Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.     

Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into various tissue types, including bone, cartilage, tendon, adipocytes, and marrow stroma, making them potentially useful for human cell and gene therapies. Our objective was to demonstrate the utility of glass needle-mediated microinjection as a method to deliver macromolecules (e.g. dextrans, DNA) to hMSCs for cell and molecular biological studies. hMSCs were isolated and cultured using a specific fetal bovine serum, prescreened for its ability to promote cell adherence, proliferation, and osteogenic differentiation. Successful delivery of Oregon Green-dextran via intranuclear microinjection was achieved, yielding a postinjection viability of 76 +/- 13%. Excellent short-term gene expression (63 +/- 11%) was achieved following microinjection of GFP-containing vectors into hMSCs. Higher efficiencies of short-term gene expression ( approximately 5-fold) were observed when injecting supercoiled DNA, pYA721, as compared with the same DNA construct in a linearized form, YA721. Approximately 0.05% of hMSCs injected with pYA721 containing both the GFP and neomycin resistance genes formed GFP-positive, drug-resistant colonies that survived >120 days. Injection of linearized YA721 resulted in 3.6% of injected hMSC forming drug-resistant colonies, none of which expressed GFP that survived 60-120 days. These studies demonstrate that glass needle-mediated microinjection can be used as a method of delivering macromolecules to hMSCs and may prove to be a useful technique for molecular and cell biological mechanistic studies and future genetic modification of hMSCs.     

Immunomodulation by mesenchymal stem cells:Interplay between mesenchymal stem cells and regulatory lymphocytes

Mesenchymal stem cells(MSCs) possess immunomodulatory properties, which confer enormous potential for clinical application. Considerable evidence revealed their efficacy on various animal models of autoimmune diseases, such as multiple sclerosis, systemic lupus erythematosus and uveitis. MSCs elicit their immunomodulatory effects by inhibiting lymphocyte activation and proliferation, forbidding the secretion of proinflammatory cytokines, limiting the function of antigen presenting cells, and inducing regulatory T(Treg) and B(Breg) cells. The induction of Treg and Breg cells is of particular interest since Treg and Breg cells have significant roles in maintaining immune tolerance. Several mechanisms have been proposed regarding to the MSCs-mediated induction of Treg and Breg cells. Accordingly, MSCs induce regulatory lymphocytes through secretion of multiple pleiotropic cytokines, cell-to-cell contact with target cells and modulation of antigen-presenting cells. Here, we summarized how MSCs induce Treg and Breg cells to provoke immunosuppression.     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

Substance P enhances mesenchymal stem cells-mediated immune modulation

Since clinical application of MSCs requires long-term ex vivo culture inducing senescence in MSCs and reducing the therapeutic activity of transplanted MSCs, numerous efforts have been attempted to sustain the active state of MSCs. Substance P (SP) is a neuropeptide that functions to activate the cellular physiological responses of MSCs, including proliferation, migration, and secretion of specific cytokines. In this study, we explored the potential of SP to restore the weakened immune modulating activity of MSCs resulting from long-term culture by measuring T cell activity and interleukin-2 (IL-2) secretion of CD4⁺ Jurkat leukemic T cells and primary CD4⁺ T cells. As the number of cell passages increased, the immunosuppressive function of MSCs based on T cell activity decreased. This weakened activity of MSCs could be restored by SP treatment and nullified by co-treatment of an NK1 receptor blocker. Higher levels of transforming growth factor beta 1 (TGF-β1) secretion were noted in the medium of SP-treated late passage MSC cultures, but IL-10 levels did not change. SP-treated MSC-conditioned medium decreased T cell activity and IL-2/Interferon gamma (IFN-g) secretion in T cells even in the activation by lipopolysaccharide (LPS) or CD3/CD28 antibodies, both of which were successfully blocked by inhibiting the TGF beta signaling pathway. This stimulatory effect of SP on late passage MSCs was also confirmed in direct cell–cell contact co-culture of MSCs and CD4⁺ Jurkat T cells. Collectively, our study suggests that SP pretreatment to MSCs may recover the immunosuppressive function of late passage MSCs by potentiating their ability to secrete TGF-β1, which can enhance the therapeutic activity of ex vivo expanded MSCs in long-term culture.     

