Pressure and Distortion Regulate Human Mesenchymal Stem Cell Gene Expression

While the concept that physical forces such as tension and compression are involved in mature tissue modeling is widely accepted, the role of these specific types of mechanical loading in the differentiation and maturation of uncommitted cell types like human mesenchymal stem cells (hMSCs) is currently unknown. We observed that hMSCs have the fundamental ability to distinguish between dynamic tensile and compressive loading by regulating distinct gene expression patterns and that these differences in gene expression can be related to conformational changes in cell shape and volume. Dynamic tension was found to regulate both fibroblastic and osteogenic associated genes while dynamic compression up-regulated genes associated with chondrogenesis. Identifying genes involved in the mechanotransduction of different modes of physical loading in hMSC may greatly enhance the ability to rationally design tissue regeneration systems to restore proper tissue function.     

Matrix Stiffness Modulates Mesenchymal Stem Cell Sensitivity to Geometric Asymmetry Signals

Human stem cells hold significant potential for the treatment of various diseases. However, their use as a therapy is hampered because of limited understanding of the mechanisms by which they respond to environmental stimuli. Efforts to understand extracellular biophysical cues have demonstrated the critical roles of geometrical and mechanical signals in determining the fate of stem cells. The goal of this study was to explore the interplay between cell polarity and matrix stiffness in stem cell lineage specification. We hypothesize that confining cells to asymmetric extracellular matrix islands will impart polarity at a single-cell level and will interact with mechanical signals to define the lineage of stem cells. To test these hypotheses, we employed microcontact printing to create patterned symmetric and asymmetric hydrogel islands of soft and hard surface stiffness. Human mesenchymal stem cells (hMSCs) were confined to these islands at the single-cell level and given the ability to differentiate along adipogenic or osteogenic routes. Our results demonstrated that cell polarity defines the lineage specification of hMSCs only on islands with low stiffness. Insight gained from this study provides a rational basis for designing stem cell cultures to enhance tissue engineering and regenerative medicine strategies.     

CD146 Expression Influences Periapical Cyst Mesenchymal Stem Cell Properties

Recent studies have identified a new human dental derived progenitor cell population with multi-lineage differentiation potential referred to as human periapical cyst mesenchymal stem cells (hPCy-MSCs). In the present study, we compared two subpopulations of hPCy-MSCs characterised by the low or high expression of CD146 to establish whether this expression can regulate their stem cell properties. Using flow cytometry, we evaluated the stem cell marker profile of hPCy-MSCs during passaging. Furthermore, CD146^Low and CD146^High cells were sorted by magnetic beads and subsequently both cell populations were evaluated for differences in their proliferation, self-renewal, stem cell surface markers, stemness genes expression and osteogenic differentiation potential.We found that hPCy-MSCs possessed a stable expression of several mesenchymal stem cell surface markers, whereas CD146 expression declined during passaging.In addition, sorted CD146^Low cells proliferated significantly faster, displayed higher colony-forming unit-fibroblast capacity and showed higher expression of Klf4 when compared to the CD146^High subset. Significantly, the osteogenic potential of hPCy-MSCs was greater in the CD146^Low than in CD146^High population. These results demonstrate that CD146 is spontaneously downregulated with passaging at both mRNA and protein levels and that the high expression of CD146 reduces the proliferative, self-renewal and osteogenic differentiation potential of hPCy-MSCs. In conclusion, our study demonstrates that changes in the expression of CD146 can influence the stem cell properties of hPCy-MSCs.     

可溶性人干细胞因子基因导入真核细胞及其表达

本文构建了编码可溶性人ＳＣＦ基因的逆转录病毒载体ｐＬＸＳＮ－ＳＣＦ，应用Ｌｉｐｏｆｅｃｔｉｎ基因转移法将重组质粒导入Ψ２和ＰＡ３１７病毒包装细胞，经Ｇ４１８选择性培养基筛选，获得产重组病毒的包装细胞ＰＡ３１７－ＳＣＦ，其病毒效价为２．４×１０５～８．５×１０５ＣＦＵ／ｍｌ，继而感染人造血干祖细胞和ＮＩＨ３Ｔ３细胞。应用ＰＣＲ、ＡＰＡＡＰ免疫组化染色和化学发光一直接酶标法检测上述细胞中人ＳＣＦ基因的转染和表达，结果表明逆转录病毒载体介导转染的ｒｈ～ＳＣＦ基因在真核细胞中获得有效的表达。并将转有外源基因的人骨髓细胞进行体外液体长期培养（ＬＴＣ），观察造血干祖细胞增殖分化期间基因的表达情况。     

