Identification of surface markers for prospective isolation of human endometrial stromal colony-forming cells

BACKGROUND: Human endometrium is a highly regenerative tissue. We hypothesizedthat the source of endometrial stromal and vascular regenerationis a resident stromal stem/progenitor cell population. Putativehuman endometrial stromal stem/progenitor cells have been identifiedusing clonal assays, a retrospective functional stem cell assay.Therefore, the aim of this study was to screen potential stemcell markers for the prospective isolation of human endometrialstromal stem/progenitor cells and to determine their capacityto identify colony-forming stromal cells. METHODS: Single-cell suspensions of human endometrial stromal cells weresorted using fluorescence-activated cell sorting into positiveand negative populations based on STRO-1, CD133, CD90 or CD146expression for clonal assays. All markers were immunolocalizedin human endometrium. RESULTS: Small populations (2–9%) of human endometrial stromalcells expressed each of the markers. Only CD146⁺ cells wereenriched for colony-forming cells, and CD90^hi cells showed atrend for greater enrichment compared with CD90^lo cells. STRO-1and CD146 were localized to perivascular cells of the endometrium.CD90 was strongly expressed by functionalis stroma and perivascularcells, but only weakly expressed in the basalis stroma. CD133was expressed by epithelial cells of the endometrium, ratherthan by stroma or perivascular cells. CONCLUSIONS: This study identified CD146 as a marker of colony-forming humanendometrial stromal cells supporting the concept that humanendometrium contains a population of candidate stromal stem/progenitorcells.     

Rapid and efficient in vitro generation of pancreatic islet progenitor cells from nonendocrine epithelial cells in the adult human pancreas

The absence of efficient and directed methods for the differentiation of adult pancreatic progenitor cell populations to pancreatic islet cells has raised doubts concerning the regeneration potential inherent in the adult pancreas. Relatively low levels of islet cell differentiation have been reported using adult pancreatic cells in vivo and in vitro. In the present study, we initially enriched for a nonendocrine epithelial component of the adult human pancreas and defined conditions that are permissive to islet cell differentiation in vitro. Sequential progression of cell differentiation in the permissive conditions allowed for incremental evaluation of changes occurring in the cell population. Optimization of the differentiation process, for the efficient production of islet endocrine cells, was accomplished by identifying specific factors and culture conditions that increased islet progenitor production 250-fold. Ultimately, 85% percent of the nonendocrine epithelial cells isolated from human pancreatic tissue and cultured in the optimized conditions for 8 days, readily re-expressed pancreatic duodenal homeobox-1 (Pdx1). Sixty-five percent of these Pdx1-expressing cells were capable of additional islet endocrine cell differentiation. This represents a significant advancement in the differentiation of an adult pancreatic progenitor cell population in vitro and suggests that the nonendocrine compartment of the human pancreas remains an important cell resource for the generation of transplantable islets to treat diabetes.     

Characterisation of Human Limbal Side Population Cells Isolated Using an Optimised Protocol From an Immortalised Epithelial Cell Line and Primary Limbal Cultures

The challenges in limbal stem cell biology largely remain in the process of identification, isolation and expansion of these adult corneal epithelial stem cells of the eye. Due to the absence of specific limbal stem cell markers, identification and isolation of putative limbal stem cells is a complicated task. The side population assay is an isolation method that utilises the ability of stem cells to efflux the DNA-binding dye Hoechst 33342 (or other vital dyes) combined with dual wavelength flow cytometry and is a valuable strategy to enrich for limbal stem cells. This assay has been used to successfully identify stem/ progenitor cell populations in a variety of tissues and cell lines. Here we optimise this assay to identify SP cell populations in both primary human limbal epithelial cultures and in an established human corneal epithelial cell line. The limbal SP fraction showed higher expression of ATP-binding cassette sub-family G member 2 (ABCG2), ΔNp63—a common limbal stem cell marker and the stem cell marker Sox2 compared to non-SP cells (NSP).     

Progenitor cell characterization and location in the developing human liver

Tissue-derived stem cells may offer future liver disease therapies. The developing human liver provides an excellent model to examine normal hepatic progenitor cell maturation, but candidate populations are poorly characterized. We sought to identify putative progenitor phenotypes in first-trimester human liver, by characterizing the architectural relationship between developing epithelial, mesenchymal, and hematopoietic lineages. Bipotential hepatoblasts were identified by co-expression of hepatocytic (cytokeratin 18, albumin) and biliary(cytokeratin 19) specific markers and epithelial-specific E-cadherin. Restriction of dlk/pref-1 expression to hepatoblasts identifies this as a novel human marker allowing for hepatoblast sorting for in vitro analysis. Furthermore, the liver stem cell and haematopoietic marker Thy-1 was co-expressed with markers of hematopoietic (CD34) and mesenchymal (vimentin) lineage restriction on portal vein endothelium. Therefore, this structure may constitute a novel progenitor compartment with hemangioblast-like properties.     

