Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

Stem-cell-based therapies have gradually become a hot topic due to their high plasticity and self-renewing ability; clinical investigations with stem cell products in regenerative medicine are addressing a wide spectrum of conditions using a variety of stem cell types. These pluripotent cells including embryonic stem cells (ESCs), termed induced pluripotent stem cells (iPSCs), were first tested but inhibited in their clinical applications owing to ethical and tumorigenic problems. As a promising candidate for tissue regeneration, mesenchymal stem cells (MSCs) are fibroblast-like, with high plasticity and self-renewing ability and are able to develop into diverse cell lineages. 1 Among the MSCs from different adult tissues, placental-derived mesenchymal stem cells (PDMSCs), which reside in the fetal membranes of the term placenta, are easily accessible and less invasive. Their abundance, high proliferative potency, short population doubling time, strong immunosuppression and lack of ethical concerns make them indispensable in stem cell research and therapy. 2 Specific growth factors, cytokines and extracellular matrix components may have an important role in the determination of stem cell fate by switching from self-renewal to a differentiation stage. During lineage alteration to a specific tissue cell type, it was thought that MSCs progressively and developmentally became lineage restricted. 3 Yet some evidences have suggested that when terminally differentiated mammalian cells are cultured under special conditions, they will revert to a more primitive phenotype. [4][5][6] More recently, in the presence of human embryonic stem cell medium supplemented with valproic acid, stem cells derived from amniotic fluid could be fully reprogrammed to pluripotency  7 This process was defined as dedifferentiation and is considered as one of the mechanisms to reroute cell fate. 8 Furthermore, a downregulation of lineagespecific genes and an upregulation of stem genes occurred immediately after initiation of the dedifferentiation process. 8 This phase was characterized by repression of somatic genes via methylation, increased cell proliferation, altered morphology, signal transduction changes, reactivation of telomerase activity and the mesenchymal-to-epithelial transition (MET). 9,10 MET includes the loss of mesenchymal characteristics, such as motility, and the acquisition of epithelial characteristics such as cell polarity and the expression of cell adhesion molecules. 11 In addition, bone marrow mesenchymal stem cells (BMMSCs) which were induced into osteocytes, chondrocytes and adipocytes, can dedifferentiate into a primitive population on the withdrawal of stimulating culture medium. [12][13][14] This new population correlated with cell cycle arrest and associated genes, had enhanced cell survival, greater efficacy in differentiation and improved therapeutic potential in vitro and in vivo compared with uncommitted BMMSCs. 15,16 On the other hand, a number of studies showed enhanced mitochondrial biogenesis in various stem cell differentiation models including ESCs and iPSCs. 17,18 The immature mitochondrial phenotype in ESCs consists of fewer mitochondria, poorly developed cristae and a perinuclear location of mitochondria. 19,20 These characteristics have been regarded as potential markers of pluripotency in ESCs; 20 however, it has not been clearly established whether the morphology and the mitochondrial network is pluripotency dependent or stem cell specific. In addition, it has been suggested that mitochondrial dynamics and oxidative phosphorylation (OXPHOS) activity can influence each other during the biological process. 21 Consequently, we suggest that the altered OXPHOS activity will accompany the differentiation and dedifferentiation processes.
In the present study, we aimed to comprehensively analyze the transition in morphology, surface antigens, metabolism and multilineage potency during PDMSCs differentiation and dedifferentiation to clarify whether unrestricted lineage exists in differentiated PDMSCs. We showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The new population proliferated faster than unmanipulated PDMSCs, and could be differentiated into adipocytes, osteocytes and hepatocytes. Gene expression profiling showed a panel of genes with significantly up-or downregulated expression between adipogenically differentiated and dedifferentiated cells. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to regenerate, proliferate and differentiate during the differentiation, dedifferentiation and redifferentiation processes. 22,23 These observations indicated that the dedifferentiated PDMSCs were distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies.

