Characterization of cell signaling,morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.     

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.     

Maintenance of human adipose derived stem cell (hASC) differentiation capabilities using a 3D culture

In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.     

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages

Adipose-derived stem cells (ASCs) have been successfully applied in treating bone defects both in animals and humans and promoted osteogenesis in vivo significantly. However, the mechanism of in vivo osteogenesis of ASCs was still little known, we hypothesized that this was mediated in part by interaction between implanted ASCs and local vein endothelial cells. In this study, human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVEC) were isolated and characterized. Cells were then either cultured alone or cocultured. Alkaline phosphatase (ALP) staining, quantitative measurement of ALP activity and Alizarin staining of hASCs cultured alone, HUVEC cultured alone and cells cocultured demonstrated that osteogenic differentiation of cocultured cells increased obviously. Osteocalcin (OC) expression of hASCs cocultured with HUVEC showed an obvious raise than hASCs cultured alone. HUVEC cultured alone showed BMP-2 secretion and increased with culturing time. Real-time PCR of the cocultured cells showed four osteogenic differentiation related genes raised with culturing time, while two adipogenic differentiation related genes showed a slightly decrease with culturing time. Results of our study with different culture models showed that in vitro osteogenesis of hASCs was enhanced by coculture with HUVEC which secreted BMP-2. This study not only provided us with an in vitro model of studying interaction between cells, but also helped us to understand the in vivo therapeutic mechanisms of ASCs.     

Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferator-activated receptor (PPAR-γ2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.     

Human adipose-derived stem cells cultured in keratinocyte serum free medium: Donor’s age does not affect the proliferation and differentiation capacities

Background

Although donor age-related effects of characteristics of mesenchymal stem cells (MSC), such as a decrease in the proliferation and differentiation capacity and an increase of senescence and apoptosis, are evident, such effects are generally less prominent in adipose-derived stem cells (ASC). Using a hormone and growth factor rich medium (KFSM), this study cultured ASC from abdominal subcutaneous fat of 27 adult females in three age groups: 30-39 y, 40-49 y and 50-60 y, and investigated the growth and differentiation characteristics.

Results

The derived ASC had an immunophenotype similar to that of bone marrow derived MSC (BMSC). They could be stably expanded with an average population doubling time of 21.5 ± 2.3 h. Other than a higher pre-adipogenic commitment and a lower adipogenic differentiation capability in ASC derived from the old age group, other characteristics including proliferation rate, doubling time, telomere length, as well as the osteogenic and chondrogenic differentiation capacity were the same regardless of the donor’s age.

Conclusions

The study demonstrates a promising proliferation and differentiation capabilities of ASC regardless of the donor’s age. The compromised adipogenic potential in the older donors could be a benefit for their application in regeneration therapy.     

Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2

Background aimsHuman bone marrow mesenchymal stromal cells are useful in regenerative medicine for various diseases, but it remains unclear whether the aging of donors alters the multipotency of these cells. In this study, we examined age-related changes in the chondrogenic, osteogenic and adipogenic potential of mesenchymal stromal cells from 17 donors (25–81 years old), including patients with or without systemic vascular diseases.MethodsAll stem cell lines were expanded with fibroblast growth factor-2 and then exposed to differentiation induction media. The chondrogenic potential was determined from the glycosaminoglycan content and the SOX9, collagen type 2 alpha 1 (COL2A1) and aggrecan (AGG) messenger RNA levels. The osteogenic potential was determined by monitoring the alkaline phosphatase activity and calcium content, and the adipogenic potential was determined from the glycerol-3-phosphate dehydrogenase activity and oil red O staining.ResultsSystemic vascular diseases, including arteriosclerosis obliterans and Buerger disease, did not significantly affect the trilineage differentiation potential of the cells. Under these conditions, all chondrocyte markers examined, including the SOX9 messenger RNA level, showed age-related decline, whereas none of the osteoblast or adipocyte markers showed age-dependent changes.ConclusionsThe aging of donors from young adult to elderly selectively decreased the chondrogenic potential of mesenchymal stromal cells. This information will be useful in stromal cell–based therapy for cartilage-related diseases.     

Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca²⁺ concentration ([Ca²⁺]_out) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca²⁺]_out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca²⁺]_out transiently increased the intracellular Ca²⁺ concentration ([Ca²⁺]_in) due to activation of the calcium-sensing receptor (CaSR). The Ca²⁺-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca²⁺]_out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca²⁺]_out enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca²⁺]_in and stimulation of Ca²⁺/calmodulin-dependent NFAT-signaling pathways.     

Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR

BackgroundInsulin-like growth factor 2 (IGF2), an essential component of the stem cell niche, has been reported to modulate the proliferation and differentiation of stem cells. Previously, a continuous expression of IGF2 in tissues was reported to maintain the self-renewal ability of several types of stem cells. Therefore, in this study, we investigated the expression of IGF2 in adipose tissues and explored the effects of IGF2 on adipose-derived stromal cells (ADSCs) in vitro.MethodsThe expression pattern of IGF2 in rat adipose tissues was determined by gene expression and protein analyses. The effect of IGF2 on proliferation, stemness-related marker expression and adipogenic and osteogenic differentiation was systematically investigated. Furthermore, antagonists of IGF2-specific receptors—namely, BMS-754807 and picropodophyllin—were added to explore the underlying signal transduction mechanisms.ResultsIGF2 levels displayed a tendency to decrease with age in rat adipose tissues. After the addition of IGF2, isolated ADSCs displayed higher proliferation and expression of the stemness-related markers NANOG, OCT4 and SOX2 and greater differentiation potential to adipocytes and osteoblasts. Additionally, both type 1 insulin-like growth factor receptor (IGF-1R) and insulin receptor (IR) participated in the IGF2-mediated promotion of stemness in ADSCs.ConclusionsOur findings indicate that IGF2 could enhance the stemness of rat ADSCs via IGF-1R and IR and may highlight an effective method for the expansion of ADSCs for clinical application.     

Effect of Labeling with Iron Oxide Particles or Nanodiamonds on the Functionality of Adipose-Derived Mesenchymal Stem Cells

Stem cells are increasingly the focus of translational research as well as having emerging roles in human cellular therapy. To support these uses there is a need for improved methods for in vivo cell localization and tracking. In this study, we examined the effects of cell labeling on the in vitro functionality of human adipose-derived mesenchymal stem cells. Our results provide a basis for future in vivo studies investigating implanted cell fate and longevity. In particular, we investigated the effects of two different particles: micron-sized (∼0.9 µm) fluorescently labeled (Dragon Green) superparamagnetic iron oxide particles (M-SPIO particles); and, carboxylated nanodiamonds of ∼0.25 µm in size. The effects of labeling on the functionality of adipose-derived MSCs were assessed by in vitro morphology, osteogenic and adipogenic differentiation potential, CD marker expression, cytokine secretion profiling and quantitative proteomics of the intra-cellular proteome. The differentiation and CD marker assays for stem-like functionality were not altered upon label incorporation and no secreted or intra-cellular protein changes indicative of stress or toxicity were detected. These in vitro results indicate that the M-SPIO particles and nanodiamonds investigated in this study are biocompatible with MSCs and therefore would be suitable labels for cell localization and tracking in vivo.     

Human adipose-derived stem cells display myogenic potential and perturbed function in hypoxic conditions

Here, we enriched a human cell population from adipose tissue that exhibited both mesenchymal plasticity, self-renewal capacity, and a cell-surface marker profile indistinguishable from that of bone marrow-derived mesenchymal stem cells. In addition to adipogenic and osteogenic differentiation, these adipose-derived stem cells displayed skeletal myogenic potential when co-cultured with mouse skeletal myocytes in reduced serum conditions. Physical incorporation of stem cells into multinucleated skeletal myotubes was determined by genetic lineage tracing, whereas human-specific antibody staining was employed to demonstrate functional contribution of the stem cells to a myogenic lineage. To investigate the effects of hypoxia, cells were maintained and differentiated at 2% O(2). In contrast with reports on bone marrow-derived stem cells, both osteogenic and adipogenic differentiation were significantly attenuated. In summary, the relative accessibility of adipose-derived mesenchymal stem cells from human donors provides opportunity for molecular investigation of mechanistic dysfunction in disease settings and may introduce new prospects for cell-based therapy.     

Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells

A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.     

MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network

MicroRNAs are a group of endogenous regulators that participate in several cellular physiological processes. However, the role of miR-137 in the osteogenic differentiation of human adipose-derived stem cells (hASCs) has not been reported. This study verified a general downward trend in miR-137 expression during the osteogenic differentiation of hASCs. MiR-137 knockdown promoted the osteogenesis of hASCs in vitro and in vivo. Mechanistically, inhibition of miR-137 activated the bone morphogenetic protein 2 (BMP2)-mothers against the decapentaplegic homolog 4 (SMAD4) pathway, whereas repressed lysine-specific histone demethylase 1 (LSD1), which was confirmed as a negative regulator of osteogenesis in our previous studies. Furthermore, LSD1 knockdown enhanced the expression of BMP2 and SMAD4, suggesting the coordination of LSD1 in the osteogenic regulation of miR-137. This study indicated that miR-137 negatively regulated the osteogenic differentiation of hASCs via the LSD1/BMP2/SMAD4 signaling network, revealing a new potential therapeutic target of hASC-based bone tissue engineering.     

