3.1 The global changes of metabolomic and proteomic landscape during hDPSCs differentiation
The primary human hDPSCs were cultured with standard osteo/odontogenic medium for two weeks and their differential capacity was examined. The cultured hDPSCs were positive for CD29, CD90 and negative for CD45, CD34 accorded with the criteria for mesenchymal stem cells (Fig. 1A). The induced ALP activity and calcium nodules accumulation were detected with osteo/odontogenic medium (Fig. 1B). Some osteo/odontogenic genes such as DMP1, DSPP, OCN, and OSX were examined to be upregulated in the induction medium (Fig. 1C).
Then, the fluctuations of intracellular metabolites were characterized during hDPSCs differentiation in different phases (day 0, 1, 3, 7, 14) by untargeted metabolomics relative-quantitative analysis with UHPLC-MS/MS (Fig. 2A). Metabolic analyses showed that a total of 194 differentially expressed metabolites (DEMs) was displayed in intracellular concentrations during hDPSCs differentiation. The metabolite profiles were related to pathways including glycolysis, mitochondria oxidation, fatty acid metabolism, amino acid and nucleotide biosynthesis. Remarkably, some of the essential energy-generating and biosynthetic pathways exhibited significant upregulation during early stage of differentiation (0–7 day) and subsequently reduced or stable in the late stage (14 day). Some metabolites associated with lipid acid degradation were largely decreased during osteo/odontogenic differentiation. Results of PCA of metabolomic data showed that biological replicates were mostly clustered together, and samples from different time points were separately distributed, except that the three groups of 0, 1, and 3 D showed partly overlapped, which support the two key time points 7 and 14 day in analyzing the cellular differentiation process (Fig. 2B). The KEGG metabolic pathways were analyzed to be enriched in purine metabolism, pyrimidine metabolism, galactose metabolism, some amino acid metabolism, glycerophospholipid metabolism, and TCA cycle (P < 0.01) (Fig. 2C).
In parallel to metabolomic measurement, the widespread proteomic patterns in hDPSCs after induction for 0, 7, 14 days were investigated by DIA proteomic analysis. The repeatability of protein samples was eligible (Fig. 3B). A total of 4706 proteins were quantified to be DEPs after induction in hDPSCs. These DEPs classified by GO annotation in biological process category were mainly associated with cellular process, single-organism process and metabolic process (Fig. 3C). In the metabolic process, the DEPs were participated in pathways similar as in metabolome including glycolysis, oxidative phosphorylation, fatty acid metabolism, nucleotides and amino acid biosynthesis (Fig. 3A). Most of them showed consistent tendency with the metabolic profiles. The integrated metabolomic-proteomic analysis revealed a widespread and dynamic remodeling of metabolism during hDPSCs osteo/odontogenic differentiation, and many of them were related to glycolysis, TCA cycle, fatty acid metabolism, nucleotide metabolism, and amino acid synthesis.
3.2 Upregulation of glycolysis and TCA cycle during hDPSCs differentiation
The glucose consumption and lactate release were extremely increased throughout the whole stage of osteo/odontogenic differentiation. Levels of upper glycolytic intermediates (alpha-D-glucose, glucose-6-phosphate, fructose-1,6-bisphosphate) displayed increase with the osteo/odontogenic induction in hDPSCs. The protein levels of relevant enzymes such as phosphoglucomutase-1 (PGM1), hexokinase-1 (HK1), pyruvate kinase (PKLR and PKM), pyruvate dehydrogenase (PDHA1), displayed an increase to activate the glycolysis process. An early upregulation of TCA cycle in mitochondria was found during osteo/odontogenic differentiation of hDPSCs. The TCA cycle intermediates, including citrate, cis-aconitate, succinate, and malate, displayed a coordinated increase peaking around 7 days in early differentiation of hDPSCs (Fig. 4A). These alterations of metabolites were consistent with the increased expression of related TCA enzymes. Specifically, the isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH) displayed an increase in protein abundance. Some TCA enzyme subunit increased along with the TCA cycle activation, such as components of succinate dehydrogenase (SDHA), succinate-CoA ligase (SUCLG1), and 2-oxoglutarate dehydrogenase complex (OGDH) (Fig. 4B). The increased energy-generation and biosynthesis displayed with large accumulation of ATP molecules.
