Multilineage Cell Types Research Articles

Dental applications of induced pluripotent stem cells and their derivatives.

Periodontal tissue regeneration is the ideal tactic for treating periodontitis. Tooth regeneration is the potential strategy to restore the lost teeth. With infinite self-renewal, broad differentiation potential, and less ethical issues than embryonic stem cells, induced pluripotent stem cells (iPSCs) are promising cell resource for periodontal and tooth regeneration. This review summarized the optimized technologies of generating iPSC lines and application of iPSC derivatives, which reduce the risk of tumorigenicity. Given that iPSCs may have epigenetic memory from the donor tissue and tend to differentiate into lineages along with the donor cells, iPSCs derived from dental tissues may benefit for personalized dental application. Neural crest cells (NCCs) and mesenchymal stem or stomal cells (MSCs) are lineage-specific progenitor cells derived from iPSCs and can differentiate into multilineage cell types. This review introduced the updated technologies of inducing iPSC-derived NCCs and iPSC-derived MSCs and their application in periodontal and tooth regeneration. Given the complexity of periodontal tissues and teeth, it is crucial to elucidate the integrated mechanisms of all constitutive cells and the spatio-temporal interactions among them to generate structural periodontal tissues and functional teeth. Thus, more sophisticated studies in vitro and in vivo and even preclinical investigations need to be conducted.

Extracellular vesicle-encapsulated miR-22-3p from bone marrow mesenchymal stem cell promotes osteogenic differentiation via FTO inhibition

BackgroundBone marrow mesenchymal stem cells (BMSCs) exhibit the capacity to self-renew and differentiate into multi-lineage cell types, including osteoblasts, which are crucial regulators of fracture healing. Thus, this study aims to investigate the effect of microRNA (miR)-22-3p from BMSC-derived EVs on osteogenic differentiation and its underlying mechanism.MethodsExtracellular vesicles (EVs) were isolated from BMSCs and taken up with BMSCs. Dual-luciferase reporter gene assay was used to verify the binding relationship between miR-22-3p and FTO. Loss- and gain-of-function experiments were performed to determine the roles of EV-delivered miR-22-3p and FTO in osteogenic differentiation as well as their regulatory role in the MYC/PI3K/AKT axis. To determine the osteogenic differentiation, ALP and ARS stainings were conducted, and the levels of RUNX2, OCN, and OPN level were determined. In vivo experiment was conducted to determine the function of EV-delivered miR-22-3p and FTO in osteogenic differentiation, followed by ALP and ARS staining.ResultsmiR-22-3p expression was repressed, while FTO expression was elevated in the ovariectomized mouse model. Overexpression of miR-22-3p, EV-delivered miR-22-3p, increased ALP activity and matrix mineralization of BMSCs and promoted RUNX2, OCN, and OPN expressions in BMSCs. miR-22-3p negatively targeted FTO expression. FTO silencing rescued the suppressed osteogenic differentiation by EV-delivered miR-22-3p inhibitor. FTO repression inactivated the MYC/PI3K/AKT pathway, thereby enhancing osteogenic differentiation both in vivo and in vitro.ConclusionIn summary, miR-22-3p delivered by BMSC-derived EVs could result in the inhibition of the MYC/PI3K/AKT pathway, thereby promoting osteogenic differentiation via FTO repression.

Fasudil enhanced differentiation of BMSCs in vivo and vitro, involvement of P38 signaling pathway

Bone mesenchymal stem cells (BMSCs) are a well-known donor graft source due to their potential for self-renewal and differentiation into multi-lineage cell types, including osteoblasts that are critical for fracture healing. Fasudil (FAS), a Rho kinase inhibitor, has been proven to induce the differentiation of bone marrow stem cells (BMSCs) into neuron-like cells. However, its role in the osteogenesis of BMSCs remain uncertain. Herein, we for the first time studied the effects of FAS on osteogenic differentiation in a mouse fracture model and further explored the involved mechanisms in mouse BMSCs. The results showed that FAS stimulated bone formation in the fracture mouse model. Additionally, at 30 μM, FAS significantly promotes alkaline phosphatase activity, mineralization, and the expression of osteogenic markers COL-1, RUNX2 and OCN in murine BMSCs. Blocking of P38 by SB202190 significantly reversed the effects of FAS, in vitro, suggesting that P38, but not ERK or JNK activation is required for FAS-induced osteogenesis. Collectively, our results indicate that FAS may be a promising agent for promoting fracture healing.

