Pancreatic islet differentiation of human embryonic stem cells by microRNA overexpression

Development of stem cell‐based therapies for the treatment of type 1 diabetes would provide a renewable supply of human β‐cells. Human embryonic stem cells (ESCs) are considered to be one of the stem cell populations with sufficient proliferative capacity to achieve this goal. Currently, differentiation protocols directing ESCs toward a pancreatic fate employ a variety of expensive cytokines and inhibitors. With the known significance of microRNAs in islet development, we present a novel and cost‐effective strategy in which miR‐375 overexpression promotes pancreatic endocrine differentiation in hESCs in the absence of any extrinsic factors. miR‐375 has been shown to be a key regulator of pancreatic development and function in zebrafish, mouse and human. In this study, hESCs were transduced with lentiviral vectors containing human miR‐375 precursor and aggregated to form human embryoid bodies (hEBs) for up to 21 days. Morphological assessment, immunocytochemistry and DTZ staining confirmed that miR‐375‐induced hEBs have similar characteristics to those of mature islets. In addition, the dynamic expression profile of endodermal marker Foxa2 and endocrine‐specific genes, including HNF4α, Pdx1, Pax6, Nkx6.1, Glut2 and insulin, were detected by quantitative real‐time PCR. Finally, insulin release upon glucose stimulation was detected in our differentiated clusters. The data presented here demonstrate the feasibility of using microRNAs to direct differentiation into the pancreatic lineage. Copyright © 2013 John Wiley & Sons, Ltd.     

BMP‐2‐transduced human bone marrow stem cells enhance neo‐bone formation in a rat critical‐sized femur defect

Synthetic graft materials are considered as possible substitutes for cancellous bone, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human bone marrow mesenchymal stem cells (hBMSCs) and osteoinductive bone morphogenetic protein‐2 (BMP‐2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical‐sized bone defect. hBMSCs were either stimulated with rhBMP‐2 or transduced with BMP‐2 by gene transfer. The effect of both protein stimulation and gene transfer was compared for osteogenic outcome. X‐rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post‐operatively. In addition, bone‐labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical‐sized defect in 33% of the rats treated with the Ad‐BMP‐2‐transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only 10% of the rats treated with rhBMP2/hBMSCs, 12 weeks post‐treatment, the bone defect was closed in all treated rats of the Ad‐BMP‐2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad‐BMP‐2‐ and rhBMP‐2‐treated rats. The amount of neocartilage was less pronounced in both BMP‐2‐related groups. In summary, scaffolds with BMP‐2‐transduced hBMSCs performed better than those with the rhBMP2/hBMSCs protein. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive proteins may represent a promising alternative for bone regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Combined treatment with platelet‐rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose‐derived stem cells in three‐dimensional collagen scaffolds

Osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis and progressive joint destruction. Bioengineered scaffolds are widely studied for regenerative surgery strategies in osteochondral defect management, also combining the use of stem cells, growth factors and hormones. The utility in tissue engineering of human adipose‐derived stem cells (ASCs) isolated from adipose tissue has been widely noted. Autologous platelet‐rich plasma (PRP) represents an alternative strategy in regenerative medicine for the local release of endogenous growth factors and hormones. Here we compared the effects of three‐dimensional (3D) collagen type I scaffold culture and combined treatment with PRP and human recombinant insulin on the chondro‐/osteogenic differentiation of ASCs. Histochemical and biomolecular analyses demonstrated that chondro‐/osteogenic differentiation was increased in ASC‐populated 3D collagen scaffolds compared with two‐dimensional (2D) plastic dish culture. Chondro‐/osteogenic differentiation was further enhanced in the presence of combined PRP (5% v/v) and insulin (100 nm ) treatment. In addition, chondro‐/osteogenic differentiation associated with the contraction of ASC‐populated 3D collagen scaffold and increased β1/β3‐integrin expression. Inhibition studies demonstrated that PRP/insulin‐induced chondro‐/osteogenic differentiation is independent of insulin‐like growth factor 1 receptor (IGF‐1R) and mammalian target of rapamycin (mTOR) signalling; IGF‐R1/mTOR inhibition even enhanced ASC chondro‐/osteogenic differentiation. Our findings underline that 3D collagen scaffold culture in association with platelet‐derived growth factors and insulin favour the chondro‐/osteogenic differentiation of ASCs, suggesting new translational applications in regenerative medicine for the management of osteochondral defects. Copyright © 2016 John Wiley & Sons, Ltd.     

MicroRNAs can effectively induce formation of insulin‐producing cells from mesenchymal stem cells

MicroRNAs regulate insulin secretion, pancreatic development and beta cell differentiation. However, the function of microRNAs in the formation of insulin‐producing cells (IPCs) from adult stem cells is poorly understood. We examine the microRNA expression profile in nestin‐positive umbilical cord‐derived mesenchymal stem cells (N‐UCMSCs) and nestin‐positive pancreatic mesenchymal stem cells using a deep sequencing approach. We also selected specific microRNAs for overexpression in N‐UCMSCs and found that miR‐375 and miR‐26a induced IPCs differentiation from N‐UCMSCs by downregulating target genes including mtpn, sox6, bhlhe22 and ccnd1. Small interfering RNAs were also used to knock down these genes in N‐UCMSCs to induce the formation of IPCs. These results suggest that endogenous microRNAs involved in the formation of IPCs from adult stem cells show promise for advancing the development of an effective cell transplant therapy for diabetes. Copyright © 2017 John Wiley & Sons, Ltd.     

