Implications of adipose-derived stromal cells in a 3D culture system for osteogenic differentiation: an in vitro and in vivo investigation

Background contextHealthy mammalian cells in normal tissues are organized in complex three-dimensional (3D) networks that display nutrient and signaling gradients. Conventional techniques that grow cells in a 2D monolayer fail to reproduce the environment that is observed in vivo. In recent years, 3D culture systems have been used to mimic tumor microenvironments in cancer research and to emulate embryogenesis in stem cell cultures. However, there have been no studies exploring the ability for adipose-derived stromal (ADS) cells in a 3D culture system to undergo osteogenic differentiation.PurposeTo characterize and investigate the in vitro and in vivo potential for human ADS cells in a novel 3D culture system to undergo osteogenic differentiation.Study designBasic science and laboratory study.MethodsHuman ADS cells were isolated and prepared as either a 2D monolayer or 3D multicellular aggregates (MAs). Multicellular aggregates were formed using the hanging droplet technique. Cells were treated in osteogenic medium in vitro, and cellular differentiation was investigated using gene expression, histology, and microCT at 1-, 2-, and 4-week time points. In vivo investigation involved creating a muscle pouch by developing the avascular muscular interval in the vastus lateralis of male athymic rats. Specimens were then pretreated with osteogenic medium and surgically implanted as (1) carrier (Matrigel) alone (control), (2) carrier with human ADS cells in monolayer, or (3) human ADS cells as MAs. In vivo evidence of osteogenic differentiation was evaluated with micro computed tomography and histologic sectioning at a 2-week time point.ResultsHuman ADS cells cultured by the hanging droplet technique successfully formed MAs at the air-fluid interface. Adipose-derived stromal cells cultured in monolayer or as 3D MAs retain their ability to self-replicate and undergo multilineage differentiation as confirmed by increased runx2/Cbfa2, ALP, and OCN and increased matrix mineralization on histologic sectioning. Multicellular aggregate cells expressed increased differentiation potential and extracellular matrix production over the same human ADS cells cultured in monolayer. Furthermore, MAs reseeded onto monolayer retained their stem cell capabilities. When implanted in vivo, significantly greater bone volume and extracellular matrix were present in the implanted specimens of MAs confirmed on both microCT and histological sectioning.ConclusionsThis is the first study to investigate the capability of human ADS cells in a 3D culture system to undergo osteogenic differentiation. The results confirm that MAs maintain their stem cell characteristics. Compared with analogous cells in monolayer culture, the human ADS cells as MAs exhibit elevated levels of osteogenic differentiation and increased matrix mineralization. Furthermore, the creation of uniform spheroids allows for improved handling and manipulation during transplantation. These findings strongly support the concept that 3D culture systems remain not only a viable option for stem cell culture but also possibly a more attractive alternative to traditional culture techniques to improve the osteogenic potential of human adipose stem cells.     

成人表皮干细胞和汗腺细胞的体外分离和培养

背景：人表皮干细胞和汗腺细胞的分离培养及鉴定,为探讨人表皮干细胞再生汗腺的可行性打下基础。目的：探讨体外分离培养人表皮干细胞及汗腺细胞的有效方法。方法：采集不同年龄段的泌尿外科患者术后包皮组织,充分清洗消毒后去除皮下组织,将其修剪成0.5cm×0.5cm的皮片,用Ⅳ型胶原纯化、富集成人表皮干细胞,倒置相差显微镜观察细胞形态：使用免疫荧光染色对成人表皮干细胞表型进行分析;CCK-8检测细胞的增殖曲线;采用胶原酶消化法从人全层无损伤皮肤中分离提取汗腺细胞,并进行扩增和鉴定。结果：倒置相差显微镜下见分离培养的成人表皮干细胞呈卵圆形、细胞之间紧密相连,呈铺路石状,免疫荧光染色显示细胞表达CK19、β1整合素。倒置相差显微镜下见汗腺细胞呈扁平多角形,表达汗腺细胞标志细胞CK7、18、19及CEA。结论：本实验结果说明胶原酶消化法分离培养成人表皮干细胞及汗腺样细胞是可行的。     