Results
Morphology and adipocyte markers of adipogenic PDMSCs reverted to a primitive state after dedifferentiation. PDMSCs at passage 3 displayed a long spindle shape (Figure 1a). After adipogenesis for 14 days, PDMSCs   Figures 1d and e). Then, the dedifferentiated PDMSCs was passaged ( Figure 1f) and cultured in general PDMSC medium for another 14 days. At the molecular level, the expression of the adipocyte-specific genes FABP4 and PPARG were significantly upregulated at day 14 of adipogenesis when compared with undifferentiated PDMSCs (Po0.001). After withdrawing adipogenic medium, the markers were significantly downregulated to a similar level as PDMSCs, and there was no difference between each passage of dedifferentiated PDMSCs (Figure 1g).
Surface antigen expression of adipogenic PDMSCs at the mRNA and protein levels reverted to a primitive state after dedifferentiation. As previously reported, PDMSCs positively express mesenchymal markers such as CD29, CD44, CD90 and CD105, but negatively express the hematopoietic markers CD14 and HLA-DR. 24 The expression levels in PDMSCs were set to 1 for normalization. Positive markers for CD29, CD44, CD90 and CD105 mRNA expression levels were significantly decreased in AL cells and then increased after dedifferentiation in general PDMSC medium ( Figure 2a). The CD29 mRNA level in DePDMSCs at passage 3 was significantly higher than the level in PDMSCs at passage 3 (Po0.05). The CD105 mRNA level in AL cells was significantly downregulated compared with PDMSCs at passage 3 (Po0.001), and the mRNA expression level in DePDMSCs at passage 2 was lower than PDMSCs at passage 3 (Po0.05). However, the expression returned to a level comparable to PDMSCs at passage 3 after culture in the negative makers were not altered. Then, after dedifferentiation for 21 days, DePDMSCs at passage 3 had a comparable expression pattern of positive and negative surface antigens to PDMSCs at passage 3. The results in our study showed that CD44, CD90 and CD105 were significantly altered during adipogenic differentiated PDMSCs (Po0.05) but reverted to a comparable level after dedifferentiation; the hematopoietic markers were not altered throughout the differentiation and dedifferentiation processes ( Figure 2d). Immunocytochemistry (n = 3; Figure 2e) of the PDMSCs at passage 3 revealed highly positive expression for SOX2, weakly positive expression for OCT4 and negative expression for CD34, NANOG, SSEA4 and TRA-1-60R. After adipogenic differentiation for 14 days, AL cells showed downregulated expression for the positive embryonic stem cell markers (OCT4 and SOX2), while they returned to a level similar to uncommitted PDMSCs after dedifferentiation for 21 days. Conversely, the negative markers were not altered during the adipogenic differentiation and dedifferentiation processes.
The mitochondrial network of PDMSCs undergoes changes during adipogenic differentiation and dedifferentiation. Although the globular shape and perinuclear localization of mitochondria in ESCs and iPSCs has been regarded as a potential marker of pluripotency, 20 it has not been clearly established whether the morphology of the mitochondrial network is pluripotency dependent or stem cell specific. Staining of the mitochondrial network revealed that expanding PDMSCs display a developed network composed of thread-like mitochondria spread throughout the cytoplasm ( Figure 3a); adipogenic differentiation resulted in cells with more and larger round-shaped mitochondria (Figure 3b). After dedifferentiation, DePDMSCs also displayed a Given that mitochondrial biogenesis also requires the synthesis and import of many mitochondrial proteins, we next analyzed the abundance of several mitochondria specific genes at the mRNA level throughout the processes. These regulators included ATP5A1, COX4I1, MT-CO1, MT-CO2, TFAM, TOMM34, LONP1 and PPAR-a. We showed a trend towards mRNA abundance of all mitochondria specific genes in AL cells when compared with PDMSCs at passage 3, but they were downregulated in DePDMSCs at passage 3 ( Figure 3d). ATP5A1, TFAM and TOMM34 in AL cells showed a significantly increased level compared with undifferentiated PDMSCs or DePDMSCs (Po0.01). COX4I1, MT-CO2 and LONP1 in AL cells also showed a significantly increased level compared with undifferentiated PDMSCs or DePDMSCs (Po0.05). However, MT-CO1 and PPAR-a demonstrated a higher trend but there was no difference in AL cells when compared with PDMSCs and DePDMSCs. These data further support the notion that an enhanced or decreased mitochondrial biogenesis process occurs during the differentiation and dedifferentiation processes.
