Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid（PLLA） were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide（MTT） assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

Alendronate affects osteoprotegerin/receptor of activator of nuclear factor κB-ligand expression in human marrow stroma cells in vitro

Objective To evaluate the effect of alendronate on osteoprotegerin(OPG)and receptor of activator of nuclear factor κB-ligand(RANKL)expression in human marrow stroma cells(hMSCs)in vitro.Methods hMSCs were isolated from human marrow,cultured in vitro,and randomly divided into two groups:alendronate group,hMSCs culture fluid containing 1×10-7mol/L alendronate;control group,no special treatment but culturing hMSCs in DMEM.Two weeks after treatment,the expressions of OPG and RANKL were evaluated by RT-PCR and Western blot.Results hMSCs became uniform spindle-shaped fibroblasts.As cells proliferated,they formed colonies and showed whirlpool arrangement.After one week’s treatment,hMSCs in alendronate group had reduced processes and gradually showed disc shape,which did not happen in control group but kept fibroblast shape and just increased in density.In RT-PCR,the ratio of OPG/RANKL in alendronate group and control group was 8.77±1.16 and 4.58±1.27,respectively.In Western blot,the ratio of OPG/RANKL in alendronate group and control group was 2.58±0.47 and 1.52±0.32,respectively.The ratio of OPG/RANKL was higher in alendronate group than in control group(P<0.01).Conclusion Alendronate enhances OPG expression and inhibits RANKL expression of hMSCs in vitro.

Murine corneal stroma cells suppress bone marrow-derived dendritic cells maturation in vitro

Background Prostaglandin E2 （PGE2） is a key modulator of dendritic cells （DCs） function, and cornea-derived transforming growth factor beta 2 （TGF-β2） promotes the generation of phenotypically and functionally immature DCs. Therefore, this study was carried out to investigate whether PGE2 is involved in the suppressive effect on DCs maturation mediated by corneal stroma cells （CSCs） and whether PGE2 and TGF-β2 have additive effects in this immunosuppressive mechanism. Methods Bone marrow-derived DCs （BM-DCs）, splenic T cells and CSCs culture supernatant were obtained from mice via various protocols. After that, the level of PGE2 in CSCs culture supernatant was analyzed by enzyme-linked immunosorbent assay. Then, immature BM-DCs pretreated by E-prostanoid 2 receptor antagonist AH6809 or dimethyl sulfoxide were induced to mature in the presence of lipopolysaccharide, with or without CSCs culture supernatant. In parallel experiments, neutralizing TGF-β2 antibody or normal goat IgG was added into the supernatant. Next, the cellular surface markers for DCs maturation, including CD80, CD86, and major histocompatibility complex class Ⅱ （MHC Ⅱ）, were analyzed by flow cytometry; the capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran uptake. Results Higher concentration of PGE2 was detected in CSCs culture supernatant than in the fresh medium. In addition, compared with control group, after treated with the supernatant in the mature stage, BM-DCs displayed lower expression of CD80, CD86 and MHC Ⅱ, lower T cell stimulatory capacity and higher endocytosis function. However, after the application of AH6809, BM-DCs partially regained T cell stimulatory capacity and expression of CD86 and MHC Ⅱ, but partially lost endocytic activity. Moreover, after the application of AH6809 and neutralizing TGF-β2 antibody, the result of statistical analysis indicated that there was a statistical difference of interaction in the expression of MHC Ⅱ and T cell stimulatory capacity. Conclusions PGE2 contributes to the suppressive effect on BM-DCs maturation mediated by CSCs in vitro, and PGE2 and TGF-β2 have additive effects on the immunosuppression of BM-DCs.

Adipose-derived stromal cells: Their identity and uses in clinical trials, an update

In adults, adipose tissue is abundant and can be easily sampled using liposuction. Largely involved in obesity and associated metabolic disorders, it is now described as a reservoir of immature stromal cells. These cells, called adipose-derived stromal cells (ADSCs) must be distinguished from the crude stromal vascular fraction (SVF) obtained after digestion of adipose tissue. ADSCs share many features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but they also display some specific features, including a greater angiogenic potential. Their angiogenic properties as well as their paracrine activity suggest a putative tumor-promoting role for ADSCs although contradictory data have been published on this issue. Both SVF cells and ADSCs are currently being investigated in clinical trials in several fields (chronic inflammation, ischemic diseases, etc. ). Apart from a phase Ⅲ trial on the treatment of fistula,most of these are in phaseⅠand use autologous cells. In the near future, the end results of these trials should provide a great deal of data on the safety of ADSC use.

Effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells

Objective:We investigated the effects of intermittent negative pressure on osteogenesis in human bone marrowderived stroma cells(BMSCs) in vitro. Methods:BMSCs were isolated from adult marrow donated by a hip osteoarthritis patient with prosthetic replacement and cultured in vitro. The third passage cells were divided into negative pressure treatment group and control group. The treatment group was induced by negative pressure intermittently(pressure:50 kPa,30 min/times,and twice daily). The control was cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast microscopy,the determination of alkaline phosphatase(ALP) activities,and the immunohistochemistry of collagen type I. The mRNA expressions of osteoprotegerin(OPG) and osteoprotegerin ligand(OPGL) in BMSCs were analyzed by real-time polymerase chain reaction(PCR). Results:BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure,the activity of ALP increased significantly,and the expression of collagen type I was positive. In the treatment group,the mRNA expression of OPG increased significantly(P<0.05) and the mRNA expression of OPGL decreased significantly(P<0.05) after 2 weeks,compared with the control. Conclusion:Intermittent negative pressure could promote osteogenesis in human BMSCs in vitro.

Anti-inflammatory potential of human corneal stroma-derived stem cells determined by a novel in vitro corneal epithelial injury model

BACKGROUND An in vitro injury model mimicking a corneal surface injury was optimised using human corneal epithelial cells(hCEC).AIM To investigate whether corneal-stroma derived stem cells(CSSC) seeded on an amniotic membrane(AM) construct manifests an anti-inflammatory, healing response.METHODS Treatment of hCEC with ethanol and pro-inflammatory cytokines were compared in terms of viability loss, cytotoxicity, and pro-inflammatory cytokine release, in order to generate the in vitro injury. This resulted in an optimal injury of 20%(v/v) ethanol for 30 s with 1 ng/mL interleukin-1(IL-1) beta. Co-culture experiments were performed with CSSC alone and with CSSC-AM constructs.The effect of injury and co-culture on viability, cytotoxicity, IL-6 and IL-8 production, and IL1 B, TNF, IL6, and CXCL8 mRNA expression were assessed.RESULTS Co-culture with CSSC inhibited loss of hCEC viability caused by injury. Enzyme linked immunosorbent assay and polymerase chain reaction showed a significant reduction in the production of IL-6 and IL-8 pro-inflammatory cytokines, and reduction in pro-inflammatory cytokine mRNA expression during co-culture with CSSC alone and with the AM construct. These results confirmed the therapeutic potential of the CSSC and the possible use of AM as a cell carrier for application to the ocular surface.CONCLUSION CSSC were shown to have a potentially therapeutic anti-inflammatory effectwhen treating injured hCEC, demonstrating an important role in corneal regeneration and wound healing, leading to an improved knowledge of their potential use for research and therapeutic purposes.

High expression of human clotting factor IX cDNA in the bone marrow stroma cells of hemophilia B patient

Hemophilia B, a serious bleeding disorder, is an inherited X chromosome-linked diseasecaused by the deficiency or inactiveness of human clotting factor IX (FIX). The conven-tional clinical treatment of plasma infusion is expensive and associated with a high risk

Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

辐射合成的温敏材料上兔角膜基质细胞的培养

目的:探索以电子加速器合成具有温度敏感特性的聚异丙基丙烯酰胺凝胶(PNIPAAm)及接枝有PNIPAAm水凝胶的培养皿的方法,及兔角膜基质细胞在温敏材料PNIPAAm水凝胶上的生长条件及特性,以及利用PNIPAAm水凝胶获得的细胞片。方法:55%N-异丙基丙烯酰胺(NIPAAm)单体和0.5%的N,N’-亚甲基双丙烯酰胺的异丙醇溶液70μL,加入到35 mm培养皿上,电子加速器下辐射合成温敏材料(PNIPAAm),后续处理后,接种兔角膜基质细胞体外培养。结果:按本实验中的单体配方以及辐射合成方案,能够在培养皿表面合成PNIPAAm,角膜基质细胞能在部分接枝了PNIPAAm的培养皿上生长,并能成片脱离。结论:使用辐射合成温敏培养皿能够获得单层及多层无载体的角膜基质细胞片。