表皮细胞体外增殖与增殖表型细胞含量变化规律的实验研究

目的研究人表皮细胞(humanepidermalcells,hECs)体外扩增与增殖表型细胞含量变化规律。方法收集2～3岁健康幼儿包皮共20例,对标本拍照,软件分析照片中标本面积,分离、收集表皮细胞,计算原代(P0)细胞获得率(5例)。记录不同代次细胞生长曲线,细胞直径,克隆形成率(5例)。对不同代次细胞的角蛋白19(keratin,K19)和外皮蛋白(involucrin)的mRNA表达情况及阳性细胞率分别进行RTPCR检测(5例)和流式细胞仪(FACS)分析(5例)。结果原代细胞平均获得率为(164±0297)×106cm2。hECs最大可扩增(700±37)倍,第5代(P5)的扩增倍数为(243±13)倍。P0克隆形成率为(45±4)%,随传代依次降低,至第5代(P5)时为(9±2)%,至第6代(P6)时消失。RTPCR检测发现K19mRNA在P5及P5之前表达,P6未见表达;Involucrin各代均表达。FACS检测各代hECs中K19阳性细胞百分率逐渐降低,P0时为(6697±314)%,P6为(465±138)%;外皮蛋白表达阳性细胞百分率逐渐增高,P0时为(1165±162)%,P6为(9703±266)%。结论体外扩增至第5代的幼儿包皮来源的表皮细胞,维持着增殖细胞表型,符合组织工程表皮种子细胞的要求。表皮细胞体外扩增能力逐渐降低与增殖表型细胞含量减少有关。     

人胎儿表皮干细胞的体外分离培养及基因转染

目的：探讨人胎儿表皮干细胞体外分离培养的方法以及作为体外基因转染靶细胞的可行性。方法：利用Ⅳ型胶原快速贴附法分离人胎儿表皮干细胞，以人胎儿成纤维细胞条件培养液配制表皮干细胞培养基，通过角蛋白19（K19）和整合素β1免疫组化染色、细胞周期分析及克隆形成率测定，对培养细胞进行鉴定。采用脂质体介导法，以含血管内皮细胞生长因子165（VEFG165）基因片段的真核表达载体pcDNA3.1(pcDNA3.1/VEGF165)转染培养细胞；采用病毒载体介导法，以含报告基因绿色荧光蛋白（GFP）的重组腺相关病毒载体（raav/GFP)转染培养细胞。应用免疫组化染色及荧光显微镜观察检测转染效果。结果：人胎儿表皮干细胞呈明显克隆性生长、克隆形成率高，G1期细胞比例明显高于普通基底层角质细胞，K19和整合素β1免疫组化染色呈强阳性。pcDNA3.1/VEGF165转染的表皮干细胞VEGF165免疫组化染色阳性，raav/GFP转染的表皮干细胞呈现强荧光。结论：利用Ⅳ型胶原快速贴附法及人胎儿成纤维细胞条件培养基，可初步实现人胎儿表皮干细胞的分离培养。以质体为介导或以腺相关病毒为载体进行人胎儿表皮干细胞的体外基因转染是可行的。     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

膀胱黏膜上皮细胞体外培养的实验研究

目的 探索组织工程化尿道黏膜类似组织体外培养条件。方法取雄性大耳白兔幼兔的膀胱壁组织，分别用Ⅱ型胶原酶和DispaseⅡ处理后，胰蛋白酶消化获得单细胞悬液，接种后用含有或不含有表皮生长因子的培养液培养并传代，动态观察细胞形态变化及生长增殖的不同情况．并对细胞进行超微结构观察。结果用Ⅱ型胶原酶处理的标本在有表皮生长因子存在的情况下，细胞生长良好，混杂少量的成纤维细胞，在体外可传10代以上，成活30天以上。结论Ⅱ型胶原酶处理的幼兔膀胱黏膜上皮细胞纯度较高，可在体外培养一定时间并保持增殖能力．表皮生长因子是必要条件。     