Limbal epithelial side-population cells have stem cell-like properties, including quiescent state

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell-like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell-like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.     

In vitro differentiation of liver progenitor cells derived from healthy dog livers

Naturally occurring liver disease in dogs resemble human liver disease in great detail; including the activation of liver progenitor cells (LPC) in acute and chronic liver disease. The aim of the present study was to isolate, culture, and characterize progenitor cells derived from healthy mature dog livers. A nonparenchymal cell fraction enriched with small hepatocytes was isolated and cultured in Hepatozyme-serum-free media (SFM) to stimulate the growth of colony-forming small epithelial cells. After 2 weeks of culturing, clonal expansion of keratin 7 (K7) immunopositive small cells with a large nucleus/cytoplasm ratio emerged in the hepatocyte monolayer. These colonies expressed genes of several hepatocyte (CYP1A1, ALB, and KRT18), cholangiocyte/LPC (KRT7 and KRT19), and progenitor cell markers (alpha-fetoprotein, CD44, prominin1, KIT, THY1, and neural cell adhesion molecule 1), indicating their immature and bipotential nature. Gene-expression profiles indicated a more pronounced hepatic differentiation in Hepatozyme-SFM compared to William's Medium E (WME). Furthermore, colony-forming cells differentiated toward intermediate hepatocyte-like cells with a more pronounced membranous K7 immunostaining. In conclusion, colony-forming small epithelial cells in long-term canine liver cell cultures express LPC markers and have differentiating capacities. These cells may therefore be considered as progenitor cells of the liver.     

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.     

肿瘤干细胞的分离培养及生物学特性

背景：肿瘤干细胞是一类具有自我更新能力、不定分化潜能的种子细胞,可以形成不同分化程度的肿瘤细胞, 是恶性肿瘤不断生长、复发转移、无法被传统的放化疗方法彻底杀死的根源。 目的：总结肿瘤干细胞的分离培养及其生物学特性。 方法：由作者采用电子检索的方式,在万方数据库中检索以“肿瘤干细胞,分离培养,生物学特性” 为中文关键词,计算机检索1990-01/2010-12有关肿瘤干细胞的分离培养及生物学特性的文章,经检索共查到相关文献45篇。经阅读标题、摘要、全文后,排除内容重复、普通综述、Meta分析类文章后,筛选纳入22篇文献进行评价。 结果与结论：在肺癌、胰腺癌、结肠癌、肝细胞癌、神经系统肿瘤等多种脏器肿瘤中,CD133被作为鉴定肿瘤干细胞的特异性标记物之一。巢蛋白、波形蛋白和CD117在神经系统肿瘤干细胞中有表达。CD44及内皮素转化酶在肺癌肿瘤干细胞中的表达具有意义。CD44和CD24可作为分选人胰腺癌细胞株PANC-1中肿瘤干细胞的表面标志。而CD166在大肠癌肿瘤干细胞中高表达。     

胰腺干细胞负向筛选分子的鉴定

背景：利用胰腺干/祖细胞体外增殖分化形成β细胞是细胞替代疗法治疗糖尿病的潜在细胞来源之一。由于缺少有效的筛选分子,胰腺干细胞的分离及特性研究进展缓慢。 目的：观察植物凝集素花生凝集素、菜豆凝集素和热稳定抗原对胰腺细胞的标记情况,以期找到胰腺干细胞的负向筛选分子。 方法：采用荧光组织化学方法对植物凝集素(花生凝集素、菜豆凝集素)和细胞表面分子热稳定抗原抗体在成体小鼠胰腺、胚胎胰腺及切除后再生胰腺组织中进行化学及荧光组织化学检测;流式细胞仪分析负向筛选分子对胰腺细胞分群情况的影响;应用细胞培养方法对不同群细胞进行体外培养和观察。 结果与结论：花生凝集素对胚胎胰腺中分化的腺泡细胞能较好标记,但不标记未分化的胰腺干/祖细胞及前体细胞;菜豆凝集素对未分化和分化的胚胎期胰腺细胞均有标记,热稳定抗原能标记正在分化的胚胎胰腺祖细胞、前体细胞和已分化胰腺细胞。在成体胰腺中,花生凝集素和菜豆凝集素均能标记胰腺腺泡和部分导管细胞,但不能标记胰岛细胞;热稳定抗原能特异标记腺泡细胞、导管细胞、血管内皮细胞、血细胞及再生胰腺新生区的导管样结构。PNA^-/lowHSA^-/low细胞占总细胞量的1.6%,对其培养观察发现,其中富含体积较小、三角形或多角形的上皮细胞,这些细胞可连续传代。提示协同使用花生凝集素与热稳定抗原可以筛除大部分分化的胰腺细胞及部分非胰腺细胞,从而富集胰腺干细胞。     

Prospective Isolation and Functional Analysis of Stem and Differentiated Cells from the Mouse Mammary Gland

Prospective isolation and in vitro and in vivo analysis of primary mouse mammary epithelial cells has been used to separate cell subpopulations and identify stem, progenitor and differentiated cell compartments. Progress has been made from cell separation strategies based on a single marker of the luminal epithelial or myoepithelial compartments to use of markers that allow simultaneous isolation of non-epithelial, basal/myoepithelial and luminal epithelial cells. Transplant analysis has shown that mammary stem cells are found in the basal/myoepithelial compartment, whereas in vitro colony progenitors are found in the luminal compartment. A basal population enriched for stem cell activity can be purified from the myoepithelial cells and the most recent data shows that the luminal population can now be prospectively split into estrogen receptor positive and estrogen receptor negative cells. Future work aims to molecularly characterise these populations to identify new drug targets, which can be used to specifically kill breast cancer stem cells.     

