Adipocyte Lineage Differentiation Research Articles

Adipocyte lineage differentiation potential of MSCs isolated from reaming material.

Mesenchymal stem cells (MSCs) obtained from various sources have been used for different therapeutic applications including tissue regeneration. Reamer/irrigator/aspirator (RIA) has been increasingly used in recent years for the derivation of MSCs. Here in this investigation we have comparatively analyzed MSCs obtained from iliac crest bone marrow (ICBM) and RIA for their morphology, cluster determinant (CD) markers, and adipogenic differentiation capacity. MSCs were isolated, cultured, and purified from both sources and then flow cytometric studies were performed to study their characteristics. The differentiation potential of RIA and ICBM was examined by an Oil Red O staining protocol. Moreover, the tissue-specific markers related to adipogenesis were analyzed by real-time polymerase chain reaction (RT-PCR). The cells were cultured in the relevant induction medium and then adipogenic lineage differentiation was tested and confirmed for all MSC preparations. Additionally, analysis by flow cytometer was indicative of RIA derived MSCs (RIA-MSCs) having a more homogenous population than ICBM derived MSCs. The RIA-MSCs differentiation toward adipogenic lineage was more efficient compared with ICBM-MSCs. Direct comparative analysis of RIA to ICBM-MSCs indicated that the RIA-MSCs had a higher potential toward adipocyte lineage differentiation compared with ICBM-MSCs.

PR048: Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells

PR048: Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells

Nitric oxide balances osteoblast and adipocyte lineage differentiation via the JNK/MAPK signaling pathway in periodontal ligament stem cells

BackgroundCritical tissues that undergo regeneration in periodontal tissue are of mesenchymal origin; thus, investigating the regulatory mechanisms underlying the fate of periodontal ligament stem cells could be beneficial for application in periodontal tissue regeneration. Nitric oxide (NO) regulates many biological processes in developing embryos and adult stem cells. The present study was designed to investigate the effects of NO on the function of human periodontal ligament stem cells (PDLSCs) as well as to elucidate the underlying molecular mechanisms.MethodsImmunofluorescent staining and flow cytometry were used for stem cell identification. Western blot, reverse transcription polymerase chain reaction (RT-PCR), immunofluorescent staining, and flow cytometry were used to examine the expression of NO-synthesizing enzymes. The proliferative capacity of PDLSCs was determined by EdU assays. The osteogenic potential of PDLSCs was tested using alkaline phosphatase (ALP) staining, Alizarin Red staining, and calcium concentration detection. Oil Red O staining was used to analyze the adipogenic ability. Western blot, RT-PCR, and staining were used to examine the signaling pathway.ResultsHuman PDLSCs expressed both inducible NO synthase (iNOS) and endothelial NO synthase (eNOS) and produced NO. Blocking the generation of NO with the NOS inhibitor l-NG-monomethyl arginine (l-NMMA) had no influence on PDLSC proliferation and apoptosis but significantly attenuated the osteogenic differentiation capacity and stimulated the adipogenic differentiation capacity of PDLSCs. Increasing the physiological level of NO with NO donor sodium nitroprusside (SNP) significantly promoted the osteogenic differentiation capacity but reduced the adipogenic differentiation capacity of PDLSCs. NO balances the osteoblast and adipocyte lineage differentiation in periodontal ligament stem cells via the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) signaling pathway.ConclusionsNO is essential for maintaining the balance between osteoblasts and adipocytes in PDLSCs via the JNK/MAPK signaling pathway.Graphical NO balances osteoblast and adipocyte lineage differentiation via JNK/MAPK signaling pathway

ROCK inhibitor Y‐27632 maintains the proliferation of confluent human mesenchymal stem cells

The transplantation of cell sheets of mesenchymal stem cells (MSCs) is expected to be the next generation of periodontal regenerative therapy. An adequate method of multilayering MSCs has yet to be established. When cell sheets proliferate, they usually contract and detach from culture dishes and then the proliferation of cells in the contracted areas is arrested. ROCK-mediated contractile force causes cell contraction. Although multilayer formation medium (MFM) stimulated the proliferation of growth-arrested confluent MSCs, MSCs detached from the culture dish. Therefore, we investigated the effects of ROCK inhibitor Y-27632 on the proliferation and detachment of confluent MSCs, and examined the ability of cells to differentiate within the cell sheets. Confluent MSCs were cultured in MFM containing transforming growth factor-β1, ascorbic acid and fetal bovine serum either with or without Y-27632. Cell proliferation was examined by bromodeoxyuridine incorporation assays and total DNA measurement. Sheet contractions were examined by light microscopy and stereomicroscopy. Multilayer formations and focal adhesion assembly were observed with confocal microscopy. Characteristic of cells were examined by flow cytometric analysis. Osteoblast lineage differentiation was observed with alkaline phosphatase and alizarin red S staining. Adipocyte lineage differentiation was observed with oil red O staining. The addition of Y-27632 to MFM prevented the cell sheets from detaching and did not inhibit MSC growth. The cell numbers cultured with MFM/Y-27632 were significantly higher than that obtained with MFM-only on day 4. Cell sheets detached from the culture dish on day 4, and the number of bromodeoxyuridine-positive cells in the detached area decreased. Cells in the cell sheets had similar characteristics to primary MSCs, and differentiated into osteoblast and adipocyte lineages. Y-27632 both prevented the MSC sheets from detaching and maintained the multilayered proliferation of confluent MSCs by MFM, and then cells in the sheets had differentiation potency.

BMP4 increases expression of HMGA2 in mesenchymal stem cells

BMP4 has been linked to early steps of adipocyte lineage differentiation but only little is known about its corresponding downstream pathways. Herein, we have investigated whether or not the expression of high mobility group protein HMGA2, another protein linked to proliferation and differentiation within the process of adipogenesis, may be influenced by BMP4 signaling in adipose tissue derived stem cells. Compared to FGF1, a strong inducer of HMGA2 in immortalized pre-adipocytes, BMP4 was found moderately to induce the HMGA2 mRNA expression in serum starved adipose tissue derived stem cells and myometrial cells. In contrast, no such activity was noted in canine bone marrow derived mesenchymal stem cells. As to adipocyte lineage differentiation the functions of BMP4 and HMGA2 mechanistically overlap. Thus, we propose that in adipose tissue BMP4 acts in part by activating HMGA2 making this architectural transcription factor one of the major downstream players in that system.