肿瘤间质比在口腔鳞状细胞癌患者预后评估中的价值分析

目的:分析肿瘤间质比(tumor-stroma ratio,TSR)在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)患者预后中的价值。方法:收集2015年1月~2017年12月在蚌埠医学院第一附属医院接受根治性切除术的OSCC患者为研究对象,并收集临床及病理资料。TSR以50%为界限,将其分为高间质比组(≥50%)及低间质比组(<50%)。分析TSR和OSCC患者的无病生存期及总生存期之间的关系。结果:术后随访及临床资料完整的98例患者中,高间质比患者42例,低间质比患者56例。高间质比组OSCC患者的5年总生存率以及5年无病生存率分别为31.0%(13/42)、26.2%(11/42);低间质比组OSCC患者的5年总生存率以及5年无病生存率分别为73.2%(41/56)、67.9%(38/56)。肿瘤发病部位与患者的5年总生存率(χ^(2)=1.327,P=0.932)及5年无病生存率(χ^(2)=3.113,P=0.683)无关;肿瘤临床分期、TSR与患者的5年总生存率5年无病生存率显著相关(P<0.001)。单因素COX分析结果显示,年龄、肿瘤T分期、淋巴结转移、TSR与OSCC患者总体生存率以及无病生存率有关。而通过多因素COX研究显示,肿瘤T分期、淋巴结转移以及TSR是影响OSCC患者总生存率与无病生存率的独立危险因素(P<0.05)。结论:TSR可作为OSCC患者术后预后的影响因素,可为OSCC患者术后辅助治疗方法的选择提供参考依据。

角膜基质再生与修复的研究进展

角膜在眼球屈光系统中起着重要作用,角膜基质是角膜的主要组成部分,角膜基质的损伤可能导致永久性的视力损害,角膜移植术是目前治疗角膜基质疾病最有效的方法,但供体缺乏,长期免疫抑制治疗以防止排斥反应,以及移植物存活的限制阻碍了其进一步发展。此外,还可利用角膜基质内源性再生能力使角膜基质的胶原细胞外基质在合适的条件下能自我更新。然而,角膜基质复杂的超微结构难以在体外模拟。因此,再生医学被应用来克服这些挑战。这些方法涉及多个领域,包括干细胞诱导分化、组织工程技术、基因编辑等。本文就相关的角膜基质再生与修复技术、研究进展和存在的问题进行了归纳总结,并为未来角膜基质再生与修复的临床应用提供可能的途径。

Long non-coding RNA: The functional regulator of mesenchymal stem cells

Mesenchymal stem cells(MSCs) are a subset of multipotent stroma cells residing in various tissues of the body. Apart from supporting the hematopoietic stem cell niche, MSCs possess strong immunoregulatory ability and multiple differentiation potentials. These powerful capacities allow the extensive application of MSCs in clinical practice as an effective treatment for diseases.Therefore, illuminating the functional mechanism of MSCs will help to improve their curative effect and promote their clinical use. Long noncoding RNA(LncRNA) is a novel class of noncoding RNA longer than 200 nt. Recently,multiple studies have demonstrated that LncRNA is widely involved in growth and development through controlling the fate of cells, including MSCs. In this review, we highlight the role of LncRNA in regulating the functions of MSCs and discuss their participation in the pathogenesis of diseases and clinical use in diagnosis and treatment.

