草酸钙晶体对大鼠肾小管上皮细胞损伤作用的实验研究

目的 通过研究一水草酸钙晶体粘附于体外培养的Wistar大鼠肾小管上皮细胞后引起的细胞功能变化，探讨尿路结石形成的机制。方法 分离、培养正常雄性Wistar大鼠的肾小管上皮细胞，在原代培养第5天时，将细胞分成3组（空白对照组，草酸组及一水草酸钙组），分别加入正常培养液，含有草酸钠（1、3、5mmol／L）的培养液及含有草酸钠（1、3、5mmol／L）和一水草酸钙晶体（5mmol／L）的培养液，实验8h后利用扫描电镜观察一水草酸钙晶体对肾小管上皮细胞的粘附情况，并分别用比色法测定细胞的钙镁ATP酶和钠钾ATP酶的活性，观察比较细胞功能的变化情况。结果 实验8h后，扫描电镜观察提示一水草酸钙组中有大量一水草酸钙晶体贴附于肾小管上皮细胞表面。①在三个草酸梯度浓度中，草酸组细胞钙镁ATP酶和钠钾ATP酶活性均未见明显下降，与对照组无显著性差异（P〉0．05）；②在三个草酸梯度浓度中，一水草酸钙组细胞钙镁ATP酶和钠钾ATP酶活性均明显下降。与对照组及草酸组有显著性差异（P〈0．05）；③在不同草酸浓度的一水草酸钙组间细胞钙镁ATP酶和钠钾ATP酶活性无显著性差异（P〉0．05）。结论 在高草酸环境下，对体外培养的Wistar大鼠的肾小管上皮细胞产生损伤作用的主要是一水草酸钙晶体而非游离草酸根离子。一水草酸钙晶体粘附于肾小管上皮细胞以后，可以造成细胞的损伤，这一机制可能有助于肾结石的发生。     

细胞外ATP对体外培养乳鼠脊髓神经元生长的影响

目的 探讨三磷酸腺苷(adenosine triphosphate，ATP)对培养乳鼠脊髓神经元活性及生长的影响。方法 将不同浓度的ATP加入培养的大鼠乳鼠脊髓神经元培养液中，使终浓度分别为0．1、1．0和10．0mmol／L，倒置相差显微镜观察细胞生长及形态。用四唑盐比色法(MTT)和乳酸脱氢酶(LDH)法分别检测不同浓度组神经元的活性和细胞损伤程度。用图像分析仪分析不同浓度ATP对神经元突起生长的影响。结果 0．1mmol／LATP可提高细胞活性，但对细胞突起生长无影响；1．0mmol／LATP提高神经元活性，促进突起生长；10．0mmol／LATP降低神经元活性，抑制突起生长。结论 细胞外ATP能剂量依赖性地影响培养神经元活性，适宜浓度的ATP能提高神经元活性并促进神经元突起的生长。     

高浓度谷氨酸对原代培养人脑胶质瘤细胞生长抑制和凋亡的影响

目的探讨高浓度谷氨酸对人脑胶质瘤原代培养细胞的生长抑制和凋亡诱导作用。方法在体外对新鲜的人脑胶质瘤标本进行原代培养；不同浓度的谷氨酸作用不同时间后，进行形态学观察噻唑蓝（MTT）法检测生长抑制率；流式细胞仪Armexin V-FITC／PI双染法检测凋亡率。结果原代人脑胶质瘤细胞短期培养成功。高浓度谷氨酸对培养细胞有明显的抑制作用，并且存在一定的剂量依赖关系。当谷氨酸的浓度为25～50mmol／L时，可明显诱导培养细胞凋亡，并且凋亡率随药物浓度增加和时间延长而增高；当谷氨酸浓度达100mmol／L以上时，细胞则主要以坏死为主。结论以酶消化法可成功对人脑胶质瘤细胞进行体外原代培养；在一定的浓度范围内，高浓度谷氨酸能明显的抑制原代培养人脑胶质瘤细胞生长，并诱导其凋亡。     

