Insulin-Producing Cells Derived from Human Embryonic Stem Cells: Comparison of Definitive Endoderm- and Nestin-Positive Progenitor-Based Differentiation Strategies

Human embryonic stem cells (hESCs) are pluripotent and capable of undergoing multilineage differentiation into highly specialized cells including pancreatic islet cells. Thus, they represent a novel alternative source for targeted therapies and regenerative medicine for diabetes. Significant progress has been made in differentiating hESCs toward pancreatic lineages. One approach is based on the similarities of pancreatic β cell and neuroepithelial development. Nestin-positive cells are selected as pancreatic β cell precursors and further differentiated to secrete insulin. The other approach is based on our knowledge of developmental biology in which the differentiation protocol sequentially reproduces the individual steps that are known in normal β cell ontogenesis during fetal pancreatic development. In the present study, the hESC cell line PKU1.1 was induced to differentiate into insulin-producing cells (IPCs) using both protocols. The differentiation process was dynamically investigated and the similarities and differences between both strategies were explored. Our results show that IPCs can be successfully induced with both differentiation strategies. The resulting IPCs from both protocols shared many similar features with pancreatic islet cells, but not mature, functional β cells. However, these differently-derived IPC cell types displayed specific morphologies and different expression levels of pancreatic islet development-related markers. These data not only broaden our outlook on hESC differentiation into IPCs, but also extend the full potential of these processes for regenerative medicine in diabetes.     

小鼠胚胎干细胞对黑色素瘤细胞体外生物学行为的影响

探讨体外共培养环境中小鼠胚胎干细胞对小鼠黑色素瘤B16细胞的影响。建立C57BL/6小鼠胚胎干细胞系,通过小鼠胚胎干细胞与肿瘤细胞体外共培养模型观察小鼠胚胎干细胞对肿瘤细胞的形态及生长行为的影响,MTT法与transwell小室法分别检测共培养后肿瘤细胞粘附性、迁移性及侵袭性的变化。共培养中小鼠胚胎干细胞能够侵入并推开小鼠黑色素瘤细胞形成自己的生长空间,与对照组比较共培养后肿瘤细胞的粘附性、迁移性及侵袭性均显著降低(P<0.05,P<0.01)。结果表明体外共培养体系中小鼠胚胎干细胞能够侵袭肿瘤细胞,并降低细胞粘附、迁移及侵袭相关恶性生物学行为。     

Pancreatic endocrine and exocrine cell ontogeny from renal capsule transplanted embryonic stem cells in streptozocin-injured mice.

In this study, we describe pancreatic cell ontogeny in renal capsule-transplanted embryonic stem cells (ES) after injury by streptozocin (STZ), showing pancreatogenesis in situ. Seven-week-old female BALB/c nude mice were treated with either a single 175- or 200-mg/kg STZ dose, a regimen that induces substantial beta-cell damage without overt hyperglycemia, and transplanted 24 hr later with 1 x 10(5) ES. Immunohistochemistry was performed on ES tissue at 15, 21, and 28 days after transplantation using antibodies against stage- and lineage-specific pancreatic markers. After 21 days, PDX-1+ pancreatic foci first appeared in the renal capsule and expressed both amylase and endocrine hormones (insulin, glucagon, and somatostatin). These foci increased in size by day 28 because of acinar and duct cell proliferation, whereas endocrine cells remained non-dividing, and made up 2-4% of ES tumor volume. PDX-1, Nkx6.1, Ngn3, and ISL-1 protein localization patterns in pancreatic foci were comparable with embryonic pancreatogenesis. A prevalence of multihormonal endocrine cells, a characteristic of adult beta-cell regeneration, indicated a possible divergence from embryonic islet cell development. The results indicate that beta-cell damage, without overt hyperglycemia, induces a process of fetal-like pancreatogenesis in renal capsule-transplanted ES, leading to beta-cell neogenesis.     

Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media

The unique pluripotential characteristic of human embryonic stem cells heralds their use in fields such as medicine, biotechnology, biopharmaceuticals, and developmental biology. However, the current availability of sufficient quantities of embryonic stem cells for such applications is limited, and generating sufficient numbers for downstream therapeutic applications is a key concern. In the absence of feeder layers or their conditioned media, human embryonic stem cells readily differentiate to form embryoid bodies, indicating that trophic factors secreted by the feeder layers are required for long-term proliferation and maintenance of pluripotency. Adding further complexity to the elucidation of the factors required for the maintenance of pluripotency is the variability of different fibroblast feeder layers (of mouse or human origin) to effectively support human embryonic stem cells. Currently, the deficiency of knowledge concerning the exact identity of factors within the pathways for self-renewal illustrates that a number of factors may be required to support pluripotent, undifferentiated growth of human embryonic stem cells. This study utilized a proteomic analysis (multidimensional chromatography coupled to tandem mass spectrometry) to isolate and identify proteins in the conditioned media of three mitotically inactivated fibroblast lines (human fetal, human neonatal, and mouse embryonic fibroblasts) used to support the undifferentiated growth of human embryonic stem cells. One-hundred seventy-five unique proteins were identified between the three cell lines using a 相似文献   

Galectin-4 Reduces Migration and Metastasis Formation of Pancreatic Cancer Cells

Galectin-4 (Gal-4) is a member of the galectin family of glycan binding proteins that shows a significantly higher expression in cystic tumors of the human pancreas and in pancreatic adenocarcinomas compared to normal pancreas. However, the putative function of Gal-4 in tumor progression of pancreatic cancer is still incompletely understood. In this study the role of Gal-4 in cancer progression was investigated, using a set of defined pancreatic cancer cell lines, Pa-Tu-8988S (PaTu-S) and Pa-Tu-8988T (PaTu-T), as a model. These two cell lines are derived from the same liver metastasis of a human primary pancreatic adenocarcinoma, but differ in their growth characteristics and metastatic capacity. We demonstrated that Gal-4 expression is high in PaTu-S, which shows poor migratory properties, whereas much lower Gal-4 levels are observed in the highly metastatic cell line PaTu-T. In PaTu-S, Gal-4 is found in the cytoplasm, but it is also secreted and accumulates at the membrane at sites of contact with neighboring cells. Moreover, we show that Gal-4 inhibits metastasis formation by delaying migration of pancreatic cancer cells in vitro using a scratch assay, and in vivo using zebrafish (Danio rerio) as an experimental model. Our data suggest that Gal-4 may act at the cell-surface of PaTu-S as an adhesion molecule to prevent release of the tumor cells, but has in addition a cytosolic function by inhibiting migration via a yet unknown mechanism.     

High-throughput RNAi screening identifies a role for TNK1 in growth and survival of pancreatic cancer cells

To identify novel targets in pancreatic cancer cells, we used high-throughput RNAi (HT-RNAi) to select genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately 32 kinases were designated as hits, of which 22 kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase nonreceptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we showed that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NF-κB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells but seems to be involved in growth and survival. The application of functional genomics by using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.     

兔胚胎神经干细胞的分离、培养和鉴别

目的：研究兔胎脑神经干细胞体外生长特性,为探讨神经干细胞的临床应用及神经系统的发育奠定基础。方法：采用含碱性成纤维细胞生长因子（bFGF）和表皮细胞生长因子（EGF）的N2无血清培养技术,取18天龄兔胚胎脑组织,分离神经干细胞,并观察分离的细胞体外培养、增殖、分化潜能,免疫组化鉴定。结果：从18天龄兔胎脑皮质和纹状体中成功分离出具有自我更新和多分化潜能的神经干细胞,在无血清培养时细胞呈半贴壁状态生长,形成神经球,可传代。细胞呈Nestin免疫反应阳性;在含血清培养基中培养时则分化,分化后的细胞表达神经元细胞、星形胶质细胞和少突胶质细胞的特异性抗原。结论：来自兔胎脑神经干细胞能在体外培养、增殖并保持传代能力。无血清N2EGF、bFGF培养基有利于兔胎脑神经干细胞的存活和增殖,含血清培养基能诱导兔胎脑神经干细胞分化。     

In vitro cultivation and differentiation of fetal liver stem cells from mice

During embryonic development, pluripotent endoderm tissue in the developing foregut may adopt pancreatic fate or hepatic fate depending on the activation of key developmental regulators. Transdifferentiation occurs between hepatocytes and pancreatic cells under specific conditions. Hepatocytes and pancreatic cells have the common endodermal progenitor cells. In this study we isolated hepatic stem/progenitor cells from embryonic day (ED) 12-14 Kun-Ming mice with fluorescence-activated cell sorting (FACS). The cells were cultured under specific conditions. The cultured cells deploy dithizone staining and immunocytochemical staining at the 15th, 30th and 40th day after isolation. The results indicated the presence of insulin-producing cells. When the insulin-producing cells were transplanted into alloxaninduced diabetic mice, the nonfasting blood glucose level was reduced. These results suggested that fetal liver stem/progenitor cells could be converted into insulin-producing cells under specific culture conditions. Fetal liver stem/progenitor cells could become the potential source of insulin-producing cells for successful cell transplantation therapy strategies of diabetes.     