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.     

mitoTEMPO改善糖尿病小鼠脂肪干细胞的氧化损伤

目的:探讨线粒体靶向抗氧化剂mitoTEMPO对糖尿病小鼠脂肪干细胞(Adipose-derived stem cells,ADSCs)氧化损伤的影响。方法:采用60%高脂饮食喂养雄性C57小鼠(4周龄)连续8周,并在高脂喂养第2周,对小鼠进行连续5天腹腔注射低剂量链脲佐菌素(streptozotocin,STZ)(25 mg·kg-1)制备2型糖尿病小鼠模型。喂养2周后,检测小鼠血浆葡萄糖水平等指标符合2型糖尿病标准者纳入实验。分别从正常小鼠与STZ诱导的糖尿病小鼠的腹股沟处皮下脂肪组织分离培养脂肪干细胞(ADSCs),并将其各分为4组:DMEM培养的正常ADSCs组(nADSCs组),DMEM培养的糖尿病ADSCs组(dADSCs组),TEMPO治疗的糖尿病ADSCs组(T-dADSCs组),mitoTEMPO治疗的糖尿病ADSCs组(mitoT-dADSCs组)。采用细胞计数试剂盒-8(CCK-8)检测细胞存活能力;油红-O和茜素红染色分别检测成脂细胞分化与成骨细胞分化能力;划痕实验和Transwell试验分别测定细胞迁移能力;DCF和mito SOX染色荧光成像分别检测细胞内和线粒体中的活性氧簇(Reactive oxygen species, ROS)水平。结果:①与nADSCs组相比,d ADSCs组的细胞活力明显下降(P0.05)、成骨细胞分化与成脂细胞分化程度明显下降(P0.05)、脂肪干细胞迁移能力明显下降(P0.05)、细胞内和线粒体中ROS水平明显升高(P0.05)。②与dADSCs组相比,T-dADSCs和mitoT-dADSCs组的细胞内和线粒体中的ROS水平明显降低(P0.05);与dADSCs组相比,mitoT-dADSCs组的成骨细胞分化与成脂细胞分化能力明显提升(P0.05),基本达到nADSCs组的分化水平;与dADSCs组相比,mitoT-dADSCs治疗组的细胞迁移能力显著升高(P0.05)、T-dADSCs组的细胞迁移能力增长无明显差异。结论:mitoTEMPO可以减轻糖尿病时线粒体内活性氧簇蓄积造成的脂肪干细胞的氧化应激损伤与功能紊乱。     

Retropatellar fat pad–derived stem cells from older osteoarthritic patients have lesser differentiation capacity and expression of stemness genes

Background aimsThe use of retropatellar fat pad–derived mesenchymal stromal cells (RFMSCs) for cell-based therapy, particularly for cartilage repair, has been reported by several investigators in recent years. However, the effects of the donor's age and medical condition on the characteristics of RFMSCs have not been well established. The aim of this study was to determine whether age and medical condition can reduce the multipotential of stem cells isolated from the retropatellar fat pad.MethodsThe RFMSCs were isolated from patients with osteoarthritic knee cartilage (degenerative group; 40–60 years old) and compared with patients without degenerative knee disease (young group; <40 years old) in terms of their growth kinetics, immunophenotype, differentiation ability and stemness gene expression.ResultsData showed that RFMSCs from both groups have similar growth kinetics and immunophenotype profile at passage 3. However, RFMSCs from the degenerative group showed lower adipogenic, osteogenic and chondrogenic differentiation ability compared with RFMSCs derived from the young group. The stemness gene expression level of RFMSCs derived from the degenerative group was lower than that in the young group. RFMSCs from both groups met the minimum criteria of mesenchymal stromal cells and have the potential for cartilage regeneration. However, RFMSCs from the degenerative group showed lower regeneration capability.ConclusionsThese results indicate that older age and osteoarthritic condition did affect the multipotential of stem cells derived from the retropatellar fat pad under the current prescribed condition. More studies will be conducted to clarify whether the age or medical condition contributed more to the loss of differentiation capacity and stemness gene expression of RFMSCs.