肝细胞生长因子对骨髓基质干细胞与纤维连接蛋白涂层黏附行为的影响

目的 观察肝细胞生长因子（HGF）对骨髓基质干细胞与纤维连接蛋白涂层黏附行为的影响。方法 将不同浓度的肝细胞生长因子加入预涂纤维连接蛋白的96孔板，观察细胞的黏附特性、增殖活性及形态学改变。结果 在肝细胞生长因子的诱导下，骨髓基质干细胞在纤维连接蛋白预包被的96孔板底分布均匀，多呈纤维母细胞样外观；肝细胞生长因子诱导的骨髓基质干细胞对纤维连接蛋白的黏附性是一种剂量-依赖性方式，50％的有效剂量（ED50）大约是20μg／L，当达到最大黏附力时剂量为100μg／L；与空白对照组相比，50～200μg／L的肝细胞生长因子能促进骨髓基质干细胞的增殖，其中以50μg／L组细胞增殖活性最强（P〈0．05）。结论 在最适浓度下，肝细胞生长因子能显著提高骨髓基质干细胞与纤维连接蛋白涂层的黏附行为。     

糖尿病小鼠胰腺干细胞转分化为胰岛样细胞

目的 观察链脲佐菌素所致糖尿病小鼠胰腺干细胞是否能转分化为胰岛样细胞.方法 以链脲佐菌素建立糖尿病小鼠模型,分离培养其胰腺导管上皮细胞,经体外扩增及诱导培养后,以细胞免疫化学方法检测PDX1表达,行STZ染色和葡萄糖刺激的胰岛素释放试验鉴定其功能.结果 糖尿病小鼠胰腺干细胞经体外培养和诱导分化后,PDX1阳性,并形成胰岛样细胞团;胰岛样细胞对高糖刺激(15.0 mmol/L)的胰岛素释放较低糖(5.6 mmol/L)时增加了1.4倍(37.2±11.2比25.9±7.6,t =2.830,P＜0.05),DTZ染色阳性.结论 链脲佐菌素所致糖尿病小鼠胰腺干细胞在体外培养条件下可转分化为胰岛素分泌细胞.     

地塞米松对骨髓基质干细胞体外增殖的影响

目的 观察地塞米松对小鼠骨髓基质干细胞体外增殖的作用。方法 取雄性6周龄ICR小鼠股骨骨髓基质细胞进行体外培养，在细胞培养不同时间点加入10^-8mol／L地塞米松（实验组）或保持基础培养条件（对照组），用CellTiter方法分别检测两组的细胞增殖情况，并予比较。结果 在细胞培养不同阶段开始应用地塞米松的实验组中，细胞增殖能力较对照组降低，随着培养时间延长实验组细胞数量明显少于对照组。实验组中细胞形态较对照组更趋成熟。结论 地塞米松在促进骨髓干细胞定向分化早期抑制骨髓基质干细胞体外增殖。     

培养基钙浓度对复方壳多糖组织工程皮肤基底膜构建的形态学研究

目的寻找最适于组织工程皮肤生长及基底膜构建的体外培养基钙浓度。方法按照鲁元刚等建立的方法制备复方壳多糖组织工程皮肤，根据培养基钙浓度不同，分为1．00、1．45、1．65和1．95mmol／L四组。在37℃、5％CO2及90％以上湿度孵箱内浸没培养3d后，将培养物置于不锈钢筛网上进行气一液界面三维培养，每天换液1次。培养7、15d，行HE、PAS染色观察组织工程皮肤的组织学特性；并利用免疫组织化学染色对基底膜的主要成分——Ⅳ型胶原进行观察，评价基底膜构建情况。结果各组组织工程皮肤组织学观察类似正常皮肤，有分化良好的表皮和致密真皮，但1．95mmol／L组表皮分化较其他组更好；1．00、1．45及1．65mmol／L组角化层外侧常伴未角化的角质形成细胞，1．95mmol／L组表皮细胞角化完全，且与颗粒层细胞结合牢固。PAS染色显示各组真、表皮间有紫红色带状结构；免疫组织化学染色显示真皮与表皮间Ⅳ型胶原反应呈阳性，且1．95mmol／L组阳性反应强于1．00mmol／L组。结论钙浓度为1．95mmol／L的培养基最适合于复方壳多糖组织工程皮肤生长及基底膜构建。     

