Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non‐specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti‐restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC–SMC co‐cultures, we investigated the ability of DS–SILY₂₀, a decorin mimic, to promote EC health. DS–SILY₂₀ increased EC proliferation and migration by 1.5‐ and 2‐fold, respectively, which corresponded to increased phosphorylation of ERK‐1/2. Interestingly, IL‐6 secretion and the production of both E‐selectin and P‐selectin were reduced in the presence of 10 μm DS–SILY₂₀, even in the presence of the potent pro‐inflammatory cytokine platelet‐derived growth factor (PDGF). In hyperplastic and quiescent EC–SMC co‐cultures, DS–SILY₂₀ treatment reduced the secretion of IFNγ, IL‐1β, IL‐6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E‐selectin and P‐selectin expression was further reduced following DS–SILY₂₀ treatment in both co‐culture models. These results indicate that DS–SILY₂₀ promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Temporal profiling of the growth and multi‐lineage potentiality of adipose tissue‐derived mesenchymal stem cells cell‐sheets

Cell‐sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold‐free constructs. Adipose tissue‐derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow‐derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC‐CSs) is non‐existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC‐CSs development; and (b) the multipotentiality of ASC‐CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid‐supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC‐CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT–PCR and histology at multiple time points. ASC‐CSs remained healthy up to 3 weeks of hyperconfluent culture. One week‐old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non‐hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC‐CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.     

Co‐culture of adipose‐derived stem cells and endothelial cells in fibrin induces angiogenesis and vasculogenesis in a chorioallantoic membrane model

Neovascularization of adipose tissue equivalents is a crucial step in successful adipose tissue engineering, since insufficient vascularization results in graft resorption in an in vivo situation. A possible cellular approach to overcome this limitation is the co‐implantation of adipose‐derived stem cells (ASCs) with endothelial cells to stimulate the formation of a vascular network. We investigated the potential of ASCs derived from human abdominal fat tissue co‐cultured with endothelial progenitor cells (EPCs) from human peripheral blood to stimulate neovascularization of fibrin constructs on the chorioallantoic membrane (CAM) of fertilized chicken eggs, in direct comparison to human umbilical vein endothelial cells (HUVECs). After 9 days of incubation, cell–fibrin constructs were explanted and histologically evaluated with respect to ingrowth of avian blood vessels into the construct and formation of human blood vessels by co‐implanted endothelial cells. When administered on the CAM, ASCs successfully guided host vasculature into the construct (angiogenesis) and guided formation of capillary‐like structures by co‐implanted human endothelial cells (vasculogenesis), with HUVECs being superior to EPCs, leading to a perfused avian and human capillary network within the fibrin construct. However, the results also showed that perfused human blood vessels were only observed near the CAM compared to unperfused capillary‐like structures near the top of the construct, indicating that perfusion of the cell–fibrin construct takes longer than 9 days. In conclusion, as blood vessel formation is an essential step during adipogenic differentiation, the data support our hypothesis that cellular communication between transplanted ASCs and endothelial cells is beneficial for vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.     

Extracellular matrix enhances differentiation of adipose stem cells from infrapatellar fat pad toward chondrogenesis

The objective was to improve proliferation and chondrogenic potential of adipose stem cells (ASCs) by expansion on extracellular matrix (ECM) deposited by either ASCs or synovium‐derived stem cells (SDSCs). ASCs isolated from porcine infrapatellar fat pad were separately expanded on conventional plastic flasks, ASC‐deposited ECM and SDSC‐deposited ECM. ASCs were centrifuged to form pellets and cultured in a serum‐free chondrogenic medium with either TGFβ3 or TGFβ3 combined with BMP‐6. Cell number yielded on ECM expansion did not show a significant difference in deposition between ASCs and SDSCs but was 6–10 times that grown on non‐coated flasks. ECM‐expanded ASCs exhibited a lower level of intracellular reactive oxygen species (ROS) compared to those grown on non‐coated flasks. Typical chondrogenic markers, including type II collagen and glycosaminoglycans (GAGs), were intensively distributed in the pellets from ECM‐expanded ASCs instead of those from flask‐grown cells. ASCs expanded on ECM, either from ASCs or SDSCs, exhibited a similar chondrogenic index (GAG:DNA), which was significantly higher than that from ASCs grown on non‐coated flasks. The combination of TGFβ3 and BMP‐6 increased 36% more in ASC chondrogenic index than the treatment with TGFβ3 alone. Interestingly, ECM pretreatment also decreased expanded ASC hypertrophic marker genes. ECM deposited by either ASCs or SDSCs did not exhibit enhanced adipogenic differentiation of ASCs. Our study indicates that the sequential application of ECM for cell expansion and combined TGFβ3 with BMP‐6 for chondrogenic differentiation may be a promising approach for ASC‐based cartilage tissue engineering and regeneration. Copyright © 2011 John Wiley & Sons, Ltd.     

