Cryopreservation of isolated human hepatocytes for transplantation: State of the art

Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.     

Conversion of mammalian Müller glial cells into a neuronal lineage by in vitro aggregate-culture

Mammalian Müller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Müller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Müller glial cells could only differentiate into neurons under non-adhesive conditions. However, Müller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Müller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Müller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa.     

Effects of autologous stem cell transplantation on ventricular electrophysiology in doxorubicin-induced heart failure

To investigate whether stem cell transplantation affects ventricular electrophysiology in vivo, either autologous bone marrow mesenchymal stem cells or skeletal myoblast cells were transplanted via a catheter into a doxorubicin-treated failing heart. Four weeks after transplantation, electrophysiological investigation showed that transplantation of either cell type prolonged the local activation time and increased the activation time dispersion. In the stem cell transplantation groups, a positive correlation was demonstrated between activation time dispersion and the number of stem cell-derived cells in the pacing site. It is concluded that transplantation of either mesenchymal stem cells or skeletal myoblast cells might exacerbate abnormalities of local ventricular conduction in the doxorubicin-treated failing heart.     

Improved activity of streptozotocin-selected insulinoma cells following microencapsulation and transplantation into diabetic mice

We have recently shown that repeated streptozotocin (STZ) treatment induces the selection of insulinoma cells (RINmS) with both improved resistance to diabetogenic toxins and functional activity, compared to parental RINm cells. The aim of the present study was to estimate the potential of RINmS cells to maintain their engineered characteristics during in vivo hyperglycemic conditions. It was found that microencapsulation and transplantation into diabetic mice preserved a three-fold higher level of insulin content in selected RINmS cells when compared to the parental ones. Retrieval of transplanted encapsulated cells from the peritoneal cavity of diabetic mice had a significantly higher insulin content and a more intense insulin response to secretogogues in selected RINmS cells when compared to retrieved RINm cells. In conclusion, our results show that RINmS cells do not lose their improved functional characteristics after encapsulation and transplantation into diabetic mice.     

Leucine stimulates insulin secretion via down-regulation of surface expression of adrenergic α2A receptor through the mTOR (mammalian target of rapamycin) pathway: implication in new-onset diabetes in renal transplantation

The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients.     

Association of the ACE-II genotype with the risk of nephrotic syndrome in Pakistani children

Nephrotic syndrome is a common pediatric glomerular disease associated with heavy proteinuria. Since, the angiotensin converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism is a putative genetic risk factor for NS, in this study, ACE (I/D) polymorphism was analyzed in 268 NS and 223 control samples by a PCR-based method. The genotypic and allelic frequencies were determined and the association between ACE I/D polymorphism and NS was evaluated. The frequency distribution of the II, ID and DD genotypes was 82 (30.6%), 128 (47.8%) and 58 (21.6%) in the NS patients and 9 (4.0%), 171 (76.7%) and 43 (19.3%) in the control samples respectively. In the Pakistani pediatric NS population, the II genotypic and allelic frequencies were found to be significantly associated with the disease (OR = 6.755; C.I = 3-14.9). No significant association was found between this polymorphism and the response to standard steroid therapy. Thus, in contrast to reports from other parts of the world, the II genotype was found to be significantly associated with NS in the Indian and Malay populations and in the Pakistani population described here. To our knowledge, this is the first report from Pakistan describing the association of the ACE I/D polymorphism with pediatric NS. On the basis of these results, it is suggested that analysis of the ACE (I/D) polymorphism should be performed for the early diagnosis in the high risk NS patients in South Asia.     

太湖新银鱼移植对(鳖)早期摄食和生长的影响

研究选择长江中游西洞庭湖水系太湖新银鱼移植水体(黄石水库)和未移植水体(蒙泉水库),研究太湖新银鱼(Neosalanx taihuensis Chen)移植对浮游动物食性鱼类(Hemiculter leucisculus Basilewsky)早期生长和摄食的影响。2009年7月下旬和8月中旬共采集稚鱼157尾,其中7月下旬采集稚鱼在14—23日龄之间,两水体间生长差异不显著;8月中旬采集稚鱼在20—49日龄之间,黄石水库生长率显著小于蒙泉水库。对样品耳石日轮分析发现25日龄之前两水体稚鱼生长率相似,之后黄石水库稚鱼较蒙泉水库生长慢。食性分析发现25日龄前两水体稚鱼食物组成相似,主要摄食轮虫、小型枝角类和桡足类;25日龄后黄石水库稚鱼食性没有显著变化,而蒙泉水库稚鱼则转食大型枝角类、昆虫及鱼卵和仔鱼。两水体气候条件、营养状况、鱼类区系组成上基本相同,是否有太湖新银鱼移植是两水体间的主要差别。太湖新银鱼春群在1—5月间繁殖,而的繁殖在6月下旬之后。因此在早期生活史阶段与太湖新银鱼的食物竞争会主要发生在转食大型浮游动物之后。太湖新银鱼摄食使黄石水库大型浮游动物饵料资源短缺,稚鱼在25日龄后不能转食,是导致黄石水库幼鱼在25日龄后生长减慢的重要因素。     

