Differentiation Of hADSCs Research Articles

The combination of miR-122 overexpression and Let-7f silencing induces hepatic differentiation of adipose tissue-derived stem cells.

Human adipose tissue-derived stem cells (hADSCs) have been considered as a promising source for cell therapy of liver diseases due to their accessibility, abundance, and expression of hepatocyte markers. Currently, the important role of certain microRNAs (miRNAs) has been reported during hepatic differentiation of stem cells. However, the combination effect of miRNAs on hepatic differentiation of these cells needs to be more investigated. The present study seeks to determine whether the combination of miRNAs can enhance hepatic differentiation of hADSCs in the absence of any other stimulation. First, lentiviral transduction was used to overexpress miR-122 and silence d let-7f in hADSCs for up to 21 days. Then, hepatic functionality was evaluated by analyzing specific hepatocyte genes and biochemical markers at different time points of differentiation induction. Stable miR-122 overexpression and let-7f silencing together in hADSCs resulted in increased expression of hepatocyte markers including ALB, AFP, CK18, CK19, and HNF4a. In addition, urea and albumin production, immunocytochemistry, and glycogen staining confirmed that the treated cells differentiated toward hepatocyte-like cells. Therefore, our findings demonstrate the possibility of using microRNAs to induce hADSCs into functional hepatocyte-like cells.

Effects of RPE-conditioned medium on the differentiation of hADSCs into RPE cells, and their proliferation and migration

Age-related macular degeneration (AMD) is associated with the dysfunction and death of the retinal pigment epithelium (RPE). Recently, there has been increasing interest in stem cell-derived RPE cells for cell replacement therapies, such as those for AMD. The present study investigated whether RPE-conditioned medium (RPECM) could promote the differentiation of human adipose tissue-derived mesenchymal stromal cells (hADSCs) into RPE cells, and enhance the proliferation and migration of these cells. Reverse-transcription quantitative polymerase chain reaction analysis demonstrated that RPECM induced hADSCs to differentiate into cells expressing RPE markers, including retinoid isomerohydrolase (RPE65), cytokeratin (CK8) and Bestrophin, which were identified to be significantly upregulated by ~10-fold, 3.5-fold and 2.4-fold, respectively, compared with the control group [hADSCs cultured in ADSC-conditioned medium (ADSCCM)]. The immunocytochemistry and western blot analysis results demonstrated that the protein levels of RPE65, CK8 and Bestrophin were significantly increased in RPECM-treated hADSCs. In addition, Cell Counting Kit-8 analysis demonstrated that RPECM promoted the proliferation of induced cells. RPECM also increased the expression level of the cell proliferative marker Ki-67. Furthermore, to evaluate the migration potential, cell migration assays were performed. These assays demonstrated that following RPECM treatment hADSCs migrated more quickly compared with the control group. The results of the present study suggest that RPECM induces hADSCs to differentiate into RPE cells with higher proliferative and migratory potentials, which may aid in applications for hADSCs in RPE regenerative therapy.

The enhancement of differentiating adipose derived mesenchymal stem cells toward hepatocyte like cells using gelatin cryogel scaffold

Liver tissue engineering creates a promising methodology for developing functional tissue to restore or improve the function of lost or damaged liver by using appropriate cells and biologically compatible scaffolds. The present paper aims to study the hepatogenic potential of human adipose derived mesenchymal stem cells (hADSCs) on a 3D gelatin scaffold in vitro. For this purpose, mesenchymal stem cells were isolated from human adipose tissue and characterized by flowcytometry analysis and mesodermal lineage differentiation capacity. Then, porous cryogel scaffolds were fabricated by cryogelating the gelatin using glutaraldehyde as the crosslinking agent. The structure of the scaffolds as well as the adhesion and proliferation of the cells were then determined by Scanning Electron Microscopy (SEM) analysis and MTT assay, respectively. The efficiency of hepatic differentiation of hADSCs on 2D and 3D culture systems has been assessed by means of morphological, cytological, molecular and biochemical approaches.Based on the results of flowcytometry, the isolated cells were positive for hMSC specific markers and negative for hematopoietic markers. Further, the multipotency of these cells was confirmed by adipogenic and osteogenic differentiation and the highly porous structure of scaffolds was characterized by SEM images. Biocompatibility was observed in the fabricated gelatin scaffolds and the adhesion and proliferation of hADSCs were promoted without any cytotoxicity effects. In addition, compared to 2D TCPS, the fabricated scaffolds provided more appropriate microenvironment resulting in promoting the differentiation of hADSCs toward hepatocyte-like cells with higher expression of hepatocyte-specific markers and appropriate functional characteristics such as increased levels of urea biosynthesis and glycogen storage. Finally, the created 3D gelatin scaffold could provide an appropriate matrix for hepatogenic differentiation of hADSCs, which could be considered for liver tissue engineering applications.

Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting.

Smooth muscle differentiated human adipose derived stem cells (hADSCs) provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D) bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA) was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy (mean cell viability was 88.16 ± 3.98 vs. 61.76 ± 15% for microtissues and single-cells, respectively). These results provide a novel way to enhance the smooth muscle differentiation of hADSCs and a simple method to maintain better cell viability in 3D bioprinting.

Synergy Between Choroid Plexus Epithelial Cell-Conditioned Medium and Knockout Serum Replacement Converts Human Adipose-Derived Stem Cells to Dopamine-Secreting Neurons

Human adipose-derived stem cells (hADSCs) have great capacity to differentiate into mesodermal origins as well as nonmesodermal lineages, including neural cells. This valuable feature paves the way for the therapeutic application of hADSCs for neurodegenerative maladies such as Parkinson's disease (PD). We tested the capacity of choroid plexus epithelial cell-conditioned medium (CPEC-CM) alone or cocktailed with knockout serum (KS) to induce dopaminergic (DAergic) differentiation of hADSCs. To this end, hADSCs from lipoaspirate were phenotypically characterized and shown to maintain mesodermal multipotency so that selected media easily differentiated them into osteoblasts, chondrocytes, and adipocytes. To begin inducing hADSC neuronal differentiation, we isolated CPECs from rat brain and expanded them in culture to obtain CPEC-CM. We then treated hADSCs with optimized quantities of collected CPEC-CM, KS, or both. The ADSCs treated with either CPEC-CM or CPEC-CM and KS displayed morphological changes typical of neuron-like phenotypes. As revealed by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR (qPCR), and immunostaining analyses, hADSCs cotreated with CPEC-CM and KS expressed significantly higher levels of neuronal and DAergic markers in comparison with single-treated groups. Moreover, the hADSCs began expressing dopamine-biosynthesizing enzymes mainly after cotreatment with CPEC-CM and KS. Consequently, only cotreated hADSCs were capable of synthesizing and releasing dopamine detectable by high-performance liquid chromatography (HPLC). Finally, hADSCs growing in an ordinary medium were found positive for astrocytic marker glial fibrillary acidic protein (GFAP), but stopped GFAP expression on either single or cotreatments. These combined results suggest that CPEC-CM and KS can synergize to remarkably augment DAergic induction of hADSCs, an effect that has implications for cell replacement therapy for PD and related disorders.

Hanging drop culture enhances differentiation of human adipose-derived stem cells into anterior neuroectodermal cells using small molecules

Inspired by in vivo developmental process, several studies were conducted to design a protocol for differentiating of mesenchymal stem cells into neural cells in vitro. Human adipose-derived stem cells (hADSCs) as mesenchymal stem cells are a promising source for this purpose. At current study, we applied a defined neural induction medium by using small molecules for direct differentiation of hADSCs into anterior neuroectodermal cells. Anterior neuroectodermal differentiation of hADSCs was performed by hanging drop and monolayer protocols. At these methods, three small molecules were used to suppress the BMP, Nodal, and Wnt signaling pathways in order to obtain anterior neuroectodermal (eye field) cells from hADSCs. After two and three weeks of induction, the differentiated cells with neural morphology expressed anterior neuroectodermal markers such as OTX2, SIX3, β-TUB III and PAX6. The protein expression of such markers was confirmed by real time, RT-PCR and immunocytochemistry methods According to our data, it seems that the hanging drop method is a proper approach for neuroectodermal induction of hADSCs. Considering wide availability and immunosuppressive properties of hADSCs, these cells may open a way for autologous cell therapy of neurodegenerative disorders.

Icaritin, a novel plant-derived osteoinductive agent, enhances the osteogenic differentiation of human bone marrow- and human adipose tissue-derived mesenchymal stem cells.

