Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

细胞角蛋白19启动子调控的双报告载体的构建及其在肝干/祖细胞分化研究中的应用

肝干/祖细胞是肝细胞和胆管上皮细胞(biliary epithelial cells,BECs)共同的前体细胞,为了对这一前体细胞的分化情况进行研究,利用报告基因来监测肝干/祖细胞的分化走向．首先,通过PCR方法从肝癌细胞系HepG2的全基因组中克隆了细胞角蛋白19(CK19)启动子片段,构建了CK19启动子调控的海肾荧光素酶和红色荧光蛋白(red fluorescent protein,RFP)双报告载体(pSicoR-CK19-hrl-mrfp)．其次,将上述慢病毒载体转染肝干/祖细胞后,通过流式细胞分选获得稳定转染的细胞株．再次,将上述细胞株与表达“上皮形态发生素”(Epimorphin,EPM)的PT67细胞共培养后,整合有pSicoR-CK19-hrl-mrfp表达载体的肝干/祖细胞不仅形态发生了变化,而且排列为二维环状结构,另外还检测到由CK19启动子启动表达的海肾荧光素酶和RFP,细胞形态和基因表型都证明肝干/祖细胞经诱导已经分化成为BECs．与此形成对照的是肝干/祖细胞与不表达EPM的PT67细胞共培养后,没有观测到上述的变化．所以,CK19启动子调控的双报告载体不仅可以实时地显示肝原始细胞在不同的诱导环境下的分化走向,而且还可以定量地检测CK19启动子活性的变化情况．总之,这一载体的成功构建将为研究肝干/祖细胞的分化提供了便捷的工具,同时也有助于筛选可诱导肝干/祖细胞定向分化的分子．     

Isolation of EpCAM+/CD133− hepatic progenitor cells

Progenitor cell-derived hepatocytes are critical for hepatocyte replenishment. Therefore, we established a line of human hepatic progenitor (HNK1) cells and determined their biological characteristics for experimental and therapeutic applications. HNK1 cells, isolated from human noncirrhotic liver samples with septal fibrosis, showed high expression of the hepatic progenitor cell (HPC) markers EpCAM, CK7, CK19, alpha-fetoprotein (AFP), CD90 (Thy1), and EFNA1. Expression of CD133 was very low. Ductular reactions at the periphery of cirrhotic nodules were immunohistochemically positive for these HPC markers, including EFNA1. Sodium butyrate, a differentiation inducer, induced hepatocyte-like morphological changes in HNK1 cells. It resulted in down-regulation of the hepatic progenitor cell markers EpCAM, CK7, CK19, AFP, and EFNA1 and up-regulation of mature hepatocyte markers, including albumin, CK8, and CK18. Furthermore, sodium butyrate treatment and a serial passage of HNK1 cells resulted in enhanced albumin secretion, ureagenesis, and CYP enzyme activity, all of which are indicators of differentiation in hepatocytes. However, HNK1 cells at passage 50 did not exhibit anchorage-independent growth capability and caused no tumors in immunodeficient mice, suggesting that they had no spontaneous malignant transformation ability. From this evidence, HNK1 cells were found to be EpCAM⁺/CD133^- hepatic progenitor cells without spontaneous malignant transformation ability. We therefore conclude that HNK1 cells could be useful for experimental and therapeutic applications.     

Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation

Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein. CONCLUSION: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.     

Characterization of cells in the developing human liver

Human hepatic progenitor cells (HPCs) have been shown to co-express the hematopoietic stem cell (HSC) markers, CD117 and CD34. These cells differentiate not only into hepatocytes and cholangiocytes but also into pancreatic ductal and acinar cells under certain conditions. The fetal liver (FL) is rich in precursor/stem cells; however, little is known about (i) the markers expressed by liver cells during fetal development and (ii) whether an equivalent to the adult liver stem-like progenitors exists in the FL. Here, (i) FL tissue obtained from human 5-18-week-old fetuses were evaluated by means of flow cytometry, immunocyto-, and histochemistry for the emergence of cells expressing and co-expressing known hematopoietic, hepatic, and pancreatic cell markers, and (ii) isolated putative HPCs were phenotypically and molecularly characterized. We report that (i) red blood and endothelial cell precursors were most abundant in early gestation. Cells expressing HSC and pancreatic markers were found in the first trimester, while cells expressing hepatic markers appeared in the second trimester. Very few committed cells were present in FLs obtained early in the first trimester. In addition, cells expressing pancreatic markers co-expressed the HSC marker CD117. (ii) Isolated CD117+/CD34+/CD90- cells in vitro expressed both the genes and proteins for the hepatic markers such as albumin, alpha feto protein (AFP), alpha1-antitrypsin, and cytokeratin 19 (CK19). Our study suggests that hepatoblast and ductal plate/bile duct development mainly occurs during the second trimester. FLs in gestation weeks 5-9 had the highest numbers of precursor cells and the least committed cells. Cells that differentiate into Alb+ or CK19+ can be isolated from early FLs and may be appropriate progenitors for establishing novel systems to investigate basic mechanisms for cell therapy.     

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low‐affinity receptor p75NTR, glial cell‐derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRα‐1), integrin β1, α‐enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E‐cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression. J. Cell. Physiol. 225: 180–185, 2010. © 2010 Wiley‐Liss, Inc.     

Derivation and Characterization of Hepatic Progenitor Cells from Human Embryonic Stem Cells

The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell–derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.     