钙浓度变化对体外培养人表皮角质细胞增殖的影响

目的:比较不同钙浓度培养的人表皮角质细胞(HEKs)增殖能力的差异,确定体外培养表皮角质细胞的最适钙浓度.方法:根据培养液中CaCl2浓度不同,将HEKs分为无钙培养组和0.1、0.3、0.5、1.0 mmol/L CaCl2培养组.细胞培养3 h后,加钙离子荧光染料Fluo-3-AM,显微镜观察细胞内的荧光强度,以反映细胞内钙离子浓度的变化;细胞培养2 d后加Hochest33258核染液,荧光显微镜下观察细胞生长形态;培养3 d时镜下观察细胞的生长情况,并分别用流式细胞术和MTT法分析细胞的增殖情况.结果:钙培养可使细胞内钙离子浓度增加,表现为细胞内Fluo-3-AM荧光强度增强,且随着CaCl2浓度的增加,荧光强度亦随之增加;在钙浓度为0.3mmol/L时,细胞的增殖指数最高,为(51.98±14.31)%,增殖活性最强;0.1mmol/L组次之;随着钙浓度的增加,细胞的增殖指数下降,增殖活性受到抑制,0.5～1.0mmol/L钙培养组的增殖指数和增殖活性均明显低于无钙培养组(P均<0.01).结论:不同钙浓度培养影响表皮角质细胞的增殖能力,钙浓度为0.3 mmol/L时细胞的增殖能力最强.     

异丙酚对布比卡因诱导的PC12细胞毒性的作用

目的 探讨异丙酚对布比卡因诱导的PC12细胞毒性的作用及其可能机制。方法PCI2细胞接种于96孔培养板中培养，随机分为4组：对照组、布比卡因组、异丙酚组和布比卡因＋异丙酚组，分别加入Hanks液、布比卡因（终浓度为0．03、0．06、0．09、0．12、0．15mmol／L）、异丙酚（终浓度为1、2,4、8mmol／L）以及同时加入布比卡因（终浓度为0．09mmol／L）和异丙酚（终浓度为1、2,4、8mmol／L）。培养24h时，采用二甲基噻唑二甲基四唑溴盐比色微量分析法测定各孔的细胞活力和上清液乳酸脱氢酶（LDH）的活性，应用流式细胞仪检测硫氧还蛋白-1（Trx-1）和硫氧还蛋白还原酶-1（TrxR-1）表达率。结果 与对照组比较，布比卡因组PC12细胞活力降低（P〈0．01），且呈浓度依赖性；异丙酚组PC12细胞活力差异无统计学意义（P〉0．05）。与布比卡因组的0．09mmol／L亚组比较，布比卡因＋异丙酚组的2—8mmol／L亚组PC12细胞活力增加（P〈0．05）。与对照组比较，布比卡因组和布比卡因＋异丙酚组LDH活性升高，Trx-1和TrxR-1表达率降低（P〈0．01）；与布比卡因组比较，异丙酚组和布比卡因＋异丙酚组LDH活性降低，Trx-1和TrxR-1表达率升高（P〈0．01）。结论 布比卡因对PC12细胞具有浓度依赖性的细胞毒性作用。异丙酚可通过保护内源性硫氧还蛋白系统减轻布比卡因诱导的PCI2细胞的毒性。     

