The Identification of Suppressor Cells in the Murine Fetal Liver*

ABSTRACT: Fetal suppressor cells are potentially important for maintenance of pregnancy and for the immune regulation of the identification of self. We have identified and partially characterized a population of murine fetal liver cells which are active suppressors of a variety of immune responses, including mixed lymphocyte responses (MLR), responses to mitogens plaque-forming cell responses to heterologous erythrocytes. The suppression is non-H-2-restricted and can act via a factor. The cell is preactivated in fetal liver and may represent a T-cell precursor. The suppression of MLR is maximal (approximately 100%) if cells are added within the first 48 h of culture initiation. The characteristics of this suppressor cell are compared to other known MLR and perinatal suppressor systems.     

Human Fetal Liver Cells Induce Colonies in Spleen of Lethally Irradiated Mice

Murine hematopoietic tissues contain cells which, upon injection into lethally irradiated mice, produce nodules on the surface of their spleen (colony-forming unit—spleen; CFU-S). The exact hierarchical level of the hematopoietic progenitors which give rise to CFU-S is not fully established; however, cell populations highly enriched for repopulating stem cells appear to contain a high percentage of CFU-S. The experiments reported here involved the injection of human fetal liver cells into mice, under conditions similar to those of the CFU-S test. These data demonstrate that human fetal liver cells are able to induce spleen colonies (tentatively called human CFU-S) when injected into lethally irradiated mice. The number of CFU-S was increased by prior purification of human fetal liver cells. When mice were injected with human fetal liver cells inactivated by irradiation, no human CFU-S were observed. Positive staining of cells found in spleen colonies, using monoclonal antibodies specific for various human determinants, indicated the human origin of part of them. The presence of human cells within the colonies was further confirmed by in situ hybridization using a probe specific for human DNA. A mean of 30–40% of analyzed colonies was thus shown to contain some patches of human cells. These data confirm that human hematopoietic cells are able to seed, proliferate, and differentiate in a murine microenvironment.     

大鼠肝移植术后受体骨髓间充质干细胞输注肝内示踪与标记方法比较

目的采用羧基荧光素二醋酸盐琥珀酰亚胺酯（carboxyfluorescin diacetate succinimidyl ester,CFSE）与溴化脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine,Brdu）两种细胞标记物观测肝移植术后受体骨髓间充质干细胞（MSCs）肝内分布情况,并对两种方法进行比较。方法在已构建的DA-Lewis大鼠原位肝移植模型及提取培养Lewis大鼠MSCs的基础上分别用CFSE和Brdu对Lewis来源的MSCs进行标记．术中经门静脉输注．通过荧光显微镜和免疫组织化学法观测术后2mo内各时间点受体肝组织内MSCs的分布情况。结果CFSE细胞标记率约为（94．1±1．4）％。受体肝组织第1、7、14天内有CFSE标记的MSCs聚集,对照组第5天肝组织内发布的CFSE标记MSCs消失。Brdu细胞标记率约为（90．3±3．5）％。受体肝组织第14天、1个月、2个月可见Brdu阳性的MSCs分布,对照组相应时间点肝组织内未发现Brdu阳性的MSCs。结论CFSE和Brdu均为MSCs的良好标记物。CFSE标记细胞后荧光迅速减弱,适用于短期示踪。Brdu标记细胞后可维持较长时间．适用于长期示踪。两种标记方法均是MSCs较为简单有效的方法,实验显示受体MSCs大部分分布于移植肝脏。     

Therapeutic Potential of Adult Bone Marrow Stem Cells in Liver Disease and Delivery Approaches

Hematopoietic stem cells (HSCs) and mesenchymal stem cell (MSCs) are two main subtypes of bone marrow stem cells. Extensive studies have been carried out to investigate the therapeutic potential of BMSCs in liver disease. A number of animal and human studies demonstrated that either HSCs or MSCs could be applied to therapeutic purposes in certain liver diseases. The diseased liver may recruit migratory stem cells, particularly from the bone marrow, to generate hepatocyte-like cells either by transdifferentiation or cell fusion. Transplantation of BMSCs has therapeutic effects of restoration of liver mass and function, alleviation of fibrosis and correction of inherited liver diseases. There are still controversial results over the potential effects of BMSCs on liver diseases, and some of the discrepancies are thought to be lied in the differences of experimental protocols, differences in individual research laboratory, and the uncertainties of the techniques employed. Several potential approaches for BMSCs delivery in liver diseases have been proposed in animal studies and human trials. BMSCs can be delivered via intraportal vein, systemic infusion, intraperitoneal, intrahepatic, intrasplenic. The optimal stem cells delivery should be easy to perform, less invasive and traumatic, minimum side effects, and with high cells survival rate. In this review, we focus on the up-to-date evidence of therapeutic effects of BMSCs on liver disease, the characteristics of various delivery approaches, and the considerations for future studies.     

