用于人类脂肪干细胞的纯化和体外长期培养的聚合物涂层

采用紫外固化法制备了基于丙烯酸酯类水凝胶的聚合物涂层(PC),并用X射线光电子能谱(XPS)、水接触角(WCA)和原子力显微镜(AFM)分别对PC进行了化学组成和表面性能的表征.在PC表面进行了人类脂肪干细胞(h ASC)的体外长期培养扩增,得到的第3代细胞的生物学表征结果表明,干细胞在PC表面能正常黏附生长,流式细胞仪检测发现干细胞对特征标记物CD49d,CD73,CD105的阳性显性比例较高,对HLA-DR和CD31几乎不显性,说明扩增的干细胞具有h ASC特征.对PC上扩增的干细胞进行诱导分化,并用油红O、茜素红和阿利新蓝分别进行染色分析,结果表明,该干细胞保留了h ASC的多能特性:能分化为成脂、成骨和成软骨细胞.含有单体甲基丙烯酰氧乙基三甲基氯化铵(DMC)、甲基丙烯酸环己酯(CHMA)和甲基丙烯酸-2-(二乙氨基)乙酯(DEAEMA)的PC2(质量比为3∶1∶2)在用于h ASC体外长期培养时,比其它PC和TCP更有利于细胞的黏附和增殖,纯化细胞,保持其多能性.实时荧光定量PCR(RT-q PCR)的分析表明PC2上得到的细胞更容易向成骨和成软骨细胞分化.     

Isolation and Characterization of Adipose-derived Mesenchymal Stem Cells (ADSCs) from Cattle

Adipose-derived mesenchymal stem cells (ADSCs) were isolated from the adult adipose tissue of 2-year-old cattle, and then characterized by immunofluorescence and RT-PCR. We found that primary bADSCs could be expanded for 25 passages. Expression of β-integrin, CD44, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 bADSCs were successfully induced to differentiate into osteoblasts and adipocytes. The results indicate the potential for multi-lineage differentiation of bADSCs that may represent an ideal candidate for cellular transplantation therapy.     

Investigation of Coculture of Human Adipose-Derived Stem Cells and Mature Adipocytes

The purpose of this study was to evaluate the differentiation potential of human adipose-derived stem cells (hADSCs) into adipocytes by coculturing them with human mature adipocytes. The transwell culture system was utilized for indirect coculture of hADSCs and human mature adipocytes at four different hADSCs-to-mature adipocytes ratios, i.e., 1:5, 1:1, 2:1, and 5:1. After 8?days of coculture, the Oil Red O and Trypan Blue stainings were performed for the evaluation of adipogenic differentiation of hADSCs. In addition, flow cytometric analysis and Hoechst 33342/PI double staining were performed after 20?days of coculture. The Oil Red O and Trypan Blue stainings showed that hADSCs with high viability could not differentiate into mature adipocytes after 8 or 20?days of coculture. However, flow cytometric analysis indicated that CD105 expression of hADSCs decreased after 20?days of coculture. These results indicated that hADSCs cocultured with human adult adipocytes could not successfully differentiate into adipocytes.     

Controlling the Poly(ε‐caprolactone) Degradation to Maintain the Stemness and Function of Adipose‐Derived Mesenchymal Stem Cells in Vascular Regeneration Application

Biodegradable poly(ε‐caprolactone) (PCL) scaffolds with adipose‐derived mesenchymal stem cells (ADSCs) have been used in vascular regeneration studies. An evaluation method of the effect of PCL degradation products (DP) on the viability, stemness, and differentiation capacities of ADSCs is established. ADSCs are cultured in medium containing different concentrations of PCL DP before evaluating the effect of PCL DP on the cell apoptosis and proliferation, cell surface antigens, adipogenic and osteogenic differentiation capacities, and capacities to differentiate into endothelial cells and smooth muscle cells. The results demonstrate that PCL DP exceed 0.05 mg mL^?1 may change the stemness and differentiation capacities of ADSCs. Therefore, to control the proper concentration of PCL DP is essential for ADSCs in vascular regeneration application.     

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.     

Human salivary gland stem cells ameliorate hyposalivation of radiation-damaged rat salivary glands

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren''s syndrome or radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. In the present study, we isolated tissue-specific stem cells from the human submandibular salivary gland (hSGSCs). To efficiently isolate and amplify hSGSCs in large amounts, we developed a culture system (lasting 4–5 weeks) without any selection. After five passages, we obtained adherent cells that expressed mesenchymal stem cell surface antigen markers, such as CD44, CD49f, CD90 and CD105, but not the hematopoietic stem cell markers, CD34 and CD45, and that were able to undergo adipogenic, osteogenic and chondrogenic differentiation. In addition, hSGSCs were differentiated into amylase-expressing cells by using a two-step differentiation method. Transplantation of hSGSCs to radiation-damaged rat salivary glands rescued hyposalivation and body weight loss, restored acinar and duct cell structure, and decreased the amount of apoptotic cells. These data suggest that the isolated hSGSCs, which may have characteristics of mesenchymal-like stem cells, could be used as a cell therapy agent for the damaged salivary gland.     

Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic β-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.     

Photocontrolled Multidirectional Differentiation of Mesenchymal Stem Cells on an Upconversion Substrate

The effective guidance of mesenchymal stem cell (MSC) differentiation on a substrate by near‐infrared (NIR) light is particularly attractive for tissue engineering and regenerative medicine. However, most of current substrates cannot control multidirectional differentiation of MSCs like natural tissues. Herein, a photocontrolled upconversion‐based substrate was designed and constructed for guiding multidirectional differentiation of MSCs. The substrate enables MSCs to maintain their stem‐cell characteristics due to the anti‐adhesive effect of 4‐(hydroxymethyl)‐3‐nitrobenzoic acid modified poly(ethylene glycol) (P1) attached on the upconversion substrate. Upon NIR irradiation, the P1 is released from the substrate by photocleavage. The detachment of P1 can change cell–matrix interactions dynamically. Moreover, MSCs cultured on the upconversion substrate can be specifically induced to differentiate to adipocytes or osteoblasts by adjusting the NIR laser. Our work provides a new way of using NIR‐based upconversion substrate to modulate the multidirectional differentiation of MSCs.     

CD99 type II is a determining factor for the differentiation of primitive neuroectodermal cells

CD99 is a 32-kDa cell surface molecule present on thymocytes, peripheral T cells, many other hematopoietic stem cells and somatic cells were implicated in cell-cell adhesion and cell-activation phenomena. Two major subtypes have been identified so far, designated CD99 type I and type II. We have investigated the correlation between the degree of neural differentiation and the expression of CD99 subtypes in three differentially differentiated cell lines such as CADO-ES1, RD-ES, and SH-N-SY5Y, in order of differentiation. In addition, we induced differentiation of the RD-ES cell line by N6,2'-dibutyryl-cAMP (db-cAMP). Six days after treatment with db-cAMP, RD-ES cell line has changed its morphology from uniform round cells to cells with neurites, and initially CD99 type II-overexpressed RD-ES cells showed significant down-regulation of CD99 type II, whereas CD99 type I expression remained constant. When RD- ES cells were transfected with the cDNA encoding for CD99 type I-green fluorescence protein (GFP) and type II-GFP, CD99 type II transfected RD-ES cell line remained unchanged with morphology of undifferentiated form. Our data suggest that CD99 type II acts as a negative regulator in the neural differentiation of precursor cells that might occur during nerve system development.     

Osteogenic Effects of VEGF‐Overexpressed Human Adipose‐Derived Stem Cells with Whitlockite Reinforced Cryogel for Bone Regeneration

Bone is a vascularized tissue that is comprised of collagen fibers and calcium phosphate crystals such as hydroxyapatite (HAp) and whitlockite (WH). HAp and WH are known to elicit bone regeneration by stimulating osteoblast activities and osteogenic commitment of stem cells. In addition, vascular endothelial growth factor (VEGF) is shown to promote osteogenesis and angiogenesis which is considered as an essential process in bone repair by providing nutrients. In this study, VEGF‐secreting human adipose‐derived stem cells (VEGF‐ADSCs) are developed by transducing ADSCs with VEGF‐encoded lentivirus. Additionally, WH‐reinforced gelatin/heparin cryogels (WH‐C) are fabricated by loading WH into gelatin/heparin cryogels. VEGF‐ADSC secrete tenfold more VEGF than ADSC and show increased VEGF secretion with cell growth. Also, incorporation of WH into cryogels provides a mineralized environment with ions secreted from WH. When the VEGF‐ADSCs are seeded on WH‐C, sustained release of VEGF is observed due to the specific affinity of VEGF to heparin. Finally, the synergistic effect of VEGF‐ADSC and WH on osteogenesis is successfully confirmed by alkaline phosphatase and real‐time polymerase chain reaction analysis. In vivo bone formation is demonstrated via implantation of VEGF‐ADSC seeded WH‐C into mouse calvarial bone defect model, resulted in enhanced bone development with the highest bone volume/total volume.     

Photodegradable Hydrogels for Capture,Detection, and Release of Live Cells

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T‐cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV‐induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95 % purity of CD4 and CD8 T‐cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti‐CD4 and anti‐TNF‐α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence “halo” after immunofluorescent staining and could be retrieved by site‐specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.     

Photodegradable Hydrogels for Capture,Detection, and Release of Live Cells

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T‐cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV‐induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95 % purity of CD4 and CD8 T‐cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti‐CD4 and anti‐TNF‐α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence “halo” after immunofluorescent staining and could be retrieved by site‐specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.     

