从人胚胎胰腺中分离获得巢蛋白表达阳性的细胞

目的 探讨从人胚胎胰腺组织分离巢蛋白（Nestin)阳性细胞并进行体外传代培养的方法。方法 取引产的16、18、20周人胚胎胰腺组织，分离胰岛样细胞簇（Ialet-like cell clusters,ICCs），进行体外培养。用免疫组织化学和逆转录-聚合酶链反应（RT-PCR）方法检测巢蛋白、胰十二指肠同源盒基因-1（Pancreatic and duodenal bomeobox gene-1,PDX-1/IPF-1)以及胰岛内分泌激素胰岛素和胰升糖素（Glucagon)的表达。结果 ICCs经体外培养可获得巢蛋白表达阳性细胞，这种细胞可进一步形成球状细胞簇，除有巢蛋白阳性细胞外，还有PDX-1，胰岛素和胰升糖素表达阳性的细胞。并且细胞可连续传代，结论 从人胚胎胰腺组织中可分离获得巢蛋白表达阳性细胞，这种细胞可在体外增殖并有向胰岛内分泌细胞分化的能力。     

Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture

We have reproduced a previously described method for the in vitro generation of endocrine cells in adult human pancreatic tissue culture. The aim of this study was to characterize the nature of pancreatic progenitor cells and to identify the factors necessary for their differentiation in this model. During monolayer expansion, two types of cells proliferated sequentially; first cytokeratin 19 (CK19)-positive ductal epithelial cells and then nestin-positive fibroblastoid cells. After the bromodeoxyuridine-labeled cells were traced in differentiated islet buds, some of the proliferating ductal cells had differentiated into endocrine cells, whereas nestin-positive cells could not give rise to endocrine tissue. Serum-free culture was found to be an absolute requirement for the endocrine differentiation to occur. Also, overlay of the cells with Matrigel was essential, whereas nicotinamide had a potentiating effect. The in vitro-generated islet buds released insulin in response to glucose nearly as efficiently as native islets. When transplanted under the kidney capsule of nude mice, only one of five grafts demonstrated further growth with foci of both endocrine and exocrine differentiation. Our results support the previous notion that pancreatic progenitor cells represent a subpopulation of ductal epithelial cells. No evidence was found for the development of endocrine cells from nestin-positive stem cells.     

Differential expression of the intermediate filament protein nestin during renal development and its localization in adult podocytes

Nestin, an intermediate filament protein, is widely used as stem cell marker. Nestin has been shown to interact with other cytoskeleton proteins, suggesting a role in regulating cellular cytoskeletal structure. These studies examined renal nestin localization and developmental expression in mice. In developing kidney, anti-nestin antibody revealed strong immunoreactivity in vascular cleft of the S-shaped body and vascular tuft of capillary loop-stage glomerulus. The nestin-positive structures also were labeled by endothelial cell markers FLK1 and CD31 in immature glomeruli. Nestin was not detected in epithelial cells of immature glomeruli. In contrast, in mature glomerular, nestin immunoreactivity was observed only outside laminin-positive glomerular basement membrane, and co-localized with nephrin, consistent with podocyte nestin expression. In adult kidney, podocytes were the only cells that exhibited persistent nestin expression. Nestin was not detected in ureteric bud and its derivatives throughout renal development. Cell lineage studies, using a nestin promoter-driven Cre mouse and a ROSA26 reporter mouse, showed a strong beta-galactosidase activity in intermediate mesoderm in an embryonic day 10 embryo and all of the structures except those that were derived from ureteric bud in embryonic kidney through adult kidney. These studies show that nestin is expressed in progenitors of glomerular endothelial cells and renal progenitors that are derived from metanephric mesenchyme. In the adult kidney, nestin expression is restricted to differentiated podocytes, suggesting that nestin could play an important role in maintaining the structural integrity of the podocytes.     

Pancreatic islet production of vascular endothelial growth factor--a is essential for islet vascularization, revascularization, and function

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and beta-cell mass, mice with beta-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of beta-cell secretagogues to islets, we showed that reduced insulin output is not a result of beta-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.     

