Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.

The present study was carried out to investigate and compare the in vitro differentiation potential of mesenchymal stem cells (MSCs) isolated from human dental tissues (pulp, papilla, and follicle) of the same donor. MSCs were isolated from dental tissues (pulp, papilla, and follicle) following digestion method and were analyzed for the expression of pluripotent markers and cell surface markers. All three types of MSCs were evaluated for their potential to differentiate into mesenchymal lineages. Further, the MSCs were differentiated into pancreatic β cell-like cells using multistep protocol and characterized for the expression of pancreatic lineage specific markers. Functional properties of differentiated pancreatic β cell-like cells were assessed by dithizone staining and glucose challenge test. All three types of MSCs showed fibroblast-like morphology upon culture and expressed pluripotent, and mesenchymal cell surface markers. These MSCs were successfully differentiated into mesenchymal lineages and transdifferentiated into pancreatic β cell-like cells. Among them, dental follicle derived MSCs exhibits higher transdifferentiation potency toward pancreatic lineage as evaluated by the expression of pancreatic lineage specific markers both at mRNA and protein level, and secreted higher insulin upon glucose challenge. Additionally, follicle-derived MSCs showed higher dithizone staining upon differentiation. All three types of MSCs from a single donor possess similar cellular properties and can differentiate into pancreatic lineage. However, dental follicle derived MSCs showed higher potency toward pancreatic lineage than pulp and papilla derived MSCs, suggesting their potential application in future stem cell based therapy for the treatment of diabetes.

Background The dentin is a tissue, which is formed by odontoblasts at the pulp interface of the teeth that supports the enamel. Odontoblasts, the cranial neural crest cells are derived from ectodermal mesenchymal stem cells (MSCs) and are long and polarized cells. They are present at the outer surface of dentin and play a prominent role about dentin formation. Recently, attention has been focused on induction of odontoblast using various type of MSCs and effects of the 17ß-estradiol supplementation. In this study, we establish an efficient odonto/osteoblast differentiation protocol using 17ß-estradiol supplementation while comparing the odonto/osteoblast ability of various dental MSCs. Methods Same donor derived four types of dental MSCs namely dental pulp stem cells (DPSCs), stem cells from apical papilla (SCAP), dental follicle stem cells (DFSCs), and periodontal ligament stem cells (PDLSCs) were evaluated for their stemness characteristics and potency towards odonto/osteoblast (Induced odonto/osteoblast) differentiation. Then 17ß-estradiol supplementation of 0 and 10 µM was applied to the odonto/osteoblast differentiation media for 14 days respectively. Furthermore, mRNA and protein levels of odonto/osteoblast markers were evaluated. Results All of the experimental groups displayed stemness characteristics by showing adipocyte and chondrocyte differentiation abilities, expression for cell surface markers and cell proliferation capacity without any significant differences. Moreover, all dental derived MSCs were shown to have odonto/osteoblast differentiation ability when cultured under specific conditions and also showed positive expression for odontoblast markers at both mRNA and protein level. Among all, DPSCs revealed the higher differentiation potential than other dental MSCs. Furthermore, odonto/osteoblast differentiation potential was enhanced by supplementing the differentiation media with 17ß-estradiol (E2). Conclusions Thus, DPSCs possess higher odonto/osteogenic potential than the SCAPs, DFSCs, PDLSCs and their differentiation capacity can by further enhanced under E2 supplementation.

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We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro. Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro, the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro. Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.

