ren Li

Upregulation of p16INK4A promotes cellular senescence of bone marrow-derived mesenchymal stem cells from systemic lupus erythematosus patients.

Previous studies have indicated that bone marrow-derived mesenchymal stem cells (MSCs) from patients with systemic lupus erythematosus (SLE) exhibited impaired proliferation, differentiation, and immune modulation capacities. Thus, MSCs may be associated with the pathogenesis of SLE. The aim of this study was to determine whether MSCs from SLE patients were senescent and to determine the mechanism underlying this phenomenon. MSCs from both untreated and treated SLE patients showed characteristics of senescence. The expression of p16(INK4A) was significantly increased, whereas levels of CDK4, CDK6 and p-Rb expression were decreased in the MSCs from both untreated and treated SLE patients. Knockdown of p16(INK4A) expression reversed the senescent features of MSCs and upregulated TGF-β expression. In vitro, when purified CD4+ T cells were incubated with p16(INK4A)-silenced SLE MSCs, the percentage of regulatory T cells was significantly increased. Further, we have found that p16(INK4A) promotes MSC senescence via the suppression of the extracellular signal regulated kinase (ERK) pathway. p16(INK4A) knockdown up-regulated ERK1/2 activation. Our results demonstrated that MSCs from SLE patients were senescent and that p16 (INK4A) plays an essential role in the process by inhibiting ERK1/2 activation.

TNF-α triggers osteogenic differentiation of human dental pulp stem cells via the NF-κB signalling pathway.

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) characterised by self‐renewal and multi‐lineage differentiation, including chondrocytes, adipocytes, neural cells and osteoblasts, which make it an attractive choice for tissue engineering purposes. Tumour necrosis factor α (TNF‐α) had the positive effect on the mineralisation of bone marrow MSCs and stromal cells derived from human adipose tissue. However, the effect of TNF‐α on DPSCs is unclear. We found that TNF‐α activated the NF‐κB pathway during the osteogenic differentiation of DPSCs. TNF‐α also increased mineralisation and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), runt‐related transcription factor 2 (RUNX2) and collagen type I (COL I) during this process. PDTC, an NF‐κB inhibitor, blocked the osteogenic differentiation induced by TNF‐α. No effect of TNF‐α on proliferation of DPSCs or cell cycle was detected. In summary, TNF‐α promotes mineralisation and mineralisation‐related gene expression through the NF‐κB signalling pathway in DPSCs, which may provide a foundation for autologous transplantation of DPSCs.

Insulin‐like growth factor 1 can promote proliferation and osteogenic differentiation of human dental pulp stem cells via mTOR pathway

Insulin‐like growth factor 1 (IGF‐1) is a multifunctional peptide that can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). However, it remains unclear whether IGF‐1 can promote osteogenic differentiation of human dental pulp stem cells (DPSCs). In our study, DPSCs were isolated from the impacted third molars, and treated with IGF‐1. Osteogenic differentiation abilities were investigated. We found that IGF‐1 activated the mTOR signaling pathway during osteogenic differentiation of DPSCs. IGF‐1 also increased the expression of runt‐related transcription factor 2 (RUNX2), osteocalcin (OCN), osterix (OSX) and collagen type I (COL I) during this process. Rapamycin, an mTOR inhibitor, blocked osteogenic differentiation induced by IGF‐1. Meanwhile, CCK‐8 assay and flow cytometry results demonstrated that 10–200 ng/mL IGF‐1 could enhance proliferation ability of DPSCs and 100 ng/mL was the optimal concentration. In summary, IGF‐1 could promote proliferation and osteogenic differentiation of DPSCs via mTOR pathways, which might have clinical implications for osteoporosis.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Downregulate Inflammatory Responses by Shifting the Treg/Th17 Profile in Experimental Colitis

Background/Aims: The aim of this study was to evaluate the effect and mechanisms of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on immune responses in murine colitis. Methods: Mice with dextran sulfate sodium (DSS)-induced colitis were injected intraperitoneally with hUC-MSCs or human bone marrow-derived MSCs. The cytokine levels from lamina propria mononuclear cells (LPMCs) and colon tissue were measured using ELISA. Treg and Th17 cells were analyzed using flow cytometry. The proliferation of LPMCs was assessed using Cell Counting Kit-8. Results: hUC-MSCs ameliorate DSS-induced colitis via the downregulation of colon inflammatory responses. Furthermore, hUC-MSCs adjusted modulation of Treg/Th17 cells in the spleen and mesenteric lymph nodes. hUC-MSCs also inhibited LPMCs in vitro.Conclusion: hUC-MSCs may be an alternative source of stem cells and are worthy of study in long-term clinical trials.

Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs)

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-β-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16INK4A were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16INK4A expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16INK4A short interfering RNA. The DNA damage response and p16INK4A pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy.

p16INK4A mediates age-related changes in mesenchymal stem cells derived from human dental pulp through the DNA damage and stress response

Mesenchymal stem cells derived from human dental pulp (DP-MSCs) are characterized by self-renewal and multi-lineage differentiation, which play important roles in regenerative medicine. Autologous transfers, as non-immunogenic, constitute the safest approach in cellular transplantations. However, their use may be limited by age-related changes. In the study, we compared DP-MSCs isolated from human in five age groups: 5-12 y, 12-20 y, 20-35 y, 35-50 y, and >50 y. We tested the effect of age on proliferation, differentiation, senescence-associated β-galactosidase (SA-β-gal), cell cycle and programmed cell death. DP-MSCs showed characteristics of senescence as a function of age. Meanwhile, the expression of p16(INK4A) and γ-H2A.X significantly increased with age, whereas heat shock protein 60 (HSP60) was decreased in the senescent DP-MSCs. Reactive oxygen species (ROS) staining showed the number of ROS-stained cells and the DCFH fluorescent level were higher in the aged group. Further we examined the senescence of DP-MSCs after modulating p16(INK4A) signaling. The results indicated the dysfunction of DP-MSCs was reversed by p16(INK4A) siRNA. In summary, our study indicated p16(INK4A) pathway may play a critical role in DP-MSCs age-related changes and the DNA damage response (DDR) and stress response may be the main mediators of DP-MSCs senescence induced by excessive activation of p16(INK4A) signaling.

Clinical and Biological Significance of Never in Mitosis Gene A-Related Kinase 6 (NEK6) Expression in Hepatic Cell Cancer

Nek6 is a cell cycle regulatory gene, which can control cell proliferation and survival. Recent studies suggested that desregulation of Nek6 expression plays a key role in oncogenesis. This study was aimed to investigate the potential roles of Nek6 in hepatocellular carcinoma (HCC) development. Immunohistochemistry and Western blot analysis was performed for Nek6 in 80 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were performed to determine the prognostic significance of Nek6 in HCC. In addition, Nek6 expression vector was used to detect its role in cell cycle control. Nek6 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Nek6 was associated with histological grade and the level of alpha fetal protein, and Nek6 was positively correlated with proliferation marker Ki-67. Univariate analysis showed that Nek6 expression was associated with poor prognosis. Multivariate analysis indicated that Nek6 and Ki-67 protein expression was an independent prognostic marker for HCC. While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Nek6 was upregulated. Overexpression Nek6 in Huh7 cell could promote the cell cycle. In conclusion, Nek6 is involved in the pathogenesis of hepatocellular carcinoma. It may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.

Suppression of the PI3K—Akt pathway is involved in the decreased adhesion and migration of bone marrow-derived mesenchymal stem cells from non-obese diabetic mice

T1DM (type 1 diabetes mellitus) is an autoimmune disease characterized by T‐cell‐mediated damage of islet β‐cells. The pathology of NOD (non‐obese diabetic) mouse involves the insulitis induced by infiltration of T‐cells, a similar pathogenic mechanism in T1DM patient. BM‐MSCs (bone marrow mesenchymal stem cells) are multipotent progenitor cells that can be isolated from a number of sources. Recent studies have shown that transplantation of MSCs to the NOD mice could prevent the process and have the therapeutic effects on T1DM. In our studies, we have found that migration and adhesion of BM‐MSCs from NOD mice were suppressed compared with the BM‐MSCs from ICR (imprinting control region) mice, accompanying with the abnormal distribution of FAK (focal adhesion kinase) and F‐actin (filamentous actin). Further, we have found that the activation of PI3K (phosphoinositide 3‐kinase)–Akt pathway was suppressed in BM‐MSCs from NOD mice. When the PI3K—Akt pathway was inhibited by LY294002, the adhesion and migration of BM‐MSCs from ICR mice were suppressed as well. These results indicated that the suppression of PI3K—Akt pathway is involved in the decreased adhesion and migration of BM‐MSCs from NOD mice.