OXPHOS is the main source of energy in eukaryotic cells. The results of a Human OXPHOS Magnetic Bead Panel ( Figure 3e) showed a significantly increased trend for the nicotinamide nucleotide transhydrogenase (NNT) protein during the adipogenic process, but it decreased to the original level after dedifferentiation (Po0.01). Complex I was DePDMSCs can grow more quickly than uncommitted PDMSCs and expanding PDMSCs. We further detected the proliferation ability of DePDMSCs and compared this with uncommitted PDMSCs and expanding PDMSCs. We found that the proliferation rates ( Figure 4a) of PDMSCs at passage 3, PDMSCs at passage 6 and DePDMSCs at passage 3 were slow during the first 2-3 days (latent phase) and then accelerated rapidly during 4-6 days (logarithmic phase) and thereafter slowed down (stationary phase). The doubling time of PDMSCs at passage 3 in the logarithmic phase was 2.16 ± 0.16d, PDMSCs at passage 6 was 3.46 ± 0.25d and DePDMSCs at passage 3 was 1.65 ± 0.26d ( Figure 4b). The proliferation ability results showed that DePDMSCs at passage 3 grew more quickly than PDMSCs at passage 3 (Po0.05). The proliferation ability of DePDMSCs at passage 3 and PDMSCs at passage 3 was higher than PDMSCs at passage 6 (Po0.001).
The multilineage differentiation ability of DePDMSCs was comparable to PDMSCs. To clarify whether the new dedifferentiated population was able to obtain multilineage differentiation ability, we further induced the new population via adipogenic, osteogenic and hepatogenic differentiation and compared them with PDMSCs. After PDMSCs and DePDMSCs were induced in adipogenic medium for 14 days, they acquired the typical characteristics of adipocytes, and Oil red O staining showed lipid accumulation (Figures 5a and d). DePDMSCs retained adipogenic potency and the mRNA expression levels of FABP4 and PPARG in adipogenic DePDMSCs were higher than adipogenic PDMSCs. Human adipose tissue was used as a positive control ( Figure 5e). Immunocytochemistry of FABP4 and PPARG showed negative expression in PDMSCs and DePDMSCs at day 0, and strongly positive expression at day 14 of adipogenesis ( Figure 5f).
After PDMSCs and DePDMSCs were induced in osteogenic medium for 14 days, they acquired the typical characteristics of osteocytes, and alizarin red staining showed calcium accumulation (Figures 6a and d). DePDMSCs retained osteogenic potency and the mRNA expression levels of RUNX2 and osteocalcin in osteogenic DePDMSCs were comparable to osteogenic PDMSCs. Osteoblasts were used as a positive control (Figure 6e). Immunocytochemistry of RUNX2 and osteocalcin showed negative expression in PDMSCs and DePDMSCs at day 0, and strongly positive expression at day 14 of osteogenesis ( Figure 6f). Hepatogenic PDMSCs and DePDMSCs could also uptake and release ICG; however, the expanding PDMSCs and DePDMSCs could not uptake ICG (Figure 7j). Periodic acid-Schiff staining (PAS) staining showed that both PDMSCs and DePDMSCs could store more glycogen after hepatogenic induction for 21d (Figure 7k).
