脂肪间充质干细胞在肝损伤修复中的作用

脂肪间充质干细胞(Adipose derived mesenchymal stem cells,ADMSCs)是存在于脂肪组织中的一类多能干细胞,其来源丰富,容易获得,具有多向分化潜能。自2001年被发现以来,ADMSCs已被用于多种疾病的实验及临床研究中。近年来,利用ADMSCs移植治疗肝脏疾病的替代疗法日益受到重视,本文对ADMSCs的生物学特性、分化潜能及其在肝损伤性疾病治疗方面的研究进展进行阐述,为基础及临床研究提供参考。     

模拟微重力下骨髓间充质干细胞成骨分化潜能及分子机理研究

本文旨在研究模拟微重力环境对骨髓间充质干细胞成骨分化的影响,重点研究mTOR信号通路是否介导这一过程。首先通过茜素红染色检验骨髓间充质干细胞的成骨分化情况;免疫荧光法对微丝和黏着斑蛋白Vinculin进行染色观察细胞骨架和细胞粘附性改变;蛋白免疫印迹实验检测mTOR信号通路关键信号分子p85α,Akt,p-Akt,S6K1,RhoA等的表达水平变化。结果显示模拟微重力会抑制骨髓间充质干细胞的成骨分化,造成细胞微丝结构和分布异常,细胞形态改变,黏着斑数量减少。这种抑制与mTOR信号通路的改变有关。本研究为空间环境下骨质丢失的分子机制提供了理论基础。     

两种不同淋巴细胞分离液对脐带血单核细胞的提取及后期诱导培养CIK细胞的影响

目的：研究两种不同的淋巴细胞分离液对脐带血单核细胞的分离效果及后期对CIK细胞的诱导培养的影响。方法：分别使用国产TBD以及进口GE healthcare牌淋巴细胞分离液分离提取脐带血单核细胞,用悬浮细胞培养法诱导培养脐带血单核细胞形成CIK细胞,再用血球计数板及台盼兰染色法检测细胞密度及活率。结果：国产的TBD牌所分离的单核细胞分层较清楚,并且方便提取,而使用进口GE healthcare的淋巴细胞分离液提取的单核细胞数量较多,但经过后期诱导培养CIK细胞发现两种分离液提取的细胞培养到后期数量逐渐接近。结论：国产TBD牌淋巴细胞分离液在用于脐带血淋巴细胞分离提取时较进口GE healthcare牌经济,并且细胞提取方便,后期培养效果无明显差别。     

DA-2006促进骨髓基质干细胞分化为成骨细胞效果及机制分析

目的 :探讨体外培养条件下DA-2006促进骨髓基质干细胞分化为成骨细胞的效果与机制。方法:取兔股骨,骨髓腔进行冲洗,采用全骨髓法分离纯化培养BMSCs,培养至第3代后,分别利用诱导培养液(组A),添加DA-2006的培养液(组B)以及普通培养液(对照组)培养BMSCs,采用噻唑蓝(MTT)检测3组骨髓基质干细胞增殖能力,利用荧光定量PCR技术检测骨碱性磷酸酶(ALP)、骨钙素(OC)、骨桥蛋白(OPN)等成骨性标志基因的表达情况。结果 :经过诱导后,组B细胞具有良好的形态,扩增能力迅速。在生长率方面,诱导组B较强于诱导组A,对照组团块化程度最低。对照组的表达量最低,同时组B的ALP、OC、OPN表达量明显高于组A(P<0.05)。结论 :在含DA-2006的细胞培养液诱导环境下,骨髓基质干细胞的成骨分化强于经典的地塞米松诱导效果,其机制的发挥可能与上调ALP、OC与OPN的表达水平有关。     

脂肪干细胞研究近况

脂肪干细胞是一类可以自我更新、繁殖产生更多的干细胞,进而可以分化成许多有特定功能的细胞系,具有一般干细胞的特点,可作为多种组织工程的种子细胞,有非常重要的研究和应用价值.本文着重对脂肪干细胞在诱导分化方面的最新发展进行了阐述,希望能为脂肪干细胞的进一步的研究提供理论基础.     

神经干细胞体外诱导分化的研究进展

神经干细胞是近年来脑科学研究的热点,其自我更新和多向分化的潜能为中枢神经系统疾病的治疗提供了可能,但如何使神经干细胞向特定的方向分化却一直是研究的难点.本文对神经干细胞体外的诱导分化因素及分化过程中的相关蛋白质组的研究做一综述.     