间充质干细胞与组织工程血管

间充质干细胞在再生医学领域中具有重要的应用价值,其增殖能力强,免疫原性低,具有良好的分化潜能,是理想的组织工程血管种子细胞。本文系统总结了应用间充质干细胞构建组织工程血管的研究进展,介绍了间充质干细胞分离纯化和鉴定方法,分类阐述了间充质干细胞构建组织工程血管的几种方法,并探讨了其优缺点。     

基于形态学的骨髓基质干细胞溶液图像分割研究

采用现在研究比较多的骨髓基质干细胞的培养液图像作为研究对象,实现骨髓基质干细胞分割。首先采用灰度形态学中的高低帽算法组合应用对图像进行预处理,结构函数取半径为30的球;预处理完成后,采用两次取阈值法将前景(骨髓基质干细胞)和背景(溶液)粗略地区分出来;然后采用二值形态学方法对所得到图像进行去噪处理,去噪后所得到的图像能将前景和背景较好区分出来。在此基础上求取细胞的个数和面积。     

Gene Expression Pattern of Human Chorion-Derived Mesenchymal Stem Cells during Adipogenic Differentiation

Purpose: The aim of this study was to identify the adipocyte-specific gene expression patterns in chorion-derived mesenchymal stem cells during adipogenic differentiation. Materials and Methods: Chorionic cells were isolated from the third trimester chorions from human placenta at birth and identified morphologically and by fluorescence-activated cell sorting analysis. After inducing adipogenic differentiation for 28 days, cells at days 3, 10, 21 and 28 were analyzed by Oil red O staining and RNA extraction in order to assess the expression levels of adipocyte marker genes, including CCAAT-enhancer binding protein α (C/EBPα), peroxisome proliferator- activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4) and Glycerol-3-phosphate dehydrogenase (GPD2). Cells not induced for differentiation were compared with the induced cells as a control group. Results: Chorion-derived cells showed the same pattern as fibroblasts, and expressed CD73, CD105, and CD166 antigens, but not CD45, CD34, and HLA-DR antigens. On day 3 after differentiation, cells began to stain positively upon Oil red O staining, and continuously increased in lipid granules for 4 weeks. The expression level of C/EBPα increased 4.6 fold on day 3 after induction, and continued to increase for 4 weeks. PPARγ was expressed at a maximum of 2.9 fold on day 21. FABP4 and GPD2 were significantly expressed at 4.7- and 3.0-fold, respectively, on day 21, compared to controls, and further increased thereafter. Conclusion: Human chorion-derived mesenchymal stem cells exhibited the sequential expression pattern of adipocyte marker genes during differentiation, corresponding to adipogenesis.     

Plasticity of Human Mesenchymal Stem Cell Phenotype and Expression Profile under Neurogenic Conditions

Human bone marrow MSC cultured in neurogenic medium containing EGF and FGFb demonstrated alteration of the phenotype and expression of neuronal precursor/early neuron markers nestin and NSE. Signals of expression of neuronal and oligodendroglial markers MAP-2, dm-20, and MBP were detected after prolongation of incubation in neurogenic medium to 2 weeks. Cells with neuronal morphology were immunopositive to early neuronal marker β-III-tubulin. Replacement of neurogenic medium for α-MEM with 10% fetal calf serum induced reversion of the phenotype to that typical for human MSC. This indicates high plasticity of the phenotype and expression profile of neuronal markers in MSC cultured under neurogenic conditions or possibility of dedifferentiation of MSC reaching the stage of neuronal precursors/early neurons.     