Androgen and prostatic stroma

<abstract>Aim: To investigate the effect of androgen on the proliferation, differentiation and regression of canine prostatic stromal cells in vivo and human stromal cells in vitro. Methods: Twenty-two dogs, including 15 normal prostate dogs and 7 prostatic hyperplasia dogs, had their serum concentration of testosterone and estrodiol determined by radioimmunoassay before and after castration. The expression of androgen receptor (AR) and estrogen receptor (ER) in the prostate were analysed by immunohistochemistry and semi-quantitative RT-PCR before and after castration. Light microscopy, transmission electron microscopy and TUNEL assay were carried out successively before and after castration to evaluate the prostatic histomorphology. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT). The proliferation of the cell culture was detected by MTT assay. The expression of TGFβ, bFGF, AR, and smooth muscle cell (SMC) specific proteins (myosin and/or smoothelin) were detected using immunohistochemistry and RT-PCR. The differentiation from fibroblasts to smooth muscle cells was deduced by measuring the expression of SMC specific proteins. Results: Before castration, the serum concentrations of testosterone and estrodiol were not statistically different between normal and hyperplasia groups. Following castration, the serum concentration of testosterone decreased rapidly in 2 days, and the concentration of estrodiol had no significant change compared with the pre-castration data. In the prostate, AR was presented in both the epithelial and stromal cells and the AR mRNA level was higher in hyperplasia than in normal prostate tissues (P<0.05). While ER predominantly existed in the prostate stromal cells and the ER mRNA had no difference between the hyperplasia and the normal group. Within the early phase of castration (<d7), the expression of AR was increased rapidly. Then it gradually dropped to a lower level than that of the pre-castration by the end of d90. The expression of ER remained unchanged in the whole course. The prostatic stromal cells, including SMCs and fibroblasts, diminished and underwent serial pathological changes of atrophy and apoptosis after castration. The atrophic cells were filled with huge intracellular lipofuscin. The expression of SMC myosin declined after castration, coincident with the increase in TGFβ mRNA level and decline in bFGF mRNA level. In vitro, DHT caused a weak increase in the proliferation and expression of SMC-specific proteins (P<0.05). However, DHT and bFGF together stimulated the proliferation of stromal cells significantly more than either agent alone (P<0.01). The combination of DHT and TGFβ greatly enhanced the expression of SMC-specific proteins (P<0.01) more strongly than either alone (P<0.01). Conclusions: The whole prostate gland is an androgen-sensitive organ with both the epithelium and stroma under the control of androgen. Androgen may direct the proliferation, differentiation and regression of stromal cells by regulating the expression of TGFβ, bFGF, AR and smooth muscle cell specific proteins.

Therapeutic strategies for targeting the ovarian tumor stroma

Epithelial ovarian cancer is the most lethal type of gynecologic malignancy. Sixty percent of women who are diagnosed with ovarian cancer present with advancedstage disease that involves the peritoneal cavity and these patients have a 5-year survival rate of less than 30%. For more than two decades, tumor-debulking surgery followed by platinum-taxane combination chemotherapy has remained the conventional first-line treatment of ovarian cancer. Although the initial response rate is 70%-80%, most patients with advancedstage ovarian cancer eventually relapse and succumb to recurrent chemoresistant disease. A number of molecular aberrations that drive tumor progression have been identified in ovarian cancer cells and intensive efforts have focused on developing therapeutic agents that target these aberrations. However, increasing evidence indicates that reciprocal interactions between tumor cells and various types of stromal cells also play important roles in driving ovarian tumor progression and that these stromal cells represent attractive therapeutic targets. Unlike tumor cells, stromal cells within the tumor microenvironment are in general geneticallystable and are therefore less likely to become resistant to therapy. This concise review discusses the biological significance of the cross-talk between ovarian cancer cells and three major types of stromal cells(endothelial cells, fibroblasts, macrophages) and the development of new-generation therapies that target the ovarian tumor microenvironment.

Pancreatic stellate cell: Pandora's box for pancreatic disease biology

Pancreatic stellate cells(PSCs) were identified in the early 1980 s, but received much attention after 1998 when the methods to isolate and culture them from murine and human sources were developed. PSCs contribute to a small proportion of all pancreatic cells under physiological condition, but are essential for maintaining the normal pancreatic architecture. Quiescent PSCs are characterized by the presence of vitamin A laden lipid droplets. Upon PSC activation, these perinuclear lipid droplets disappear from the cytosol, attain a myofibroblast like phenotype and expresses the activation marker, alpha smooth muscle actin. PSCs maintain their activated phenotype via an autocrine loop involving different cytokines and contribute to progressive fibrosis in chronic pancreatitis(CP) and pancreatic ductal adenocarcinoma(PDAC). Several pathways(e.g., JAK-STAT, Smad, Wnt signaling, Hedgehog etc.), transcription factors and mi RNAs have been implicated in the inflammatory and profibrogenic function of PSCs. The role of PSCs goes much beyond fibrosis/desmoplasia in PDAC. It is now shown that PSCs are involved in significant crosstalk between the pancreatic cancer cells and the cancer stroma. These interactions result in tumour progression, metastasis, tumour hypoxia, immune evasion and drug resistance. This is the rationale for therapeutic preclinical and clinical trials that have targeted PSCs and the cancer stroma.