A decrease in the activity of choline acetyltransferase, the enzyme responsible for acetylcholine synthesis in the cholinergic neurons cause neurological disorders involving a decline in cognitive abilities, such as Alzheimer’s disease. Mesenchymal stem cells (MSCs) can be used as an efficient therapeutic agents due to their neuronal differentiation potential. Different source derived MSCs may have different differentiation potential under different inductions. Various in vitro protocols have been developed to differentiate MSCs into specific neurons but the comparative effect of different protocols utilizing same source derived MSCs, is not known. To address this issue, dental pulp derived MSCs (DPSCs) were differentiated into cholinergic neurons using three different protocols. In protocol I, DPSCs were pre-induced with serum-free ADMEM containing 1 mM of β-mercaptoethanol for 24 h and then incubated with 100 ng/ml nerve growth factor (NGF) for 6 days. Under protocol II, DPSCs were cultured in serum-free ADMEM containing 15 µg/ml of D609 (tricyclodecan-9-yl-xanthogenate) for 4 days. Under protocol III, the DPSCs were cultured in serum-free ADMEM containing 10 ng/ml of basic fibroblast growth factor (bFGF), 50 µM of forskolin, 250 ng/ml of sonic hedgehog (SHH), and 0.5 µM of retinoic acid (RA) for 7 days. The DPSCs were successfully trans-differentiated under all the protocols, exhibited neuron-like morphologies with upregulated cholinergic neuron-specific markers such as ChAT, HB9, ISL1, BETA-3, and MAP2 both at mRNA and protein levels in comparison to untreated cells. However, protocol III-induced cells showed the highest expression of the cholinergic markers and secreted the highest level of acetylcholine.

Previously, we reported the successful regeneration of injured peripheral nerves using human dental pulp stem cells (DPSCs) or differentiated neuronal cells from DPSCs (DF-DPSCs) in a rat model. Here, we attempted to evaluate oxidative stress and supraspinal neuro-inflammation in rat brain after sciatic nerve injury (SNI). We divided our experimental animals into three SNI groups based on time. The expression of a microglial (Iba1) marker and reactive oxygen species (ROS) was lower in DPSCs and higher in DF-DPSCs. In contrast, the expression of an astroglial (GFAP) marker was higher in DPSCs and lower in DF-DPSCs at 2 weeks. However, the expression of ROS, Iba1 and GFAP gradually decreased at 8 and 12 weeks in the SNI DPSCs and DF-DPSCs groups compared to the SNI control. Furthermore, anti-inflammatory cytokine (IL-4 and TGF-β) expression was lower at 2 weeks, while it gradually increased at 8 and 12 weeks after surgery in the SNI DPSCs and DF-DPSCs groups. Similarly, SNI DPSCs had a high expression of pAMPK, SIRT1 and NFkB at the onset of SNI. However, 12 weeks after surgery, pAMPK and SIRT1 expression levels were higher and NFkB was down-regulated in both DPSCs and DF-DPSCs compared to the control group. Finally, we concluded that DPSCs responded early and more efficiently than DF-DPSCs to counterbalance peripheral nerve injury (PNI)-induced oxidative stress and supraspinal neuro-inflammation in rat brain.

Stem/progenitor cell-based regenerative medicine using the osteoblast differentiation of mesenchymal stem cells (MSCs) is regarded as a promising approach for the therapeutic treatment of various bone defects. The effects of the osteogenic differentiation of stem/progenitor cells on osteoclast differentiation may have important implications for use in therapy. However, there is little data regarding the expression of osteoclastogenic proteins during osteoblastic differentiation of human periosteum-derived cells (hPDCs) and whether factors expressed during this process can modulate osteoclastogenesis. In the present study, we measured expression of RANKL in hPDCs undergoing osteoblastic differentiation and found that expression of RANKL mRNA was markedly increased in these cells in a time-dependent manner. RANKL protein expression was also significantly enhanced in osteogenic-conditioned media from hPDCs undergoing osteoblastic differentiation. We then isolated and cultured CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood (UCB) mononuclear cells (MNCs) and found that these cells were well differentiated into several hematopoietic lineages. Finally, we co-cultured human trabecular bone osteoblasts (hOBs) with CD34+ HSCs and used the conditioned medium, collected from hPDCs during osteoblastic differentiation, to investigate whether factors produced during osteoblast maturation can affect osteoclast differentiation. Specifically, we measured the effect of this osteogenic-conditioned media on expression of osteoclastogenic markers and osteoclast cell number. We found that osteoclastic marker gene expression was highest in co-cultures incubated with the conditioned medium collected from hPDCs with the greatest level of osteogenic maturation. Although further study will be needed to clarify the precise mechanisms that underlie osteogenic-conditioned medium-regulated osteoclastogenesis, our results suggest that the osteogenic maturation of hPDCs could promote osteoclastic potential