In Search of Liver Cancer Stem Cells

Recent research efforts in stem cell and cancer biology have put forth a “stem cell model of carcinogenesis” which stipulates that the capability to maintain tumor formation and growth specifically resides in a small population of cells called cancer stem cells. The stem cell-like characteristics of these cells, including their ability to self-renew and differentiate; and their limited number within the bulk of the tumor mass, are believed to account for their capability to escape conventional therapies. In the past few years, the hypothesis of stem cell-driven tumorigenesis in liver cancer has received substantial support from the recent ability to identify and isolate a subpopulation of liver cancer cells that is not only able to initiate tumor growth, but also serially establish themselves as tumor xenografts with high efficiency and consistency. In this review, stem cell biology that contributes to explain tumor development in the particular context of liver cancer will be discussed. We will begin by briefly considering the knowledge available on normal liver stem cells and their role in tissue renewal and regeneration. We will then summarize the current scientific knowledge of liver cancer stem cells, discuss their relevance to the diagnosis and treatment of the disease and consider the outstanding challenges and potential opportunities that lie ahead of us.     

Morphological Changes in the Kidneys of Rats with Postischemic Acute Renal Failure after Intrarenal Administration of Fetal Mesenchymal Stem Cells from Human Bone Marrow

Chronic experiments on outbred albino rats were performed to compare the dynamics of histological signs for postischemic renal injury (90-min thermal ischemia) after intraparenchymal injection of cultured fetal MSC from human bone marrow. Functional indexes of the ischemic kidney were predetermined. In the early period after ischemia (day 4), administration of human bone marrow MSC was followed by the increase in blood flow in the microcirculatory bed and decrease in the degree of alteration in renal tubules. An increase in the area of zones with histological signs for normal function of tubules was accompanied by the improvement of biochemical indexes for renal function. In the delayed period, a protective effect of cell therapy was manifested in the prevention of death of renal tubules. Mild calcification of the necrotic tubular epithelium served as a marker of this process. Human bone marrow MSC were labeled with the fluorescent probe Calcein. These cells migrated from the site of injection, spread in the interstitium, and retained viability for 7 days. During this period, some cells were incorporated into the lumen of renal tubules. Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 3-9, 2009     

A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC.     

Role of Gap Junctions in Embryonic and Somatic Stem Cells

Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.     

URSA小鼠模型中NKT细胞功能失调研究

利用原因不明复发性自发性流产 (URSA )小鼠模型 (CBA雌鼠×DBA/ 2J雄鼠 ) ,测定免疫性流产孕鼠不同孕期子宫蜕膜及胎盘内NKT细胞的数量及其分泌的细胞因子的格局 ,以调查NKT细胞数量和功能失调在URSA中的可能作用。研究结果显示 ,与正常孕鼠相比 ,流产模型鼠妊娠早期子宫和蜕膜中NKT细胞数量减少 ,分泌IFN γ能力降低。我们推测 ,妊娠早期子宫胎盘和蜕膜NKT细胞数量和功能失调可能是导致URSA的机制之一。     

诱导多潜能干细胞

通过病毒载体导入4个外源转录因子Oct4、Sox2、c-Myc、Klf或者Oct4、Sox2、Nanog、Lin28入体细胞,可以诱导产生具有胚胎干细胞特性相似的诱导多潜能干细胞(induced pluripotent stem cells,iPS).iPS在疾病治疗和药物研究等领域具有非常重要的应用前景,但是目前存在诱导效率低以及致肿瘤性等缺点,采用改良方法诱导产生iPS是将来研究的重点.     

成体干细胞

近年来,干细胞研究已成为生物医学界研究的热点,各国科学家在基础理论、实验技术、应用研究等领域都取得了很大的进展,我国第一个干细胞制剂的临床试验研究也已经启动。为了使广大读者较全面地了解该领域的最新进展和干细胞移植技术的应用前景,我刊特组织“干细胞的可塑性与临床移植研究”的专题综述,重点介绍干细胞研究最前沿的理论问题,并对干细胞的应用研究做了较好的总结。     

移植耐受诱导的异基因胎肝移植小鼠的免疫功能重建

本文用门静脉注射异型脾细胞加腹腔注射环磷酰胺方法，成功地诱导了成年Ｂａｌｂ／ｃ小鼠（Ｈ－２ｄ）对Ｃ５７ＢＬ／６（Ｈ－２ｂ）小鼠的免疫耐受。致死照射的耐受Ｂａｌｂ／ｃ小鼠用Ｃ５７ＢＬ／６（Ｂ６）小鼠的胎肝细胞移植后，无移植排斥产生。嵌合状态分析的结果表明，在胎肝移植后９０ｄ和２４０ｄ，重建的Ｂａｌｂ／ｃ小鼠的脾细胞分别有７４．４％和８３．７％来自于供体Ｂ６小鼠．证明Ｂ６小鼠胎肝造血干细胞已经在致死照射的Ｂａｌｂ／ｃ小鼠体内稳定植入。免疫功能检测的结果表明，在胎肝移植后９０ｄ，照射Ｂａｌｂ／ｃ小鼠的免疫功能已经重建。     

Liver Anti-Fibrosis Therapy with Mesenchymal Stem Cells Secreting Hepatocyte Growth Factor

