenchymal Stem Cells

Background: The lack of appropriate mesenchymal stem cells (MSCs) selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs. Genetic engineering coupled with high-throughput proteomic studies of MSC surface markers arises as a promising strategy to identify stem cell-specific markers. However, the technical limitations are the key factors making it less suitable to provide an appropriate starting material for the screening platform. A more accurate, easily accessible approach is required to solve the issues. Methods: This study established a high-throughput screening strategy with forward versus side scatter gating to identify the adipogenesis-associated markers of bone marrow-derived MSCs (BMSCs) and tonsil-derived MSCs (TMSCs). We classified the MSC-derived adipogenic differentiated cells into two clusters: lipid-rich cells as side scatter (SSC)-high population and lipid-poor cells as SSC-low population. By screening the expression of 242 cell surface proteins, we identified the surface markers which exclusively found in lipid-rich subpopulation as the specific markers for BMSCs and TMSCs. Results: High-throughput screening of the expression of 242 cell surface proteins indicated that CD49f and CD146 were specific for BMSCs and TMSCs. Subsequent immunostaining confirmed the consistent specific expression of CD49f and CD146 and in BMSCs and TMSCs. Enrichment of MSCs by CD49f and CD146 surface markers demonstrated that the simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis of mesenchymal stem cells. Furthermore, the fate decision of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f+CD146+ immunophenotype. Conclusions: We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins. This provided a valuable tool to investigate MSC-specific markers. Additionally, we showed a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.


Introduction
Mesenchymal stem cells (MSCs) are multipotent adult stem cells located throughout vascularized tissues in the body [1,2].They are one of the most widely used cell sources for cell-based therapy and regenerative medicine owing to their self-renewal, multipotency, easily accessible and free of ethical issues [2][3][4].Conventional methods for isolating MSCs generate a heterogeneous mixture of cells with various lineage commitments, reflected by differences in protein profiles, immunomodulatory capacities, and differentiation potentials [5][6][7], which might have a dramatic impact on the effectiveness of stem cell Cells 2024, 13, 55 2 of 18 research and clinical applications.Numerous studies were performed to find a good approach to reduce inter-culture heterogeneity, including enrichment of a group of MSC subpopulations with specific features [8,9].However, the lack of appropriate cell selection methods has given the challenges for standardized harvesting, processing, and phenotyping procedures of MSCs to gain greater clinical opportunities.
The cluster of differentiation (CD) antigen (also known as CD marker) is the most commonly used group of surface proteins to identify and investigate cell surface protein immunophenotypes.A classical set of CD markers has been committed as one of the minimal criteria for the identification of human MSCs, wherein MSCs must express CD105, CD73, CD90, and lack the expression of CD45, CD34, CD14 or CD11b, CD79α, or CD19, and HLA-DR surface molecules [10].As of yet, the stemness-related surface markers remain to be found.Several groups used various antibody cocktails against cell surface markers to enrich a group of MSC subpopulations with higher differentiation potentials [11][12][13][14].Alas, no single surface marker is capable of identifying cells that satisfy the minimal criteria of MSCs from various tissue sources.
In this regard, an increasing number of comparative studies have been conducted to analyze the similarities and differences of immunophenotype across stem cell populations, aiming to discover distinct surface markers of stem cells [15][16][17][18][19][20][21].However, the adaptation of surface protein expression profiles depend on the surrounding environment and the mix of various lineage-committed cells in populations has hindered the success [22].In order to provide a fairly homogeneous cell population for screening, some studies used genetic engineering to generate reporter cell lines that specifically mark a target cell type, followed by high-throughput technologies such as genomics, transcriptomics, and proteomics to discover unique surface markers for these special target cells.For example, clustered regularly interspaced short palindromic repeat (CRISPR) technology was performed to generate highly specified reporter cell lines that mark the chondroprogenitors [23], skeletal muscle progenitors [24], and dopamine progenitors [25].This knock-in approach facilitated the screening of some lineage committed markers, although the technical limitations are the key factors making it less suitable to provide an appropriate starting material for a screening platform.Indeed, a more accurate, easily accessible approach is needed to solve these mentioned issues.
Light scattering in flow cytometry analysis provides information about the size via forward scatter (FSC) and internal complexity or granularity of cells via side scatter (SSC).FSC versus SSC (FSC/SSC) gating is commonly used to discriminate subpopulations of blood cells.However, it is insufficient to identify subpopulations of MSCs owing to the lack of appreciable difference in size or internal complexity of cells regarding potentially different functionality across subpopulations.Recently, flow cytometry studies have described adipocytes as an SSC-high population [14,26,27].Mesenchymal stem cells, as is well known, are one of the common precursors for adipocytes.Adipogenic-induced MSCs are characterized by intracellular accumulation of lipid droplets, which increases their internal complexity.Therefore, conducting a high-throughput, single-cell analysis of adipogenicinduced MSCs with FSC/SSC gating will facilitate the separation of differentiated MSCs.Taken this, we established a high-throughput screening approach with FSC/SSC gating to characterize the cell surface proteome of adipogenic differentiated MSCs.
Bone marrow-derived MSCs (BMSCs) are one of the most well-studied types of MSCs.BMSCs are isolated from adult bone marrow aspirate through an invasive procedure.Meanwhile, tonsil-derived MSCs (TMSCs) are generally obtained from discarded tissue after tonsillectomy.Given that both MSCs are multipotent and have a perivascular origin, TMSCs are considered as an alternative source for MSC population owing to their easy accessibility and rapid self-renewal capacity [28].In this study, we evaluated the surface protein profiles of BMSCs and TMSCs to provide a comparative and comprehensive characterization of MSCs from different tissue sources.