For the treatment of diseases affecting bones using bone regenerative medicine, there is an urgent need to develop safe, inexpensive drugs that can strongly induce bone formation. In the present study, we systematically investigated the effects of icaritin, a metabolic product of icariin, on the osteogenic differentiation of human bone marrow‑derived mesenchymal stem cells (hBMSCs) and human adipose tissue‑derived stem cells (hADSCs) invitro. After treatment with icaritin at concentrations of 10‑8-10‑5M, hBMSCs and hADSCs were examined for alkaline phosphatase activity, osteocalcin (OC) secretion, matrix mineralization and expression levels of bone‑related mRNA and proteins. Data showed that icaritin at concentrations 10‑7-10‑5M significantly increased alkaline phosphatase activity, OC secretion at different time points, and calcium deposition at day21. In addition, icaritin upregulated the mRNA expression of genes for bone morphogenetic proteins (BMP‑2,‑4and‑7), bone transcription factors (Runx2 and Dlx5) and bone matrix proteins (ALP, OC and Col‑1). Moreover, icaritin increased the protein levels of BMPs, Runx2 and OC, as detected by western blot analysis. These findings suggest that icaritin enhances the osteogenic differentiation of hBMSCS and hADSCs. Icaritin exerts its potent osteogenic effect possibly by directly stimulating the production of BMPs. Although the osteogenic activity of icaritin invitro was inferior to that of rhBMP‑2, icaritin displayed better results than icariin. Moreover, the low cost, simple extraction procedure, and an abundance of icaritin make it appealing as a bone regenerative medicine.

Overexpression of Prox1 Induces the Differentiation of Human Adipose-Derived Stem Cells into Lymphatic Endothelial-Like Cells In Vitro.

Constant levels of homeobox transcription factor Prox1 expression are required throughout the life of lymphatic endothelial cells (LECs) to maintain their differentiated identity. Recent studies have demonstrated that using human LECs for cell transplantation therapy may improve secondary lymphedema in a nude rat model. However, the application is currently limited by the low yield of LECs. In this study, Prox1 was overexpressed in human adipose tissue-derived stem cells (hADSCs) by using the transfection of lentiviral vectors to induce the differentiation of hADSCs to LECs. After 14 days of Prox1 overexpression, flow cytometry analysis found that the expression of LEC-specific markers such as Podoplanin and VEGFR3, along with the endothelial cell (EC) marker CD31, on Prox1-overexpressed hADSCs was significantly increased; however, the expression of mesenchymal stem cell markers, such as CD29, CD44, and CD90, was substantially reduced. In addition, the mRNA levels of the LEC-specific markers, such as Prox1, Podoplanin, LYVE1, and VEGFR3, in Prox1-overexpressed hADSCs were significantly increased at day 7 and maintained a continuously increased expression level for 28 observation days, according to real-time reverse transcriptase-polymerase chain reaction results. Western blotting and immunofluorescence staining results further confirmed that overexpression of Prox1 in hADSCs significantly increased the protein levels of Podoplanin, LYVE1, and VEGFR3, as well as those of the EC markers such as VWF and CD144, at day 14. Moreover, these differentiated cells were found to form tube-like structures in matrigel, measured by the tube formation assay. These findings suggested that overexpression of Prox1 in hADSCs successfully induced the differentiation of hADSCs into stable lymphatic endothelial-like cells. This study achieved a long-lasting expression of Prox1 in lymphatic endothelial-like cells, and it provided a potentially useful approach for developing novel therapies for limb lymphedema and lymphatic system-related diseases.

Ligand Independent and Subtype-Selective Actions of Thyroid Hormone Receptors in Human Adipose Derived Stem Cells.

Thyroid hormone (TH) receptors (TRs α and β) are homologous ligand-dependent transcription factors (TFs). While the TRs display distinct actions in development, metabolic regulation and other processes, comparisons of TRα and TRβ dependent gene regulation mostly reveal similar mechanisms of action and few TR subtype specific genes. Here, we show that TRα predominates in multipotent human adipose derived stem cells (hADSC) whereas TRβ is expressed at lower levels and is upregulated during hADSC differentiation. The TRs display several unusual properties in parental hADSC. First, TRs display predominantly cytoplasmic intracellular distribution and major TRα variants TRα1 and TRα2 colocalize with mitochondria. Second, knockdown experiments reveal that endogenous TRs influence hADSC cell morphology and expression of hundreds of genes in the absence of hormone, but do not respond to exogenous TH. Third, TRα and TRβ affect hADSC in completely distinct ways; TRα regulates cell cycle associated processes while TRβ may repress aspects of differentiation. TRα splice variant specific knockdown reveals that TRα1 and TRα2 both contribute to TRα-dependent gene expression in a gene specific manner. We propose that TRs work in a non-canonical and hormone independent manner in hADSC and that prominent subtype-specific activities emerge in the context of these unusual actions.