Pyruvate Kinase M2 Regulates Apoptosis of Intestinal Epithelial Cells in Crohn’s Disease

BackgroundPyruvate kinase M2 (PKM2), a key glycolytic enzyme, is involved in multiple cellular processes including apoptosis. Recently increased fecal PKM2 has been found in Crohn’s disease (CD), but little is known regarding its function in the pathophysiology of the disease.AimThe intestinal expression of PKM2 and its involvement in CD was investigated.MethodsPyruvate kinase M2 expression in mucosal biopsies from patients with CD and normal controls was detected by immunohistochemistry. A murine model of colitis induced by trinitrobenzenesulphonic acid (TNBS) was established and expression of PKM2, B cell lymphoma-extra large (Bcl-xl), active caspase-3 as well as cleaved poly (ADP-ribose) polymerase (PARP) was examined for association of PKM2 with intestinal epithelial cell (IEC) apoptosis. Furthermore, we treated human IEC line HT-29 by tumor necrosis factor-α (TNF-α) and used RNA interference to analyze the role of PKM2 in IEC apoptosis.ResultsIntestinal expression of PKM2 was higher in patients with CD compared with normal controls mainly locating in IECs. In TNBS-induced colitis, up-regulation of PKM2 was accompanied by the elevated expression of Bcl-xl, active caspase-3, and cleaved PARP. PKM2 was co-localized with active caspase-3 in IECs marked by E-cadherin, suggesting its role in IEC apoptosis. Expression of PKM2 and Bcl-xl in TNF-α-induced HT-29 cells was increased, while TNF-α had no effect on cellular localization of PKM2. Furthermore, knockdown of PKM2 by siRNA could inhibit expression of Bcl-xl but enhance apoptosis in TNF-α-treated HT-29 cells.ConclusionThe up-regulation of PKM2 might protect IECs against apoptosis possibly through Bcl-xl in CD, indicating its important role in the pathophysiology of CD.

Roles of Wnt/β-catenin signaling in retinal neuron-like differentiation of bone marrow mesenchymal stem cells from nonobese diabetic mice.

Recent studies have shown that mesenchymal stem cells (MSCs) are expected to become promising therapeutic agents for the treatment of diabetic retinopathy (DR); moreover, we previously demonstrated that bone marrow (BM)-MSCs from nonobese diabetic (NOD) mice (an ideal DR model) had abnormal migration and adhesion. So, we hypothesized that NOD-MSCs also have abnormal retinal neuron-like differentiation potential. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. Western blot analysis and immunofluorescence both showed that the level of retinal neuron-like markers, such as glial fibrillary acidic protein, neuron-specific nuclear protein, tyrosine hydroxylase, Thy-1, glutamine synthetase, and rhodopsin was lower in NOD-MSCs than in imprinting control region MSCs. Furthermore, we explored the precise mechanisms controlling this change in NOD-MSCs. The expression levels of some important member proteins in Wnt/β-catenin signaling were determined and suggested the downregulation of Wnt/β-catenin signaling with retinal neuron-like differentiation of NOD-MSCs. Incubation of NOD-MSCs in medium supplemented with human recombinant Wnt1 resulted in a significant upregulation of retinal neuron-like markers, and the effects of Wnt1 were dose-dependent. Taken together, our study indicated that the inhibition of Wnt/β-catenin signaling in NOD-MSCs after induction could contribute to the abnormal retinal neuron-like differentiation. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for DR.Recent studies have shown that mesenchymal stem cells (MSCs) are expected to become promising therapeutic agents for the treatment of diabetic retinopathy (DR); moreover, we previously demonstrated that bone marrow (BM)-MSCs from nonobese diabetic (NOD) mice (an ideal DR model) had abnormal migration and adhesion. So, we hypothesized that NOD-MSCs also have abnormal retinal neuron-like differentiation potential. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. Western blot analysis and immunofluorescence both showed that the level of retinal neuron-like markers, such as glial fibrillary acidic protein, neuron-specific nuclear protein, tyrosine hydroxylase, Thy-1, glutamine synthetase, and rhodopsin was lower in NOD-MSCs than in imprinting control region MSCs. Furthermore, we explored the precise mechanisms controlling this change in NOD-MSCs. The expression levels of some important member proteins in Wnt/β-catenin signaling were determined and suggested the downregulation of Wnt/β-catenin signaling with retinal neuron-like differentiation of NOD-MSCs. Incubation of NOD-MSCs in medium supplemented with human recombinant Wnt1 resulted in a significant upregulation of retinal neuron-like markers, and the effects of Wnt1 were dose-dependent. Taken together, our study indicated that the inhibition of Wnt/β-catenin signaling in NOD-MSCs after induction could contribute to the abnormal retinal neuron-like differentiation. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for DR.