Cell Culture
We used several lines obtained from different donors (2 BMSCs and 5 TMSCs).The donor information was provided in Supplementary Table S3.The BMSCs were purchased from PromoCell (Heidelberg, Germany).Tonsil-derived MSCs were thawed from a cell stock obtained from the patients using a study protocol approved by the Institutional Review Board (IRB) of Mokdong Hospital, Ewha Womans University (ECT 11-53-02) [29].Written informed consent was obtained from all donors.

Tri-Lineage Differentiation
The BMSCs and TMSCs were plated on a 6-well plate at a density of 100,000 cells per well.After 3 days, the cells were differentiated using StemPro TM differentiation kits (Thermo Fisher Scientific) for 2 weeks.The differentiation potentials were analyzed by staining with Oil red O (adipogenesis) and Alizarin red S (osteogenesis).
The BMSCs and TMSCs were chondrogenically differentiated in the standard pellet culture to detect proteoglycan.A total of 300,000 cells were centrifuged at 500× g for 10 min at room temperature to form a pellet.After 1 day, pellets were chondrogenically induced using the StemPro TM differentiation kit for 3 weeks.Then, the pellets were sectioned and stained with Safranin-O to demonstrate the presence of proteoglycan.
The micromass culture model was used as previously described [30,31] to detect lineage marker protein expressions by western blotting.Briefly, BMSCs and TMSCs were resuspended in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B at a concentration of 1 × 10 7 cells/mL.Thirty microliter droplets of the cell suspension were spotted in a 6-well culture plate.The cells were adhered to culture dishes by incubating for 1 h at 37 • C in a humidified atmosphere containing 5% CO 2 , followed by the addition of 2 mL DMEM and culturing for another day.The cells were differentiated using StemPro TM differentiation kits for 2 weeks.Then, cells were harvested and subjected to western blotting to detect lineage marker protein expressions.

High-Throughput Screening of Cell Surface Marker Profile
Flow cytometry analysis was performed to investigate the surface antigen profile of undifferentiated-and adipogenic-induced BMSCs or TMSCs.Briefly, 300,000 cells were stained with each antibody from the BD Lyoplate™ Human Cell Surface Marker Screening Panel (BD Biosciences, San Jose, CA, USA), a system consisting of 242 purified monoclonal antibodies and corresponding isotype controls, followed by an Alexa-647 conjugated secondary antibody.Non-specific fluorescence was determined using equal aliquots of unstained cell preparations.Data were obtained by analyzing 10,000 events on an ACEA NovoCyte 3000 flow cytometer (Agilent Technologies, Santa Clara, CA, USA).
Cells were classified into lipid-rich cells as the SSC-high population and lipid-poor cells as the SSC-low population for adipogenic-induced BMSCs or TMSCs.Data were obtained and analyzed by an ACEA NovoCyte 3000 flow cytometer as described above.A comparative study of surface protein profile between undifferentiated and adipogenicdifferentiated cells and between lipid-rich and lipid-poor cells was performed to identify the specific surface markers of MSCs.The proteins exclusively expressed by lipid-rich cells are considered adipogenesis-associated markers.