Mussel Adhesion-Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose-Derived Stem Cells.

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution-mediated transfection are limited due to low transfection efficiency and insufficient duration of cell-siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio-inspired polymer-mediated reverse transfection system is developed consisting of implantable poly(lactic-co-glycolic acid) (PLGA) scaffolds functionalized with siRNA-lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA-coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA-sLNP-PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose-derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA-sLNP-PLGA scaffolds enhanced bone formation in a mouse model of critical-sized bone defect. Therefore, the bio-inspired reverse transfection system can provide an all-in-one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.

Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells.

Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells.

Molecular composition of GAG-collagen I multilayers affects remodeling of terminal layers and osteogenic differentiation of adipose-derived stem cells

The effect of molecular composition of multilayers, by pairing type I collagen (Col I) with either hyaluronic acid (HA) or chondroitin sulfate (CS) was studied regarding the osteogenic differentiation of adhering human adipose-derived stem cells (hADSCs). Polyelectrolyte multilayer (PEM) formation was based primarily on ion pairing and on additional intrinsic cross-linking through imine bond formation with Col I replacing native by oxidized HA (oHA) or CS (oCS). Significant amounts of Col I fibrils were found on both native and oxidized CS-based PEMs, resulting in higher water contact angles and surface potential under physiological condition, while much less organized Col I was detected in either HA-based multilayers, which were more hydrophilic and negatively charged. An important finding was that hADSCs remodeled Col I at the terminal layers of PEMs by mechanical reorganization and pericellular proteolytic degradation, being more pronounced on CS-based PEMs. This was in accordance with the higher quantity of Col I deposition in this system, accompanied by more cell spreading, focal adhesions (FA) formation and significant α2β1 integrin recruitment compared to HA-based PEMs. Both CS-based PEMs caused also an increased fibronectin (FN) secretion and cell growth. Furthermore, significant calcium phosphate deposition, enhanced ALP, Col I and Runx2 expression were observed in hADSCs on CS-based PEMs, particularly on oCS-containing one. Overall, multilayer composition can be used to direct cell-matrix interactions, and hence stem cell fates showing for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal protein layers, which seems to enable cells to form a more adequate extracellular matrix-like environment. Statement of SignificanceNatural polymer derived polyelectrolyte multilayers (PEMs) have been recently applied to adjust biomaterials to meet specific tissue demands. However, the effect of molecular composition of multilayers on both surface properties and cellular response, especially the fate of human adipose derived stem cells (hADSCs) upon osteogenic differentiation has not been studied extensively, yet. In addition, no studies exist that investigate a potential cell-dependent remodeling of PEMs made of extracellular matrix (ECM) components like collagens and glycosaminoglycans (GAGs). Furthermore, there is no knowledge whether the ability of cells to remodel PEM components may provide an added value regarding cell growth and differentiation. Finally, it has not been explored yet, how intrinsic cross-linking of ECM derived polyelectrolytes that improve the stability of PEMs will affect the differentiation potential of hADSCs. The current work aims to address these questions and found that the type of GAG has a strong effect on properties of multilayers and osteogenic differentiation of hADSCs. Additionally, we also show for the first time that PEMs made of biogenic polyelectrolytes undergo significant remodeling of terminal layers as completely new finding, which allows cells to form an ECM-like environment supporting differentiation upon osteogenic lineage. The finding of this work may open new avenues of application of PEM systems made by layer by layer (LbL) technique in tissue engineering and regenerative medicine.

Catechol-Functionalized Hyaluronic Acid Hydrogels Enhance Angiogenesis and Osteogenesis of Human Adipose-Derived Stem Cells in Critical Tissue Defects.

