Culture and properties of adipose-derived mesenchymal stem cells: characteristics in vitro and immunosuppression in vivo

Objective: To compare the two sources of adipose and bone marrow derived mesenchymal stem cells (BMSCs and AMSCs) in immune regulation and to evaluate the therapeutic effects of AMSCs on Con A induced hepatitis and the possible mechanism involved in it. Methods: We isolated bone marrow and adipose derived mesenchymal stem cells respectively and compared their differences on T lymphocyte activation, proliferation and suppression. We also test the anti-apoptosis ability of AMSCs on LO2 cell line. The effects of intravenous infusion of AMSCs on liver damage were also tested and we detected donor AMSCs in liver of recipient and their effects on the activity of intrahepatic NKT cells. Results: BMSCs and AMSCs were similar in cell phenotype and the difference existed only in the expression of CD106. The results showed that the capacity of suppressing T cells proliferation and activation was weakened in AMSCs. AMSCs ameliorated liver damage and this effect was time and dose dependent. We detected donor AMSCs in liver of recipient which suggested tissue damage could be a clue for AMSCs migration. We also found AMSCs suppress the activity of intrahepatic NKT cells, but this suppress effects was not restricted in liver only, but the whole body. Conclusion: Cell origin and abundance are decisive factors in stem cells applications and with the same premise of AMSCs and BMSCs, adipose tissue is a more promising origin source of stem cells. The immunoregulatory features of MSCs might play an important role in various MSCs cellular therapies.     

Autocrine fibroblast growth factor 2 increases the multipotentiality of human adipose-derived mesenchymal stem cells

Multipotent mesenchymal stem cells (MSCs), first identified in the bone marrow, have subsequently been found in many other tissues, including fat, cartilage, muscle, and bone. Adipose tissue has been identified as an alternative to bone marrow as a source for the isolation of MSCs, as it is neither limited in volume nor as invasive in the harvesting. This study compares the multipotentiality of bone marrow-derived mesenchymal stem cells (BMSCs) with that of adipose-derived mesenchymal stem cells (AMSCs) from 12 age- and sex-matched donors. Phenotypically, the cells are very similar, with only three surface markers, CD106, CD146, and HLA-ABC, differentially expressed in the BMSCs. Although colony-forming units-fibroblastic numbers in BMSCs were higher than in AMSCs, the expression of multiple stem cell-related genes, like that of fibroblast growth factor 2 (FGF2), the Wnt pathway effectors FRAT1 and frizzled 1, and other self-renewal markers, was greater in AMSCs. Furthermore, AMSCs displayed enhanced osteogenic and adipogenic potential, whereas BMSCs formed chondrocytes more readily than AMSCs. However, by removing the effects of proliferation from the experiment, AMSCs no longer out-performed BMSCs in their ability to undergo osteogenic and adipogenic differentiation. Inhibition of the FGF2/fibroblast growth factor receptor 1 signaling pathway demonstrated that FGF2 is required for the proliferation of both AMSCs and BMSCs, yet blocking FGF2 signaling had no direct effect on osteogenic differentiation. Disclosure of potential conflicts of interest is found at the end of this article.     

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.     

Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue

Mesenchymal stem cells (MSCs) isolated from bone marrow (BM), cartilage, and adipose tissue (AT) possess the capacity for self-renewal and the potential for multilineage differentiation, and are therefore perceived as attractive sources of stem cells for cell therapy. However, MSCs from these different sources have different characteristics. We compared MSCs of adult Sprague Dawley rats derived from these three sources in terms of their immunophenotypic characterization, proliferation capacity, differentiation ability, expression of angiogenic cytokines, and anti-apoptotic ability. According to growth curve, cell cycle, and telomerase activity analyses, MSCs derived from adipose tissue (AT-MSCs) possess the highest proliferation potential, followed by MSCs derived from BM and cartilage (BM-MSCs and C-MSCs). In terms of multilineage differentiation, MSCs from all three sources displayed osteogenic, adipogenic, and chondrogenic differentiation potential. The result of realtime RT-PCR indicated that these cells all expressed angiogenic cytokines, with some differences in expression level. Flow cytometry and MTT analysis showed that C-MSCs possess the highest resistance toward hydrogen peroxide -induced apoptosis, while AT-MSCs exhibited high tolerance to serum deprivation-induced apoptosis. Both AT and cartilage are attractive alternatives to BM as sources for isolating MSCs, but these differences must be considered when choosing a stem cell source for clinical application.     