Fluorescence-Activated Cell Sorting (FACS) of MSCs
The BMSCs and TMSCs were obtained at passage 3. The cells were centrifuged, and the pellet was gently pipetted.Then, cells were stained with antibody cocktail against CD49f (BD Biosciences, #747725) and CD146 (BD Biosciences, #563619) at a concentration of 1 µg per 1 × 10 6 cells on ice.Flow cytometric acquisition and cell sorting were performed using a BD FACSAria III Cell Sorter (BD Biosciences).The purity of sorted cell subsets was determined by post-sorting analysis.Sorted cells were expanded up to 3 passages to generate a sufficient number of cells for subsequent experiments.

siRNA
siRNA specific for human CD49f, CD146, and non-targeting control siRNA were purchased from Santa Cruz Biotechnology (#sc-35918, #sc-43129, and #sc-37007).Transient transfection was performed by Lipofectamine TM 2000 transfection reagent (Thermo Fisher Scientific) for 6 h.Cells were differentiated using StemPro TM differentiation kits approximately 24 h post-transfection.Differentiation potentials of knockdown cells were analyzed by western blotting as described above.

Quantification and Statistical Analysis
Data are presented as the mean ± standard error of the mean (SEM).Statistical differences between groups were evaluated by one-way analysis of variance (ANOVA) with a Tukey-Kramer multiple comparisons test (* p < 0.05, ** p < 0.01, and *** p < 0.001).GraphPad Prism 9.0 statistical software (GraphPad Software, Inc., San Diego, CA, USA) was used for the analysis.A p-value < 0.05 was considered significant.

Donor's General Characteristics
First, we examined the general characteristics to ensure that the cells meet the current minimal criteria for MSCs.Both BMSCs and TMSCs differentiated into adipogenic, osteogenic, and chondrogenic lineages (Figure 1A) and expressed the classical set of surface markers defining MSCs (Figure 1B).

CD49f and CD146 Surface Proteins Exhibit a Distinct Expression Pattern on Adipogenic Differentiated MSCs
Immunofluorescence staining was used to confirm the distinct expression pattern of identified surface proteins CD49f and CD146 on adipogenic differentiated BMSCs and TMSCs.Lipid droplets were labeled by Bodipy.CD49f and CD146 were clearly observed on lipid-rich cells by colocalization of these markers with Bodipy (Figure 3A,B).We did not detect CD49f or CD146 on lipid-poor cells (DAPI only).This was consistent with the data obtained from the surface protein screen.A similar pattern was observed in BMSCs and TMSCs isolated from various donors (Supplementary Figure S1).
Double immunofluorescence staining of CD49f and CD146 revealed that these two markers were almost colocalized.Notably, simultaneous CD49f and CD146 double-stained cells were mainly observed in adipogenic-induced BMSCs (Figure 3A).Meanwhile, the loss of CD49f expression in TMSCs upon differentiation was shown by the absence of CD49f stained cells that were abundant in undifferentiated TMSCs.However, weak expression of CD49f was observed among CD146 stained cells (Figure 3B).