非接触式高压静电场微胶囊制备方法

为制备脂肪干细胞微胶囊,设计并研制基于高压静电成囊原理的非接触式微囊发生装置。以海藻酸钠在氯化钙溶液中成囊的方法制备细胞微胶囊,并分析电场电压、推进速度、电极距离和针头型号等参数对所制微囊粒径的影响。采用非接触式微囊发生器能制备出表面光滑、形状规则、大小均匀的微胶囊。用该装置制备出微囊化脂肪干细胞,经过一周培养,细胞在微胶囊中存活良好,增殖明显。     

骨髓间充质干细胞对糖尿病视网膜病变大鼠血糖及视网膜功能影响

目的 :探讨尾静脉注射骨髓间充质干细胞对糖尿病视网膜病变大鼠血糖及视网膜功能的影响。方法 :48只健康大鼠随机选取10只为正常对照组,38只建立糖尿病视网膜病变大鼠模型后,20只采用尾静脉注射间充质干细胞,18只注射等量安慰剂。观察间充质干细胞在肝、脾、肾、胰腺和眼球的分布以及在视网膜的分化特点,监测注射后1、2、3、4wk血糖变化,观察血-视网膜屏障破坏修复情况。结果 :BMSCS在肝、脾、肾、胰腺、肺等组织中均有分布,视网膜外核层可见到大量密集的BMSCs重叠存在。实验组大鼠的血糖水平均随干预时间延长逐渐下降,注射后第1 wk血糖差异即有统计学意义。EB渗漏量较基线明显减少。结论 :BMSCs可降低糖尿病视网膜病变大鼠血糖,使血-视网膜屏障得到一定程度修复。     

探讨电镜包埋时间对脂肪组织超微结构的影响

目的 :本文主要分析和探讨透射电镜制样技术中的包埋时间对脂肪组织超微结构观察的影响。方法 :自C57BL/6J小鼠完成白色脂肪和棕色脂肪组织取材,常规电镜样品处理,放置于磷酸盐缓冲液后分成三组进行包埋,分别于第2天、第7天和第14天进行组织包埋、切片,观察不同包埋时间下白色和棕色脂肪组织光镜和电镜下组织细胞结构的变化。结果 :脂肪组织放置于缓冲液中,第二天包埋后脂肪组织内的白色脂肪细胞和棕色脂肪细胞呈圆形或椭圆形,胞膜完整光滑,胞浆内脂滴呈圆形。随着包埋时间的延后,脂肪细胞呈不规则形,部分区域见细胞崩解,细胞膜可见断裂、褶皱和串珠样改变,脂滴形态改变。结论 :脂肪组织制作透射电镜标本时取材后应及时进行包埋处理,不宜在缓冲液内长时间浸泡。     

绵羊毛囊结构的组织学研究

选择年龄为1年的绵羊,在颈部取皮肤组织进行石蜡切片,HE染色,观察绵羊发育过程中毛囊的形态学结构。结果表明：绵羊毛囊在毛生长周期中的形态各不同,生长期的特点是毛球细胞快速增殖,毛乳头被包在毛球内;退化期毛囊底部密封,形成上皮束;静止期整个毛囊的组织结构变短,毛球细胞接近干细胞。本实验对研究绵羊毛纤维生长的机制具有一定的意义。     

Basal cell carcinoma stem cells exhibit osteogenic and chondrogenic differentiation potential

Specific cell subpopulations identified as cancer stem cells (CSCs) can be found in basal cell carcinoma (BCC). Generally, CSCs have a marked trans-differentiation potential that could potentially be used in differentiation therapies. However, there are no studies regarding BCC CSCs multipotency. The aim of the study was to analyze the characteristic of CSCs of BCC with emphasis on their differentiation potential upon specific induction. Specific staining and cell morphology were used for differentiation confirmation, along with the expression analysis of osteogenic (ALP, BSP, Runx2, OCN, BMP2), chondrogenic (COL1 and COL2A1), adipogenic (PPAR-γ) and neurogenic (Nestin and MAP2) markers. BCC CSCs differentiated into osteogenic and chondrogenic lineages, as judged by staining and high expression of specific markers (from 2-to 92-fold higher upon induction). Concomitantly with differentiation, the levels of cancer stem cell markers decreased in the cultures. Adipo-differentiation and neuro-differentiation were unsuccessful. In conclusion, BCC CSCs exhibit the capacity to trans-differentiate, a characteristic that may potentially be useful in the development of new strategies for the treatment of aggressive BCCs.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

Ultrastructural analysis of human bone marrow mesenchymal stem cells during in vitro osteogenesis and chondrogenesis

The main purpose of this article was to describe the morphology of mesenchymal stem cells (MSCs) differentiated in vitro towards osteogenic and chondrogenic lineages and to focus on the ultrastructural features associated with these processes. Human mononuclear cells (hMNC) were isolated, expanded, and analyzed for the expression of specific cell surface markers to demonstrate their stem cell characteristics. Human mononuclear cells were differentiated in vitro in an osteogenic and in a chondrogenic sense for 7, 14, 21, and 28 days. Subsequently, they were processed using electron microscopic analysis (FEISEM). Alizarin red and alcian blue staining were carried out to demonstrate the deposition of mineral salts and proteoglycans in the extracellular matrix. Undifferentiated MSCs showed a cell surface covered by filopodia and ondulopodia. During differentiation, the MSCs changed their shape from a round to a fibroblastic-like shape. At the end of the differentiation, several filaments with a parallel orientation in the osteogenic samples as well as a network organization in the chondrogenic samples were detected in the extracellular spaces. This study demonstrated that there are morphological features associated with the undifferentiated and differentiated states of the MSCs, which could be utilized as new parameters for identifying and classifying these cells.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