氯胺酮对谷氨酸引起神经元样嗜铬细胞瘤细胞株凋亡的影响

目的 观察氯胺酮对谷氨酸引起神经元样嗜铬细胞瘤(PC12)细胞株凋亡的影响。方法PC12细胞株分别以2×103/孔和1×105/ml密度接种于96孔细胞培养板和60 mm细胞培养皿中,置于培养基,培养基中加10 nmol/L神经生长因子(7S-NGF),96孔细胞培养板和60 mm细胞培养皿的细胞均随机分为五组,A组暴露于20 mmol/L谷氨酸中,B组暴露于20 mmol/L谷氨酸 0.1 mmol/L氯胺酮(氯胺酮比谷氨酸提前1 min 加入)中,C组暴露于20 mmol/L 谷氨酸 1.0 mmol/L氯胺酮中,D组暴露于20 mmol/L 谷氨酸 100 μmol/L D-2-氨基-5-膦酸基戊酸(D-AP5)(细胞与D-AP5提前孵育2h)中,E组暴露于等容积的不含7S-NGF的新鲜培养液中,采用MTT法测定细胞培养板中PC细胞的细胞活力,采用死端比色TUNEL系统细胞检测培养皿中PC12细胞株的凋亡率。结果 A组、B组、C组和D组细胞活力分别为:37％±6％、65％±7％、99％±10％、90％±22％,与A组比较,B组、C组、D组细胞活力均升高(P<0.05或0.01)。A组、C组、D组、E组凋亡细胞率分别为66％±10％、20％±6％、22±7％、3.2％±1.8％,与A组比较,C组、D组、E组细胞凋亡率明显降低(P<0.01)。结论 氯胺酮通过抑制谷氨酸引起的神经元样PC12细胞株凋亡而发挥神经保护作用。     

The construction of an oxalate-degrading intestinal stem cell population in mice: a potential new treatment option for patients with calcium oxalate calculus

About 80% of all urological stones are calcium oxalate, mainly caused by idiopathic hyperoxaluria (IH). The increased absorption of oxalate from the intestine is the major factor underlying IH. The continuous self-renewal of the intestinal epithelium is due to the vigorous proliferation and differentiation of intestinal stem cells. If the intestinal stem cell population can acquire the ability to metabolize calcium oxalate by means of oxc and frc transgenes, this will prove a promising new therapy option for IH. In our research, the oxalate-degrading genes of Oxalobacter formigenes (Oxf)—the frc gene and oxc gene—were cloned and transfected into a cultured mouse-derived intestinal SC population to give the latter an oxalate-degrading function. Oxf was isolated and cultivated and the oxalate-degrading genes—frc and oxc—were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed and transferred into the mouse stem cell population. After selection with G418, the expression of the genes was identified. The oxalate-degrading function of transfected cells was determined by transfection into the intestinal stem cell population of the mouse. The change in oxalate concentration was determined with an ion chromatograph. The recombinant plasmid containing oxc and frc genes was transfected into the stem cell population of the mouse and the expression of the genes found normal. The cell population had acquired an oxalate-degrading function. The oxc and frc genes could be transfected into the intestinal stem cell population of the mouse and the cells acquired an oxalate-degrading function.     