Over the last few decades, stem cell therapies have been highlighted for their potential to heal damaged tissue and aid in tissue reconstruction. However, materials used to deliver and support implanted cells often display limited efficacy, which has resulted in delaying translation of stem cell therapies into the clinic. In our previous work, we developed a mussel-inspired, catechol-functionalized hyaluronic acid (HA-CA) hydrogel that enabled effective cell transplantation due to its improved biocompatibility and strong tissue adhesiveness. The present study was performed to further expand the utility of HA-CA hydrogels for use in stem cell therapies to treat more clinically relevant tissue defect models. Specifically, we utilized HA-CA hydrogels to potentiate stem cell-mediated angiogenesis and osteogenesis in two tissue defect models: critical limb ischemia and critical-sized calvarial bone defect. HA-CA hydrogels were found to be less cytotoxic to human adipose-derived stem cells (hADSCs) in vitro compared to conventional photopolymerized HA hydrogels. HA-CA hydrogels also retained the angiogenic functionality of hADSCs and supported osteogenic differentiation of hADSCs. Because of their superior tissue adhesiveness, HA-CA hydrogels were able to mediate efficient engraftment of hADSCs into the defect regions. When compared to photopolymerized HA hydrogels, HA-CA hydrogels significantly enhanced hADSC-mediated therapeutic angiogenesis (promoted capillary/arteriole formation, improved vascular perfusion, attenuated ischemic muscle degeneration/fibrosis, and reduced limb amputation) and bone reconstruction (mineralized bone formation, enhanced osteogenic marker expression, and collagen deposition). This study proves the feasibility of using bioinspired HA-CA hydrogels as functional biomaterials for improved tissue regeneration in critical tissue defects.

Electrospun silk fibroin/poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration.

BackgroundTissue engineering has become a promising therapeutic approach for bone regeneration. Nanofibrous scaffolds have attracted great interest mainly due to their structural similarity to natural extracellular matrix (ECM). Poly(lactide-co-ε-caprolactone) (PLCL) has been successfully used in bone regeneration, but PLCL polymers are inert and lack natural cell recognition sites, and the surface of PLCL scaffold is hydrophobic. Silk fibroin (SF) is a kind of natural polymer with inherent bioactivity, and supports mesenchymal stem cell attachment, osteogenesis, and ECM deposition. Therefore, we fabricated hybrid nanofibrous scaffolds by adding different weight ratios of SF to PLCL in order to find a scaffold with improved properties for bone regeneration.MethodsHybrid nanofibrous scaffolds were fabricated by blending different weight ratios of SF with PLCL. Human adipose-derived stem cells (hADSCs) were seeded on SF/PLCL nanofibrous scaffolds of various ratios for a systematic evaluation of cell adhesion, proliferation, cytotoxicity, and osteogenic differentiation; the efficacy of the composite of hADSCs and scaffolds in repairing critical-sized calvarial defects in rats was investigated.ResultsThe SF/PLCL (50/50) scaffold exhibited favorable tensile strength, surface roughness, and hydrophilicity, which facilitated cell adhesion and proliferation. Moreover, the SF/PLCL (50/50) scaffold promoted the osteogenic differentiation of hADSCs by elevating the expression levels of osteogenic marker genes such as BSP, Ocn, Col1A1, and OPN and enhanced ECM mineralization. In vivo assays showed that SF/PLCL (50/50) scaffold improved the repair of the critical-sized calvarial defect in rats, resulting in increased bone volume, higher trabecular number, enhanced bone mineral density, and increased new bone areas, compared with the pure PLCL scaffold.ConclusionThe SF/PLCL (50/50) nanofibrous scaffold facilitated hADSC proliferation and osteogenic differentiation in vitro and further promoted new bone formation in vivo, suggesting that the SF/PLCL (50/50) nanofibrous scaffold holds great potential in bone tissue regeneration.

Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro.

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

Cogels of Hyaluronic Acid and Acellular Matrix for Cultivation of Adipose-Derived Stem Cells: Potential Application for Vocal Fold Tissue Engineering.

