Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p?<?0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.     

脂肪干细胞与间充质干细胞体外诱导分化为心肌细胞的差别

本文旨在探讨脂肪干细胞(adipose-derived stem cells, ASCs)和骨髓间充质干细胞(mesenchymal stem cells, MSCs)在组织含量、体外培养和诱导分化为心肌细胞方面的差别.ASCs从新西兰白兔皮下脂肪组织提取,MSCs从大鼠四肢长骨骨髓提取,体外培养扩增,免疫细胞学方法鉴定.采用细胞集落形成法检测组织中干细胞的含量.将不同代的干细胞用不同浓度的5-氮胞苷诱导,观察其形态变化,免疫细胞化学方法检测诱导后细胞是否转化为心肌细胞.结果显示,体外培养的ASCs呈短梭形,分布均匀,生长迅速,细胞形态单一、稳定.MSCs原代生长非常缓慢,呈簇生长,细胞纯度偏低,容易混杂其它细胞类型,传代细胞容易分化和老化.脂肪组织中ASCs含量显著高于骨髓中MSCs含量,且前者含量受年龄影响小.5-氮胞苷诱导ASCs分化为心肌细胞的有效浓度为6～9μmol/L,而MSCs在3～15μmol/L 5-氮胞苷诱导下可见心肌细胞形成.ASCs诱导分化的心肌细胞呈球形细胞团,MSCs分化的心肌细胞呈条形或棒状,其心肌细胞分化率低于ASCs.幼年动物MSCs的组织含量和心肌细胞分化率均高于老年动物,而ASCs受动物年龄影响较小.结果表明,ASCs在组织含量、细胞纯度、生长速度和心肌细胞分化率等方面均明显优于骨髓MSCs,在心肌细胞再生方面较MSCs具有更大的优势.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease

Adipose-derived mesenchymal stem cells(ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient's own subcutaneous adipose tissue, a high cell yield(i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, antiapoptotic effects, and control of immune cells via cellcell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.     

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.     

MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2

Elucidation of the molecular mechanisms governing human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-100 on the osteogenesis of hASCs. Overexpression of miR-100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR-100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR-100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR-100. Taken together, our findings imply that miR-100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.     

ARPE-19 conditioned medium promotes neural differentiation of adipose-derived mesenchymal stem cells

BACKGROUNDAdipose-derived stem cells (ASCs) have been increasingly explored for cell-based medicine because of their numerous advantages in terms of easy availability, high proliferation rate, multipotent differentiation ability and low immunogenicity. In this respect, they have been widely investigated in the last two decades to develop therapeutic strategies for a variety of human pathologies including eye disease. In ocular diseases involving the retina, various cell types may be affected, such as Müller cells, astrocytes, photoreceptors and retinal pigment epithelium (RPE), which plays a fundamental role in the homeostasis of retinal tissue, by secreting a variety of growth factors that support retinal cells.AIMTo test ASC neural differentiation using conditioned medium (CM) from an RPE cell line (ARPE-19).METHODSASCs were isolated from adipose tissue, harvested from the subcutaneous region of healthy donors undergoing liposuction procedures. Four ASC culture conditions were investigated: ASCs cultured in basal Dulbecco''s Modified Eagle Medium (DMEM); ASCs cultured in serum-free DMEM; ASCs cultured in serum-free DMEM/F12; and ASCs cultured in a CM from ARPE-19, a spontaneously arising cell line with a normal karyotype derived from a human RPE. Cell proliferation rate and viability were assessed by crystal violet and MTT assays at 1, 4 and 8 d of culture. At the same time points, ASC neural differentiation was evaluated by immunocytochemistry and western blot analysis for typical neuronal and glial markers: Nestin, neuronal specific enolase (NSE), protein gene product (PGP) 9.5, and glial fibrillary acidic protein (GFAP).RESULTSDepending on the culture medium, ASC proliferation rate and viability showed some significant differences. Overall, less dense populations were observed in serum-free cultures, except for ASCs cultured in ARPE-19 serum-free CM. Moreover, a different cell morphology was seen in these cultures after 8 d of treatment, with more elongated cells, often showing cytoplasmic ramifications. Immunofluorescence results and western blot analysis were indicative of ASC neural differentiation. In fact, basal levels of neural markers detected under control conditions significantly increased when cells were cultured in ARPE-19 CM. Specifically, neural marker overexpression was more marked at 8 d. The most evident increase was observed for NSE and GFAP, a modest increase was observed for nestin, and less relevant changes were observed for PGP9.5. CONCLUSIONThe presence of growth factors produced by ARPE-19 cells in tissue culture induces ASCs to express neural differentiation markers typical of the neuronal and glial cells of the retina.     