骨髓和脂肪来源的间充质干细胞免疫调节作用的比较

目的比较脂肪源间充质干细胞(AMSCs)和骨髓来源间充质干细胞(BMSCs)的免疫调节能力的差异。方法用流式细胞仪分析AMSCs和BMSCs的表型。通过MSCs和淋巴细胞共培养体系,检测MSCs对T细胞增殖、周期、活化、凋亡以及Th细胞分化的影响。结果表型分析结果显示AMSCs和BMSCs的表型基本一致。AMSCs和BMSCs都能抑制T细胞的增殖和早期活化,并在调节辅助性T细胞亚群的分化上作用类似。但在MSC对活化后T细胞的凋亡影响方面,BMSCs能够抑制活化T细胞的凋亡,而AMSC没有这种作用。结论 AMSCs和BMSCs对T淋巴细胞的影响基本类似,其对活化T细胞凋亡影响的差异还有待进一步的机制研究。     

An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells

It is controversial whether a biomaterial itself, rather than addition of any exogenous growth factor, could induce mesenchymal stem cells (MSCs) to differentiate into chondrogenic lineage, further to regenerate cartilage. Previous studies have shown that collagen-based hydrogel could induce MSCs to differentiate into chondrocytes in vivo but the in vitro studies only have a few reports. The evidence that biomaterials could induce chondrogenesis is not adequate. In this study, we tried to address whether type I collagen hydrogel has chondro-inductive capability in vitro and how this scaffold induces MSCs to generate cartilage tissue without exogenous growth factors in the culture medium. We encapsulated neonatal rabbit bone marrow mesenchymal stem cells (BMSCs) in type I collagen hydrogel homogeneously or implanted cell aggregates in hydrogel, and cultured them in nonchondrogenic inductive media. After at least 28 days culture, cells in the homogeneous group were tending to chondrogenic differentiation while cell density was high, and cells in the aggregate group have almost gone through chondrogenesis and formed neo-cartilage tissue with abundant specific extracellular matrix (ECM) deposition. These results indicate collagen hydrogel has inherent inductivity for the chondrogenic differentiation of BMSCs, and the optimum specification and tissue formation were accompanied with local high cell density. This research suggests a feasible strategy to induce the chondro differentiation of BMSCs independent of exogenous growth factors, which may greatly contribute to clinical cartilage regeneration. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2717-2725, 2012.     

Human mesenchymal stem cells derived from bone marrow display a better chondrogenic differentiation compared with other sources

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.     

Potential therapeutic applications of mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are a non-homogeneous population of plastic-adherent cells which were initially isolated from post-natal bone marrow. They have the capacity to differentiate to multiple mesodermal lineages including bone, cartilage and adipose tissue. In stringent culture conditions, MSCs can also be induced to differentiate into different cell types of endoderm and neuroectoderm lineages. To date, no specific marker identifies MSCs, although a number of cell surface antigens have been described which enrich for MSCs. Mesenchymal stromal cells possess a number of properties which have generated considerable interest in diverse cellular therapeutic applications. The capacity of MSCs to differentiate into multiple different cell lineages has seen them actively explored for tissue repair, particularly in cardiac, orthopaedic and neurological applications. A large body of data indicates that MSCs possess immunomodulatory properties. Mesenchymal stromal cells are immunosuppressive, interacting with T lymphocytes, antigen presenting cells, B lymphocytes, and natural killer cells. In addition, they are immunoprivileged, allowing transplantation across allogeneic barriers. These immunomodulatory properties have seen infusion of MSCs for the treatment of steroid refractory graft versus host disease, a life threatening complication of haemopoietic cell transplantation, with promising results. Furthermore, these immune functions may lead to roles in the facilitation of engraftment, induction of tolerance and as therapy in autoimmune disease.     

Mesenchymal cell populations: development of the induction systems for Schwann cells and neuronal cells and finding the unique stem cell population

Mesenchymal cell populations, referred to as mesenchymal stem cells or multipotent stromal cells (MSCs), which include bone marrow stromal cells (BMSCs), umbilical cord stromal cells and adipose stromal cells (ASCs), participate in tissue repair when transplanted into damaged or degenerating tissues. The trophic support and immunomodulation provided by MSCs can protect against tissue damage, and the differentiation potential of these cells may help to replace lost cells. MSCs are easily accessible and can be expanded on a large scale. In addition, BMSCs and ASCs can be harvested from the patient himself. Thus, MSCs are considered promising candidates for cell therapy. In this review, I will discuss recently discovered high-efficiency induction systems for deriving Schwann cells and neurons from MSCs. Other features of MSCs that are important for tissue repair include the self-renewing property of stem cells and their potential for differentiation. Thus, I will also discuss the stemness of MSCs and describe the discovery of a certain stem cell type among adult MSCs that can self-renew and differentiate into cells of all three germ layers. Furthermore, I will explore the prospects of using this cell population for cell therapy.     