人脐带、胎盘间充质干细胞样细胞中“干”性蛋白表达的研究

检测原代分离培养的人脐带、胎盘间充质干细胞样细胞中CD29、CD34、CD44、CD133、HLA-DR、vWF、MRP1、ABCG2 、P75NTR及Nestin的表达.采用组织块贴壁培养法分离培养人脐带、胎盘间充质干细胞样细胞,通过培养扩增后用流式细胞仪检测CD29、CD34、CD44、CD133、HLA-DR及vWF的表达,应用免疫荧光技术检测人脐带、胎盘间充质干细胞样细胞中MRP1、ABCG2、P75 NTR、Nestin的表达.在培养的人脐带、胎盘间充质干细胞样细胞中,流式细胞仪检测示CD29、CD44表达阳性,CD34、CD133、HLA-DR及vWF表达阴性;免疫荧光示MRP1、ABCG2 、P75NTR及Nestin在人脐带、胎盘间充质干细胞样细胞中表达阳性且荧光定位在细胞的胞浆.人脐带、胎盘间充质干细胞样细胞表达间充质干细胞的免疫表型;MRP1、ABCG2、P75NTR、Nestin在人脐带、胎盘间充质干细胞样细胞中表达阳性,提示人脐带与胎盘来源的间充质干细胞样细胞“干”性蛋白的表达情况相似.     

Gene Delivery Approaches for Mesenchymal Stem Cell Therapy: Strategies to Increase Efficiency and Specificity

A significant number of clinical trials have been undertaken to explore the use of mesenchymal stem cells (MSCs) for the treatment of several diseases such as Crohn’s disease, diabetes, bone defects, myocardial infarction, stroke etc., Due to their efficiency in homing to the tissue injury sites, their differentiation potential, the capability to secrete a large amount of trophic factors and their immunomodulatory effects, MSCs are becoming increasingly popular and expected to be one of the promising therapeutic approaches. However, challenges associated with the isolation of pure MSC populations, their culture and expansion, specific phenotypic characterization, multi-potential differentiation and challenges of efficient transplantation limit their usage. The current strategies of cell-based therapies emphasize introducing beneficial genes, which will improve the therapeutic ability of MSCs and have better homing efficiency. The continuous improvement in gene transfer technologies has broad implications in stem cell biology. Although viral vectors are efficient vehicles for gene delivery, construction of viral vectors with desired genes, their safety and immunogenicity limit their use in clinical applications. We review current gene delivery approaches, including viral and plasmid vectors, for transfecting MSC with beneficial genes. The review also discusses the use of a few emerging technologies that could be used to improve the transfer/induction of desirable genes for cell therapy.     

Interdependence of Pulsed Ultrasound and Shear Stress Effects on Cell Morphology and Gene Expression

Fluid shear stress is a key biomechanical regulatory factor in a several biological systems including bone tissue. Bone cells are also regulated by exogenous acoustic vibration, which has therapeutic benefits. In this study, we determined the effects of shear stress and pulsed ultrasound (US), alone and in series on osteoblast morphology and gene expression. We observed that shear stress (19 dyne/cm²) elongated SaOS-2 cells at 3, 6, 24, and 48 h decreasing their shape index from control values of 0.51 ± 0.01, 0.60 ± 0.05, 0.59 ± 0.04, and 0.45 ± 0.01 to 0.45 ± 0.04, 0.47 ± 0.03, 0.39 ± 0.02, and 0.33 ± 0.01, respectively. This morphological effect was inhibited at 24 and 48 h but not at 3 and 6 h by a 20 min pre-exposure to pulsed US (1.5 MHz, 30 mW/cm²). Shear stress significantly decreased Bone Morphogenetic Protein-4 (BMP-4) mRNA levels at 1, 2, 3, 6, and 24 h by 32.5 ± 1.8%, 30.8 ± 3.5%, 49.6 ± 2.8%, 23.5 ± 5.0%, 24.4 ± 2.3%, respectively. A 20 min pulsed US exposure had no significant effect. However, a 20 min pre-exposure to pulsed US caused significant 39.6 ± 3.0% and 25.6 ± 2.7% decreases in BMP-4 levels in shear stress treated cells at 3 and 24 h, respectively. These results show for the first time that pulsed US alters the mechanotransductive effects of shear stress indicating a more comprehensive understanding of therapeutic US will be obtained when it is studied in conjunction with in vivo, regulatory biomechanical forces.     