Evidence for epithelial-mesenchymal transitions in adult liver cells

Both myofibroblastic hepatic stellate cells (HSC) and hepatic epithelial progenitors accumulate in damaged livers. In some injured organs, the ability to distinguish between fibroblastic and epithelial cells is sometimes difficult because cells undergo epithelial-mesenchymal transitions (EMT). During EMT, cells coexpress epithelial and mesenchymal cell markers. To determine whether EMT occurs in adult liver cells, we analyzed the expression profile of primary HSC, two HSC lines, and hepatic epithelial progenitors. As expected, all HSC expressed HSC markers. Surprisingly, these markers were also expressed by epithelial progenitors. In addition, one HSC line expressed typical epithelial progenitor mRNAs, and these epithelial markers were inducible in the second HSC line. In normal and damaged livers, small ductular-type cells stained positive for an HSC marker. In conclusion, HSC and hepatic epithelial progenitors both coexpress epithelial and mesenchymal markers, providing evidence that EMT occurs in adult liver cells.     

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl(4). Livers were removed 9 to 13 days post-CCl(4) treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b(+) cells fail to propagate while c-kit(+)-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit(+)-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion.     

胎肝中肝干细胞的免疫组织化学研究

目的采用免疫组织化学方法显示不同时期人胚胎肝脏的干细胞,分析肝干细胞的形态与分布特点及发育过程中干细胞在肝脏中的迁徙,探讨肝脏的发生发育及肝内干细胞的来源。方法不同发育时期胎儿肝脏,取材、固定、制成石蜡切片,ABC法检测肝干细胞特异性的表面标记物CD34、CK19、C-11和OV6。结果胎肝内汇管区周边界板处有卵圆样细胞表达CD34、C-11、CK19和OV6,阳性细胞紧密排列成管,呈鞘样包绕着早期汇管区,部分包绕着初级汇管区,随着次级汇管区的成熟,卵圆样干细胞逐渐局限于赫令氏管周围;此外,胚胎发育的不同阶段均可见CD34、OV6阳性的单核样细胞分散在肝索、肝血窦之内,多见于汇管区的问充质组织之内,肝血管内鲜见。结论胚胎发育早期汇管区周边界板处含有丰富的干细胞,可能是肝脏发育的起点,这些干细胞逐渐分化为胆管上皮样细胞,然后分化为肝细胞和胆管上皮细胞;造血干细胞是肝内的另一干细胞来源,造血干细胞在肝内受到诱导作用分化为小部分的肝实质细胞。     

Putative liver progenitor cells: conditions for long-term survival in culture

Oval cells proliferate extensively in the livers of animals exposed to oncogenic insults, are bipotent and are believed to be related to the so far unidentified liver stem cell. In normal liver, cells antigenica lly related to oval cells and expressing liver and epithelial markers are considered to be liver progenitor cells. We isolated, by fluorescence-activated cell sorting or magnetic bead sorting, cells expressing the oval cell antigens OC.2 or OC.3 from the liver of normal newborn or day 12 embryonal age rats. Magnetic bead sorting of positive cells was as efficient as fluorescence-activated cell sorting. A two-chamber culture system was devised in which cells were plated onto transwell filters coated with type IV collagen and cultured in a serum-free Ham's F12 medium supplemented with free fatty acids and bovine serum albumin. Under these conditions, cells remained viable for up to 6 weeks and their antigenic phenotype was unchanged throughout. Approximately 30% of sorted cells expressed epithelial and/or liver-specific markers. Growth factors mitogenic for epithelial cells and hepatocytes did not elicit cell proliferation. These results provide an important background for further studies designed to determine the biological significance of OC.2⁺ and OC.3⁺ cells in normal liver, to test the liver stem cell hypothesis and to develop protocols for the expansion in vitro of normal liver progenitors. This revised version was published online in November 2006 with corrections to the Cover Date.     

胎肝干细胞的分离、培养与鉴定

目的体外扩增培养大鼠胎肝干细胞,研究其形态、生物学特性及表面标志物,探讨胎肝干细胞的性质。方法分离培养胎龄12-16d的胎肝细胞,SABC法检测原代、传代后及细胞克隆中的肝干细胞特异表面标志物OV-6、CK-19及nestin的表达。结果原代、传代培养的胎肝细胞部分表达OV-6、CK-19及nestin;培养3d开始出现小细胞团,1个月即形成肉眼可见的细胞集落,5-7d传代一次;细胞克隆几乎全部为干细胞标志阳性细胞。结论胎肝干细胞可通过克隆筛选法进行体外扩增,胎肝内存在nestin阳性干细胞,可能是一种更为原始的干细胞,在胚胎发育中起重要作用。     

Evidence for a common progenitor of epithelial and mesenchymal components of the liver

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a ‘bona fide'' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.     

Portal venous endothelium in developing human liver contains haematopoietic and epithelial progenitor cells

Future treatments for chronic liver disease are likely to involve manipulation of liver progenitor cells (LPCs). In the human, data characterising the regenerative response is limited and the origin of adult LPCs is unknown. However, these remain critical factors in the design of cell-based liver therapies. The developing human liver provides an ideal model to study cell lineage derivation from progenitors and to understand how foetal haematopoiesis and liver development might explain the nature of the adult LPC population. In 1st trimester human liver, portal venous endothelium (PVE) expressed adult LPC markers and markers of haematopoietic progenitor cells (HPCs) shared with haemogenic endothelium found in the embryonic dorsal aorta. Sorted PVE cells were able to generate hepatoblast-like cells co-expressing CK18 and CK19 in addition to Dlk/pref-1, E-cadherin, albumin and fibrinogen in vitro. Furthermore, PVE cells could initiate haematopoiesis. These data suggest that PVE shares phenotypical and functional similarities both with adult LPCs and embryonic haemogenic endothelium. This indicates that a temporal relationship might exist between progenitor cells in foetal liver development and adult liver regeneration, which may involve progeny of PVE.