3.3 Stimulation of fatty acid degradation during hDPSCs differentiation
The fatty acid synthesis displayed no significant alterations during hDPSCs differentiation. The levels of acetyl-CoA and fatty acid palmitaldehyde and palmitic acid were progressively decreased, and the expression of fatty acid synthase (FASN) was no difference along the differentiation. On the other hand, the fatty acid degradation displayed highly upregulation when hDPSCs was differentiated. The L-palmitoylcamitine degraded from palmitic acid was accumulated along the hDPSCs differentiation. The final degraded product CoA was significantly increased (Fig. 5A). Meanwhile, the participated degradation enzymes, such as long-chain-fatty-acid-CoA ligase 1 (ACSL1), ligase 3 (ACSL3), hydroxyacyl-coenzyme A dehydrogenase (HADH), and medium-chain specific acyl-CoA dehydrogenase (ACADM) were significantly increased (Fig. 5B).
3.4 Alterations in de novo nucleotide biosynthesis during hDPSCs differentiation
Consistent with the glucose metabolic pattern in early differential stage, nucleotide metabolism increased biosynthesis of purine and pyrimidine fulfilled the demand of nucleotide abundance during cell differentiation and signal transduction. Purine metabolism related metabolites in adenine synthesis (including AMP, ADP, ATP, adenosine and cAMP) and guanine synthesis (including GDP, dGDP, dGTP) were significantly upregulated during the early differentiation process before Day 7, while inosine and guanosine manifested an upward trend during the whole osteo/odontogenic differentiation (Fig. 6A). With regard to pyrimidine metabolism, the nucleotides and their precursors in cytosine synthesis (CMP, cytidine), uridine synthesis (UMP, deoxyuridine, uridine) and thymine synthesis (thymidine) were upregulated throughout the whole stage of osteo/odontogenic differentiation, while UTP, UDP, UDP-D-glucose and CMP levels decreased after Day 7 (Fig. 6B).
The change of metabolites is usually accompanied with enzyme levels regulation (Fig. 6C). In purine metabolism, levels of adenylate kinase isoenzyme 1 (AK1) was significantly upregulated on Day 14, while adenylate kinase isoenzyme 2 (AK2) reached its bottom on Day 7. In pyrimidine metabolism, deoxyuridine triphosphatase (DUT) was significantly downregulated, which may implying expanded intracellular pools of dUTP during differentiation. Besides, there are several enzymes that participate in both purine and pyrimidine metabolism including NDK and NT5E. Nucleoside diphosphate kinase (NDK) has two isoforms including isoform A (NME1) and B (NME2), was remarkably reduced since early differential stage that implying reducing phosphate exchange between nucleoside triphosphates to nucleoside diphosphates. There is also a co-transcription of NME1 and NME2, named as NME1-NME2, which showed a similar downregulating tendency. And 5'-nucleotidase ecto (NT5E), which catalyzes the conversion of extracellular nucleotides to membrane-permeable nucleosides, was upregulated.
3.5 Amino acid metabolism remodeling during hDPSCs differentiation
Amino acid metabolism exhibited complex behaviors and interactions with glycolysis, TCA cycle and lipid metabolism during hDPSCs differentiation. The enriched regulatory pathway included alanine, aspartate, glutamate, arginine, and glutathione metabolism (Fig. 2C). Most of amino acid were stimulated to biosynthesis during hDPSCs differentiation. As an antioxidant, glutathione is capable to prevent damage caused by reactive oxygen species to maintain cellular physiological activity. Glutathione (GSH) is a tripeptide consisted of a gamma peptide linkage formed by glutamate, cysteine, and glycine that interacts with glutamine and glucose metabolism. The intracellular concentration of glutathione in hDPSCs was significantly upregulated at the early differentiation stage before 7 days induction. Meanwhile, its relevant metabolic intermediates such as 5-L-glutamyl-L-alanine, L-pyroglutamic acid, L-glutamate, L-gamma-glutamylcysteine, and glycine were manifested an upward trend during early differentiation stage (Fig. 7A). Glutathione disulfide (GSSG) is converted to the reduced glutathione by NADPH and generated NADP + catalyzed by glutathione reductase (GSR). The production of NADP + was increased and the expression of GSR was upregulated during hDPSCs differentiation. The increased amount of NADP + maybe consumed to supply cellular activity catalyzed by isocitrate dehydrogenase (IDH1), glucose-6-phosphate 1 (G6PD), and phosphogluconate dehydrogenase (PGD). The conversion of glutathione to glutamate or glycine was dynamic changed with differential expression of catalyzed enzymes microsomal glutathione S-transferase 3 (GST), glutathione S-transferase kappa 1 (GSTK1), and cytosol aminopeptidase (LAP3) (Fig. 7B).