CD49f and CD146 Crosstalk Modulates Differentiation Potentials of MSCs
CD49f and CD146 play important roles in stem cell proliferation and differentiation [41][42][43][44][45].However, crosstalk between these markers is unidentified.We sorted subpopulations from undifferentiated MSCs and examined their differentiation potentials upon adipogenesis and osteogenesis to investigate the possible roles of CD49f and CD146 in mesenchymal stem cell differentiation.
followed by CD49f CD146 and CD49f CD146 cells at 13.9 ± 1.1% and 31.3 ± 1.5% respectively, with no cells exhibited the CD49f − CD146 + immunophenotype (Figure 4B).A postsort analysis showed that the subpopulation purity was greater than 97%.Sorted subpopulations were sub-cultured for 3 passages to obtain a sufficient number of cells for subsequent experiments.The sub-culturing partially restored the expression levels of these markers (Figure 4A,B).The adipogenic and osteogenic differentiation capabilities of subpopulations were elucidated by western blotting with lineage marker protein expressions (Figure 5).CD49f and CD146 identified subsets with varying differentiation potentials in BMSCs.CD146 The adipogenic and osteogenic differentiation capabilities of subpopulations were elucidated by western blotting with lineage marker protein expressions (Figure 5).CD49f and CD146 identified subsets with varying differentiation potentials in BMSCs.CD146 enrichment enhanced adipogenesis and osteogenesis according to higher expression levels of adipogenic markers PPARγ, ADIPOQ, and FABP4 (Figure 5A), and osteogenic markers COL1A1 and OCN (Figure 5B) in CD146 enriched cells than in CD146 depleted cells.Meanwhile, the CD49f + CD146 − subpopulation showed low potentials in adipogenesis and osteogenesis (Figure 5A,B).In TMSCs, a significantly higher expression of adipogenic indicators were observed in CD49f + CD146 + cells than in CD49f + CD146 − cells.However, a similar amount of these markers was observed in CD49f + CD146 + and CD49f − CD146 − cells (Figure 5C).Validation by immunofluorescence staining, we observed many CD146 + cells within the CD49f + CD146 + sorted subpopulation did not co-localize with Bodipy (Supplementary Figure S2).This indicated that these cells were not adipocytes (white arrows).We also observed a low expression level of ADIPOQ in TMSCs (Figure 5C).For osteogenic potential, CD49f + CD146 + cells showed higher levels of OCN expression than other groups; however, COL1A1 expression levels did not significantly change among the groups (Figure 5D).many CD146 + cells within the CD49f + CD146 + sorted subpopulation did not co-localize with Bodipy (Supplementary Figure S2).This indicated that these cells were not adipocytes (white arrows).We also observed a low expression level of ADIPOQ in TMSCs (Figure 5C).For osteogenic potential, CD49f + CD146 + cells showed higher levels of OCN expression than other groups; however, COL1A1 expression levels did not significantly change among the groups (Figure 5D).

Knockdown of CD49f or CD146 Attenuates Adipogenesis and Osteogenesis Capabilities of BMSCs
CD49f or CD146 expression levels of BMSCs were knocked down by transfecting them with siRNA against CD49f or CD146 to further elucidate whether CD49f and CD146 regulated the onset of mesenchymal stem cell differentiation.We observed a significant reduction of CD49f and CD146 in BMSCs (Figure 6A,B).Knockdown of either CD49f or CD146 attenuated adipogenesis (Figure 6C) and osteogenesis (Figure 6D) capabilities of BMSCs, as shown by lower expression levels of adipogenic markers PPARγ, ADIPOQ, and FABP4, and osteogenic markers COL1A1 and OCN in CD49f-siRNA (siCD49f) or CD146-siRNA (siCD146) transfected cells than in non-targeting control siRNA (siCon) transfected cells.
and osteogenic (D) indicators in TMSCs after 14 d of differentiation.C, control; D, differentiation.Bar charts show differentiation indicator quantitation normalized to GAPDH.The results are an average value from three independent experiments and presented as mean ± SD.Alphabet letters indicate statistically significant differences.Bars with different letters are considered statistically significant with p < 0.05.Full-length blots are presented in Supplementary Information: Full-length western blot images.

Knockdown of CD49f or CD146 Attenuates Adipogenesis and Osteogenesis Capabilities of BMSCs
CD49f or CD146 expression levels of BMSCs were knocked down by transfecting them with siRNA against CD49f or CD146 to further elucidate whether CD49f and CD146 regulated the onset of mesenchymal stem cell differentiation.We observed a significant reduction of CD49f and CD146 in BMSCs (Figure 6A,B).Knockdown of either CD49f or CD146 attenuated adipogenesis (Figure 6C) and osteogenesis (Figure 6D) capabilities of BMSCs, as shown by lower expression levels of adipogenic markers PPARγ, ADIPOQ, and FABP4, and osteogenic markers COL1A1 and OCN in CD49f-siRNA (siCD49f) or CD146-siRNA (siCD146) transfected cells than in non-targeting control siRNA (siCon) transfected cells.