Reovirus safety study for proliferation and differentiation of human adipose-derived mesenchymal stem cells

Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells’ integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.     

脂肪干细胞定向分化的影响因素

脂肪干细胞是一类由脂肪组织分离出来的间充质干细胞,具有自我更新和多向分化潜能,可长期自我更新并在特定的条件下分化形成多种终末细胞。本文总结近年来关于脂肪干细胞定向分化的研究成果、介绍脂肪干细胞的分离培养技术、影响脂肪干细胞成脂成骨定向分化能力的主要因素,以及在各种终末分化细胞之间"转分化"的作用条件,从而为寻找治疗肥胖的新靶点以及骨组织工程学提供新思路。     

The effect of quercetin on hepatic differentiation of human adipose-derived mesenchymal stem cells

Human adipose-derived mesenchymal stem cells (MSCs) can be stimulated to differentiate into hepatic cells. MSC differentiation was induced by fibroblast growth factor-4, hepatocyte growth factor, oncostatin M, and dexamethasone. The influence of quercetin on MSC hepatic differentiation in culture was assayed, and 1 or 10 μmole/L quercetin added into the induction medium enhanced the manifestation of MSC hepatic differentiation. Urea secretion, cytokeratin 19 expression, and α-fetoprotein synthesis were increased. Quercetin modulated CYP1A–cytochrome P450 activity in the differentiated cells. MSCs differentiated in the presence of quercetin exhibited higher viability and resistance to oxidative stress.     

Dynamic compression and co-culture with nucleus pulposus cells promotes proliferation and differentiation of adipose-derived mesenchymal stem cells

Adipose-derived stem cells (ASCs) are a set of multi potent stem cells potentially used in cartilage tissue engineering. We hypothesized that the effect of dynamic compression and co-culture with nucleus pulposus cells (NPCs) promotes ASC proliferation and chondrogenic differentiation. A controlled dynamic compression loading device was utilized to stimulate ASCs obtained from Sprague Dawley (SD) rats and identified by flow cytometry. The proliferation index was measured by carboxyfluorescein succinimidyl ester (CFSE) staining. Dynamic compression, as well as co-culture enhanced chondrogenic differentiation of ASCs as indicated by the expression of SOX-9, type-II collagen and aggrecan, which were measured by real-time PCR and Western blot. In our study, we found dynamic compression promoted the proliferation of ASCs and induced its differentiation into NP-like cells. Combination of dynamic compression and co-culture showed an additive effect on NP-like cell differentiation.     

Electrospun poly-l-lactic acid/polyvinyl alcohol nanofibers improved insulin-producing cell differentiation potential of human adipose-derived mesenchymal stem cells

Combination of adipose-derived mesenchymal stem cells (ADSCs) and synthetic materials in terms of pancreatic tissue engineering can be considered as a treatment of diabetes. This study aimed to evaluate the differentiation of human ADSCs to pancreatic cells on poly-l -lactic acid/polyvinyl alcohol (PLLA/PVA) nanofibers as a three-dimensional (3D) scaffold. Mesenchymal stem cells (MSCs) were characterized for mesenchymal surface markers by flow cytometry. Then ADSCs were seeded on 3D scaffolds and treated with pancreatic differentiation medium. Immunostaining assay showed that ADSCs were very efficiently differentiated into a relatively homogeneous population of insulin-producing cells. Moreover, real-time RT-PCR results revealed that pancreas-specific markers were highly expressed in 3D scaffolds compared with their expression in tissue culture plates and this difference in expression level was significant. In addition, insulin and C-peptide secreted in response to varying concentrations of glucose in the 3D scaffold group was significantly higher than that in 2D culture. The results of the present study confirmed that PLLA/PVA scaffold seeded with ADSCs could be a suitable option in pancreatic tissue engineering.     

