Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

Background

Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs).

Methods

To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16^INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential.

Results

Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs.

Conclusions

In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.     

miR-424 negatively regulates adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

目的 研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响.方法 用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化.在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导.用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达.结果 hAMSCs成脂分化后,miR-424表达下调.与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高.成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组.PPARγ和成脂相关标记物的表达量也出现下调.结论 miR-424可负向调节hAMSCs向脂肪细胞分化.     

miR-424负向调节人脂肪源间充质干细胞向成脂细胞分化

目的研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响。方法用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化。在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导。用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达。结果 hAMSCs成脂分化后,miR-424表达下调。与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高。成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组。PPARγ和成脂相关标记物的表达量也出现下调。结论 miR-424可负向调节hAMSCs向脂肪细胞分化。     

差速贴壁法分离培养脂肪源间充质干细胞

目的:探讨差速贴壁法从大鼠腹股沟脂肪垫分离纯化脂肪源间充质干细胞(adipose tissue-derived mesenchymal stem cells,ADMSCs)的可行性。方法:采用差速贴壁培养法分离ADMSCs,并与普通培养法得到的ADMSCs进行表面分子CD44阳性率对比。在第2代ADMSCs中加入条件培养基进行诱导,根据条件培养基的不同分成3组:①成骨诱导组:加入成骨培养基;②成脂诱导组:加入成脂培养基;③对照组:仅加入基础培养基。成骨诱导组和对照组进行碱性磷酸酶(ALP)检测,成脂诱导组和对照组进行油红O染色检测。结果:差速贴壁培养法获得CD44阳性率更高的ADMSCs。成骨诱导组的ALP大大高于对照组,成脂诱导组油红O染色阳性,对照组油红O染色均为阴性。结论:差速贴壁培养法从大鼠腹股沟脂肪垫中分离得到高纯度ADMSCs。     

脂肪组织源性干细胞的生物学特性与多向分化潜能

回顾脂肪组织源性干细胞的生物学特性与多向分化潜能研究新进展。源于中胚层的脂肪组织源性干细胞具有自我更新及多向分化潜能，在体外适当的诱导条件下可向脂肪细胞、软骨细胞、成骨细胞、骨骼肌、心肌细胞、内皮细胞及神经细胞分化，在组织工程及基因治疗中具有良好的应用前景。对脂肪组织源性干细胞生物学特性及多向分化潜能的研究．将为脂肪组织源性干细胞成为现代组织工程学研究的理想种子细胞并最终用于以细胞为基础的临床治疗提供前提和保障。     

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5?mM CaCl₂ and 7.5?mM CaCl₂ protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5?mM CaCl₂ and 7.5?mM CaCl₂ promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.     

In vitro protection of adipose tissue-derived mesenchymal stem cells by erythropoietin

Mobilization of stem cells and their differentiation into cardiomyocytes are known to have protective effects after myocardial infarction. The integrity of transplanted mesenchymal stem cells for cardiac regeneration is dependent on cell–cell or cell–matrix interaction, which is adversely affected by reactive oxygen species in an ischemic environment. Treatment with erythropoietin was shown to protect human adipose tissue derived mesenchymal stem cells in an ischemic injury in vitro model. The analyses indicated that expression of erythropoietin receptors played a pivotal role in erythropoietin mediated cell survival. In this study, the anti-apoptotic effect of erythropoietin on stem cells was analyzed in apoptosis-induced human mesenchymal stem cells. Apoptosis was induced in cultured adult human adipose tissue derived mesenchymal stem cells by hydrogen peroxide. A group of cultured cells was also treated with recombinant human erythropoietin in a concentration of 50 ng mL⁻¹. The degree of apoptosis was analyzed by flow-cytometry and immunohistochemical staining for Caspase 3. The average percentages of apoptotic cells were significantly higher in H₂O₂-induced stem cells than in cells co-cultured with erythropoietin (63.03 ± 4.96% vs 29 ± 3.41%, p < 0.01). We conclude that preconditioning with erythropoietin suppresses apoptosis of mesenchymal stem cells and enhances their survival.     