Tumor stem cell, or its niche, which plays a primary role in tumorigenesis?

Cancer research over the past decades has focused on neoplastic cells, or a fraction of them, i.e. tumor stem cells, as the ultimate causes of tumorigenesis. However, during recent years, scientists have come to realize that tumorigenesis is not a solo act of neoplastic cells, but rather a cooperative process in which the roles of numerous types of non-neoplastic cells should be recognized. These tumor-residing non-neoplastic cells constitute the so-called tumor-associated stroma, which in certain cases even greatly surpasses the neoplastic cellular compartment that was previously thought of as a sole determiner leading to a seemingly autonomous growth pattern. In this review, we summarize several recent research highlights that have unveiled many previously unappreciated roles for microenvironmental factors, especially during the initiation stage of tumorigenesis. It is becoming increasingly clear that the stroma’s regulatory effects constitute not only an essential force for maintaining tumor growth, but also primary causes initiating tumorigenesis.

Pancreatic cancer and its stroma: A conspiracy theory

Pancreatic cancer is characterised by a prominent desmoplastic/stromal reaction that has received little attention until recent times. Given that treatments focusing on pancreatic cancer cells alone have failed to significantly improve patient outcome over many decades, research efforts have now moved to understanding the pathophysiology of the stromal reaction and its role in cancer progression. In this regard, our Group was the first to identify the cells(pancreatic stellate cells, PSCs) that produced the collagenous stroma of pancreatic cancer and to demonstrate that these cells interacted closely with cancer cells to facilitate local tumour growth and distant metastasis. Evidence is accumulating to indicate that stromal PSCs may also mediate angiogenesis, immune evasion and the well known resistance of pancreatic cancer to chemotherapy and radiotherapy. This review will summarise current knowledge regarding the critical role of pancreatic stellate cells and the stroma in pancreatic cancer biologyand the therapeutic approaches being developed to target the stroma in a bid to improve the outcome of this devastating disease.

Shattering the castle walls: Anti-stromal therapy for pancreatic cancer

Despite the availability of potent chemotherapy regimens, such as 5-fluorouracil, folinic acid, irinotecan, and oxaliplatin(FOLFIRINOX) and nab-paclitaxel plus gemcitabine, treatment outcomes in metastatic pancreatic cancer(PC) remain unsatisfactory. The presence of an abundant fibrous stroma in PC is considered a crucial factor for its unfavorable condition. Apparently, stroma acts as a physical barrier to restrict intratumoral cytotoxic drug penetration and creates a hypoxic environment that reduces the efficacy of radiotherapy. In addition, stroma plays a vital supportive role in the development and progression of PC, which has prompted researchers to assess the potential benefits of agents targeting several cellular(e.g., stellate cells) and acellular(e.g., hyaluronan) elements of the stroma. This study aims to briefly review the primary structural properties of PC stroma and its interaction with cancer cells and summarize the current status of antistromal therapies in the management of metastatic PC.

Pancreatic cancer stroma:understanding biology leads to new therapeutic strategies

Pancreatic ductal adenocarcinoma(PDA)is among the deadliest cancers in the United States and in the world.Late diagnosis,early metastasis and lack of effective therapy are among the reasons why only 6%of patients diagnosed with PDA survive past 5 years.Despite development of targeted therapy against other cancers,little progression has been made in the treatment of PDA.Therefore,there is an urgent need for the development of new treatments.However,in order to proceed with treatments,the complicated biology of PDA needs to be understood first.Interestingly,majority of the tumor volume is not made of malignant epithelial cells but of stroma.In recent years,it has become evident that there is an important interaction between the stromal compartment and the less prevalent malignant cells,leading to cancer progression.The stroma not only serves as a growth promoting source of signals but it is also a physical barrier to drug delivery.Understanding the tumor-stroma signaling leading to development of desmoplastic reaction and tumor progression can lead to the development of therapies to decrease stromal activity and improve drug delivery.In this review,we focus on how the current understanding of biology of the pancreatic tumor microenvironment can be translated into the development of targeted therapy.