Numerous genes and multiple signaling pathways cooperate to regulate the adipogenic differentiation and dedifferentiation processes. Gene expression profiling was performed to obtain a deeper molecular insight into AL cells and their subsequent dedifferentiation to primitive cell types with multilineage potency. GeneChips were generated for PDMSCs, AL cells and DePDMSCs from three donors. We selected 2140 out of 49 395 probe sets that represented genes with differential expression between AL cells and PDMSCs after removing double entries and probe sets with no title (Supplementary Table S1). Among them, the expression levels of 952 genes were upregulated and 1 188 were downregulated on day 14 of adipogenesis. Using the same criteria, we selected 2486 out of 49 395 probe sets that represented genes with differential expression between AL cells and DePDMSCs (Supplementary Table S2). Among them, the expression levels of 1590 genes were upregulated and 896 were downregulated on day 21 of dedifferentiation. On the basis of KEGG pathway enrichment analysis, the upregulated and downregulated differentially expressed genes were mainly enriched in the multiple crucial KEGG pathways listed in Tables 1a and b. We then identified nine differentially expressed genes in DePDMSCs compared with PDMSCs (Supplementary Table S3), all genes except for fibroblast growth factor 7 (FGF7) were upregulated in DePDMSCs when compared with PDMSCs. KEGG pathway enrichment analysis demonstrated that three of the differentially expressed genes were involved in multiple pathways (Table 1c). To confirm the microarray analysis results, RT-QPCR analysis (Supplementary Figure S1) showed that the expression levels of most genes in DePDMSCs were consistent with the microarray data except for FGF7. Herein, the expression of cell adhesion molecule 1 (CADM1) was upregulated in DePDMSCs more than sevenfold when compared with PDMSCs (Po0.001). Meanwhile, the expression of matrix metallopeptidase 10 (MMP10) in DePDMSCs was upregulated for more than sevenfold (Po0.01), the expression of zinc finger protein 711 (ZNF711) in DePDMSCs was upregulated for more than threefold (Po0.05). Among them, CADM1 was included in the CAMs signaling pathway and could be beneficial to identify the central nodes within the signaling web of ECM on functional basis. 25

Discussion
In contrast to the lineage restriction in differentiated cells, it has been reported that cell fate is interconvertible. 26,27 Just through ceiling culture and without the addition of cytokines, isolated adipocytes can undergo dedifferentiation and acquire multilineage differentiation potency. 28,29 However, it is still unclear whether this phenomenon is a hallmark of cell differentiation programs or displays specificities depending on the stem cell types.
Despite being morphologically and phenotypically similar to uncommitted PDMSCs, DePDMSCs represent a previously undescribed distinct population of stem cells with several distinguished features. A previous study showed that the expression levels of ESCs markers were gradually upregulated after dedifferentiation of adipogenic amniotic fluid stem cells. 30 Generally speaking, stem cells became static and had nearly no proliferative activity after differentiation. It is generally accepted that decreased cell-division ability accompanies long culture times and increased age. 31 Considering the intriguing results in our study, AL cells may return to the cell cycle and proliferate quickly after dedifferentiation, indicating that this proliferative restriction is not permanent and dedifferentiation activates cell cycle progression genes for subsequent proliferation and transdifferentiation. 15 Recent studies performed on ESCs and iPSCs showed enhanced mitochondrial biogenesis after differentiation, 18 but whether the mitochondrial biogenesis in various stem cell models is similar to ESCs or iPSCs is not clear. Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, the regulation of the cell signaling and programmed cell death. 32 Some reports discovered a similar phenomenon in MSCs, but this was limited to BMMSCs. [33][34][35][36][37][38] Studies of adipogenic or osteogenic differentiation of BMMSCs also showed an increase in mitochondrial biogenesis and function (an increased mRNA abundance for MT-CO1, MT-CO2, COX4I1 and ATP5A1) during early steps of the differentiation process. 36,39 Furthermore, evidence also indicates that mitochondrial biogenesis is strongly associated with differentiation, and is accompanied with increased expression of subunits from complexes I, II and III, and a higher mitochondrial activity by significantly increased oxygen consumption. 40 As a consequence, differentiated cells displayed increased mitochondrial mass, a more developed mitochondrial network, and a shift toward OXPHOS to meet their energy demands. [41][42][43] Besides, NNT functions as a high-capacity source of mitochondrial nicotinamide adenine dinucleotide phosphate (NADPH), the mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states, ultimately resulting in increased cellular oxidative stress and impaired morphology and mitochondrial function. 