Stem cells based tissue engineering has been one of the potential promising therapies in the research on the repair of tissue diseases including the vocal fold. Decellularized extracellular matrix (DCM) as a promising scaffold has be used widely in tissue engineering; however, it remained to be an important issue in vocal fold regeneration. Here, we applied the hydrogels (hyaluronic acid [HA], HA-collagen [HA-Col], and HA-DCM) to determine the effects of hydrogel on the growth and differentiation of human adipose-derived stem cells (hADSCs) into superficial lamina propria fibroblasts. hADSCs were isolated and characterized by fluorescence-activated cell sorting. The results indicated that HA-DCM hydrogel enhanced cell proliferation and prolonged cell morphology significantly compared to HA and HA-Col hydrogel. Importantly, the differentiation of hADSCs into fibroblasts was also promoted by cogels of HA-Col and HA-DCM significantly. The differentiation of hADSCs towards superficial lamina propria fibroblasts was accelerated by the secretion of HGF, IL-8, and VEGF, the decorin and elastin expression, and the synthesis of chondroitin sulfate significantly. Therefore, the cogel of HA-DCM hydrogel was shown to be outstanding in apparent stimulation of hADSCs proliferation and differentiation to vocal fold fibroblasts through secretion of important growth factors and synthesis of extracellular matrix.

Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells

Event Abstract Back to Event Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells Jisoo Shin1, Jung-Ho Cho1, Yoonhee Jin1, Hyun-Ji Park1, Jong Seung Lee1, Kisuk Yang1 and Seung-Woo Cho1 1 Yonsei University, Department of Biotechnology, Korea Introduction: Controlling stem cell fate by regulating gene expression using small interfering RNA (siRNA) has been recently studied. However, conventional siRNA transfection methods are not efficient to induce guided stem cell differentiation due to low intracellular transfer of siRNA and short target silencing. To overcome these limitations, we developed bio-inspired polymer surfaces for reverse transfection of siRNA. We efficiently immobilized siRNA onto the catechol-modified surface of biodegradable polymer scaffolds to transfect siRNA into human stem cells. We tested whether this system could offer enhanced, long-term siRNA silencing to stem cells for promoting osteogenic differentiation and bone formation of stem cells. Materials and Methods: To prepare bio-inspired scaffolds for siRNA reverse transfection, biodegradable poly(lactide-co-glycolide) (PLGA) film was coated with catechol group, known as a main adhesive component of mussel pads. Then siRNA complexed with lipidoid nanoparticles was immobilized onto the polymer surfaces for non-viral gene delivery. Human adipose-derived stem cells (hADSCs) were seeded onto this scaffold system to reverse transfect siRNA targeting an osteogenic differentiation inhibitor to enhance osteogenic differentiation of hADSCs. Finally, hADSCs differentiated onto the siRNA-immobilized scaffolds were transplanted into critical-sized calvarial bone defects for in vivo bone formation. Results and Discussion: Immobilization efficiency of siRNA-lipidoid complexes on the catechol-modified surfaces was more than 90%. Target knockdown efficacy in hADSCs cultured on the surfaces modified with siRNA-lipidoid was around 90% with no significant cytotoxicity. The expression of osteogenic markers (OPN and ColI) was significantly upregulated in hADSCs differentiated on the surfaces with siRNA targeting osteogenic differentiation suppressor in vitro. Finally, we confirmed transplantation of hADSCs with siRNA-lipidoid immobilized PLGA scaffolds significantly enhanced in vivo bone formation in critical-sized calvarial bone defects confirmed by micro-CT and immunohistochemistry analysis. Conclusion: In summary, we developed bio-inspired polymer surfaces for siRNA reverse transfection platform exhibiting highly effective intracellular siRNA transfer and long-lasting target silencing. The polymer surfaces developed in this study could provide efficient genetic manipulation for improving stem cell engineering and tissue regeneration. This work was supported by a grant (HI14C1588) from the Korea Health Technology R&D Project funded by the Ministry of Health and Welfare, Republic of Korea; This work was supported by the Brain Korea 21 plus (BK21PLUS) program. J. Shin is a fellowship awardee by BK21 plus program Keywords: Bone Regeneration, stem cell, Surface modification, siRNA delivery Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials in constructing tissue substitutes Citation: Shin J, Cho J, Jin Y, Park H, Lee J, Yang K and Cho S (2016). Bio-inspired polymer surfaces for reverse transfection of siRNA to enhance osteogenic differentiation and bone formation of human adipose-derived stem cells. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00670 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Google Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Google Scholar Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho PubMed Jisoo Shin Jung-Ho Cho Yoonhee Jin Hyun-Ji Park Jong Seung Lee Kisuk Yang Seung-Woo Cho Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Calcium/Cobalt Alginate Beads as Functional Scaffolds for Cartilage Tissue Engineering.