间充质干细胞-透明质酸-多聚赖氨酸治疗脊髓损伤的实验研究

目的研究间充质干细胞—透明质酸—多聚赖氨酸复合物治疗脊髓损伤的可行性,评价其治疗效果并探讨其可能机制。方法从人骨髓中分离、培养人骨髓间充质干细胞（human bone marrow mesenchymal stem cell,hBMSC）;制作大鼠脊髓半横断模型,按照实验分组分别将hBMSC、透明质酸-多聚赖氨酸（hyaluronic acid-poly-L-lysine,HA-PLL）、hBMSC-HA-PLL复合物注入损伤区域,单纯损伤组作为对照。术后按照不同时间点评价损伤和移植后的大鼠运动功能。8周后杀死大鼠,观察不同移植组体内轴突和血管生长的情况,对不同细胞、材料及复合物移植对大鼠脊髓损伤修复效果进行评估。结果 hBMSC移植组和hBMSC-HA-PLL移植组的大鼠运动功能的改善显著好于单纯损伤及HA-PLL移植组。电镜结果证实复合物移植组可显著促进轴突和血管生长,新生的轴突和血管结构较为完整。结论 hBMSC具有促进神经功能恢复的作用,将其与HA-PLL相结合,可以促进大鼠脊髓损伤修复,其机制可能包括材料框架作用和hBMSC在体内对大鼠神经细胞的营养作用以及促进微血管的生成。     

诱导人脐带间充质干细胞向心肌细胞分化及对小鼠心肌梗死的治疗作用

目的研究脐带间充质干细胞(UC-MSC)体外分化为心肌细胞的可行性以及观察UC-MSC体内移植对心肌梗死模型小鼠的治疗效果。方法 10μmol/L 5-氮胞苷(5-aza)体外诱导UC-MSC 14 d,通过RT-PCR、免疫荧光染色鉴定其分化效果;采用腹腔注射盐酸异丙肾上腺素(ISO)每只3.0 mg/(kg/d),制作心肌梗死模型鼠;在注射ISO 48 h后,实验组将DAPI标记的UC-MSC经两次尾静脉移植给心肌梗死模型鼠,移植后第4周和第8周,分别采集实验小鼠的心脏、脾脏,以未移植细胞组的小鼠心肌损伤模型作为对照,通过心脏指数和脾脏指数测量,免疫荧光和碱性复红-苦味酸(HBFP)染色鉴定其体内分化和修复作用。结果 RT-PCR分析表明诱导的UC-MSC表达心肌特异性基因:心肌α-actin、TBX5、GATA4和NKx2.5,免疫荧光染色显示诱导细胞呈心肌α-actin和NKx2.5阳性,且呈双核现象。尾静脉移植后第4周和第8周,模型受体鼠心脏均发现有DAPI阳性细胞迁移至心肌组织且呈现心肌α-actin阳性,HBFP染色及心脏和脾脏指数结果显示移植UC-MSC对心肌损伤的模型鼠有明显的修复和治疗效果。结论 UC-MSC在体外经5-aza诱导可定向分化为心肌细胞,尾静脉体内移植UC-MSC对心肌损伤小鼠有明显的治疗效果。     

脐血干细胞移植治疗肝硬化的临床研究

目的:观察脐血干细胞移植治疗失代偿期肝硬化的临床疗效。方法:对60例失代偿期肝硬化患者进行自身对照的临床研究。在无菌条件下取健康产妇足月生产的脐带血,分离纯化脐血干细胞,通过肝动脉途径,将纯化的脐血干细胞移植入患者肝脏内,于移植后2周、4周、8周进行肝功能、凝血指标检测,并于4周及8周行腹部B超及胃镜检查,观察患者移植后不同时间症状改善情况及术后不良反应的发生情况。结果:60例接受脐血干细胞移植的肝硬化患者术中术后无明显不良反应发生。移植后,患者临床症状改善明显,食欲不振、乏力、腹水等减轻甚至消失;血清学检测:白蛋白水平较术前明显升高,凝血酶原时间、总胆红素较术前明显下降;术后8周复查胃镜,食道静脉曲张没有明显变化。结论:脐血干细胞移植治疗失代偿期肝硬化是一种安全、有效的方法,尤其是在提高白蛋白水平及改善凝血功能方面有很好的疗效,可作为肝移植治疗的过渡或补充治疗。