源于体外培养胰岛中表达Ngn3的细胞是胰腺内分泌前体细胞的新证据

目的 评估表达Ngn3的细胞对成体胰岛维护和更新的作用及其意义。方法 用6—8周龄的C57BL／6小鼠分离胰岛。胆总管内插管成功后,切除胰腺组织并用浓度为2．5mg／ml胶原蛋白酶V灌注消化。手工拣取胰岛后,置于60mm培养皿内,每个培养皿内含10—12个胰岛,用RPMI-1640(12．5mmol／L HEPES,5．2mmol／L葡萄糖和2％胎牛血清)培养液培养。用A6抗体,胰岛素抗体,胰高血糖素抗体,Nestin抗体,Ngn3抗体和5′—溴—2—脱氧尿嘧啶(5-bromo-2′-deoxy-uridine,Brdu)抗体进行胰岛细胞免疫荧光染色。结果 在增殖的胰岛细胞中,只有不到15％的细胞表达Ngn3,其中约有30％以上的细胞同时表达A6。在培养的胰岛细胞中存在表达Ngn3的细胞,这一结果与其他在胚胎发育和成体胰岛中的研究结果一致。本研究发现培养的胰岛细胞中存在同时表达A6和Ngn3的细胞。A6被认为是可分化为胆管上皮细胞的肝脏前体细胞的标记物。胰岛内表达A6的细胞可能来源于成体胰管,并迁移至胰岛内。细胞表达A6的同时表达Ngn3,表明这些细胞在胰岛内可分化为内分泌细胞。结论 培养的胰岛细胞中发现共同表达Ngn3和A6的细胞,表明胰岛内的4种内分泌细胞可能来源于成体胰岛中的同一内分泌细胞系。A6和Ngn3是对胰岛内成体干细胞研究很有帮助的标记物。这可以让我们进一步了解IPC的增殖和分化,并且有可能通过胰岛内成体干细胞来治疗糖尿病。     

Munc13-1 deficiency reduces insulin secretion and causes abnormal glucose tolerance

Munc13-1 is a diacylglycerol (DAG) receptor that is essential for synaptic vesicle priming. We recently showed that Munc13-1 is expressed in rodent and human islet beta-cells and that its levels are reduced in islets of type 2 diabetic humans and rat models, suggesting that Munc13-1 deficiency contributes to the abnormal insulin secretion in diabetes. To unequivocally demonstrate the role of Munc13-1 in insulin secretion, we studied heterozygous Munc13-1 knockout mice (+/-), which exhibited elevated glucose levels during intraperitoneal glucose tolerance tests with corresponding lower serum insulin levels. Munc13-1(+/-) mice exhibited normal insulin tolerance, indicating that a primary islet beta-cell secretory defect is the major cause of their hyperglycemia. Consistently, glucose-stimulated insulin secretion was reduced 50% in isolated Munc13-1(+/-) islets and was only partially rescued by phorbol ester potentiation. The corresponding alterations were minor in mice expressing one allele of a Munc13-1 mutant variant, which does not bind DAG (H567K/+). Capacitance measurements of Munc13-1(+/-) and Munc13-1(H567k/+) islet beta-cells revealed defects in granule priming, including the initial size and refilling of the releasable pools, which become accentuated by phorbol ester potentiation. We conclude that Munc13-1 plays an important role in glucose-stimulated insulin secretion and that Munc13-1 deficiency in the pancreatic islets as occurs in diabetes can reduce insulin secretion sufficient to cause abnormal glucose homeostasis.     

Generation of functional islet-like clusters after monolayer culture and intracapsular aggregation of adult human pancreatic islet tissue

BACKGROUND: Cellular replacement therapy represents a promising strategy for treating type I diabetes; however, such an approach is limited due to the inadequate availability of human donor tissue. Here we investigated the extent to which human islet tissue can be expanded in monolayer culture and brought back to islet function. METHODS: Adult human pancreatic cells were proliferated with a serum-free media in monolayer cultures through multiple passages. Expanded cells were dispersed and encapsulated in alginate-poly-l-lysine microcapsules wherein the cells spontaneously coalesced into islet-like clusters. Encapsulated cell clusters were subsequently transplanted into the peritoneal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: The cultured monolayer cells secreted insulin in response to glucose stimulation and maintained endocrine gene expression. Encapsulated islet-like clusters displayed cellular architecture similar to freshly isolated and encapsulated adult human islets maintained in culture, exhibiting an immunoreactive core of insulin, glucagon, and somatostatin, as well as peripheral cytokeratin-19 staining. Encapsulated aggregates significantly reduced hyperglycemia in transplanted mice within 1 week and normoglycemia was achieved after 5 weeks. Human C-peptide was detected in transplanted mice concomitant with the reduction in hyperglycemia. Capsules recovered 8 weeks posttransplantation exhibited insulin immunoreactivity. CONCLUSIONS: Collectively, these data indicate that adult human pancreatic islet cells can be expanded by three serial passages while maintaining their endocrine properties and can yield functional islet-like cell clusters through intracapsular aggregation that reverse hyperglycemia in diabetic mice. This culture and aggregation process could serve as a platform for proliferation and differentiation studies of endocrine lineage cells.     

Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas

Studies using adult human islets and mouse embryonic stem cells have suggested that the neurepithelial precursor cell marker nestin also identifies and can be used to purify beta-cell precursors. To determine whether nestin can be used to identify beta-cell progenitors in the developing human pancreas, we characterized nestin expression from 12 to 24 gestational weeks, purified nestin+ cells using an enhancer/promoter-driven selection plasmid, and determined whether nestin+ cells can differentiate into beta-cells. Nestin was visualized in the platelet endothelial cell adhesion molecule and alpha smooth muscle actin-positive blood vessels and colocalized with vimentin in the interstitium. Nestin was not observed in pan cytokeratin (pCK)-positive ductal epithelium or insulin cells. Purified nestin+ cells also coexpressed vimentin and lacked pCK immunoreactivity. Purified adult and fetal pancreatic fibroblasts also expressed nestin. The nestin enhancer/promoter used in the selection plasmid was sufficient to drive reporter gene expression, green fluorescent protein, in human fetal pancreatic tissue. Exposure of selected nestin+ cells to nicotinamide, hepatocyte growth factor/scatter factor, betacellulin, activin A, or exendin-4 failed to induce pancreatic and duodenal homeobox gene-1 or insulin message as determined by RT-PCR. Transplantation of nestin+ cells and fetal pancreatic fibroblasts into athymic mice also failed to result in the development of beta-cells, whereas nestin- fetal pancreatic epithelial cells gave rise to functional insulin-secreting beta-cells. We conclude that nestin is not a specific marker of beta-cell precursors in the developing human pancreas.     

In vivo and in vitro characterization of insulin-producing cells obtained from murine bone marrow

Efforts toward routine islet cell transplantation as a means for reversing type 1 diabetes have been hampered by islet availability as well as allograft rejection. In vitro transdifferentiation of mouse bone marrow (BM)-derived stem (mBMDS) cells into insulin-producing cells could provide an abundant source of autologous cells for this procedure. For this study, we isolated and characterized single cell-derived stem cell lines obtained from mouse BM. In vitro differentiation of these mBMDS cells resulted in populations meeting a number of criteria set forth to define functional insulin-producing cells. Specifically, the mBMDS cells expressed multiple genes related to pancreatic beta-cell development and function (insulin I and II, Glut2, glucose kinase, islet amyloid polypeptide, nestin, pancreatic duodenal homeobox-1 [PDX-1], and Pax6). Insulin and C-peptide production was identified by immunocytochemistry and confirmed by electron microscopy. In vitro studies involving glucose stimulation identified glucose-stimulated insulin release. Finally, these mBMDS cells transplanted into streptozotocin-induced diabetic mice imparted reversal of hyperglycemia and improved metabolic profiles in response to intraperitoneal glucose tolerance testing. These results indicate that mouse BM harbors cells capable of in vitro transdifferentiating into functional insulin-producing cells and support efforts to derive such cells in humans as a means to alleviate limitations surrounding islet cell transplantation.     

骨髓间充质干细胞移植可促进移植胰岛周围新生血管形成

目的  探讨胰岛移植联合骨髓间充质干细胞（MSC）移植能否促进移植胰岛周围新生血管形成。方法  以非肥胖糖尿病（NOD）小鼠作为受体,将NOD小鼠随机分为4组,联合移植组（6只）、单独胰岛移植组（6只）、单独MSC移植组（6只）、假性移植组（3只）。观察各组NOD小鼠移植后血糖和存活率的变化;采用5-乙炔基-2’脱氧尿苷（EdU）及dUTP缺口末端标记（TUNEL）方法,在胰岛移植后1、2、4周检测单独胰岛移植组和联合移植组移植胰岛的增殖与凋亡情况;采用光学显微镜（光镜）直接观察、组织化学及免疫组化的方法观察并定量分析,移植术后2、4、8周单独胰岛移植组和联合移植组移植胰岛的周围新生血管密度。结果  MSC联合移植与胰岛单独移植均能明显改善移植后小鼠的血糖水平,提高NOD小鼠的存活率。MSC联合移植可促进胰岛细胞再生,减少细胞凋亡。联合移植组移植胰岛周围血管密度明显大于单独胰岛移植组。结论  MSC可以促进移植胰岛周围新生血管生成,增加移植胰岛的血供,保护移植胰岛的功能与活性。     

Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice

Ischemia-reperfusion injury (I/R injury) is a common cause of acute renal failure. Recovery from I/R injury requires renal tubular regeneration. Hematopoietic stem cells (HSC) have been shown to be capable of differentiating into hepatocytes, cardiac myocytes, gastrointestinal epithelial cells, and vascular endothelial cells during tissue repair. The current study tested the hypothesis that murine HSC can contribute to the regeneration of renal tubular epithelial cells after I/R injury. HSC isolated from male Rosa26 mice that express beta-galactosidase constitutively were transplanted into female nontransgenic mice after unilateral renal I/R injury. Four weeks after HSC transplantation, beta-galactosidase-positive cells were detected in renal tubules of the recipients by X-Gal staining. PCR analysis of the male-specific Sry gene and Y chromosome fluorescence in situ hybridization confirmed the presence of male-derived cells in the kidneys of female recipients. Antibody co-staining showed that beta-galactosidase was primarily expressed in renal proximal tubules. This is the first report to show that HSC can differentiate into renal tubular cells after I/R injury. Because of their availability, HSC may be useful for cell replacement therapy of acute renal failure.     