Tricin enhances osteoblastogenesis through the regulation of Wnt/β-catenin signaling in human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are capable of differentiate into multilineage cell types including bone in vitro and in vivo. Aim of the study: They have been widely developed as a therapeutic approach for bone tissue repair and regeneration. However, the efficiency of lineage specific differentiation still needs improvement. We investigated the effect of Tricin on the proliferation of human adult MSCs by alamar blue assay, the mineralization of MSCs by staining of calcium deposition, and the expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs. Moreover, we also checked the effects of Tricin on the expression of key genes in Wnt/β-catenin signaling pathway. We found Tricin could promote proliferation of MSCs, as well as their mineralization. Tricin could also enhance the expression of osteogenesis marker genes including Bone sialoprotein, osteocalcin, Alkaline phosphatase, and RUNX2. Furthermore, we found out that neither siβ-catenin nor GSK-3β had effect on Tricin treatment, but siWnt3α could neutralize the effect of Tricin. Tricin can enhance osteoblastogenesis through the regulation of Wnt/β-catenin signaling pathway in human adult MSCs in vitro.

Low Intensity Pulsed Ultrasound (LIPUS) Influences the Multilineage Differentiation of Mesenchymal Stem and Progenitor Cell Lines through ROCK-Cot/Tpl2-MEK-ERK Signaling Pathway

Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into multilineage cell types, including adipocytes and osteoblasts. Mechanical stimulus is one of the crucial factors in regulating MSC differentiation. However, it remains unknown how mechanical stimulus affects the balance between adipogenesis and osteogenesis. Low intensity pulsed ultrasound (LIPUS) therapy is a clinical application of mechanical stimulus and facilitates bone fracture healing. Here, we applied LIPUS to adipogenic progenitor cell and MSC lines to analyze how multilineage cell differentiation was affected. We found that LIPUS suppressed adipogenic differentiation of both cell types, represented by impaired lipid droplet appearance and decreased gene expression of peroxisome proliferator-activated receptor γ2 (Pparg2) and fatty acid-binding protein 4 (Fabp4). LIPUS also down-regulated the phosphorylation level of peroxisome proliferator-activated receptor γ2 protein, inhibiting its transcriptional activity. In contrast, LIPUS promoted osteogenic differentiation of the MSC line, characterized by increased cell calcification as well as inductions of runt-related transcription factor 2 (Runx2) and Osteocalcin mRNAs. LIPUS induced phosphorylation of cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, which was essential for the phosphorylation of mitogen-activated kinase kinase 1 (MEK1) and p44/p42 extracellular signal-regulated kinases (ERKs). Notably, effects of LIPUS on both adipogenesis and osteogenesis were prevented by a Cot/Tpl2-specific inhibitor. Furthermore, effects of LIPUS on MSC differentiation as well as Cot/Tpl2 phosphorylation were attenuated by the inhibition of Rho-associated kinase. Taken together, these results indicate that mechanical stimulus with LIPUS suppresses adipogenesis and promotes osteogenesis of MSCs through Rho-associated kinase-Cot/Tpl2-MEK-ERK signaling pathway.

Roles of Hypoxia During the Chondrogenic Differentiation of Mesenchymal Stem Cells

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Common injuries and degenerative diseases often lead to progressive dysfunction of articular cartilage, even joint arthroplasty finally. In recent years, cell-based therapies targeting cartilage-related diseases have aroused strong interests of doctors and researchers. Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate into multilineage cell types such as osteocytes, chondrocytes and adipocytes. The chondrogenic differentiation of MSCs is regulated by many factors, including oxygen tensions. Evidences have suggested that low oxygen tension or hypoxia is involved in the chondrogenic differentiation of MSCs. Expansion and chondrogenic induction of MSCs under hypoxia generally result in enhanced chondrogenic differentiation, although with some inconsistent result. Hypoxia inducible factors (HIFs) are a group of transcription factors. Their stability and transactivation may be essential to the effect of hypoxia on the chondrogenic differentiation of MSCs. PI3K/Akt/FoxO pathways may also be involved in enhanced chondrogenic differentiation under hypoxia. In this review, we discuss the roles of hypoxic conditions in chondrogenic differentiation of MSCs and mechanisms underlying cellular responses to hypoxia.

Effect of phorbol 12-myristate 13-acetate on the differentiation of adipose-derived stromal cells from different subcutaneous adipose tissue depots.

Human adipose-tissue-derived stromal cells (hADSCs) are abundant in adipose tissue and can differentiate into multi-lineage cell types, including adipocytes, osteoblasts, and chondrocytes. In order to define the optimal harvest site of adipose tissue harvest site, we solated hADSCs from different subcutaneous sites (upper abdomen, lower abdomen, and thigh) and compared their proliferation and potential to differentiate into adipocytes and osteoblasts. In addition, this study examined the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, on proliferation and differentiation of hADSCs to adipocytes or osteoblasts. hADSCs isolated from different subcutaneous depots have a similar growth rate. Fluorescence-activated cell sorting (FACS) analysis showed that the expression levels of CD73 and CD90 were similar between hADSCs from abdomen and thigh regions. However, the expression of CD105 was lower in hADSCs from the thigh than in those from the abdomen. Although the adipogenic differentiation potential of hADSCs from both tissue regions was similar, the osteogenic differentiation potential of hADSCs from the thigh was greater than that of hADSCs from the abdomen. Phorbol 12-myristate 13-acetate (PMA) treatment increased osteogenic differentiation and suppressed adipogenic differentiation of all hADSCs without affecting their growth rate and the treatment of Go6983, a general inhibitor of protein kinase C (PKC) blocked the PMA effect. These findings indicate that the thigh region might be a suitable source of hADSCs for bone regeneration and that the PKC signaling pathway may be involved in the adipogenic and osteogenic differentiation of hADSCs.