膀胱黏膜上皮细胞体外培养的实验研究

目的探索组织工程化尿道黏膜类似组织体外培养条件.方法取雄性大耳白兔幼兔的膀胱壁组织,分别用Ⅱ型胶原酶和Dispase Ⅱ处理后,胰蛋白酶消化获得单细胞悬液,接种后用含有或不含有表皮生长因子的培养液培养并传代,动态观察细胞形态变化及生长增殖的不同情况,并对细胞进行超微结构观察.结果用Ⅱ型胶原酶处理的标本在有表皮生长因子存在的情况下,细胞生长良好,混杂少量的成纤维细胞,在体外可传10代以上,成活30天以上.结论Ⅱ型胶原酶处理的幼兔膀胱黏膜上皮细胞纯度较高,可在体外培养一定时间并保持增殖能力,表皮生长因子是必要条件.     

Excimer laser ablation of fibrocartilage: an in vitro and in vivo study

To date, lasers have found only limited applications in orthopedics. We employed a 308 nm XeCl excimer laser for ablation of fibrocartilage, in order to investigate the feasibility of excimer laser assisted meniscectomy. Experiments were conducted both in vitro and in vivo. For the in vitro study, human menisci, obtained during surgery and autopsy, were irradiated via a 600 microns core fiber at radiant exposures ranging between 20 mj/mm2 and 80 mj/mm2, at 20 Hz. Ablation rate measurements and histological analysis of the samples were performed. The ablation rates were found to range from 3 microns/pulse to 100 microns/pulse depending on the radiant exposure and/or the applied pressure on the fiber delivery system. Thermographic analysis was also performed during pulsed excimer as well as CW Nd:Yag and CW CO2 laser irradiation. Temperatures were lower for excimer laser (Tmax less than 65 degrees) than CW ND: Yag (Tmax less than 210 degrees) or CW CO2 (Tmax less than 202 degrees) laser. For the in vitro study, medial meniscectomy was performed in 15 rabbits with the excimer laser and a CW Nd:Yag laser in the right and left knee respectively. Excimer laser irradiation was performed at 70 mj/mm2. Nd:Yag irradiation was performed via a 600 microns core fiber at power outputs between 20 to 40 W for 10 and 20 seconds duration. The healing response to injury was investigated by histological analysis of the menisci after 1 day, 1, 2, 4, and 8 weeks following the laser procedure. Excimer laser treated menisci showed less inflammatory reaction and noticeable repair with minimal inflammatory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel ex vivo culture system for studying bone repair

Repair of large bony defects still remains a challenge for surgeons. Hydroxyapatite (HA) is well known for its biocompatibility and osseoconduction properties in the osseous environment. In this study the biofunctionality of a newly developed scaffold comprising of collagen and HA, with variable macropores was examined. The biological response was evaluated using primary human osteoblast cells (HOBs). Cell infiltration, proliferation and differentiation were assessed. The results showed that HOBs were able to migrate from the collagen into the HA pores with greater cell migration and infiltration observed in those scaffolds with larger pores. Furthermore, it was shown that Alkaline Phosphatase, a differentiation marker for HOBs was enhanced as the average macropore size increased. This in vitro model provides a more relevant method of testing the biofunctionality and migration ability of cells at a trauma site following implantation in bone and cartilage.     