44,45 These co-regulators cooperate to drive mitochondrial biogenesis and oxidative switching by co-activating many transcription factors. In fact, the use of molecules to promote or inhibit mitochondrial biogenesis or function, or by interfering with the expression of mitochondrial biogenesis regulators or proteins involved in mitochondrial function, has been demonstrated to impact stemness and cell differentiation. 18,46,47 To determine whether the converted cells with similar phenotypes are just structural entities or retain their multilineage potency requires further investigation. Then, we converted DePDMSCs to adipocytes, osteocytes and hepatocytes. A previous study showed that the dedifferentiated  adipocytes had adipogenic potency, 29 the results of our study suggest greater adipogenic potency in DePDMSCs than in PDMSCs. Furthermore, another study demonstrated that the dedifferentiated cells could achieve the morphology of other lineages more easily and quickly during the process of transdifferentiation. 48 On the other hand, dedifferentiated BMMSCs can redifferentiate into neural cells, osteocytes and adipocytes, 15,16 the dedifferentiated PDMSCs in our study were able to overcome the mesodermal commitment to other lineages. However, there is no study on the detection of hepatocyte differentiation potency in dedifferentiated MSCs as far as we know. The hepatogenic differentiation ability of DePDMSCs is comparable to PDMSCs in our study. In sum, the successful fate conversion of dedifferentiated PDMSCs was not restricted to related lineages within the same germ layer but notably crossed the lineage boundaries beyond limited cellular conversion. 49 The sequential adipogenic differentiation and dedifferentiation processes resulted in novel stem cells that proliferated faster and retained multilineage potency; however, the mechanism underlying this cross-talk remained to be determined. In this context, we identified a number of differentially expressed genes that were regulated by the complex communication between signaling pathways during adipogenic differentiation and dedifferentiation. Intriguingly, we found a single gene named CADM1 was extremely higher in DePDMSCs when compared with PDMSCs. CADM1 can directly regulate mast cell net adhesion directly through CADM1-dependent adhesion, 25 furthermore, it was strongly correlated with the bone-forming capacity of human MSCs and could be used as a reliable in vitro diagnostic marker. 50 CAMs serve as a well-known signaling pathway for diverse biological processes including cellular interactions, adhesions and micro-environmental decisions. 22 In addition to that, ECM Figure 6 Continued has a critical role in the formation of adipogenically differentiated cells and the differentiated cells started to release the lipid droplets and leave bare network of ECM. 51,52 The ECM components modulate cell behavior and enable the cells to regenerate, proliferate, differentiate, grow, orientate and constrain themselves for perfectible regeneration by cell-cell and cell-ECM interactions. 23 Here, we indicated that the enhanced proliferative ability and differentiation ability of DePDMSCs may attribute to the upregulated CAMs signaling pathway and ECM components. In spite of that, further research is still required to unravel the process in other cell types and to clarify more detailed mechanisms involved in the interplay between these two processes, which progressed as a reprogramming method in ESCs or iPSCs.
In conclusion, we systematically and comprehensively demonstrated that PDMSC-derived AL cells were able to successfully dedifferentiate and acquire a more primitive phenotype under certain culture conditions. In addition, we not only detected alterations in morphology, adipocyte markers and stem cell markers, but we also observed the mitochondrial network during differentiation and dedifferentiation and found that it may serve as a marker of absent or acquired pluripotency in various stem cell models. Furthermore, the dedifferentiated cells entered the cell cycle and retained their multi-potentiality to transdifferentiate into other lineages in response to extrinsic factors. The CAMs signaling pathway and ECM components regulate cell behavior in proliferation and differentiation; consequently, we suggest that biochemical analysis of native adipogenic ECM would be a  Adipogenic differentiation and dedifferentiation of PDMSCs. To induce adipogenesis, the third passage PDMSCs at 1 × 10 5 /well density in 12-well plates were treated with adipogenic medium (OriCell hMSC Adipogenic Differentiation Medium, Cyagen Biosciences, Guangzhou, China) in 12-well plates for 2 weeks. The differentiated PDMSCs were defined as AL cells and then the adipogenic medium was removed from AL cells and replaced by general PDMSCs medium for 1 week. After that, the cells were defined as DePDMSCs at passage 1. Then, DePDMSCs continued to be cultured in general PDMSCs medium to passage 3. After 2 weeks, lipoprotein lipase (FABP4) and peroxisome proliferatoractivated receptor-g (PPARG) were identified by mRNA detection.