Mesenchymal Stem Cell Deformability and Implications for Microvascular Sequestration

Mesenchymal stem cells (MSCs) have received considerable attention in regenerative medicine, particularly in light of prospects for targeted delivery by intra-arterial injection. However, little is known about the mechanics of MSC sequestration in the microvasculature and the yield pressure (P_Y), above which MSCs will pass through microvessels of a given diameter. The objectives of the current study were to delineate the dependency of P_Y on cell size and the heterogeneity of cell mechanical properties and diameters (D_CELL) of cultured MSCs. To this end the transient filtration test was employed to elucidate the mean filtration pressure (〈P_Y〉) for an ensemble of pores of a given size (D_PORE) similar to in vivo microvessels. Cultured MSCs had a log-normal distribution of cell diameters (D_CELL) with a mean of 15.8 ± 0.73 SD μm. MSC clearance from track-etched polycarbonate filters was studied for pore diameters of 7.3–15.4 μm. The pressure required to clear cells from filters with 30–85 × 10³ pores rose exponentially with the ratio λ = D_CELL/D_PORE for 1.1 ≤ λ ≤ 2.2. The clearance of cells from each filter was characterized by a log-normal distribution in P_Y, with a mean filtration pressure of 0.02 ≤ 〈P_Y〉 ≤ 6.7 cmH₂O. For λ ≤ 1.56, the yield pressure (P_Y) was well represented by the cortical shell model of a cell with a viscous interior encapsulated by a shell under cortical tension τ₀ = 0.99 ± 0.42 SD dyn/cm. For λ > 1.56, the 〈P_Y〉 characteristic of the cell population rose exponentially with λ. Analysis of the mean filtration pressure (〈P_Y〉) of each sample suggested that the larger diameter cells that skewed the distribution of D_CELL contributed to about 20% of the mean filtration pressure. Further, if all cells had the same deformability (i.e., P_Y as a function of λ) as the average cell population, then 〈P_Y〉 would have risen an order of magnitude above the average from fivefold at λ = 1.56 to 200-fold at λ = 2.1. Comparison of 〈P_Y〉 to published microvascular pressures suggested that 〈P_Y〉 may exceed microvessel pressure drops for λ exceeding 2.1, and rise 14-fold above capillary pressure drop at λ = 3 leading to 100% sequestration. However, due to the large variance of in vivo microvascular pressures entrapment of MSCs may be mitigated. Thus it is suggested that selecting fractions of the MSC population according to cell deformability may permit optimization of entrapment at sites targeted for tissue regeneration.     

Interferon-Gamma Modification of Mesenchymal Stem Cells: Implications of Autologous and Allogeneic Mesenchymal Stem Cell Therapy in Allotransplantation

Bone marrow-derived mesenchymal stem cells (MSC) have unique immunomodulatory and reparative properties beneficial for allotransplantation cellular therapy. The clinical administration of autologous or allogeneic MSC with immunosuppressive drugs is able to prevent and treat allograft rejection in kidney transplant recipients, thus supporting the immunomodulatory role of MSC. Interferon-gamma (IFN-γ) is known to enhance the immunosuppressive properties of MSC. IFN-γ preactivated MSC (MSC-γ) directly or indirectly modulates T cell responses by enhancing or inducing MSC inhibitory factors. These factors are known to downregulate T cell activation, enhance T cell negative signalling, alter T cells from a proinflammatory to an anti-inflammatory phenotype, interact with antigen-presenting cells and increase or induce regulatory cells. Highly immunosuppressive MSC-γ with increased migratory and reparative capacities may aid tissue repair, prolong allograft survival and induce allotransplant tolerance in experimental models. Nevertheless, there are contradictory in vivo observations related to allogeneic MSC-γ therapy. Many studies report that allogeneic MSC are immunogenic due to their inherent expression of major histocompatibility (MHC) molecules. Enhanced expression of MHC in allogeneic MSC-γ may increase their immunogenicity and this can negatively impact allograft survival. Therefore, strategies to reduce MSC-γ immunogenicity would facilitate “off-the-shelf” MSC therapy to efficiently inhibit alloimmune rejection and promote tissue repair in allotransplantation. In this review, we examine the potential benefits of MSC therapy in the context of allotransplantation. We also discuss the use of autologous and allogeneic MSC and the issues associated with their immunogenicity in vivo, with particular focus on the use of enhanced MSC-γ cellular therapy.     