Soluble factors-mediated immunomodulatory effects of canine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived mesenchymal stem cells (AD-MSCs), which can differentiate into several lineages, have immunomodulatory properties similar to those of bone marrow-derived MSCs. However, the specific mechanism by which the immunomodulatory effect of MSCs occurs is not clear. In this study, we isolated canine AD-MSCs (cAD-MSCs) and induced their development into adipocyte, osteocyte, and neuron-like cells. We then investigated their phenotype and cytokine expression to determine whether they were able to exert an immunomodulatory effect and what the underlying mechanisms of this effect were. cAD-MSCs expressed CD44, CD90, and MHC class I and were also partially positive for the expression of CD34; however, they did not express CD14 and CD45. In addition, they expressed the mRNA of transforming growth factor beta (TGF-beta), IL-6, IL-8, CCL2, CCL5, vascular endothelial growth factor, hepatocyte growth factor (HGF), tissue inhibitor metalloproteinase-1/2, and cyclooxygenase-2 but not that of IL-10. Further, leukocyte proliferation induced by mitogens was suppressed when they were cocultured with irradiated cAD-MSCs, as well as with culture supernatants of cAD-MSCs alone. Moreover, TNF-alpha production significantly decreased, whereas TGF-beta, IL-6, and interferon-gamma production significantly increased in cAD-MSCs that were cocultured with leukocytes. Finally, immonomodulatory factors of MSCs, such as TGF-beta, HGF, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in cAD-MSCs that were cocultured with leukocytes; however, the production of PGE2 and IDO showed different kinetics, and leukocyte proliferation was effectively restored by PGE2 and IDO inhibitors. Taken together, these results indicate that the immunomodulatory effects of cAD-MSCs are associated with soluble factors (TGF-beta, HGF, PGE2, and IDO). Therefore, it is suggested that cAD-MSCs have a potential therapeutic use in the treatment of immune-mediated disease.     

神经型钙黏蛋白对小鼠脂肪间充质干细胞的成神经作用

目的:探讨神经型钙黏蛋白(N-cadherin)在小鼠脂肪间充质干细胞(ADMSCs)成神经分化中的作用。方法;用差速培养法获取小鼠ADMSCs,传至第5代,经成神经诱导液诱导ADMSCs 7 d后,免疫荧光检测胶质纤维酸性蛋白(GFAP)、神经元特异性烯醇化酶(NSE)和微管相关蛋白2(MAP2)的表达,半定量PCR检测细胞中N-cadherin mRNA的表达;采用N-cadherin对细胞进行基因修饰,免疫荧光检测神经丝蛋白(NF)和GFAP的表达。结果:ADMSCs经成神经诱导7 d后表达GFAP、NSE和MAP2,半定量PCR结果显示,与对照组相比成神经诱导后的细胞表达N-cadherin显著增高。转染N-cadherin的N-cadherin细胞培养24 h后发出细长的突起,与相邻细胞之间形成网状连接。免疫荧光结果显示,转染后的细胞NF和GFAP表达阳性。结论:ADMSCs在体外多种作用下,具有向神经细胞分化的潜能。N-cadherin转染后能改变ADMSCs的形态,并且在ADMSCs向神经分化中发挥一定的作用。     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

猪的脂肪和骨髓来源间充质干细胞培养特性比较

目的明确小型猪的脂肪来源间充质干细胞（A_r_MSCs）和猪的骨髓来源间充质干细胞（BM—MSCsl体外培养特性的异同。方法广西巴马小型猪,雌雄不限,猪龄4～6个月,体质量20～30kg。AT-MSCs来源于小型猪腹股沟皮下组织．BM—MSCs来源于小型猪的骨髓组织。培养AT-MSCs和BM—MSCs并观察它们的细胞形态。流式细胞仪检测Arr_MSCs和BM—MSCs的表面标志物（CD29、CD34、CD45、CD90）。分别观察Arr-MSCs和BM—MSCs的细胞生长分化能力;实时聚合酶链反应（PCR）检测基因表达。结果流式细胞仪检测结果表明,A．r_MSCs和BM—MSCs均表达CD29[分别为（99．06±0．30）％、（99．94±0．05）％]、CD90[分别为（97．404-0．40）％、（97．43±1J29）％1阳性,CD34、CD45阴性。AT-MSCs传代需培养5～7d,而BM—MSCs需培养7～10d。与BM—MSCs比较,AT—MSCs具有更强的生长分化能力。实时PCR检测基因表达结果显示．AT—MSCs和BM—MSCs均能分化心肌特异标志物a—skeletalactin和Troponin—I．二者差异无统计学意义．表明AT—MSCs和BM—MSCs均具备多项分化潜能．结论AT—MSCs是小型猪干细胞移植治疗的理想选择．     

Adipose tissue-derived mesenchymal stem cells are more potent suppressors of dendritic cells differentiation compared to bone marrow-derived mesenchymal stem cells

Both mesenchymal stem cells (MSCs) and dendritic cells (DCs) are engaged in the regulation of the immune response parallel to their numerous functions.The main objective of this study was to compare the effects of mesenchymal stem cells isolated from human adipose tissue or human bone marrow on the expression of specific cell surface markers as well as the secretion of some cytokines by monocyte-derived dendritic cells. The set of methods used includes cell cultures, magnetic beads isolation of cells, flow cytometry, ELISA and proteome profiler kit assays. The results obtained show that MSCs isolated from human adipose tissue are more potent immunomodulators of differentiation of human DCs in comparison to the bone marrow-derived MSCs. In both cases the percentages of CD14+ cells were increased in co-cultures of MSCs and DCs and at the same time down-regulated the expression of CD80, CD86 and CD83 as in all experiments the effect of adipose tissue MSCs was stronger. Similarly, the secretion of IL-10 by dendritic cells was up-regulated in co-cultures of MSCs and dendritic cells and the effect was stronger when adipose tissue-derived MSCs were used.Taken together all results presented reveal the higher potential of the adipose tissue-derived MSCs to inhibit the differentiation and expression of functionally important co-stimulatory molecules on the surface of monocyte-derived dendritic cells than the bone marrow-derived MSCs.     