Chondrogenic differentiation of human adipose mesenchimal stem cells: Influence of a biomimetic gelatin genipin crosslinked porous scaffold

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system. Microsc. Res. Tech. 77:928–934, 2014. © 2014 Wiley Periodicals, Inc.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

Behavior of mesenchymal stem cells stained with 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) in osteogenic and non osteogenic cultures

4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) is a DNA dye widely used to mark and trace stem cells in therapy. We here studied the effect of DAPI staining on the behavior of mesenchymal stem cells cultured in either a control, non-osteogenic medium or in an osteogenic differentiation medium. In the control medium, the number of stem cells/field, as well as the number of fluorescent cells/field increased up to the sixth day in both control and DAPI-treated cultures. Afterwards, both the number of fluorescent cells and their fluorescence intensity decreased. Control cells were fusiform and with some long extensions that apparently linked them to neighboring cells, while DAPI-treated cells were mostly round cells with fine and short extensions. The trypan-blue exclusion method showed 99% cell viability in both groups, however, both alkaline phosphatase activity and the thiazolyl blue formazan assay (indicative of mitochondrial metabolism) gave significantly lower values in DAPI-marked cells. The mitochondrial mass, as indicated by specific staining and flow cytometry, showed no differences between groups. Mesenchymal stem cells gave origin to mineralized nodules in the osteogenic differentiation medium and there were not DAPI-marked cells on the ninth day of culture. Alkaline phosphatase activity, viability assay and number of cells/field and of mineralized nodules/field were similar in both groups. So, DAPI treatment did not change cell viability and proliferation during osteogenic differentiation of mesenchymal stem cells. However, since these cells loose DAPI marking after 9 days in osteogenic cultures suggests that DAPI may not be an effective marker for mesenchymal stem cells implanted in bone tissue for long periods.     

Adipose-derived mesenchymal stem/stromal cells: from the lab bench to the basic concepts for clinical translation

In the last years, much work has shown that the most effective repair system of the body is represented by stem cells, which are defined as undifferentiated precursors that own unlimited or prolonged self-renewal ability, which also have the potential to transform themselves into various cell types through differentiation.All tissues that form the body contain many different types of somatic cells, along with stem cells that are called ‘mesenchymal stem (or stromal) cells’ (MSC). In certain circumstances, some of these MSC migrate to injured tissues to replace dead cells or to undergo differentiation to repair it.The discovery of MSC has been an important step in regenerative medicine because of their high versatility. Moreover, the finding of a method to isolate MSC from adipose tissue, so called ‘adipose-derived mesenchymal stem cells’ (ASC), which share similar differentiation capabilities and isolation yield that is greater than other MSC, and less bioethical concerns compared to embryonic stem cells, have created self-praised publicity to procure almost any treatment with them. Here, we review the current techniques for isolation, culture and differentiation of human ASC (hASC), and describe them in detail. We also compile some advantages of the hASC over other stem cells, and provide some concepts that could help finding strategies to promote their therapeutic efficiency.     

Microscopy analysis of bone marrow-derived osteoprogenitor cells cultured on hydrogel 3-D scaffold

Bone marrow contains progenitor cells that are able to differentiate into several mesenchymal lineages, including bone. These cells may also provide a potential therapy for bone repair. The purpose of this study was to select the osteoprogenitor cell subpopulation from bone marrow-derived mesenchymal stem cells (MSCs) and to test the ability of a hydrogel scaffold to support growth and osteogenic differentiation. MSCs isolated from rat femur bone marrow were cultured in DMEM medium supplemented with antibiotics, FCS, and L-glutamine. Osteogenic supplements (dexamethasone, sodium beta-glycerophosphate, and ascorbic acid) were added for one, two or three weeks. A selective subpopulation of osteoprogenitor cells was identified by immunohistochemistry, general morphology, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). Committed osteogenic cells were transferred to a 3-D hydrogel scaffold and cultured for an additional week. In standard culture, the osteoprogenitor cells formed cell clusters identified by Alizarin red S staining and by positive osteocalcin immunostaining. The number of osteoprogenitor cells, matrix synthesis, and mineralization increased gradually up to three weeks in culture. Mineral deposition in the matrix analyzed by EDS revealed the presence of calcium and phosphate ions at a Ca/P molar ratio of 1.73 in both the osteogenic cultures and the scaffold osteoprogenitor culture. Histological preparations revealed cell clusters within the hydrogel scaffold and SEM analysis revealed cell clusters attached to the scaffold surface. It is concluded that the hydrogel scaffold can support growth and differentiation of osteogenic cultures including mineralization and can potentially serve as a bone graft substitute containing committed osteoprogenitor cells.     

New insights into the cellular makeup and progenitor potential of palatal connective tissues

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco‐gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de‐epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de‐epithelialised MSCs (deMSCs) and re‐entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony‐forming unit‐ fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA‐DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA?, CD31?) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,—in the pericapillary regions and in remote regions of the lamina propria‐ and pericytes—surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision‐making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.