Multilineage co‐culture of adipose‐derived stem cells for tissue engineering

Stem cell interactions through paracrine cell signalling can regulate a range of cell responses, including metabolic activity, proliferation and differentiation. Moving towards the development of optimized tissue‐engineering strategies with adipose‐derived stem cells (ASCs), the focus of this study was on developing indirect co‐culture models to study the effects of mature adipocytes, chondrocytes and osteoblasts on bovine ASC multilineage differentiation. For each lineage, ASC differentiation was characterized by histology, gene expression and protein expression, in the absence of key inductive differentiation factors for the ASCs. Co‐culture with each of the mature cell populations was shown to successfully induce or enhance lineage‐specific differentiation of the ASCs. In general, a more homogeneous but lower‐level differentiation response was observed in co‐culture as compared to stimulating the bovine ASCs with inductive differentiation media. To explore the role of the Wnt canonical and non‐canonical signalling pathways within the model systems, the effects of the Wnt inhibitors WIF‐1 and DKK‐1 on multilineage differentiation in co‐culture were assessed. The data indicated that Wnt signalling may play a role in mediating ASC differentiation in co‐culture with the mature cell populations. Copyright © 2012 John Wiley & Sons, Ltd.     

Adipose‐derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co‐culture with adipose‐derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co‐cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase‐14. Moreover, we showed OEC/ASC‐induced vessel‐like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co‐culture‐induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system. Copyright © 2012 John Wiley & Sons, Ltd.     

Micropattern size‐dependent endothelial differentiation from a human induced pluripotent stem cell line

The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two‐dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue‐engineering advances; thus, methods to improve endothelial differentiation efficiency are vital to these efforts. In this study, we examine the hypothesis that the diameter of micropatterned islands influences endothelial differentiation from human induced pluripotent stem cells (hiPSCs). Comparing island diameters of 80, 140, 225 and 500 µm, we found that co‐cultures of control ECs and pericytes did not yield variable ratios of cell types; however, when hiPSCs were differentiated toward a bicellular population of ECs and pericytes on these varying micropattern feature sizes, we found that smaller islands promoted EC differentiation efficiency, yielding a derived population composed of 70% ECs, which exhibited a greater sprouting propensity. Differentiation on the largest feature size exhibited a smaller EC yield, similar to that on non‐patterned substrates. Taken together, these data demonstrate that micropatterned islands of varying diameters can be used to modulate EC differentiation efficiency. Copyright © 2015 John Wiley & Sons, Ltd.     

A mixed co‐culture of mesenchymal stem cells and transgenic chondrocytes in alginate hydrogel for cartilage tissue engineering

To regenerate articular cartilage tissue from degeneration and trauma, synovial mesenchymal stem cells (SMSCs) were used in this study as therapeutic progenitor cells to induce therapeutic chondrogenesis. To accomplish this, chondrocytes pre‐transduced with adenoviral vectors carrying the transforming growth factor (TGF) β3 gene were selected as transgenic companion cells and co‐cultured side‐by‐side with SMSCs in a 3D environment to provide chondrogenic growth factors in situ. We adopted a mixed co‐culture strategy for this purpose. Transgenic delivery of TGF‐β3 in chondrocytes was performed via recombinant adenoviral vectors. The mixed co‐culture of SMSCs and transgenic chondrocytes was produced in alginate gel constructs. Gene expression in both SMSCs and chondrocytes were characterized. Biochemical assays in vitro and in vivo showed that release of TGF‐ß3 from transgenic chondrocytes not only induced SMSC differentiation into chondrocytic cells but also preserved the chondrocytic phenotype of chondrocytes from suspected dedifferentiation. As a result, this mixed co‐culture strategy in conjunction with TGF‐ß3 gene delivery could be a promising approach in cartilage tissue engineering. Copyright © 2012 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.     