人胰腺癌细胞再增殖体外模型的建立

目的:细胞再增殖是导致胰腺癌放化疗失败的主要原因之一,但缺乏合适的用于研究胰腺癌再增殖的细胞模型。本研究拟建立简便、实用的胰腺癌细胞再增殖体外模型。方法:表达绿色荧光蛋白-荧光素酶(GFP-Luc)的慢病毒感染人胰腺癌细胞,经嘌呤霉素筛选,用荧光显微镜和流式细胞仪观察双标记细胞GFP表达情况,用生物成像检测双标记细胞Luc活性及分析细胞数量与Luc活性之间的关系。以X-线照射胰腺癌细胞制备饲养细胞,以相应的双标记肿瘤细胞为报告细胞进行共培养。对共培养细胞进行荧光显微镜观察和生物成像,以判断饲养细胞对报告细胞的生长促进作用。结果:通过表达GFP-Luc的慢病毒感染获得双标记人胰腺癌细胞,经荧光显微术、流式细胞术和生物成像术证实这些双标记的人胰腺癌细胞能有效地表达GFP和Luc活性,可作为报告细胞用于建立人胰腺癌再增殖细胞模型。将经X-线照射的饲养细胞和相应的报告细胞共培养,经荧光显微镜观察和生物成像分析,结果显示X-线照射过的饲养细胞对报告细胞的生长具有显著的促进作用。结论:成功建立了简便、实用的人胰腺癌再增殖体外模型,该模型能很好地模拟人体内胰腺癌细胞再增殖过程,为进一步研究胰腺癌细胞再增殖的分子机制提供了新的技术手段。     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

Embryonic Stem Cells: Spontaneous and Directed Differentiation

The specific structural features of embryonic stem cells and embryoid bodies and mechanisms of their differentiation in different cell types are considered. The mouse embryonic stem cells (line R1) formed multilayer colonies which enlarged as a result of fast cell division. Embryoid bodies that derived from embryonic stem cells consisted of an outer layer, an inner layer, and an internal cavity. The structure of cells of the outer and inner layers markedly differed. Spontaneous and directed differentiation of embryoid bodies is determined by some unspecific and specific factors (growth and differentiation factors and extracellular matrix proteins). Retinoic acid, the most commonly used inducer of differentiation of the embryonic stem cells, induces different types of differentiation when applied at different concentrations. The sequence of expression of tissue specific genes and proteins during differentiation of the embryonic stem cells in vitrois similar to that in vivo.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.     

一种新的人胚胎干细胞无饲养层培养方法的建立

目的:以转染碱性成纤维细胞生长因子(bFGF)的人胎肝基质细胞株(FLSC)培养人胚胎干细胞(hESC),寻找更加安全、有效的体外培养扩增方法。方法:通过ELISA方法定量检测转基因的人FLSC条件培养基中bFGF的分泌量;以商业化的mTeSR1无血清无饲养层培养基、常规小鼠胚胎成纤维细胞(MEF)条件培养基,以及转染bFGF的人FLSC条件培养基(bFGF/FLSC-CM)分别培养扩增H9细胞。通过观察hESC形态、免疫荧光染色、流式细胞检测及RT-PCR,检测hESC全能性标志物的表达。结果:ELISA方法检测bFGF/FLSC-CM中bFGF因子的分泌量为(770.09±17.28)pg/mL,而MEF-CM中bFGF因子的分泌量为(55.59±0.61)pg/mL,两者存在显著差异(P0.01);在3种培养体系下,免疫荧光检测hESC全能性标志Oct-4、Tra-1-81抗体的表达均呈阳性,流式检测细胞表面阶段特异性胚胎抗原4(SSEA-4)抗体阳性细胞的比例均在99%左右;RT-PCR检测到hESC特异的转录因子Oct-4、Nanog、Sox-2的表达。结论:以转染bFGF的人FLSC条件培养基可以有效扩增hESC,可为临床应用提供一种安全、高效、低成本的无饲养层培养方法。