The therapeutic efficacy of human adipose tissue-derived mesenchymal stem cells on experimental autoimmune hearing loss in mice

Autoimmune inner ear disease is characterized by progressive, bilateral although asymmetric, sensorineural hearing loss. Patients with autoimmune inner ear disease had higher frequencies of interferon-γ-producing T cells than did control subjects tested. Human adipose-derived mesenchymal stem cells (hASCs) were recently found to suppress effector T cells and inflammatory responses and therefore have beneficial effects in various autoimmune diseases. The aim of this study was to examine the immunosuppressive activity of hASCs on autoreactive T cells from the experimental autoimmune hearing loss (EAHL) murine model. Female BALB/c mice underwent β-tubulin immunization to develop EAHL; mice with EAHL were given hASCs or PBS intraperitoneally once a week for 6 consecutive weeks. Auditory brainstem responses were examined over time. The T helper type 1 (Th1)/Th17-mediated autoreactive responses were examined by determining the proliferative response and cytokine profile of splenocytes stimulated with β-tubulin. The frequency of regulatory T (Treg) cells and their suppressive capacity on autoreactive T cells were also determined. Systemic infusion of hASCs significantly improved hearing function and protected hair cells in established EAHL. The hASCs decreased the proliferation of antigen-specific Th1/Th17 cells and induced the production of anti-inflammatory cytokine interleukin-10 in splenocytes. They also induced the generation of antigen-specific CD4(+) CD25(+) Foxp3(+) Treg cells with the capacity to suppress autoantigen-specific T-cell responses. The experiment demonstrated that hASCs are one of the important regulators of immune tolerance with the capacity to suppress effector T cells and to induce the generation of antigen-specific Treg cells.     

Bioluminescence imaging of calvarial bone repair using bone marrow and adipose tissue-derived mesenchymal stem cells

A combined strategy using bioluminescence imaging, bone densitometry and histology was used to analyze the bone regeneration capacity of human bone marrow (hBMSC) and adipose tissue (hAMSC) mesenchymal stem cells, seeded in an osteoconductive arginine-glycine-aspartate (RGD) crosslinked hydrogel scaffold, implanted in a mouse calvarial bone defect. We show that firefly luciferase labeled stem cells can be monitored in vivo through a prolonged 90 days period, during which hBMSCs survive better than hAMSCs and that the density of scaffold bearing defects increased significantly more than that of defects without scaffolds.     

Short-term exposure to tumor necrosis factor-alpha enables human osteoblasts to direct adipose tissue-derived mesenchymal stem cells into osteogenic differentiation

Tumor necrosis factor-alpha (TNF-α) is one major inflammatory factor peaking at 24?h after bone fracture in response to injury; its role in bone healing is controversial. The aims of this study were to investigate whether the duration of exposure to TNF-α is crucial for the initiation of bone regeneration and to determine its underlying mechanism(s). We demonstrated that 24?h of TNF-α treatment significantly abrogated osteocalcin gene expression by human primary osteoblasts (HOBs). However, when TNF-α was withdrawn after 24?h, bone sialoprotein and osteocalcin gene expression levels in HOBs at day 7 were significantly up-regulated compared with the HOBs without TNF-α treatment. In contrast, continuous TNF-α treatment down-regulated bone sialoprotein and osteocalcin gene expression. In addition, in an indirect co-culture system, HOBs pretreated with TNF-α for 24?h induced significantly greater osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) than the HOBs without TNF-α treatment. TNF-α treatment also promoted endogenous bone morphogenetic protein 2 (BMP-2) production in HOBs, while blocking the BMP-2 signaling pathway with Noggin inhibited osteogenic differentiation of ASCs in the co-culture system. Furthermore, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway after TNF-α treatment occurred earlier than BMP-2 protein expression. BMP-2 production by HOBs and osteogenic differentiation of ASCs in the co-culture system with HOBs was significantly decreased when HOBs were pretreated with TNF-α in combination with the p38 MAPK-specific inhibitor (SB203580). Taken together, we provide evidence that exposure duration is a critical element in determining TNF-α's effects on bone regeneration. We also demonstrate that the p38 MAPK signaling pathway regulates the expression of BMP-2 in osteoblasts, which then acts through a paracrine loop, to direct the osteoblast lineage commitment of mesenchymal stem cells.