Human fallopian tube: a new source of multipotent adult mesenchymal stem cells discarded in surgical procedures

Background  

The possibility of using stem cells for regenerative medicine has opened a new field of investigation. The search for sources to obtain multipotent stem cells from discarded tissues or through non-invasive procedures is of great interest. It has been shown that mesenchymal stem cells (MSCs) obtained from umbilical cords, dental pulp and adipose tissue, which are all biological discards, are able to differentiate into muscle, fat, bone and cartilage cell lineages. The aim of this study was to isolate, expand, characterize and assess the differentiation potential of MSCs from human fallopian tubes (hFTs).     

脂肪组织来源的干细胞的研究进展

来自中胚层的脂肪组织与骨髓组织一样含有大量能自我更新和多向系分化潜能的细胞群称为脂肪干细胞。其取材方便，来源丰富，可在体外稳定增殖传代。并与骨髓间充质干细胞有相似的多向分化表面标志CD105、STRO-1以及CD166受体。研究发现它具有多向系分化潜能，除可以分化为问充质来源的脂肪、骨，软骨、脂肪以及骨骼肌、心肌等细胞，也可诱导分化为来源于外胚层的神经细胞以及具有功能性的血管内皮细胞，用以修复骨、软骨、心肌、骨骼肌、血管以及神经等组织。其具有造血支持作用以及可被逆转录病毒、腺病毒以及慢病毒较高的转染效率等优点，可以作为基因转移良好的靶细胞。因而，脂肪来源的干细胞其有望成为组织工程、细胞治疗以及基因转染良好的种子细胞。     

Induction of intervertebral disc-like cells from adult mesenchymal stem cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)-like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor beta (TGF beta)-mediated induction in vitro. Bone marrow-derived MSCs were differentiated in spheroid culture towards the chondrogenic lineage in the presence of TGF beta(3) dexamethasone, and ascorbate. A customized cDNA-array comprising 45 cartilage-, bone-, and stem cell-relevant genes was used to quantify gene expression profiles. After TGF beta-mediated differentiation, MSC spheroids turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, including aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein, although at levels closer to IVD tissue than to hyaline articular cartilage. Like IVD tissue, the spheroids were strongly positive for collagen type I and osteopontin. MSC spheroids expressed more differentiation markers at higher levels than culture-expanded IVD cells and chondrocytes, which both dedifferentiated in monolayer culture. In conclusion, mesenchymal stem cells adopted a gene expression profile that resembled native IVD tissue more closely than native joint cartilage. Thus, these cells may represent an attractive source from which to obtain IVD-like cells, whereas modification of culture conditions is required to approach the molecular phenotype of chondrocytes in hyaline cartilage.     

The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells

Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1±2.3 days vs. 9.0±1.9 days between each passage for BM-MSCs, respectively, P<0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1-21% O(2)) than BM-MSCs (P<0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P<0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P<0.005) and VEGF expression (P<0.001)) and osteogenesis (as revealed by osteocalcin expression (P<0.001) and micro-CT imagery for newly formed bone tissue (P<0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.     

Human Mesenchymal Stem Cells Derived from Bone Marrow Display a Better Chondrogenic Differentiation Compared with Other Sources

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.     

Multipotent mesenchymal stromal cells in otorhinolaryngology

Multipotent mesenchymal stromal cells (MSCs) are primitive cells capable of restoring damaged mesenchyme and with the ability to differentiate into mature cells of bone, cartilage, muscle, fat, nerve or fibrous tissues. MSCs are therefore good candidates for applications in regenerative medicine and cell based therapy. They regenerate through self-renewal, differentiational capacity, immune modulation and secretion of bioactive molecules. Authors present a review of MSCs applications in otorhinolaryngology. The major interest is focused on phonosurgery, sensorineural deafness and reconstruction of large tissue defects with bone, cartilage or soft tissue replacement. Current evidence of MSCs treatment efficacy in otorhinolaryngology is based on animal models. The true impact on clinical treatment will not be known until clinical studies prove functional outcomes in human medicine.     