Exosomes from human adipose-derived mesenchymal stem cells promote migration through Wnt signaling pathway in a breast cancer cell model

Recent studies have demonstrated that the Wnt/β-catenin signaling plays an important role in stem cell aging. However, the mechanisms of cell senescence induced by Wnt/β-catenin signaling are still poorly understood. Our preliminary study has indicated that activated Wnt/β-catenin signaling can induce MSC aging. In this study, we reported that the Wnt/β-catenin signaling was a potent activator of reactive oxygen species (ROS) generation in MSCs. After scavenging ROS with N-acetylcysteine, Wnt/β-catenin signaling-induced MSC aging was significantly attenuated and the DNA damage and the expression of p16^INK4A, p53, and p21 were reduced in MSCs. These results indicated that the Wnt/β-catenin signaling could induce MSC aging through promoting the intracellular production of ROS, and ROS may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling.     

Biodegradable zinc oxide composite scaffolds promote osteochondral differentiation of mesenchymal stem cells

Osteoarthritis (OA) involves the degeneration of articular cartilage and subchondral bone. The capacity of articular cartilage to repair and regenerate is limited. A biodegradable, fibrous scaffold containing zinc oxide (ZnO) was fabricated and evaluated for osteochondral tissue engineering applications. ZnO has shown promise for a variety of biomedical applications but has had limited use in tissue engineering. Composite scaffolds consisted of ZnO nanoparticles embedded in slow degrading, polycaprolactone to allow for dissolution of zinc ions over time. Zinc has well-known insulin-mimetic properties and can be beneficial for cartilage and bone regeneration. Fibrous ZnO composite scaffolds, having varying concentrations of 1–10 wt.% ZnO, were fabricated using the electrospinning technique and evaluated for human mesenchymal stem cell (MSC) differentiation along chondrocyte and osteoblast lineages. Slow release of the zinc was observed for all ZnO composite scaffolds. MSC chondrogenic differentiation was promoted on low percentage ZnO composite scaffolds as indicated by the highest collagen type II production and expression of cartilage-specific genes, while osteogenic differentiation was promoted on high percentage ZnO composite scaffolds as indicated by the highest alkaline phosphatase activity, collagen production, and expression of bone-specific genes. This study demonstrates the feasibility of ZnO-containing composites as a potential scaffold for osteochondral tissue engineering.     

Morphogenetic signals from chondrocytes promote chondrogenic and osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potentially useful cells for musculoskeletal tissue engineering. However, controlling MSC differentiation and tissue formation in vivo remains a challenge. There is a significant need for well-defined and efficient protocols for directing MSC behaviors in vivo. We hypothesize that morphogenetic signals from chondrocytes may regulate MSC differentiation. In micromass culture of MSCs, incubation with chondrocyte-conditioned medium (CCM) significantly enhanced the production of cartilage specific matrix including type II collagen. In addition, incubation of MSCs with conditioned medium supplemented with osteogenic factors induced more osteogenesis and accumulation of calcium and increased ALP activity. These findings reveal that chondrocyte-secreted factors promote chondrogenesis as well as osteogenesis of MSCs during in vitro micromass culture. Moreover, when MSCs expanded with chondrocyte-conditioned medium were encapsulated in hydrogels and subsequently implanted into athymic mice, basophilic extracellular matrix deposition characteristic of neocartilage was evident. These results indicate that articular chondrocytes produce suitable morphogenetic factors that induce the differentiation program of MSCs in vitro and in vivo.