乳腺癌肿瘤干细胞体外培养体系的建立

目的建立一种适合乳腺癌肿瘤干细胞体外培养的三维培养体系。方法根据不同的培养方法分为两组:二维细胞培养组,采用传统二维细胞培养方法培养乳腺癌MCF-7细胞;三维细胞培养组,将乳腺癌MCF-7细胞接种于三维胶原支架材料上,在摇床上动态培养5d,在光学显微镜和激光共聚焦显微镜下观察乳腺癌肿瘤细胞在胶原材料上的形态学特征。采用CCK-8细胞增殖检测的方法,比较三维培养组和二维培养组细胞增殖能力,通过流式细胞术检测两组乳腺癌肿瘤干细胞标志CD44~+/CD24~-/low的表达差异。结果光学显微镜下观察结果显示:二维培养组MCF-7细胞为单层生长平展的粘附生长,呈多边形、铺路石样的上皮细胞形态;三维培养组MCF-7来源的细胞再次二维培养后,细胞展现出三角形或者长梭形及较多细胞呈不规则形,部分细胞还伸展出长浸润性伪足。激光共聚焦显微镜观察显示,MCF-7细胞均匀地生长在三维支架材料上,局部放大后清晰可见长伪足,且细胞大小不均一,细胞形态多样。三维培养组细胞增殖速度与二维培养组相似。与二维培养组相比较,三维培养组CD44~+/CD24~-细胞比例显著增加,由38.9%增加至60.3%。结论基于三维胶原支架材料的三维培养体系能够有效维持乳腺癌肿瘤干细胞干性。     

Characterization of an in vitro intervertebral disc organ culture system

Intervertebral disc organ culture has the capacity to control mechanical and chemical boundary conditions while keeping the tissue largely intact, and allowing interventions that would be impossible or unethical on animal studies. Recent studies on ex vivo organ culture has mostly involved small animals, or been limited to development and validation studies. In this study, bovine caudal discs were used. The large animal model design ensures that sufficient tissue is available for measurement of multiple dependent variables on the same disc, and a similar aspect ratio, diffusion distance, composition and rate of proteoglycan synthesis to human lumbar discs. The first goal of this study was to refine a set of dependent variables capable of characterizing the response of the intervertebral disc to culturing and to develop a technique to measure cell viability in all three regions of the disc. The second goal was to use these variables to compare static and diurnal loading as a method of maintaining intervertebral disc structure, composition, and cell metabolism similar to the in vivo state. Static (0.2 MPa) and diurnal loading (0.1 and 0.3 MPa alternating at 12 h intervals) were applied and intervertebral discs were examined after 4 or 8 days with dependent variables including changes in geometry (disc height and diameter), composition (tissue water content, tissue proteoglycan content and proteoglycan content lost to the culture media), cell viability and metabolism (proteoglycan synthesis). Results indicate that there was a decrease in disc height and water content after culture regardless of culture duration or loading condition. Cell viability significantly decreased with culture duration in the inner annulus and nucleus; however, a significant reduction in cell viability for the diurnal versus static loading condition was only observed after 8 days in the nucleus region. No significant differences were seen in viability of the outer annulus region with time, or in any loading groups. We conclude that our system is capable of keeping bovine caudal discs alive for at least 8 days without significant changes in GAG content, or cell metabolism, and that static loading was slightly better able to maintain cell viability than diurnal loading. This system offers promise for the future studies on large intervertebral discs requiring measurements of multiple mechanical and biological dependent variables on the same tissue.     

Reinsertion of stimulated nucleus pulposus cells retards intervertebral disc degeneration: an in vitro and in vivo experimental study.