草酸和一水草酸钙结晶对人肾小管上皮细胞的影响

目的 观察草酸和一水草酸钙结晶对体外培养的人肾小管上皮细胞(HK-2)的毒性作用和蛋白表达的影响,探讨上皮细胞受损在肾结石形成过程中的可能作用.方法 体外培养正常人HK-2细胞至长满后,换成无血清培养基,加入草酸至不同终浓度,显微镜下观察结晶的形成及其对细胞的粘附.收集结晶以傅立叶红外光谱仪(FT-IR)分析其成分.CCK-8试剂盒检测1、2、5和10mmol/L草酸分别作用4、12和24 h后对HK-2细胞的毒性反应,Bradford法检测HK-2细胞表达总蛋白量的变化.结果草酸加入含Ca2+的DMEM培养基后,数分钟内显微镜F即可见结晶形成并粘附于细胞表面.FT-IR分析表明结晶成分为一水草酸钙.草酸和一水草酸钙对HK-2的毒性作用呈明显浓度依赖性,但在1～10 mmol/L草酸浓度下毒性作用不随时间延长而增加.1、2、5mmol/L草酸作用12 h和对照组HK-2表达蛋白量分别为(358±51)、(365±43)、(328±52)和(329±60)mg/L,P值均＞0.05,而10 mmol/L草酸作用12 h后,HK-2表达蛋白量为(264±76)mg/L,显著低于对照组(P＜0.05).结论 草酸和一水草酸钙可对正常人HK-2细胞产生毒性损伤,造成细胞表达蛋白的改变,可能在肾结石的形成中起重要作用.     

Oxalate is toxic to renal tubular cells only at supraphysiologic concentrations

BACKGROUND: Oxalate-induced tissue damage may play an initiating role in the pathophysiology of calcium oxalate nephrolithiasis. The concentration of oxalate is higher in the renal collecting ducts ( approximately 0.1 to 0.5 mmol/L) than in the proximal tubule ( approximately 0.002 to 0.1 mmol/L). In the present investigation, we studied the damaging effect of oxalate to renal proximal and collecting tubule cells in culture. METHODS: Studies were performed with the renal proximal tubular cell lines, LLC-PK1 and Madin Darby canine kidney II (MDCK-II), and the renal collecting duct cell lines, rat renal cortical collecting duct (RCCD1) and MDCK-I. Confluent monolayers cultured on permeable growth substrates in a two-compartment culture system were apically exposed for 24 hours to relatively low (0.2, 0.5, and 1.0 mmol/L) and high (5 and 10 mmol/L) oxalate concentrations, after which several cellular responses were studied, including monolayer morphology (confocal microscopy), transepithelial electrical resistances (TER), prostaglandin E(2) (PGE(2)) secretion, lactate dehydrogenase (LDH) release, DNA synthesis ([(3)H]-thymidine incorporation), total cell numbers, reactive oxygen species (H(2)O(2)) generation, apoptotic (annexin V and DNA fragmentation), and necrotic (propidium iodide influx) cell death. RESULTS: Visible morphologic alterations were observed only at high oxalate concentrations. TER was concentration-dependently decreased by high, but not by low, oxalate. Elevated levels of PGE(2), LDH, and H(2)O(2) were measured in both cell types after exposure to high, but not to low oxalate. Exposure to high oxalate resulted in elevated levels of DNA synthesis with decreasing total cell numbers. High, but not low, oxalate induced necrotic cell death without signs of programmed cell death. CONCLUSION: This study shows that oxalate is toxic to renal tubular cells, but only at supraphysiologic concentrations.     

Comparative functional study of clonal insulin-secreting cells cultured in five commercially available tissue culture media

The electrofusion-derived rat insulin-secreting cell line BRIN-BD11 was cultured in five different commercially available media to determine the optimum medium for the in vitro maintenance of such clonal cell lines. Cells were cultured in RPMI-1640, DMEM, McCOY'S, F-12K, or MEM culture medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 U/ml penicillin and 0.1 g/L streptomycin). Insulin secretion studies performed after 10 days revealed RPMI-1640 to be the best performing medium in terms of insulin secretory responsiveness to a range of stimuli including glucose, L-alanine, L-arginine, carbachol, and glibenclamide. Insulin release was significantly decreased (p < 0.01 to p < 0.05) in all other media compared to RPMI-1640. Only the cells cultured in RPMI-1640 and DMEM showed a significant glucose-induced insulin secretory response (p < 0.01 and p < 0.05). McCOY'S gave the next best result followed by F-12K and MEM. After the 10-day culture period, the highest insulin content was found in cells cultured in RPMI-1640 and DMEM with significantly lower levels of insulin in cells cultured in McCOY'S, F-12K, and MEM (p < 0.01 to p < 0.001). RPMI-1640 was used for further studies to investigate the effects of 5.6-16.7 mmol/L glucose in culture on the secretory responsiveness of BRIN-BD11 cells. Significant responses to a number of nonglucidic secretagogues were seen following culture at 5.6 and 16.7 mmol/L glucose, although responsiveness was less than after culture with 11.1 mmol/L glucose. At 16.7 mmol/L glucose culture, glucose-stimulated insulin release was abolished.     