Abstract

The objective of this study is to investigate the anti-fibrotic effect of combined mesencymal stem cells (MSCs) and gene therapy on liver fibrosis. When transfected by the complex with a plasmid DNA of hepatocyte growth factor (HGF) and the spermine-introduced pullulan of gene carrier, MSCs secreted HGF protein over 1 week. The HGF secreted from transfected MSC had the biological activity to promote the albumin production of hepatocytes. After intravenous injection, the HGF-secreting MSCs (HGF-MSC) accumulated in the liver. The injection of HGF-MSC decreased the fibrosis area in a rat model of liver fibrosis to a significantly great extent compared with that of original MSC. In the in vitro experiment, the higher number of HGF-transfected MSCs was migrated by stromal cell-derived factor (SDF)-1α more strongly than the original MSC. Considering the promotion of SDF-1α secretion in the liver fibrosis, it is possible that, when transplanted, genetically-engineered MSCs are accumulated in the liver due to their higher response to SDF-1α. It is concluded that the intravenous injection of genetically-engineered MSCs is a promising therapy for liver fibrosis.     

极小胚胎样干细胞的生物学特性

近年来,研究者从小鼠骨髓和其他组织脏器中分离并纯化了一类数量极其稀少的极小胚胎样干细胞(very small embryonic-like stem cells,VSELs).VSELs不仅表达多能干细胞的表面分子标记,并能向3个胚层方向分化.有学者推测,VSELs可能是在哺乳动物组织/器官的发育早期迁移并定居下来的,且能在特定情况下向组织特异的单潜能干细胞方向分化.据此,VSELs可能在成体组织的更新和损伤组织的再生修复过程中发挥重要作用.     

龟板有效成分抗紫外线损伤所致的胎鼠表皮干细胞的凋亡

目的确定紫外线损伤所致的胎鼠表皮干细胞凋亡模型,筛选抗凋亡的龟板有效成分,为进一步应用开发打下基础。方法分离培养胎鼠表皮干细胞,用流式细胞仪检测CK19和Integrin β1双标阳性细胞鉴定纯度。用紫外线直接照射细胞造成损伤模型,用龟板各成分对照培养,用流式细胞仪检测FITC-Annexin V和碘化丙锭(PI)双标,根据细胞凋亡率确定紫外线损伤模型和筛选具有抗凋亡作用的龟板有效成分。结果①第3代胎鼠表皮干细胞已达到96.57%以上纯度;②紫外线照射量为100mJ/cm2损伤15min再培养20h可获得稳定的损伤模型,总凋亡率为41.06%;③龟板有效成分2B、S6、S8、S9均有抗凋亡作用。结论龟板有效成分S8具有较好的抗紫外线损伤所致的胎鼠表皮干细胞凋亡作用,应进一步探讨其机制。     

Effect of Intrauterine Exposure of Murine Fetus to Cyclophosphamide on Development of Thymus

The objective of this study was to demonstrate thymic alterations produced by cyclophosphamide intervention during intrauterine life of murine fetus. Cyclophosphamide (CP) was administered to pregnant mice on day 11 of gestation in a single dose of 10 mg/kg body weight. Fetuses were dissected out on day 19 and studied for various effects on thymus. Thymus of fetuses exposed to cyclophosphamide showed thymic atrophy with retardation of thymic size and a remarkable shrinkage in lobular morphology. Histological studies showed a massive depletion of thymic cortex. Study of thymocytes revealed an increase in apoptotic cell count and percent DNA fragmentation along with a decrease in proliferation. Thymocytes obtained from fetuses of CP-treated mice showed a higher expression of caspase-activated DNase (CAD) indicating that the CP-dependent induction of apoptosis in thymocytes involved caspase pathway. The results of the present study may help in understanding the mechanism of the teratogenic effect of cyclophosphamide on thymus.     

胸腺在大鼠肝脏自由基代谢中的作用

本文以成年去胸腺大鼠为模型,旨在观察胸腺对肝脏自由基代谢的影响。实验结果表明,雌性去胸腺大鼠和雌、雄性老年大鼠肝匀浆及肝微粒体过氧化脂质增多,肝匀浆超氧化物歧化酶活力和还原型谷胱甘肽含量降低,但雄性去胸腺大鼠上述指标与对照组相比无明显变化。     

Effect of Transplantation of Fetal Liver Tissue on Regenerative Activity of Hepatocytes in Normal Rats

Regenerative activity of hepatocytes was studied in rats after transplantation of fetal liver tissue preexposed to low-intensity laser. Stimulation of the mitotic activity of hepatocytes by fetal liver tissue exposed to low-intensity laser is proven.     

干细胞在心肌梗死治疗中的应用

近年来，干细胞的研究进展，给心肌梗死的治疗带来了新的希望，干细胞移植也成为了当前研究的热点问题。骨髓干细胞、胚胎干细胞、骨骼肌成肌细胞等已被应用于心肌的再生。研究表明，干细胞移植能有效地改善梗死心肌的功能，但其作用机制尚不完全明了。本研究对干细胞移植治疗心肌梗死研究中取得的成就、有待解决的问题以及临床应用前景作了一个总的评述。