High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells

Pdx1 has been shown to convert hepatocytes into both exocrine and endocrine pancreatic cells in mice, but it fails to selectively convert hepatocytes into pure insulin-producing cells (IPCs). The molecular mechanisms underlying the transdifferentiation remain unclear. In this study, we generated a stably transfected rat hepatic cell line named WB-1 that expresses an active form of Pdx1 along with a reporter gene, RIP-eGFP. Our results demonstrate that Pdx1 induces the expression of multiple genes related to endocrine pancreas development and islet function in these liver cells. We do not however find any expression of the late-stage genes (Pax4, Pax6, Isl-1, and MafA) related to beta-cell development, and the cells do not secrete insulin upon the glucose challenge. Yet when WB-1 cells are transplanted into diabetic NOD-scid mice, these genes become activated and hyperglycemia is completely reversed. Detailed comparison of gene expression profiles between pre- and posttransplanted WB-1 cells demonstrates that the WB-1 cells have similar properties as that seen in pancreatic beta-cells. In addition, in vitro culture in high-glucose medium is sufficient to induce complete maturation of WB-1 cells into functional IPCs. In summary, we find that Pdx1-VP16 is able to selectively convert hepatic cells into pancreatic endocrine precursor cells. However, complete transdifferentiation into functional IPCs requires additional external factors, including high glucose or hyperglycemia. Thus, transdifferentiation of hepatocytes into functional IPCs may serve as a viable therapeutic option for patients with type 1 diabetes.     

巢蛋白和神经元素3在人胚胎胰腺中的表达和分布

目的探讨胰腺干细胞标志巢蛋白（nestin）和神经元素3（Ngn3）在人胚胎胰腺组织中的表达及其与内分泌细胞、导管细胞和外分泌细胞标志的共表达情况。方法以因病理因素行药物引产的12周-14周前人胚胎胰腺14例为材料,应用免疫荧光染色和逆转录聚合酶链反应（RT—PCR）方法检测nestin和Ngn3的表达情况,免疫荧光染色检测胰腺内分泌标志胰岛素、胰高血糖素和外分泌标志CK-PAN和CK-19。结果nestin和Ngn3在12～14周人胚胎胰腺组织均广泛表达。nestin和Ng．3阳性细胞中均无胰岛素及胰高血糖素表达;在胰岛中未见到nestin与Ngn3共表达,但在导管中可见共表达。RT—PCR结果显示12～14周人胚胎胰腺中均有nestin和Ngn3的表达。结论Ngn3和nestin在人胚胎胰腺未分化细胞中表达,而具有分泌功能的内分泌细胞无表达。     

Inhibition of p38 MAP kinase in the early posttransplantation phase redistributes blood vessels from the surrounding stroma into the transplanted endocrine tissue

Transplanted pancreatic islets attain a chronically decreased vascular density following transplantation, despite the increased concentrations of vascular endothelial growth factor (VEGF) secreted from beta-cells in response to hypoxia during culture and in the immediate posttransplantation phase. VEGF, however, exerts dual effects on endothelial cells, and in islet endothelial cells of the adult, the vascular permeability-inducing effects of VEGF seem normally more pronounced than those to induce angiogenesis. p38 MAP kinase activity has recently been shown to serve as a switch to separate these properties of VEGF; inhibition of p38 MAP kinase activity enhances VEGF-induced angiogenesis and, at the same time, abrogates VEGF-induced vascular permeability. We hypothesized that the revascularization of transplanted islets may be hampered by a predisposition of adult islet endothelial cells to react to VEGF by forming fenestrae rather than migrating and proliferating. We therefore administered the p38 MAP kinase inhibitor SB203580 by daily IP injections for the first 14 days following transplantation, and then studied the influence of this treatment on the oxygen tension, blood perfusion, and vascular density of the islet grafts 1 month posttransplantation. SB203580 treatment redistributed islet graft blood vessels from the stroma into the endocrine tissue, and this redistribution of blood vessels into the endocrine tissue was accompanied by an increased oxygenation of the islet cells. However, the total number of blood vessels in the tissue was not affected. The blood perfusion of the islet grafts was also similar in control and SB203580-treated animals. Our results suggest that effects of VEGF to preferentially induce vascular permeability may partially contribute to, but is not the main cause of, low revascularization of transplanted islets.