Discussion
We established a novel high-throughput screening strategy for identification of the adipogenesis-associated surface proteins, paving the way to elucidate the mesenchymal stem cell-specific markers.Typically, high-throughput screening of the surface protein profile of a pooled cell population provides very little information regarding potentially various identities across subpopulations of MSCs owing to stem cell heterogeneity.In this paper, adipogenic differentiated MSCs are separated by light scatter gating on account of the differences in internal complexity of cells, enables the comparative study of surface protein profiles between lipid-rich and lipid-poor cells.The primary advantage of this approach is that the need for reporter cell lines is eliminated.This improves the physiological relevance of the data and allows us to address many unanswered questions about mesenchymal stem cell immunophenotypes and identity.
A compilation of the results from the surface protein expression profile of lipid-rich and lipid-poor cells suggests that the surface markers CD49f and CD146, which are almost exclusively found in the lipid-rich subpopulation, are adipogenesis-associated markers.The expression of CD49f and CD146 on both undifferentiated and adipogenic induced cells facilitates sorting for the enrichment of MSCs.Subsequent investigations identified subsets with various differentiation potentials.
Indeed, understanding MSC surface protein profile holds great promise to predict their native physiological functions.In this study, both BMSCs and TMSCs show general properties of MSCs.Besides, BMSCs and TMSCs share similar cell surface protein repertoires with 56 common proteins following high-throughput screening of the panel consisting of 242 primary antibodies to surface proteins.Regarding the differences across MSCs, the lack of HLA-A2 and MIC A/B on TMSCs suggests different immunomodulatory properties between TMSCs and BMSCs [46,47].Additionally, these cells exhibit discrepancies in the CD49f and CD146 immunophenotype during the undifferentiated state.Of note, BMSCs and TMSCs show different patterns of ADIPOQ and CD49f expression levels in response to adipogenic differentiation stimuli.Possibly, the tissue-specific peculiarities of MSCs may result in different immunophenotypes, secretome compositions, immunomodulatory properties and paracrine activities [48,49].It is also possible that the age-related changes may result in variations in MSCs characteristics [50,51].In the current study, BMSCs derived from old donors, TMSCs were isolated from young donors (Supplementary Table S3).
CD49f [also known as integrin α6 (ITGA6)] is a member of the integrin alpha chain family of proteins.As a matrix adhesion molecule, CD49f plays important roles in the proliferation and migration of stem cells [74].Knockdown of CD49f results in the phosphorylation of focal adhesion kinase (FAK) and the reduction of NANOG, OCT4, and SOX2 in human pluripotent stem cells [44].This suggests that CD49f plays roles in inactivating FAK signaling and supports stem cell self-renewal.Additionally, CD49f expression is sensitive to environmental changes [75].Furthermore, the expression switch of CD49f shows dramatic consequences for cell proliferation/differentiation transition.In particular, CD49f increases during adipogenesis and treatment with CD49f-blocking antibody promotes pre-adipocytes to reenter the cell cycle [76].However, these studies lack protein-protein interaction analysis; therefore, we cannot definitively conclude that CD49f solely regulates the cell fate decision.In fact, our results indicate that the CD49 + CD146 − immunophenotype exhibits low adipogenesis and osteogenesis in BMSCs and TMSCs.
As proposed by Chen et al. [77], adipogenic and osteogenic differentiation of MSCs is achieved by the actions of critical signaling pathways and key transcription factors.Therefore, we assume that CD49f and CD146 are among the mediators regulating the lineage commitment of MSCs via targeting key transcription factors such as PPARγ, C/EBPs, or RUNX2.In fact, the simultaneous expression of CD49f and CD146 is required for enhanced adipogenic and osteogenic differentiation shown in this study (Figure 5A) suggests a possible crosstalk between CD49f and CD146 surface markers.In addition, the switch-off of CD49f in CD49f + CD146 + enriched TMSCs results in loss of their adipogenic potential (Figure 5C), as shown by the maintenance of CD146 + MSCs in an undifferentiated state (Figure S2).This is consistent with previous study [76].Taken together, we speculate that CD49f and CD146 act reciprocally to regulate adipogenic differentiation of MSCs.Furthermore, this study demonstrated that the differentiation potential of MSCs from different sources is regulated by distinct responses of cells to differentiation stimulations despite sharing a common CD49f + CD146 + immunophenotype.Further analysis is required to discover the underlying mechanisms regulating cell fate decision of MSCs isolated from diverse tissues.
We could not investigate the change of CD49f and CD146 surface proteins before and after osteogenesis owing to limitations in single-cell dissociation of osteogenic differentiated MSCs.Nevertheless, our data reveal that CD49f + CD146 + enriched BMSCs and TMSCs exhibit higher osteogenic differentiation potential than other subgroups.This indicated that the CD49f + CD146 + immunophenotype may identify a high adipogenesis and osteogenesis subpopulation.
The knockdown of either CD49f or CD146 by transfection with siRNA attenuated adipogenesis and osteogenesis of BMSCs, although the decrease of ADIPOQ, COL1A1, and OCN expression levels were not statistically significant.A previous study shows that CD146 mRNA continuously increases from day 3 after adipogenic induction [70].Therefore, we speculate that the transient effect of siRNA is not enough to maintain the significant decrease of adipogenesis and osteogenesis potentials in siCD49f or siCD146 transfected BMSCs.We suggest that a more stable genetic engineering system will help validate the role of CD49f and CD146 in regulating MSC differentiation.