Surgical-derived oral adipose tissue provides early stage adult stem cells

Background/purposeStem cells (SCs) are characterized by the ability to undergo self-renewal and differentiation into multi-lineage cell types. The low risk to donors, the ease of harvest, and the abundance in the human body of adipose-derived SCs make it an attractive source for adult SCs. Materials and methodsOral adipose tissue was collected by clinical surgery as certified by the Institutional Review Board. SCs were isolated and purified with collagenase containing Dulbecco's modified Eagle's medium (DMEM) and confirmed by immunofluorescence staining with early stage SCs and germinal layer markers. Telomere length was assayed following the procedure provided with the TeloTAGGG kit, and the reverse transcription polymerase chain reaction (RT-PCR) of octamer-binding transcription factor 4 (Oct4) was detected with primers 5′-GTA CTC CTC GGT CCC TTT CC-3′ and 5′-CAA AAA CCC TGG CAC AAA CT-3′. ResultsSCs derived from human oral adipose tissue (hOASCs) express Oct4 (an early stage stem cell marker) as well as bone morphogenetic protein 4 (BMP4) and nestin (both germ layer markers). Furthermore, the telomere length assay showed that hOASCs possess high-molecular-weight fragments of telomere, an indication of self-renewal capacity. Contrary to previous observations that the differentiation ability of adult SCs is affected by cell age and source of restructure, our results suggested that hOASCs possess pluripotential at the early stage and the ability to continually proliferate without curtailment of the telomere length. ConclusionHuman oral adipose tissues can be a beneficial source for isolating pluripotent adult SCs for clinical autologous applications.

Model microgravity enhances endothelium differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of differentiation into multilineage cell types under certain induction conditions. Previous studies have demonstrated that physical environments and mechanical force can influence MSC fate, indicating that these factors may be favorable inducers for clinical treatment. Our previous study found that MSCs are spread with a spindle shape when cultured in normal gravity (NG), and under modeled microgravity (MMG) for 72h, they become unspread and round and their cytoskeleton fibers are reorganized. These morphological changes affected the function of MSCs through the activity of RhoA. We examined the responses of MSCs under MMG stimulation, followed with VEGF differentiation. We found that MSCs under MMG for 72h were differentiated into endothelial-like cells by detecting the expression of endothelial-specific molecules (Flk-1 and vWF), which were also able to form a capillary network. Their endothelial differentiation potential was improved under MMG compared with that under NG. We believe that this method is a novel choice of MMG stimulation for neovascularization. This phenomenon may increase the potential of MSC differentiation, which might be a new strategy for the treatment of various vascular diseases and improve vascularization in tissue engineering.

Therapeutic application of mesenchymal stem cells in bone and joint diseases

Mesenchymal stem cells (MSCs), the non-hematopoietic progenitor cells, are multi-potent stem cells from a variety of tissues with the capability of self-renewal, proliferation, differentiation into multi-lineage cell types, as well as anti-inflammatory and immunomodulatory. These properties make MSCs an ideal source of cell therapy in bone and joint diseases. This review describes the advances of animal study and preliminary clinical application in the past few years, related to MSC-based cell therapy in the common bone and joint diseases, including osteoarthritis, rheumatoid arthritis, osteoporosis, osteonecrosis of the femoral head and osteogenesis imperfecta. It highlights the promising prospect of MSC in clinical application of bone and joint diseases.

Stable Transgene Expression in Primitive Human CD34+Hematopoietic Stem/Progenitor Cells, Using theSleeping BeautyTransposon System

Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34(+) hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.

Multilineage potential of STRO-1+ rat dental pulp cellsin vitro

The aim of the current study was to determine whether STRO-1 selection is an effective approach for purifying rat dental pulp stem cells, and especially whether such selection is beneficial on the multilineage differentiation capacity, i.e. whether selection will account for a higher rate of differentiation or lesser variability. In this study, two cell populations (STRO-1(+) and non-sorted cells) were cultured under conditions promoting neurogenic, adipogenic, myogenic and chondrogenic differentiation. Results of light microscopy, histochemistry, and immunohistochemistry showed that STRO-1(+) cells were capable of advancing into all four differentiation pathways under the influence of inductive media. Quantitative PCR and statistical analysis on specific differentiation markers confirmed that there were significant upregulations in STRO-1(+) cells compared to the other populations, during induction culture. On the basis of our results, we concluded that: (a) rat STRO-1(+) dental pulp stem cells are capable of differentiating towards multilineage cell types, including neural cells, adipocytes, myocytes and chondrocytes; (b) the STRO-1(+) population has a more defined multilineage potential compared to non-sorted cells, probably because of its more homogeneous nature. .