AICAR, a small chemical molecule, primes osteogenic differentiation of adult mesenchymal stem cells

The chemical approach to controlling stem cell fates is emerging as a powerful tool, holding great promise in tissue engineering and regenerative medicine. Various small molecules have been demonstrated capable of modulating stem cell differentiation. In this paper, we studied the effects of 5-aminoimidazole-4-carboxamide-1-?-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), on mesenchymal stem cells (MSCs). AICAR at high concentrations (1.0-2.0 mM) significantly inhibited proliferation of both human amnion-derived MSCs (hAMSCs) and rabbit bone marrow-derived MSCs (BM-MSCs). Most importantly, AICAR efficiently promoted the osteogenic differentiation of hAMSCs and BM-MSCs in both growth medium and osteogenic medium. However, Metformin, another AMPK activator, showed no such effects. Meanwhile, AICAR significantly inhibited adipogenic differentiation of hAMSCs and BM-MSCs. Our data suggests that AICAR represents a potent molecule, which can be applied in bone tissue regeneration.     

Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenic differentiation in vitro

Mesenchymal stem cells (MSCs) can be isolated from various tissues and represent an attractive cell population for tissue-engineering purposes. MSCs from bone marrow (bone marrow stromal cells [BMSCs]) are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro. Therefore, BMSCs are said to be available for allogenic cell therapy. Although the immunological characteristics of BMSCs have been the subject of various investigations, those of stem cells isolated from adipose tissue (ASCs) have not been adequately described. In addition, the influence of osteogenic differentiation in vitro on the immunological characteristics of BMSCs and ASCs is the subject of this article. Before and after osteogenic induction, the influence of BMSCs and ASCs on the proliferative behavior of resting and activated allogenic peripheral blood mononuclear cells (PBMCs) was studied as a measure of the immune response (mixed lymphocyte culture). At the same points, the expression of immunologically relevant surface markers (e.g., major histocompatibility complex (MHC)-I, MHC-II, CD40, CD40L) was measured, and correlations between the different sets of results were sought. The pattern of surface antigen expression of BMSCs is the same as that of ASCs. Analogous to BMSCs, undifferentiated cells isolated from adipose tissue lack expression of MHC-II; this is not lost in the course of the osteogenic differentiation process. In co-culture with allogenic PBMCs, both cell types fail to lead to any significant stimulation, and they both retain these characteristics during the differentiation process. BMSCs and ASCs suppress proliferation on activated PBMCs before and after osteogenic differentiation. Our results confirm that MSCs are immune modulating cells. These properties are retained even after osteogenic induction in vitro and seem to be similar in BMSCs and ASCs. Our results suggest that allogenic transplantation of BMSCs and ASCs would be possible, for example, in the context of tissue engineering.     

间充质干细胞在组织工程气管中的研究进展

组织工程学通过细胞或组织重建,修复缺损组织并保留其生物功能,成为气管替代治疗的新途径.种子细胞、生长因子及气管支架材料是组织工程气管的三大要素,学者们一直在寻求理想的种子细胞.间充质干细胞(MSCs)是一种具有高度自我更新能力和多向分化潜能的干细胞,广泛分布于骨髓、脐带、脂肪组织、心肌组织、大脑、肌肉和皮肤等,可向骨、软骨、脂肪、神经等多种细胞定向分化.MSCs具有增殖能力强、分化范围广、免疫调节等功能特点,能够修复损伤的组织,有望成为应用于组织工程气管的理想种子细胞.旨在对MSCs在组织工程气管中的应用作一综述.     

Bone Marrow Mesenchymal Cells: How Do They Contribute to Tissue Repair and Are They Really Stem Cells?

Adult stem cells typically generate the cell types of the tissue in which they reside, and thus the range of their differentiation is considered limited. Bone marrow mesenchymal stem cells (MSCs) are different from other somatic stem cells in that they differentiate not only into the same mesodermal-lineage such as bone, cartilage, and adipocytes but also into other lineages of ectodermal and endodermal cells. Thus, MSCs are a unique type of adult stem cells. In addition, MSCs home to damaged sites, differentiate into cells specific to the tissue and contribute to tissue repair. Therefore, application of MSCs in the treatment of various diseases, including liver dysfunction, myocardial infarction, and central nervous system repair, has been initiated. Because MSCs are generally harvested as adherent cells from bone marrow aspirates, however, they comprise heterogeneous cell populations and their wide-ranging differentiation ability and repair functions are not yet clear. Recent evidence suggests that a very small subpopulation of cells that assume a repair function with the ability to differentiate into trilineage cells resides among human MSCs and effective utilization of such cells is expected to improve the repair effect of MSCs. This review summarizes recent advances in the clarification of MSC properties and discusses future perspectives.