Evaluation of platelet‐rich plasma and hydrostatic pressure regarding cell differentiation in nucleus pulposus tissue engineering

Generation of a biological nucleus pulposus (NP) replacement by tissue engineering appears to be a promising approach for the therapy of early stages of intervertebral disc degeneration. Thereby, autologous mesenchymal stem cells (MSCs) represent an attractive cell source compared to cells of the NP that are already altered in their phenotype due to degenerative processes. This study compares the influence of 3D pellet culture and alginate beads, as well as that of different media compositions, by the addition of human platelet‐rich plasma (PRP) or transforming growth factor (TGF‐β₁) in interaction with hydrostatic pressure on chondrogenic differentiation of human MSCs compared to NP cells. We found that gene expression of the chondrogenic markers aggrecan, collagen type 2 and collagen type 1 and Sox9 was considerably lower in cells cultivated with PRP compared to TGF‐β₁. Immunohistology confirmed this result at protein level in pellet culture. Additionally, the pellet culture system was found to be more suitable than alginate beads. A positive influence of hydrostatic pressure could only be shown for individual donors. In summary, in comparison to TGF‐β₁, human PRP did not induce adequate chondrogenic differentiation for both culture systems and cell types used. The mixture of growth factors in PRP promoted proliferation rather than chondrogenic differentiation. Based on these results, an application of PRP in human NP tissue‐engineering approaches cannot be recommended. Copyright © 2011 John Wiley & Sons, Ltd.     

INS‐1 cell glucose‐stimulated insulin secretion is reduced by the downregulation of the 67 kDa laminin receptor

Understanding β cell–extracellular matrix (ECM) interactions can advance our knowledge of the mechanisms that control glucose homeostasis and improve culture methods used in islet transplantation for the treatment of diabetes. Laminin is the main constituent of the basement membrane and is involved in pancreatic β cell survival and function, even enhancing glucose‐stimulated insulin secretion. Most of the studies on cell responses towards laminin have focused on integrin‐mediated interactions, while much less attention has been paid on non‐integrin receptors, such as the 67 kDa laminin receptor (67LR). The specificity of the receptor‐ligand interaction through the adhesion of INS‐1 cells (a rat insulinoma cell line) to CDPGYIGSR‐, GRGDSPC‐ or CDPGYIGSR + GRGDSPC‐covered surfaces was evaluated. Also, the effects of the 67LR knocking down over glucose‐stimulated insulin secretion were investigated. Culture of the INS‐1 cells on the bioactive surfaces was improved compared to the low‐fouling carboxymethyl dextran (CMD) surfaces, while downregulation of the 67LR resulted in reduced cell adhesion to surfaces bearing the CDPGYIGSR peptide. Glucose‐stimulated insulin secretion was hindered by downregulation of the 67LR, regardless of the biological motif available on the biomimetic surfaces on which the cells were cultured. This finding illustrates the importance of the 67LR in glucose‐stimulated insulin secretion and points to a possible role of the 67LR in the mechanisms of insulin secretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Preferential tendon stem cell response to growth factor supplementation

Tendon injuries are increasingly prevalent around the world, accounting for more than 100 000 new clinical cases/year in the USA alone. Cell‐based therapies have been proposed as a therapeutic strategy, with recent data advocating the use of tendon stem cells (TSCs) as a potential cell source with clinical relevance for tendon regeneration. However, their in vitro expansion is problematic, as they lose their multipotency and change their protein expression profile in culture. Herein, we ventured to assess the influence of insulin‐like growth factor 1 (IGF‐1), growth and differentiation factor‐5 (GDF‐5) and transforming growth factor‐β1 (TGFβ1) supplementation in TSC culture. IGF‐1 preserved multipotency for up to 28 days. Upregulation of decorin and scleraxis expression was observed as compared to freshly isolated cells. GDF‐5 treated cells exhibited reduced differentiation along adipogenic and chondrogenic pathways after 28 days, and decorin, scleraxis and collagen type I expression was increased. After 28 days, TGFβ1 supplementation led to increased scleraxis, osteonectin and collagen type II expression. The varied responses to each growth factor may reflect their role in tendon repair, suggesting that: GDF‐5 promotes the transition of tendon stem cells towards tenocytes; TGFβ1 induces differentiation along several pathways, including a phenotype indicative of fibrocartilage or calcified tendon, common problems in tendon healing; and IGF‐1 promotes proliferation and maintenance of TSC phenotypes, thereby creating a population sufficient to have a beneficial effect. Copyright © 2014 John Wiley & Sons, Ltd.     

An in vitro expansion score for tissue‐engineering applications with human bone marrow‐derived mesenchymal stem cells

Human bone marrow‐derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue‐engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony‐forming unit (CFU) assay, population doubling time (PDT), senescence‐associated β‐galactosidase (SA‐β‐Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA‐β‐Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late‐passage cells than in early‐passage cells. Early‐passage cells underwent efficient osteogenic differentiation, with mid‐passage cells performing best in chondrogenic differentiation. Late‐passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.     

Magnetic labeling of pancreatic β‐cells modulates the glucose‐ and insulin‐induced phosphorylation of ERK1/2 and AKT

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β‐cells. INS‐1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1–1 µM) for 12 h. The phosphorylation of extracellular signal‐regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose‐dependent manner. SPIO labeling (200 µg Fe ml^?1) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose‐stimulated phosphorylation of ERK1/2 and AKT and insulin‐stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS‐1 cells, through modulation of the glucose‐ or insulin‐induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.