Conclusions
We established an accurate, robust, transgene-free method for screening adipogenesis associated cell surface proteins.This provides a valuable tool to investigate MSC-specific markers.Additionally, we show that a possible crosstalk between CD49f and CD146 modulates the adipogenesis of MSCs.The simultaneous expression of CD49f and CD146 is required for adipogenesis and osteogenesis potentials.Further investigation is necessary to elucidate the mechanisms controlling MSC fate determination through CD49f and CD146 across MSCs from diverse tissues.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells13010055/s1, Figure S1.The specificity of CD49f and CD146 on BMSCs (A) and TMSCs (B) isolated from various donors.Figure S2.The expression pattern of CD49f and CD146 on adipogenic-induced TMSC subpopulations.Immunofluorescent imaging of adipogenic differentiated TMSC subpopulations.Cells were stained with DAPI (blue), Bodipy (green), and CD49f or CD146 (red).Scale bar: 100 µm.We found many CD146+ cells within CD49f+CD146+ sorted subpopulation did not co-localize with Bodipy, indicating the non-adipocyte identity of these cells (white arrows).Table S1.The surface marker expression profile of BMSCs.Table S2.The surface marker expression profile of TMSCs.Table S3.Donor's information.Institutional Review Board Statement: Ethical approval and consent to participate.The study protocol to obtain TMSCs followed the Hel-sinki declaration and was approved by the Institutional Review Board (IRB) of Mokdong Hospital, Ewha Womans University.(Title of the approved project: "Isolation of multipotent mesenchymal stem cells from human palatine tonsils", approval number: ECT 11-53-02, date of approval: 22 August 2011).
Informed Consent Statement: Not applicable.

Figure 1 .
Figure 1.MSCs general characteristics.(A) Tri-lineage differentiation potential of BMSCs (upper panel) and TMSCs (lower panel) toward adipogenic, osteogenic and chondrogenic lineages was confirmed by staining with Oil red O (adipogenesis), Alizarin red S (osteogenesis), and Safranin O (chondrogenesis).Scale bar: adipogenesis and osteogenesis: 100 µm; chondrogenesis: 200 µm.(B) Flow cytometric analysis of BMSCs (upper panel) and TMSCs (lower panel).Both BMSCs and TMSCs expressed the classical set of mesenchymal stem cell surface markers, which is one of the minimal criteria for the identification of human MSCs proposed by the ISCT.

Figure 1 . 19 Figure 2 .
Figure 1.MSCs general characteristics.(A) Tri-lineage differentiation potential of BMSCs (upper panel) and TMSCs (lower panel) toward adipogenic, osteogenic and chondrogenic lineages was confirmed by staining with Oil red O (adipogenesis), Alizarin red S (osteogenesis), and Safranin O (chondrogenesis).Scale bar: adipogenesis and osteogenesis: 100 µm; chondrogenesis: 200 µm.(B) Flow cytometric analysis of BMSCs (upper panel) and TMSCs (lower panel).Both BMSCs and TMSCs expressed the classical set of mesenchymal stem cell surface markers, which is one of the minimal criteria for the identification of human MSCs proposed by the ISCT.Cells 2024, 13, x FOR PEER REVIEW 7 of 19