Articular cartilage is a highly organized tissue with complex biomechanical properties. However, injuries to the cartilage usually lead to numerous health concerns and often culminate in disabling symptoms, due to the poor intrinsic capacity of this tissue for self-healing. Although various approaches are proposed for the regeneration of cartilage, its repair still represents an enormous challenge for orthopedic surgeons. The field of tissue engineering currently offers some of the most promising strategies for cartilage restoration, in which assorted biomaterials and cell-based therapies are combined to develop new therapeutic regimens for tissue replacement. The current study describes the in vitro behavior of human adipose-derived mesenchymal stem cells (hADSCs) encapsulated within calcium/cobalt (Ca/Co) alginate beads. These novel chondrogenesis-promoting scaffolds take advantage of the synergy between the alginate matrix and Co+2 ions, without employing costly growth factors (e.g., transforming growth factor betas (TGF-βs) or bone morphogenetic proteins (BMPs)) to direct hADSC differentiation into cartilage-producing chondrocytes.

Caveolin-1 is essential in the differentiation of human adipose-derived stem cells into hepatocyte-like cells via an MAPK pathway-dependent mechanism.

Human adipose-derived stem cells (hADSCs), widely present in the adult human body, are an emerging and attractive tool for the establishment of stem cell-based therapies for the treatment of liver disease. However, the mechanism underlying hADSCs hepatic differentiation remains to be elucidated. Caveolin-1 (Cav-1), a 21–24 kDa membrane structural protein, is important in liver regeneration and development. In the present study, fluorescence immuno-cytochemistry and western blotting were used to analyze the expression levels of Cav-1 and evaluate its effects on the hepatic differentiation of hADSCs. The results revealed that primary hADSCs preserved the ability to proliferate and differentiate into hepatocyte-like cells. As demonstrated by semiquantitative reverse transcription-polymerase chain reaction, hepatocyte-inducing factors significantly increased the expression of Cav-1 in a time-dependent manner, as indicated by increased expression levels of the albumin (ALB) and α-fetoprotein (AFP) markers. In addition the expression levels of ALB and HNF1A significantly decreased following small interfering RNA-mediated knockdown of Cav-1. The mitogen-activated protein kinase (MAPK) signaling pathway was activated during hepatic differentiation and inhibited following Cav-1 knockdown. These results suggested that Cav-1 may regulate the hepatocyte-like differentiation of hADSCs by modulating mitogen-activated protein kinase kinase/MAPK signaling. The results of the present study will provide experimental and theoretical basis for further clinical studies on stem cell transplantation in the treatment of liver disease.

Bifunctional coating based on carboxymethyl chitosan with stable conjugated alkaline phosphatase for inhibiting bacterial adhesion and promoting osteogenic differentiation on titanium

In this work, alkaline phosphatase (ALP) was covalently immobilized on carboxymethyl chitosan (CMCS)-coated polydopamine (PDA)-functionalized Ti to achieve a bifunctional surface. Our results showed ∼89% reduction in Staphylococcus epidermidis adhesion on this surface compared to that on pristine Ti. The ALP-modified Ti supported cell proliferation, and significantly enhanced cellular ALP activity and calcium deposition of osteoblasts, human mesenchymal stem cells (hMSCs) and human adipose-derived stem cells (hADSCs). The extent of enhancement in the functions of these cells is dependent on the surface density of immobilized ALP. The substrate prepared using an ALP solution of 50μg/cm2 resulted in 44%, 54% and 129% increase in calcium deposited by osteoblasts, hMSCs and hADSCs, respectively, compared to those cultured on pristine Ti. The ALP-modified substrates also promoted the osteogenic differentiation of hMSCs and hADSCs by up-regulating gene expressions of runt-related transcription factor 2 (RUNX2), osterix (OSX), and osteocalcin (OC) in the two types of stem cells. The surface-immobilized ALP was stable after being subjected to 1h immersion in 70% ethanol and autoclaving at 121°C for 20min. However, the enzymatic bioactivity of the surface-immobilized ALP was reduced by about 50% after these substrates were immersed in phosphate buffered saline (PBS) or PBS containing lysozyme for 14 days.