Evaluation of tenogenic differentiation potential of selected subpopulations of human adipose‐derived stem cells

Identification of a suitable cell source and bioactive agents guiding cell differentiation towards tenogenic phenotype represents a prerequisite for advancement of cell‐based therapies for tendon repair. Human adipose‐derived stem cells (hASCs) are a promising, yet intrinsically heterogenous population with diversified differentiation capacities. In this work, we investigated antigenically‐defined subsets of hASCs expressing markers related to tendon phenotype or associated with pluripotency that might be more prone to tenogenic differentiation, when compared to unsorted hASCs. Subpopulations positive for tenomodulin (TNMD+ hASCs) and stage specific early antigen 4 (SSEA‐4+ hASCs), as well as unsorted ASCs were cultured up to 21 days in basic medium or media supplemented with TGF‐β3 (10 ng/ml), or GDF‐5 (50 ng/ml). Cell response was evaluated by analysis of expression of tendon‐related markers at gene level and protein level by real time RT‐PCR, western blot, and immunocytochemistry. A significant upregulation of scleraxis was observed for both subpopulations and unsorted hASCs in the presence of TGF‐β3. More prominent alterations in gene expression profile in response to TGF‐β3 were observed for TNMD+ hASCs. Subpopulations evidenced an increased collagen III and TNC deposition in basal medium conditions in comparison with unsorted hASCs. In the particular case of TNMD+ hASCs, GDF‐5 seems to influence more the deposition of TNC. Within hASCs populations, discrete subsets could be distinguished offering varied sensitivity to specific biochemical stimulation leading to differential expression of tenogenic components suggesting that cell subsets may have distinctive roles in the complex biological responses leading to tenogenic commitment to be further explored in cell based strategies for tendon tissues.     

Targeted delivery of adipose‐derived stem cells via acellular dermal matrix enhances wound repair in diabetic rats

Cell‐based therapeutic intervention has emerged as a new approach to accelerate wound closure. Adipose‐derived stem cells (ASCs), as a fascinating cell source, have received much attention in tissue repair and regeneration. In this study we evaluated the potential of acellular dermal matrix (ADM) scaffold serving as a carrier for the delivery of ASCs and investigated its therapeutic effects on wound healing. First, ASCs were isolated and characterized for multidifferentiation potential. ASCs–ADM grafts were then prepared, and ADM scaffold was shown to support the in vitro growth and proliferation of ASCs. Next, we analysed paracrine factors in conditioned medium and found that ASCs–ADM grafts secreted various cytokines, including VEGF, HGF, TGFβ and bFGF. Moreover, ASCs–ADM conditioned medium notably stimulated the migration and proliferation of fibroblasts. In vivo, we established an excisional wound model in diabetic rats which received phosphate‐buffered saline (PBS), ADM or ASCs–ADM grafts, respectively. Our results demonstrated that implantation of ASCs–ADM significantly enhanced tissue regeneration and increased epithelialization, resulting in accelerated wound closure. Immunofluorescence analysis further indicated that capillary density was evidently increased in the ASCs–ADM group compared with the control or ADM group. In addition, western blot analysis showed that ASCs–ADM significantly increased the expression of angiogenic factors, which was consistent with in vitro data. Taken together, our results suggest that targeted delivery of ASCs via ADM scaffold accelerate diabetic wound healing through a paracrine mechanism, with enhanced granulation tissue formation and increased re‐epithelialization and neovascularization. Copyright © 2012 John Wiley & Sons, Ltd.     

Production of human tissue‐engineered skin trilayer on a plasma‐based hypodermis

Full thickness wounds require a dermal component to achieve functional permanent skin restoration. Currently available tissue‐engineered skin substitutes lack a subcutaneous fat layer that would functionally contribute some of the mechanical and thermoregulatory properties of normal skin. To generate a trilayer engineered skin equivalent, we included bone marrow mesenchymal (BM‐MSC) or adipose tissue‐derived (ASC) stromal cells in a human plasma hydrogel exposed to adipogenic clues for three weeks. Approximately half of the cells differentiated under these conditions into mature adipocytes that survived for two years in culture with minimal medium change. In vitro generation of bona fide fully differentiated adipocytes was assessed by leptin secretion and ultrastructurally demonstrated through semithin to ultrathin sectioning and lipid staining with osmium tetroxide. Furthermore, presence of BM‐MSCs or ASCs within the subcutaneous layer contributed to the epidermal differentiation program, with more proliferating basal cells depositing basal membrane proteins and differentiating into mature keratinocytes that were able to generate a pluristratified epithelium. In conclusion, we engineered a fully differentiated human skin trilayer that could present multiple applications such as use for in vitro drug absorption tests and regenerative therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

eNOS transfection of adipose‐derived stem cells yields bioactive nitric oxide production and improved results in vascular tissue engineering