间充质干细胞与生物人工肝的研究进展

人工肝支持系统 (artificialliversupportsystem ,ALSS ;简称人工肝artificialliver)对治疗肝硬化等肝病所引起的肝功能衰竭有很大的帮助 ,它是一种用物理或生物的方法替代行使肝脏功能的装置 ,而干细胞的研究则给生物人工肝带来了一个质的飞跃 ,间充质干细胞 (mesenchymalstemcell,MSC)作为成体干细胞的一员 ,具有来源广泛、复制和增殖能力强、分化能力强、可能避免应用免疫抑制剂和恼人的伦理问题等诸多独特的优质优势 ,使之成为人工器官研究中的一个重要环节 ,将会给肝病患者带来了新的曙光     

A Gene and Neural Stem Cell Therapy Platform Based on Neuronal Cell Type-Inducible Gene Overexpression

Purpose

Spinal cord injury (SCI) is associated with permanent neurological damage, and treatment thereof with a single modality often does not provide sufficient therapeutic outcomes. Therefore, a strategy that combines two or more techniques might show better therapeutic effects.

Materials and Methods

In this study, we designed a combined treatment strategy based on neural stem cells (NSCs) introduced via a neuronal cell type-inducible transgene expression system (NSE::) controlled by a neuron-specific enolase (NSE) promoter to maximize therapeutic efficiency and neuronal differentiation. The luciferase gene was chosen to confirm whether this combined system was working properly prior to using a therapeutic gene. The luciferase expression levels of NSCs introduced via the neuronal cell type-inducible luciferase expression system (NSE::Luci) or via a general luciferase expressing system (SV::Luci) were measured and compared in vitro and in vivo.

Results

NSCs introduced via the neuronal cell type-inducible luciferase expressing system (NSE::Luci-NSCs) showed a high level of luciferase expression, compared to NSCs introduced via a general luciferase expressing system (SV::Luci-NSCs). Interestingly, the luciferase expression level of NSE::Luci-NSCs increased greatly after differentiation into neurons.

Conclusion

We demonstrated that a neuronal cell type-inducible gene expression system is suitable for introducing NSCs in combined treatment strategies. We suggest that the proposed strategy may be a promising tool for the treatment of neurodegenerative disorders, including SCI.     

整合素传递力学信号影响间充质干细胞分化的研究进展

简述硬度的可控性及其控制方法、不同硬度调控间充质干细胞(mesenchymal stem cells,MSCs)分化的相应方向和整合素在硬度调控MSCs分化的信号通路中的作用。其中,重点说明整合素在硬度调控MSCs分化的信号通路中的作用。硬度调控MSCs分化的信号通路包括:Rho/ROCK信号通路、整合素/FAK信号通路、ERK信号通路、JNK信号通路、Wnt-β-catenin信号通路和PI3K/Akt信号通路等。而整合素作为跨膜异二聚体糖蛋白,参与部分信号通路传递力学信号给MSCs。不同的整合素家族参与不同的信号通路来调控MSCs向不同方向分化,且这些信号通路间存在相互影响。研究结论为组织修复、器官再造和再生医学等方面的应用提供理论依据。     

Embryonic Stem Cell Marker Expression Pattern in Human Mesenchymal Stem Cells Derived from Bone Marrow, Adipose Tissue, Heart and Dermis