Centrifugal countercurrent chromatography to elucidate surface differences of adipose tissue-derived stem cells

The current methods of isolation of adipose tissue-derived stem cells result in a heterogeneous population that might interfere with their differentiation potential and makes it difficult to compare the results between different groups. Partition in aqueous two-phase systems is one of the few techniques that separate cells on the basis of surface properties, gentle enough to isolate fragile cell types in isotonic conditions without altering their structure, and can be easily scaled. In this study, stem cells isolated from human adipose tissue seeded and expanded in vitro were fractionated by using centrifugal countercurrent distribution in an aqueous two-phase system. The separated subpopulations revealed the high heterogeneity of adipose tissue-derived stem cell samples. Comparative partition analyses showed that aging induces a loss of heterogeneity, which is not due to a loss of cell viability associated to age. The phosphatidylserine externalization, an apoptotic feature, is the main factor in cell partition that results in a decreased hydrophobicity of the cell surface. This procedure may be suitable for separating adipose tissue-derived stem cell populations enriched in some functional and/or structural surface characteristics. The possibility of a very effective separation of different subpopulations in opposite phases would be an interesting development of the method.     

Oxidative Epigallocatechin Gallate Coating on Polymeric Substrates for Bone Tissue Regeneration

Plant derived flavonoids have not been well explored in tissue engineering applications due to difficulties in efficient formulations with biomaterials for controlled presentation. Here, the authors report that surface coating of epigallocatechin gallate (EGCG) on polymeric substrates including poly (L‐lactic acid) (PLLA) nanofibers can be performed via oxidative polymerization of EGCG in the presence of cations, enabling regulation of biological functions of multiple cell types implicated in bone regeneration. EGCG coating on the PLLA nanofiber promotes osteogenic differentiation of adipose‐derived stem cells (ADSCs) and is potent to suppress adipogenesis of ADSCs while significantly reduces osteoclastic maturation of murine macrophages. Moreover, EGCG coating serves as a protective layer for ADSCs against oxidative stress caused by hydrogen peroxide. Finally, the in vivo implantation of EGCG‐coated nanofibers into a mouse calvarial defect model significantly promotes the bone regeneration (61.52 ± 28.10%) as compared to defect (17.48 ± 11.07%). Collectively, the results suggest that EGCG coating is a simple bioinspired surface modification of polymeric biomaterials and importantly can thus serve as a promising interface for tuning activities of multiple cell types associated with bone fracture healing.     

Electrospun poly(l‐lactide) nanofibers coated with mineral trioxide aggregate enhance odontogenic differentiation of dental pulp stem cells

A combination of bioceramics and nanofibrous scaffolds holds promising potential for inducing of mineralization in connective tissues. The aim of the present study was to investigate the attachment, proliferation and odontogenic differentiation of dental pulp stem cells (DPSC) on poly(l ‐lactide) (PLLA) nanofibers coated with mineral trioxide aggregate (MTA). Polymeric scaffolds were fabricated via the electrospinning method and their surface was coated with MTA. DPSC were isolated from dental pulp and their biological behavior was evaluated on scaffolds and the control group using MTT assay. Alkaline phosphatase (ALP) activity, biomineralization and the expression of odontogenic genes were analyzed during odontogenic differentiation. Isolated DPSC showed spindle‐shaped morphology with multi‐lineage differentiation potential and were positive for CD73, CD90 and CD105. MTA‐coated PLLA (PLLA/MTA) exhibited nanofibrous structure with average fiber diameter of 756 ± 157 nm and interconnected pores and also suitable mechanical properties. Similar to MTA, these scaffolds were shown to be biocompatible and to support the attachment and proliferation of DPSC. ALP activity transiently peaked on day 14 and was significantly higher in PLLA/MTA scaffolds than in the control groups. In addition, increasing biomineralization was observed in all groups with a higher amount in PLLA/MTA. Odontogenic‐related genes, DSPP and collagen type I showed a higher expression in PLLA/MTA on days 21 and 14, respectively. Taken together, MTA/PLLA electrospun nanofibers enhanced the odontogenic differentiation of DPSC and showed the desired characteristics of a pulp capping material.     

Effect of F68 on Cryopreservation of Mesenchymal Stem Cells Derived from Human Tooth Germ

The use of stem-cell-based therapies in regenerative medicine and in the treatment of disorders such as Parkinson, Alzheimer's disease, diabetes, spinal cord injuries, and cancer has been shown to be promising. Among all stem cells, mesenchymal stem cells (MSCs) were reported to have anti-apoptotic, immunomodulatory, and angiogenic effects which are attributed to the restorative capacity of these cells. Human tooth germ stem cells (HTGSCs) having mesenchymal stem cell characteristics have been proven to exert high proliferation and differentiation capacity. Unlike bone-marrow-derived MSCs, HTGSCs can be easily isolated, expanded, and cryopreserved, which makes them an alternative stem cell source. Regardless of their sources, the stem cells are exposed to physical and chemical stresses during cryopreservation, hindering their therapeutic capacity. Amelioration of the side effects of cryopreservation on MSCs seems to be a priority in order to maximize the therapeutic efficacy of these cells. In this study, we tested the effect of Pluronic 188 (F68) on HTGSCs during long-term cryopreservation and repeated freezing and defrosting cycles. Our data revealed that F68 has a protective role on survival and differentiation of HTGSCs in long-term cryopreservation.