C57/BL6小鼠骨髓间充质干细胞的体外优化培养和酒精及其代谢物毒性效应的探讨

目的建立C57/BL6小鼠骨髓间充质干细胞(marrowmesenchymalstemcells,MSCs)的体外分离和培养体系,探讨酒精及其代谢物对MSCs的毒性。方法从C57/BL6小鼠的股骨骨髓中分离MSCs,通过优化细胞的培养方法进行有效培养、传代,最终获得纯度较高的MSCs,并对第4代细胞进行CD90和CD34免疫组织化学染色鉴定。对第2代细胞以4×105个/ml密度接种于96孔板,每孔200μl,第2天以用乙醇及乙醛进行染毒,剂量设计为乙醇5.7、17.0、50.0、100.0、150.0mmol/L,乙醛4.5、0.9、0.18、0.036、0.0072、0.00144、0.000288mmol/L,对照组为MSCs以不加乙醇及乙醛的10%胎牛血清的α-MEM培养液培养。3d后MTT检测细胞增殖活性。结果MSCs在前6代表现出较好的稳定性和旺盛的自我增殖能力,免疫组织化学染色显示第4代MSCsCD90呈阳性、CD34呈阴性。MTT检测乙醇在17.0、100.0及150.0mmol/L时出现细胞增殖抑制,与对照组比较,差异有统计学意义(P<0.05);乙醛>0.18mmol/L时随着浓度增加细胞增殖力减弱,在4.5mmol/L时出现了细胞增殖抑制,与对照组比较,差异有统计学意义(P<0.05)。结论优化培养体系能有效分离和培养MSCs,乙醇和乙醛均能抑制其增殖,表现出毒性作用,乙醛的毒性作用较乙醇强,其造成的损伤更不容忽视。     

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

Introduction

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions.

Materials and method

Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein.

Results

The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes.

Conclusion

Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.     

Feasibility of using early mesencephalic neural plate for intracerebral grafting

The purpose of this study was to elucidate the biological significance and the possibility of intracerebral grafting of neuroepithelial stem cells derived from the mesencephalic neural plate. Immunohistological studies of embryonic day 10.5 (E10.5) Wister rats revealed strong nestin expression in the mesencephalic part of the neural plate. Mesencephalic neural plates removed from E10.5 rats were processed to either tissue or cell dissociation culture. They were cultured in vitro under various conditions and were analyzed 7 days after the primary culture. When they were cultured as a tissue, cell proliferation and differentiation into neurons extending long neurites were obvious in a serum-free medium, in a medium containing 3% serum, and in a medium containing 20 ng/ml epidermal growth factor. On the other hand, in a medium containing 10 ng/ml basic fibroblast growth factor (bFGF), both vigorous cell proliferation and sphere formation were recognized. Furthermore, marked neurite growth was rarely seen in this culture. When they were plated in a dissociation culture, cell proliferation and neurosphere generation were also recognized only in a medium containing bFGF, depending on the initial cell concentration. The spheres, generated 7 days after the primary cell culture, were positively stained by nestin. These data suggested that bFGF was able to amplify the stem cell population present in the mesencephalic neural plate derived from early embryos. This might make it possible to obtain a large number of stem cells as donor material for neural transplantation on demand.     