间充质干细胞促进脐带血干细胞体外扩增的研究进展

非亲缘脐带血移植是治疗造血系统疾病的重要移植方式之一,但脐带血移植面临的最大挑战是造血干细胞(HSCs)数量不足,特别是成人患者受到脐带血干细胞数量的限制,导致造血及免疫恢复延迟,非复发死亡率升高。体外扩增脐带血HSCs(UCB-HSCs)是解决该问题的途径之一。研究发现可以通过模拟骨髓造血龛(niche)这一生态位使HSCs在体外进行自我更新增殖,而间充质干细胞(MSCs)正是造血龛的重要的组成细胞之一。本文将探讨MSCs在UCB-HSCs体外扩增中的应用。重点以MSCs促造血的特点、机制,促进脐带血干细胞增殖的各种策略以及其临床应用和前景做一综述。     

间充质干细胞旁分泌作用的研究进展

间充质干细胞（MSCs）通常利用多分化特性在组织损伤时起到修复功能。然而,近期研究表明,MSCs大多数治疗作用都是通过旁分泌来发挥作用的,其中最受关注的是可溶性蛋白分泌和细胞外膜泡（EVS）。MSCs释放的EVS可反映细胞的来源,能够影响局部微环境中其他细胞的活动。越来越多人提出利用MSCs分泌的各种因子（称为分泌体）替代MSCs细胞治疗的观点。现就MSCs旁分泌特性、分泌体发生和释放机制以及细胞来源对旁分泌影响等方面的研究进展进行综述。     

Allogeneic mesenchymal stem cells: agents of immune modulation

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.     

Telomere stability and telomerase in mesenchymal stem cells

Telomeres are repetitive genetic material that cap and thereby protect the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in high concentrations in the embryonic stem cells and in fast growing embryonic cells, and declines with age. It is still unclear to what extent there is telomerase in adult stem cells, but since these are the founder cells of cells of all the tissues in the body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important. In the present communication we focus on telomere expression and telomere length in stem cells, with a special focus on mesenchymal stem cells. We consider different mechanisms by which stem cells can maintain telomeres and also focus on the dynamics of telomere length in mesenchymal stem cells, both the overall telomere length and the telomere length of individual chromosomes.     

Transdifferentiation of mesenchymal stem cells derived from human fetal lung to hepatocyte-like cells

The great shortage of human hepatic cells makes it desirable to generate extrahepatic stem or precursor cells. In recent years, it has been reported that human multipotential mesenchymal stem cells (hMSCs) differentiate into hepatocyte-like cells. The fetal lung is one of the largest organs containing many MSCs that can be easily obtained. Whether MSCs from fetal lung can differentiate into hepatocytes or bile duct cells is an important issue in basic medicine and clinical application. We isolated fetal lung cells, and expanded and analyzed them. At passage 4, their morphologic, immunophenotyping and cytokine secretions were similar to adult bone marrow-derived MSCs. We conclude that these cells from fetal lung are MSCs, indicating that human fetal lung is an ideal source of hMSCs. hMSCs from fetal lung induced in special differentiation medium showed homogeneous and small polygonal endothelial-like morphology, expressing weak mRNA, as well as Alb and AFP. This implies that hMSCs from fetal lung can differentiate into hepatocyte-like cells.     

Secretion of immunoregulatory cytokines by mesenchymal stem cells

According to the minimal criteria of the International Society of Cellular Therapy, mesenchymal stem cells (MSCs) are a population of undifferentiated cells defined by their ability to adhere to plastic surfaces when cultured under standard conditions, express a certain panel of phenotypic markers and can differentiate into osteogenic, chondrogenic and adipogenic lineages when cultured in specific inducing media. In parallel with their major role as undifferentiated cell reserves, MSCs have immunomodulatory functions which are exerted by direct cell-to-cell contacts, secretion of cytokines and/or by a combination of both mechanisms. There are no convincing data about a principal difference in the profile of cytokines secreted by MSCs isolated from different tissue sources, although some papers report some quantitative but not qualitative differences in cytokine secretion. The present review focuses on the basic cytokines secreted by MSCs as described in the literature by which the MSCs exert immunodulatory effects. It should be pointed out that MSCs themselves are objects of cytokine regulation. Hypothetical mechanisms by which the MSCs exert their immunoregulatory effects are also discussed in this review. These mechanisms may either influence the target immune cells directly or indirectly by affecting the activities of predominantly dendritic cells. Chemokines are also discussed as participants in this process by recruiting cells of the immune systems and thus making them targets of immunosuppression. This review aims to present and discuss the published data and the personal experience of the authors regarding cytokines secreted by MSCs and their effects on the cells of the immune system.     