RNA extraction and reverse transcription. Using the RNAiso plus kit (TaKaRa, Tokyo, Japan), total RNA was isolated according to the manufacturer's instructions. The RNA was first treated with DNase (TaKaRa) in a 10 μl reaction with 5 × gDNA Easer Buffer (2 μl), gDNA Easer (1 μl) and total RNA (1 μg). The reaction was conducted at 42°C for 2 min. For the mRNAs, the PrimeScript RT reagent Kit (TaKaRa) was used for reverse transcription (RT) in a total volume of 20 μl with 4 μl 5 × PrimeScript Buffer PCR buffer, 1 μl PrimeScript RT enzyme mix I, 1 μL RT Primer Mix and 10 μl of the RNA sample. The RT reaction started with a 15-min incubation period at 37°C and ended after a 5-s enzyme-denaturing step at 85°C.

Figure 7 Continued
Adipogenic PDMSCs are not lineage restricted C Hu et al cytochrome P (CYP)1A2, CYP3A4 and albumin (ALB) were detected. The differentially expressed genes of microarray in DePDMSCs and PDMSCs were confirmed by RT-QPCR. The reference gene β-actin was used as a relative control for the expression levels. The primers for the target products were designed as in Supplementary Table S4.
Flow cytometry for surface antigen expression. Culture-expanded cells were washed with PBS-containing 0.3% (w/v) bovine serum albumin (BSA) and the concentration was adjusted to 1 × 10 6 cells/100 μl. PDMSCs at passage 3, AL cells and DePDMSCs at passage 3 were examined for mesenchymal and hematopoietic marker expression of surface antigens by incubating with the antibodies CD14-phycoerythrin (PE), CD29-fluorescein isothiocyanate (FITC), CD44-FITC, CD105-PE and HLA-DR-FITC (Abcam, Cambridge, UK). Antibodies including mouse IgG2a-FITC, mouse IgG2a-PE/Cy5.5, mouse IgG1-FITC and rat IgG2b-FITC (Abcam) were used as isotype controls. After being labeled with antibodies in the dark at room temperature for 30 min, cells were washed twice with PBS. Flow cytometry was conducted using a BD LSR II (Beckman Coulter, Los Angeles, CA, USA), and the data were analyzed using BD FACSDiva software. Cell lysis collection. Cells were rinsed with ice-cold PBS, and then ice-cold mitochondrial lysis buffer with freshly added phosphatase and protease inhibitors were added (0.2 ml per well of a 12-well plate). Adherent cells were scraped off the dish with a cell scraper and the suspension was transferred into a centrifuge tube and gently rocked for 15-30 min at 4°C. The lysate was centrifuged at 14 000 × g for 20 min at 4°C and the supernatant was immediately transferred into fresh prechilled micro-centrifuge tubes. The lysate was diluted at 1:4 for BCA assays with a spectrophotometer (Beckman Coulter Multimode Detector DTX880, Beckman Coulter). Finally, the lysate was aliquoted and stored at ⩽ − 70°C.  Before the assay, the samples were extracted using mitochondrial lysis buffer with protease inhibitors (EMD Millipore) and phosphatase inhibitors (EMD Millipore) according to the recommend protocol. Briefly, OXPHOS assay plates were washed with wash buffer, sealed and mixed on an orbital plate shaker for 10 min at room temperature. The wash buffer was decanted and 25 μl of control, mitochondria lysis buffer and samples were added in each well. Then, 25 μl beads were added into each well and incubated for 2 h at room temperature on an orbital shaker. After incubation, well contents were removed via the washing instructions provided by the protocol. Fifty microliters of detection antibodies were then added to the wells and incubated with samples for 1 h at room temperature while shaking. After incubation, well contents were removed as previously described and 50 μl streptavidinphycoerythrin was added to each well. The samples were incubated with streptavidin-phycoerythrin for 30 min at room temperature. After the incubation period, samples were washed as previously described and resuspended in Sheath Fluid. Plates were run on the Luminex MagPix machine and data were collected using the Luminex xPONENT software (v. 4.2).