Targeting Pancreatic Progenitor Cells in Human Embryonic Stem Cell Differentiation for the Identification of Novel Cell Surface Markers

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.     

Identification of cells with colony-forming activity, self-renewal capacity, and multipotency in ovarian endometriosis

Endometriosis, the growth of endometrial tissue outside the uterine cavity, is a common gynecological disorder affecting 10% to 15% of women in their reproductive years. Retrograde menstrual shedding containing endometrial stem/progenitor cells has been postulated to be involved in its pathogenesis. In this study, we identified putative endometriotic stem/progenitor cells by their colony-forming potential, self-renewal capacity, and multipotency. Purified epithelial and stromal cells isolated from ovarian endometriotic cysts formed large and small colony-forming units (CFUs) in clonogenic assay. The colony-forming activity of epithelial and stromal cells was found to differ greatly between autologous endometrium and ovarian endometrioma samples. The large CFUs could propagate more than the small CFUs. The endometriotic epithelial small CFUs expressed epithelial markers (epithelial cell adhesion molecule, cytokeratin, and α6 integrin); only occasional large CFUs expressed α6 integrin. Aside from the expression of fibroblast markers, stromal CFUs also expressed three somatic stem cell markers: sal-like 4, CD133, and Musashi-1. Endometriotic stromal cells derived from large CFUs could differentiate into four mesenchymal lineages when cultured in the respective inducing-media, as determined by histochemical staining and RT-PCR of lineage specific markers. These findings demonstrate that ovarian endometrioma contains a subset of cells displaying somatic stem cell properties.     

The Skin: A Home to Multiple Classes of Epithelial Progenitor Cells

To maintain homeostasis in the adult skin, epithelial keratinocyte stem cells are thought to divide infrequently giving rise to short-lived (transit amplifying) cells that undergo a limited number of cell divisions and ultimately terminal differentiation. This model for the epidermal stem cell niche has increased in complexity by the multiple putative progenitor keratinocyte populations that have recently been identified in distinct regions of the interfollicular epidermis and hair follicle appendages. Under normal conditions, these progenitor populations are long-lived and able to sustain the cellular input to certain epidermal structures including the interfollicular epidermis and sebaceous gland. Other putative epithelial progenitors derived from the hair follicle possess high in vitro proliferative capacity and are able to regenerate skin, hair and sebaceous lineages in transplantation studies. These new findings present the cutaneous epithelium as a highly compartmentalized structure potentially maintained by multiple classes of progenitor cells. In this review, we will discuss the implications of these new putative epithelial progenitor populations and their potential to be influenced by external stimuli for skin homeostasis and carcinogenesis.     

Adipose-derived stem cells and their potential to differentiate into the epithelial lineage

Adipose-derived stem cells (ASCs) possess a multilineage differentiation potential, can be used from an autologous origin, and are, therefore, attractive candidates for clinical applications to repair or regenerate damaged tissues and organs. Adipose tissue as a stem cell source is ubiquitously available and has several advantages compared with other sources. It is easily accessible in large quantities with a minimal invasive harvesting procedure, and the isolation of ASCs yields a high amount of stem cells, which is essential for stem cell-based therapies and tissue engineering. Differentiation of ASCs into cell types of mesodermal origin has been shown in a variety of studies. The plasticity of ASCs toward cells of the mesodermal lineage has been shown by their differentiation into chondrocytes, osteoblasts, adipocytes, and myocytes. Their potential to differentiate into lineages with nonmesodermal origin is even more exciting: ASCs are also able to differentiate into cells of ecto- and endodermal origin. Various in vitro and in vivo studies documented the induced differentiation into neural cells, hepatocytes, pancreatic islet cells, endothelial cells, and epithelial cells. Epithelial cells can embryologically arise from each of the 3 germ layers. This article summarizes and discusses the current knowledge of the potential of ASCs to differentiate into the epithelial lineage. The differentiation of ASCs into different types of epithelial cells, including hepatocytes, pancreatic cells, and endothelial cells, is highlighted together with a view on current clinical trials and future options.     

Androgenetic embryonic stem cells form neural progenitor cells in vivo and in vitro

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied, the developmental capacity of AG ESCs is much less clear. Here, we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed, with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs, we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article.