Flow cytometric discrimination of mesenchymal progenitor cells from bone marrow-adherent cell populations using CD34/44/45(-) and Sca-1(+) markers

Background Cultured bone marrow adherent cells (BMACs) have been commonly used as stem cells in bone and cartilage regeneration therapy. However, BMACs are actually a heterogeneous cell population, and clinicians might have previously transplanted more fibroblasts or other cells than actual stem cells. The purposes of this study were to (1) isolate immature mesenchymal stem cells with CD34/44/45 and Sca-1 surface-antigen patterns from BMACs using flow-activated cell sorting and (2) investigate their differentiation potential. Methods Bone marrow cells were extracted from the mouse femur and cultured. Adherent cells could be identified approximately 3 days after seeding, and nonadherent cells were removed with the medium when it was changed. BMAC samples were cultured for 3, 7, 10, 14, 21, 28, 35, and 42 days after the first seeding. We directly isolated CD34/44/45(−)Sca-1(+) mesenchymal progenitor cells (MPC1) and CD34/45(−)/44(+) Sca-1(+) mesenchymal progenitor cells (MPC2) from BMACs based on their cell surface marker patterns using a fluorescence-activated cell sorter. These subgroups — MPC1, MPC2, and the residual cells in BMACs (non-MPC population: RCs) — were then induced to differentiate into bone, cartilage, and fat using a plate culture. The cultures were examined after histochemical staining on day 14. Results In a plate culture, the MPC1 population had higher potential to differentiate into osteoblasts, chondrocytes, and lipocytes; whereas MPC2 and RCs differentiated into only two lineages: osteoblasts and lipocytes. The incidence of these multipotential cells was less than 5% among the cultured BMACs. MPC1 proliferated up to 17-fold within 3–4 weeks after separation from floating cells and did not increase thereafter. Conclusions BMACs are conventionally thought to differentiate into cartilage only in pellet culture, but we showed that MPC1 produced cartilage-like extracellular matrix in plate culture. MPC1, which are more immature cells than MPC2 and RCs, were multipotential progenitors that showed unique cartilage-differentiation potential. MPC1 had less ability to proliferate in BMAC culture, but they might have higher potential for chondrogenic differentiation.     

Improved in vitro function of islets using small intestinal submucosa

Transplantation of human pancreatic islets has been demonstrated to be a viable alternative to exogenous insulin therapy for diabetes mellitus. However, optimum results require transplantation of islets from two to three pancreas donors after a minimum number of days in culture. This implies that a substantial part of the transplanted islet mass may be nonfunctional. This study investigates the ability of an optimized technique to retain islet function using porcine-derived small intestinal submucosa (SIS) during in vitro culture. Groups of purified human islets were cultured for 3 weeks in modified standard islet culture conditions of CMRL = 1066 tissue culture medium supplemented with 25 mmol/L HEPES, penicillin/streptomycin, and a commercial insulin-transferin-selenium (ITS) supplement. Islets (50 to 200 IE/condition; n = 5 preparations) were cultured in plates containing noncoated Biopore membrane inserts alone, or on inserts that had been covered with SIS. Function was assessed by static incubation with low (4 mmol/L), or high (20 mmol/L) glucose at the end of each week. Glucose-stimulated release of human insulin was measured by radioimmunoassay (Linco, St. Charles, Missouri). Remaining islets were stained and evaluated visually. Neither culture condition resulted in significantly different basal secretion until week 3 (P =.05). However, by the end of week 2 and for the duration of the experiment thereafter, SIS-treated islets exhibited a higher SI (P <.05). At the end of the experiment, islets cultured on the SIS exhibited excellent morphology, with greater than 90% staining positive with Dithizone. Islets cultured on the inserts alone lost their initial morphology, becoming "loose" in appearance. The results of this study indicate that SIS enables enhanced function of islets in vitro as compared to non-SIS supported culture conditions.