Analysis of cell proliferation. For osteogenic differentiation, PDMSCs and DePDMSCs at passage 3 at 5 × 10 4 / well in 12-well plates were treated with osteogenic medium (OriCell hMSC Osteogenic Differentiation Medium, Cyagen Biosciences) as per the manufacturer's protocol. The medium was changed three times per week. After 2 weeks, osteogenic differentiation was evaluated by Alizarin Red S staining (Sigma) and detection of RUNX2 and OC mRNA. PDMSCs cultured in normal growth medium and immortalized human fetal osteoblastic cells (hFOB1.19 cell line, Cell Bank of the Chinese Academy of Sciences, Shanghai, China) served as controls.
Periodic acid-Schiff staining. The medium was taken out from culture plates and cells were washed with PBS three times. Then, the cells were fixed using 4% paraformaldehyde (MultiSciences Biotech) for 30 min. After being oxidized in periodic acid (Sigma) for 10 min and washed three times with PBS, cells were treated with Schiff's reagent (Sigma) for 15 min. Afterwards, cells were rinsed in PBS for 10 min and counterstained with hematoxylin (Sigma). The staining results were observed under an inverted phase-contrast microscope (ECLIPAS TS100, Nikon).
Uptake and secretion of indocyanine green (ICG). Hepatogenesis differentiation medium was replaced with L-DMEM medium containing 1 mg/ml ICG (Sigma). After incubation at 37°C for 15 min, the cells were rinsed three times with PBS and ICG uptake was measured using an inverted microscope. Dishes were refilled with general PDMSCs medium for 6 h and color changes were examined.
Gene expression profiling of adipogenic differentiated and dedifferentiated cells. After RNA extraction, all quantification and microarray experiments were performed at the Shanghai Biotechnology Corporation using Affymetrix PrimeView human gene expression (Affymetrix, Santa Clara, CA, USA). RNA integrity was analyzed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Qualified total RNA was further purified using an RNeasy micro kit (Qiagen, Hilden, Germany) and an RNase-Free DNase kit (QIAGEN). The RNA purity and concentration were determined using a Nanodrop 2000 (Nanodrop Products, Wilmington, DE, USA). Total RNA was amplified, labeled and purified using the GeneChip 3'IVT Express Kit (Affymetrix) following the manufacturer's instructions to obtain biotin-labeled cRNA. Array hybridization and washes were performed using the GeneChip Hybridization, Wash and Stain Kit (Affymetrix) in the Hybridization Oven 645 (Affymetrix) and Fluidics Station 450 (Affymetrix) following the manufacturer's instructions. Slides were scanned by the GeneChip Scanner 3000 (Affymetrix) and Command Console Software 4.0 (Affymetrix) with default settings. The scanned images were first assessed by visual inspection, and then analyzed to generate raw data files that were saved as CEL files using the default settings of GCOS 1.4. The raw data were normalized using an RMA algorithm in the Gene Spring Software 11.0 (Agilent Technologies). Expression profiling was performed for nine samples (n = 3 donors) subdivided into three groups: 3 × (PDMSCs), 3 × (AL cells) and 3 × (DePDMSCs P3). The microarray data sets have been submitted to the Gene Expression Omnibus (GEO) database and are accessible via the GEO ID: GSE73964.
Data normalization, selection criteria and analysis strategy. First, we were interested in genes whose expression was significantly up-or downregulated during the course of adipogenic differentiation. Thus, in the first step, each of the three AL cell GeneChips were compared with each of the three PDMSC GeneChips for comparative gene expression analysis. Genes were selected as differentially expressed on the basis of specific change call and fold change (FC) criteria. Changes in the P-value o0.01 and the FC limit 42 or o − 2 were calculated for the mean FC of three comparisons to allow the selection of genes that were differentially expressed during adipogenesis. Next, we compared AL cell gene expression values with the corresponding values from DePDMSCs. The differentially expressed genes were selected according to the same criteria mentioned above. Third, we compared PDMSC gene expression values with the corresponding values from DePDMSCs. All differentially expressed genes were uploaded to the Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 and analyzed according to the default set of statistical parameters. 53 DAVID and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for the evaluation and statistical analysis of genes. 54