Mesenchymal stem cells (MSCs) have been isolated from a variety of human tissues, e.g., bone marrow, adipose tissue, dermis, hair follicles, heart, liver, spleen, dental pulp. Due to their immunomodulatory and regenerative potential MSCs have shown promising results in preclinical and clinical studies for a variety of conditions, such as graft versus host disease (GvHD), Crohn’s disease, osteogenesis imperfecta, cartilage damage and myocardial infarction. MSC cultures are composed of heterogeneous cell populations. Complications in defining MSC arise from the fact that different laboratories have employed different tissue sources, extraction, and cultivation methods. Although cell-surface antigens of MSCs have been extensively explored, there is no conclusive evidence that unique stem cells markers are associated with these adult cells. Therefore the aim of this study was to examine expression of embryonic stem cell markers Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4 in adult mesenchymal stem cell populations derived from bone marrow, adipose tissue, dermis and heart. Furthermore, we tested whether human mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions. We found that bone marrow MSCs express embryonic stem cell markers Oct4, Nanog, alkaline phosphatase and SSEA-4, adipose tissue and dermis MSCs express Oct4, Nanog, SOX2, alkaline phosphatase and SSEA-4, whereas heart MSCs express Oct4, Nanog, SOX2 and SSEA-4. Our results also indicate that human adult mesenchymal stem cells preserve tissue-specific differences under in vitro culture conditions during early passages, as shown by distinct germ layer and embryonic stem cell marker expression patterns. Studies are now needed to determine the functional role of embryonic stem cell markers Oct4, Nanog and SOX2 in adult human MSCs.     

Local and Remote Effects of Mesenchymal Stem Cell Administration on Skin Wound Regeneration

Wound healing is an important medical problem. We evaluated the efficacy of locally administered mesenchymal stem cells (MSCs) isolated from human umbilical cords on the dynamics of skin wound healing. The study was conducted on the backs of Wistar rats, where two square wounds were created by removing all layers of the skin. Four groups were studied in two series of experiments: (1) a Control_NaCl group (the wounds were injected with 0.9% NaCl solution) and a Control_0 group (intact wounds on the opposite side of the same rat’s back); (2) an MSC group (injected MSCs, local effect) and a Control_sc group (intact wounds on the opposite side of the back, remote MSC effect). The area and temperature of the wounds and the microcirculation of the wound edges were measured. Histological and morphometric studies were performed on days 3 and 7 after the wounds were created. The results showed that the injection trauma (Control_NaCl) slowed the regeneration process. In both MSC groups (unlike in either control group), we observed no increase in the area of the wounds; in addition, we observed inhibition of the inflammatory process and improved wound regeneration on days 1–3 in the remote group and days 1–5 in the local (injected) group. The MSC and Control_sc groups demonstrated improved microcirculation and suppression of leukocyte infiltration on day 3. On day 7, all the studied parameters of the wounds of the Control_0 group were the same as those of the wounds that received cell therapy, although in contrast to the results of the Control_ NaCl group, fibroblast proliferation was greater in the MSC and Control_sc groups. The dynamics of the size of the wounds were comparable for both local and remote application of MSCs. Thus, even a one-time application of MSCs was effective during the first 3–5 days after injury due to anti-inflammatory processes, which improved the regeneration process. Remote application of MSC, as opposed to direct injection, is advisable, especially in the case of multiple wounds, since the results were indistinguishable between the groups and injection trauma was shown to slow healing.     

成体小鼠骨髓间充质干细胞的培养和纯化

目的建立一种简单易行的小鼠骨髓间充质干细胞(MSC)的分离培养方法,并对其表面标志进行鉴定,为其应用提供实验依据.方法采用差速贴壁法体外分离、扩增MSC,倒置显微镜观察其生长过程,HE染色观察原代培养细胞的生长特性,免疫细胞化学检测MSC表面抗体的表达及细胞的大概纯度.结果用此法进行小鼠骨髓间充质干细胞的培养过程中,血液系细胞在换液过程中被去除,成纤维细胞污染可经差速贴壁法去除.经鉴定获得的骨髓间充质细胞能达到理想的纯度,生长状态良好.结论采用差速贴壁培养法可获得一定纯度的MSC,这种方法简单、便捷、实用,并且用这种方法所分离获得的细胞生长状态好,在体外条件下能大量增殖,原代及传代生长都可形成均一的细胞集落.