Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.     

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.     

Adipogenic differentiation of human adipose-derived stem cells grown as spheroids

Understanding the process of adipogenesis is critical if suitable therapeutics for obesity and related metabolic diseases are to be found. The current study presents proof of feasibility of creating a 3-D spheroid model using human adipose-derived stem cells (hASCs) and their subsequent adipogenic differentiation. hASC spheroids were formed atop an elastin-like polypeptide-polyethyleneimine (ELP-PEI) surface and differentiated using an adipogenic cocktail. Spheroids were matured in the presence of dietary fatty acids (linoleic or oleic acid) and evaluated based on functional markers including intracellular protein, CD36 expression, triglyceride accumulation, and PPAR-γ gene expression. Spheroid size was found to increase as the hASCs matured in the adipocyte maintenance medium, though the fatty acid treatment generally resulted in smaller spheroids compared to control. A stable protein content over the 10-day maturation period indicated contact-inhibited proliferation as well as minimal loss of spheroids during culture. Spheroids treated with fatty acids showed greater amounts of intracellular triglyceride content and greater expression of the key adipogenic gene, PPAR-γ. We also demonstrated that 3-D spheroids outperformed 2-D monolayer cultures in adipogenesis. We then compared the adipogenesis of hASC spheroids to that in 3T3-L1 spheroids and found that the triglyceride accumulation was less profound in hASC spheroids than that in 3T3-L1 adipocytes, correlated with smaller average spheroids, suggesting a relatively slower differentiation process. Taken together, we have shown the feasibility of adipogenic differentiation of patient-derived hASC spheroids, which with further development, may help elucidate key features in the adipogenesis process.     

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。     

miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells

Human adipose-derived stem cells (hADSC) are capable of differentiating into an osteogenic lineage. It is believed that microRNAs (miRNAs) play important roles in regulating this osteogenic differentiation of human adipose-derived cells, although its molecular mechanism remains unclear. We investigated the miRNA expression profile during osteogenic differentiation of hADSCs, and assessed the roles of involved miRNAs during the osteogenic differentiation. We obtained and cultured human adipose-derived stems cells from donors who underwent elective liposuction or other abdominal surgery at our institution. miRNA expression profiles pre- and post-osteogenic induction were obtained using microarray essay, and differently expressed miRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of osteogenic proteins was detected using an enzyme-linked immunosorbent assay. Putative targets of the miRNAs were predicted using online software MiRanda, TargetScan, and miRBase. Eight miRNAs were found differently expressed pre- and post-osteogenic induction, among which four miRNAs (miR-17, miR-20a, miR-20b, and miR-106a) were up-regulated and four miRNAs (miR-31, miR-125a-5p, miR-125b, and miR-193a) were down-regulated. qRT-PCR analysis further confirmed the results. Predicted target genes of the differentially expressed miRNAs based on the overlap from three public prediction algorithms: MiRanda, TargetScan, and miRBase Target have the known functions of regulating stem cell osteogenic differentiation, self-renewal, signal transduction, and cell cycle control. We identified a group of miRNAs that may play important roles in regulating hADSC cell differentiation toward an osteoblast lineage. Further study of these miRNAs may elucidate the mechanism of hADSC differentiation into adipose tissue, and thus provide basis for tissue engineering.     

Sox9基因诱导脂肪干细胞向软骨细胞分化

目的:观察Sox9基因对人脂肪干细胞(ADSCs)的诱导作用.方法:分离、纯化、培养人源ADSCs,并绘制生长曲线,传代三次后的ADSCs利用脂质体转染Sox9基因,选用抗生素G418进行筛选.以空载体转染细胞作对照,分别取48h和14d转染后的细胞做Flag蛋白免疫组织化学鉴定.通过检测转染细胞中Ⅱ型胶原来确定ADSCs是否向软骨细胞分化.结果:ADSCs呈长梭形,形态与骨髓间充质干细胞相似,600μ/ml G418为最适筛选浓度.转染后第48h和14d的细胞均能表达Sox9基因融合表达的Flag蛋白.第48h和14d,转染效率分别为93%和75%.转染后14d的ADSCs表达Ⅱ型胶原,转染后48h实验组和对照组都为阴性.结论:Sox9基因能诱导脂肪干细胞向软骨细胞分化.     

Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice

Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have recently reported that adipose-derived stromal cells (ASCs) could differentiate into mesenchymal lineages in vitro. In this study, we performed neural induction using ASCs from GFP transgenic mice and were able to induce these ASCs into neuronal and glial cell lineages. Most of the neurally induced cells showed bipolar or multipolar appearance morphologically and expressed neuronal markers. Electron microscopy revealed their neuronal morphology. Some cells also showed glial phenotypes, as shown immunocytochemically. The present study clearly shows that ASCs derived from GFP transgenic mice differentiate into neural lineages in vitro, suggesting that these cells might provide an ideal source for further neural stem cell research with possible therapeutic application for neurological disorders.     

脂肪干细胞的共培养及其分化应用概述

干细胞在体外特定培养条件下可以被诱导分化成具有不同体细胞表型的细胞。除了通过不同培养条件进行体外诱导分化的方法外,用成熟体细胞与干细胞共培养同样可以诱导干细胞定向分化。以下首先简述了脂肪干细胞 (Adipose-derived stem cells,ADSCs) 的来源及其标志,然后重点就ADSCs的不同培养方法、诱导分化及最新的临床应用进行阐述,包括药物及化学诱导培养、体细胞与ADSCs二维、三维共培养等,最后提出ADSCs的问题所在并对此技术进行展望。     

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.     

Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells

The limited capacity of nervous system to promote a spontaneous regeneration and the high rate of neurodegenerative diseases appearance are keys factors that stimulate researches both for defining the molecular mechanisms of pathophysiology and for evaluating putative strategies to induce neural tissue regeneration. In this latter aspect, the application of stem cells seems to be a promising approach, even if the control of their differentiation and the maintaining of a safe state of proliferation should be troubled. Here, we focus on adipose tissue-derived stem cells and we seek out the recent advances on the promotion of their neural differentiation, performing a critical integration of the basic biology and physiology of adipose tissuederived stem cells with the functional modifications that the biophysical, biomechanical and biochemical microenvironment induces to cell phenotype. The pre-clinical studies showed that the neural differentiation by cell stimulation with growth factors benefits from the integration with biomaterials and biophysical interaction like microgravity. All these elements have been reported as furnisher of microenvironments with desirable biological, physical and mechanical properties. A critical review of current knowledge is here proposed, underscoring that a real advance toward a stable, safe and controllable adipose stem cells clinical application will derive from a synergic multidisciplinary approach that involves material engineer, basic cell biology, cell and tissue physiology.     

Genomic integrity of human induced pluripotent stem cells:Reprogramming,differentiation and applications

Ten years after the initial generation of induced pluripotent stem cells(hiPSCs)from human tissues, their potential is no longer questioned, with over 15000 publications listed on PubMed, covering various fields of research; including disease modeling, cell therapy strategies, pharmacology/toxicology screening and 3 D organoid systems. However, despite evidences that the presence of mutations in hiPSCs should be a concern, publications addressing genomic integrity of these cells represent less than 1% of the literature. After a first overview of the mutation types currently reported in hiPSCs, including karyotype abnormalities, copy number variations, single point mutation as well as uniparental disomy, this review will discuss the impact of reprogramming parameters such as starting cell type and reprogramming method on the maintenance of the cellular genomic integrity. Then, a specific focus will be placed on culture conditions and subsequent differentiation protocols and how their may also trigger genomic aberrations within the cell population of interest.Finally, in a last section, the impact of genomic alterations on the possible usages of hiPSCs and their derivatives will also be exemplified and discussed. We will also discuss which techniques or combination of techniques should be used to screen for genomic abnormalities with a particular focus on the necessary quality controls and the potential alternatives.