Human mesenchymal stem cells isolated from the umbilical cord

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. In this study human MSCs were successfully isolated from the umbilical cords. The research characteristics of these cells, e.g., morphologic appearance, surface antigens, growth curve, cytogenetic features, cell cycle, differentiation potential and gene expression were investigated. After 2weeks of incubation, fibroblast-like cells appeared to be dominant. During the second passage the cells presented a homogeneous population of spindle fibroblast-like cells. After more than 4months (approximately 26 passages), the cells continued to retain their characteristics. Flow cytometry analysis revealed that CD29, CD44, CD95, CD105 and HLA-I were expressed on the cell surface, but there was no expression of hematopoietic lineage markers, such as CD34, CD38, CD71 and HLA-DR. Chromosomal analysis showed the cells kept a normal karyotype. The cell cycle at the third passage showed the percentage of G(0)/G(1), G(2)/M and S phase were 88.86%, 5.69% and 5.45%, respectively. The assays in vitro demonstrated the cells exhibited multi-potential differentiation into osteogenic and adipogenic cells. Both BMI-1 and nucleostemin genes, expressed in adult MSCs from bone marrow, were also expressed in umbilical cord MSCs. Here we show that umbilical cords may be a novel alternative source of human MSCs for experimental and clinical applications.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 × 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.     

A simple and highly efficient method for transduction of human adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a wide range of cell types and provide a potential to transfer therapeutic protein in vivo, making them valuable candidates for gene therapy and cell therapy. However, using MSCs in in vivo is limited due to the low rate of transfection and transduction efficacy. Therefore, developing methods to efficiently transfer genes into MSCs would provide a number of opportunities for using them in the clinic. Here, we introduce a simple and robust method for efficient transduction of human adipose-derived MSCs by modification under the culture condition of human embryonic kidney cells 293 (HEK293T) and MSCs. Moreover, as a transduction enhancer, polybrene was replaced with Lipofectamine, a cationic lipid. Therefore, we showed that transduction of primary cells can be increased efficiently by modifying the culture condition.     

Growth factors and chondrogenic differentiation of mesenchymal stem cells

The main purpose of the article is to review recent knowledge about growth factors and their effect on the chondrogenic differentiation of mesenchymal stem cells under in vitro conditions. Damaged or lost articular cartilage leads to progressive debilitation, which have major impact on the life quality of the affected individuals of both sexes in all age groups. Mature hyaline cartilage has a very low self-repair potential due to intrinsic properties - lack of innervation and vascular supply. Another limiting factor is low mitotic potential of chondrocytes. Small defects are healed by migration of chondrocytes, while large ones are healed by formation of inferior fibrocartilage. However, in many cases osteoarthritis develops. Recently, cellular therapy combining mesenchymal stem cells and proper differentiation factors seems to be promising tool for hyaline cartilage defects healing.     

Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes if an appropriate cellular environment is provided. Notch signals exchanged between neighboring cells through the Notch receptor can eventually dictate cell differentiation. In our study, we show that MSC differentiation into cardiomyocytes is dependent on the Notch signal. METHODS: We created a myocardial infarction model in rat by coronary ligation, administered direct intramyocardial injection of DAPI-labeled MSC immediately, and observed the differentiation of MSCs after 14 days by immunofluorescence staining against troponin T. We cultured MSCs and cardiomyocytes in four ways, respectively, in vitro. (1) MSCs cocultured with cardiomyocytes obtained from neonatal rat ventricles in a ratio of 1:10. (2) The two types of cells were cultured in two chambers separated by a semipermeable membrane as indirect coculture group. (3) Notch receptor-soluble jagged1 protein was added to indirect coculture group. (4) Both jagged1 protein and gamma-secretase inhibitor-DAPT were added to indirect coculture group. Two weeks later, we observed the differentiation percentage, respectively, by immunofluorescence staining. RESULTS: We found the differentiation of MSCs which were close to cardiomyocytes in vivo. The differentiation percentage of the four cell culture group was 30.13+/-2.16%, 12.52+/-1.18%, 26.33+/-2.20%, and 13.08+/-1.15%. CONCLUSIONS: MSCs can differentiate into cardiomyocytes in vitro and in vivo if a cardiomyocyte microenvironment is provided. 2. Cell-to-cell interaction is very important for the differentiation of MSCs into cardiomyocytes. 3. Jagged1 protein can activate Notch signal and enhance the differentiation of MSC into cardiomyocyte, while the effect can be inhibited by DAPT.