Reinsertion of autogenous nucleus pulposus, an innovative method to delay further disc degeneration, has been proved with an experimental animal model. This study examined whether coculture of nucleus pulposus cells with annulus fibrosus cells (a) activates annulus fibrosus cells and (b) retards disc degeneration when reinserted into the disc in a rabbit model of disc degeneration. Coculture of the two cell types stimulated proliferation of each, as indicated by increased DNA synthesis measured by increases in DNA polymerase alpha expression and uptake of 5-bromo-2'deoxy-uridine assessed by an enzyme-linked immunosorbent assay. In a model of disc degeneration in rabbits, reinsertion of activated nucleus pulposus cells delayed the formation of clusters of chondrocyte-like cells, the destruction of disc architecture, and the elaboration of type-II collagen as measured immunohistochemically compared with no treatment. The direct reinsertion of activated nucleus pulposus cells into the disc offers a promising line of investigation for delaying intervertebral disc degeneration, although these results obtained with notochordal cells may not necessarily apply when mature central nucleus pulposus cells are used.     

大鼠骨髓来源树突状细胞体外培养体系的优化

目的 探讨优化大鼠骨髓来源树突状细胞(DC)体外培养体系的方法.方法 应用重组大鼠粒细胞-巨噬细胞集落刺激因子(recombinant rat GM-CSF,rrGM-CSF) 20 μg/L和重组大鼠白介素(recombinant rat interleukin,rrIL)- 4 10 μg/L诱导分化Lewis大鼠...     

压应力对表皮干细胞增殖与分化的影响

目的：初步观察压应力对表皮干细胞增殖分化的影响。方法：以大鼠的皮肤扩张动物模型为在体观察对象;4、8、12kPa的压应力予体外培养的大鼠表皮干细胞进行间歇加压（每天3次,每次2h,共1周）为离体观察对象。利用免疫组化染色、免疫荧光双染、细胞克隆形成率等技术与方法检测β1整合素、角蛋白19（K19）、角蛋白14（K14）、角蛋白10（K10）、α6整合素、CD71的表达特征,以及压应力对表皮千细胞增殖分化的影响。结果：组织学观察显示,扩张皮肤的表皮层明显增厚;表皮层的β1整合素、K19、K14阳性细胞数量于扩张的第5天开始增多,至15d达峰值后下降,扩张完成后20d趋于正常;棘层和颗粒层不仅可见上述阳性细胞,且出现α6整合素^bri／CD71^dim细胞的表达。体外培养的表皮干细胞,8kPa、12kPa的压应力作用1周后,细胞克隆形成率分别为48．67％、49．36％,明显高于4kPa组（23．17％）与对照组（23．00％）,8kPa、12kPa组可见K10阳性细胞表达。结论：表皮干细胞具有压应力敏感性,适当的压应力可诱导表皮干细胞增殖与分化。     

几丁质膜作为表皮干细胞培养支架的实验研究

Objective To investigate the feasibility of constructing a skin tissue engineering cover-ing on chitinous membrane using rat epidermal stem cells (ESCs). Methods Rat ESCs were isolated and cultured by cold digestive method and collagen type Ⅳ adherent method. Cell colonies were observed with in-verted microscope. Expressions of DNA and RNA of ESCs were detected with laser scanning confocal micro-scope. Growth curves of cells were determined with Alamar BlueTM colorimetric method. Expressions of sur-face markers of ESCs (CD29, CD71, CD49d, and CD34) were detected with flow cytometer. Positive expres-sions of CK15, CK19, and P63 of ESCs were determined by immunohistochemistry. Influence of original chi-tinous membrane leachate in different dilutions on ESCs was observed. Condition of growth of ESCs on the ve-hicle was observed. Results Isolated cultured cells were verified as ESCs, of which the doubling generation time was 48 hs. CD29 and CD49d were positive;CD71 and CD34 were negative;CKi9, CK15, and P63 were positive. Compared with that of control group, ESCs cultured in chitinous membrane leachate showed slight cell proliferation when diluted to 1:8-1:512 dilutions, but there was no statistically significant difference (P>0.05). The checkerboard-form cell colonies of ESCs could be visualized with naked eyes on the chi-tinous membrane in 2-4 weeks of culture. A multitude of ESCs were seen to grow on fibres under microscope. Conclusions Chitinous membrane may be used as ESCs culture vehicle, and biological compatibility is good.