This study evaluates the durability of a novel tissue engineered blood vessel (TEBV) created by seeding a natural vascular tissue scaffold (decellularized human saphenous vein allograft) with autologous adipose‐derived stem cells (ASC) differentiated into endothelial‐like cells. Previous work with this model revealed the graft to be thrombogenic, likely due to inadequate endothelial differentiation as evidenced by minimal production of nitric oxide (NO). To evaluate the importance of NO expression by the seeded cells, we created TEBV using autologous ASC transfected with the endothelial nitric oxide synthase (eNOS) gene to produce NO. We found that transfected ASC produced NO at levels similar to endothelial cell (EC) controls in vitro which was capable of causing vasorelaxation of aortic specimens ex vivo. TEBV (n = 5) created with NO‐producing ASC and implanted as interposition grafts within the aorta of rabbits remained patent for two months and demonstrated a non‐thrombogenic surface compared to unseeded controls (n = 5). Despite the xenograft nature of the scaffold, the TEBV structure remained well preserved in seeded grafts. In sum, this study demonstrates that upregulation of NO expression within adult stem cells differentiated towards an endothelial‐like lineage imparts a non‐thrombogenic phenotype and highlights the importance of NO production by cells to be used as endothelial cell substitutes in vascular tissue engineering applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid‐derived stem cells to insulin‐producing clusters

This study investigated the differentiation of human amniotic fluid‐derived stem cells (hAFSCs) into insulin‐producing clusters in vitro. Adenovirally‐delivered mouse Pdx1 (Ad‐Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β‐cell markers. When Ad‐Pdx1‐transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly‐l ‐ornithine, the expression of islet‐associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C‐peptide ELISA confirmed that Ad‐Pdx1‐transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β‐cells. To sustain the β‐cell‐like phenotype and investigate the effect of three‐dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1‐transduced cells were encapsulated in alginate and cultured long‐term under serum‐free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β‐cell‐like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets. Copyright © 2012 John Wiley & Sons, Ltd.     

The suitability of human adipose‐derived stem cells for the engineering of ligament tissue

Rupture of the anterior cruciate ligament (ACL) is the one of the most common sports‐related injuries. With its poor healing capacity, surgical reconstruction using either autografts or allografts is currently required to restore function. However, serious complications are associated with graft reconstructions and the number of such reconstructions has steadily risen over the years, necessitating the search for an alternative approach to ACL repair. Such an approach may likely be tissue engineering. Recent engineering approaches using ligament‐derived fibroblasts have been promising, but the slow growth rate of such fibroblasts in vitro may limit their practical application. More promising results are being achieved using bone marrow mesenchymal stem cells (MSCs). The adipose‐derived stem cell (ASC) is often proposed as an alternative choice to the MSC and, as such, may be a suitable stem cell for ligament engineering. However, the use of ASCs in ligament engineering still remains relatively unexplored. Therefore, in this study, the potential use of human ASCs in ligament tissue engineering was initially explored by examining their ability to express several ligament markers under growth factor treatment. ASC populations treated for up to 4 weeks with TGFβ1 or IGF1 did not show any significant and consistent upregulation in the expression of collagen types 1 and 3, tenascin C and scleraxis. While treatment with EGF or bFGF resulted in increased tenascin C expression, increased expression of collagens 1 and 3 were never observed. Therefore, simple in vitro treatment of human ASC populations with growth factors may not stimulate their ligament differentiative potential. Copyright © 2011 John Wiley & Sons, Ltd.     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.     

Clinical‐scale expansion of adipose‐derived stromal cells starting from stromal vascular fraction in a single‐use bioreactor: proof of concept for autologous applications

Adipose‐derived stromal cells (ASCs) are adult multipotent cells increasingly used for cell therapy due to their differentiation potential, their paracrine effect and their convenience. ASCs are currently selected from stromal vascular fractions (SVFs) of adipose tissue and expanded in 2D flasks following good manufacturing practices. This process is limited in surface area, labour‐intensive and expensive, especially for autologous applications requiring selection and expansion steps for every patient. Closed and automated bioreactors offer an alternative for scalable and cost‐effective production of ASCs. This study investigated a single‐use stirred‐tank bioreactor that can expand ASCs from SVFs on microcarriers. A preliminary microcarrier screening in static and spinner flask conditions was performed to evaluate the best candidate for adhesion, amplification and harvest. The selected microcarrier was used for process development in the bioreactor. The first experiments showed poor selectivity and growth of the ASCs from the SVF (n  =  2). The process was then adjusted by two means: (1) decreasing the platelet lysate in the medium for enhancing cell adherence; and (2) adding a shear protectant (Pluronic F68). Following these modifications, we demonstrated that the number of population doublings of ASCs from SVFs was not significantly different between the bioreactor and the 2D controls (n  =  3). In addition, the ASC characterization after culture showed that cells maintained their clonogenic potential, phenotype, differentiation potential and immunosuppressive capacities. This study provides the proof of concept that isolation and amplification of functional ASCs from SVFs can be performed in a stirred‐tank bioreactor combined with microcarriers. Copyright © 2016 John Wiley & Sons, Ltd.