Figure 2 . 2 .
Figure 2. Generic method for FACS-based high-throughput screening of adipogenesis associated markers of MSCs.(A,B) Cell surface proteome of BMSCs (A) and TMSCs (B) were analyzed by flow Figure 2. Generic method for FACS-based high-throughput screening of adipogenesis associated markers of MSCs.(A,B) Cell surface proteome of BMSCs (A) and TMSCs (B) were analyzed by flow cytometry.Adipogenic differentiated BMSCs and TMSCs were gated into lipid-rich cells as the SSC-high population and lipid-poor cells as the SSC-low population.The surface markers which exclusively found in lipid-rich subpopulation were considered as adipogenesis-associated markers.(C) Heatmap of the expression level of surface proteins on undifferentiated and adipogenic differentiated BMSCs.CD49f and CD146 were almost exclusively expressed in the lipid-rich population.The results are an average value from three independent experiments.(D) Heatmap of the expression level of surface proteins on undifferentiated and adipogenic differentiated TMSCs.CD49f and CD146 were almost exclusively expressed in the lipid-rich subpopulation.CD49f surface protein was significantly downregulated in adipogenic differentiated TMSCs.The results are an average value from three independent experiments.

Figure 5 .
Figure 5. CD49f and CD146 crosstalk modulates differentiation potentials of MSCs.(A,B) Western blotting was used to assess the protein expression levels of adipogenic (A) and osteogenic (B)

Figure 5 .
Figure 5. CD49f and CD146 crosstalk modulates differentiation potentials of MSCs.(A,B) Western blotting was used to assess the protein expression levels of adipogenic (A) and osteogenic (B) indicators in BMSCs after 14d of differentiation.C, control; D, differentiation.Bar charts show differentiation indicator quantitation normalized to GAPDH.The results are an average value from three independent experiments and presented as mean ± SD.Alphabet letters indicate statistically significant differences.Bars with different letters are considered statistically significant with p < 0.05.(C,D) Western blotting analysis was used to assess the protein expression levels of adipogenic (C) and osteogenic (D) indicators in TMSCs after 14 d of differentiation.C, control; D, differentiation.Bar charts show differentiation indicator quantitation normalized to GAPDH.The results are an average value from three independent experiments and presented as mean ± SD.Alphabet letters indicate statistically significant differences.Bars with different letters are considered statistically significant with p < 0.05.Full-length blots are presented in Supplementary Information: Full-length western blot images.

Figure 6 .
Figure 6.siRNA-mediated knockdown of CD49f or CD146 expression level in BMSCs.(A,B) Western blotting was used to analyze the protein expression levels of CD49f and CD146 after siRNAmediated knockdown in BMSCs.(C,D) The effect of CD49f or CD146 knockdown on adipogenesis (C) and osteogenesis (D) of BMSCs.Bar charts show differentiation indicator quantitation normalized to GAPDH.The results are the average value from three independent experiments and presented as mean ± SD.Alphabet letters indicate statistically significant differences.Bars with different letters are considered statistically significant with p < 0.05.Full-length blots are presented in Supplementary Information: Full-length western blot images.

Figure 6 .
Figure 6.siRNA-mediated knockdown of CD49f or CD146 expression level in BMSCs.(A,B) Western blotting was used to analyze the protein expression levels of CD49f and CD146 after siRNA-mediated knockdown in BMSCs.(C,D) The effect of CD49f or CD146 knockdown on adipogenesis (C) and osteogenesis (D) of BMSCs.Bar charts show differentiation indicator quantitation normalized to GAPDH.The results are the average value from three independent experiments and presented as mean ± SD.Alphabet letters indicate statistically significant differences.Bars with different letters are considered statistically significant with p < 0.05.Full-length blots are presented in Supplementary Information: Full-length western blot images.

Author
Contributions: A.N.-T.T., H.Y.K. and H.S.K. designed the experiments; A.N.-T.T. performed the experiments, analyzed the data and made the figures; A.N.-T.T. wrote the draft; H.Y.K., S.-Y.O. and H.S.K. checked and revised the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Korean Fund for Regenerative Medicine (KFRM) funded by the Korea government (the Ministry of Science and ICT, and the Ministry of Health and Welfare (23B0101L1).