Isolation of Mouse Pancreatic Ductal Progenitor Cells Expressing CD133 and c-Met by Flow Cytometric Cell Sorting
ABSTRACT Islet transplantation has become available across the globe since a novel protocol was reported. However, because donors are in short supply, only a minority of patients benefit from this procedure. Pancreatic progenitor cells are a promising resource for regeneration of new islets, but whether progenitor cells reside in ductal epithelium is not clear.
Mouse pancreas was examined by immunohistochemistry with cell surface markers specific for ductal cells. We developed an isolation method for ductal cells by flow cytometric cell sorting using a newly identified specific marker for ductal cells. By using an in vitro colony assay, we characterized their proliferative and multipotent capacity.
CD133 is expressed specifically in ductal epithelium. Flow cytometric analysis revealed that purified ductal cells are highly enriched in the CD133(+)CD34(-)CD45(-)Ter119(-) fraction. An analysis of clonal epithelial colonies formed by individual cells revealed that progenitor cells with multilineage differentiation capacity are present in neonatal ductal epithelium. Moreover, these progenitor cells express c-Met. In adult mice, progenitor cells that show a high proliferative capacity but appear committed to a ductal lineage are co-purified with CD133(+)CD34(-)CD45(-)Ter119(-) cells.
We established a system for isolating and culturing mouse pancreatic ductal cells that relies on flow cytometric cell sorting. Clonal analysis revealed that a population of progenitor cells is present among CD133(+) ductal cells. Isolation of these cells will facilitate future studies into the roles of pancreatic progenitor cells in regeneration and carcinogenesis.
- SourceAvailable from: Rex A Hess
[Show abstract] [Hide abstract]
- "The epididymal luminal environment relies on important absorptive and secretory functions of the epithelium, and the microvilli plays a fundamental role in these processes. Prominins 1 and 2 are encoded by paralog genes, and are believed to participate in the organization of plasma membrane protrusions in several epithelial cells (Weigmann et al., 1997; Corbeil et al., 2000, 2010; Fargeas et al., 2004; Florek et al., 2005; Gashaw et al., 2007; J aszai et al., 2007; Oshima et al., 2007; Lardon et al., 2008). Prominin 1 localizes preferentially to microvilli (Corbeil et al., 1999, 2010; Yang et al., 2012), and it has been identified in the epididymis and in the flagellum of spermatozoa present in the testicular tubuli (Fargeas et al., 2004). "
ABSTRACT: The role of oestrogens in epididymal function is still unclear. Knockout of the oestrogen receptor ESR1 (Esr1−/−) or treatment with the anti-oestrogen Fulvestrant affect epididymal milieu and sperm motility. We investigated the effect of in vivo treatment of rats with Fulvestrant on: (i) expression of genes that may be important for the architecture and function of the epididymal epithelium: prominins 1 and 2, metalloproteinase 7, claudin 7, beta-catenin and cadherin 13, and (ii) levels of oestradiol and testosterone, and expression of oestrogen and androgen receptors, in the initial segment (IS), caput, corpus and cauda epididymis. Fulvestrant (i) reduced gene expression of prominin 1 (variant 1) in the caput, reduced prominin 1 protein content in the caput epididymis and in the efferent ductules, and increased the localization of prominin 1 in microvilli of the caput and corpus; (ii) reduced gene expression of prominin 2 in the corpus and cauda epididymis; (iii) increased the metalloproteinase 7 content in the apical region of principal cells from IS/caput; (iv) reduced in the corpus epididymis, but increased in the efferent ductules, the cadherin 13 mRNA level; (v) reduced testosterone but increased oestradiol levels in the corpus and cauda; (vi) increased the androgen receptor protein content in all regions of the epididymis, and the oestrogen receptor GPER in the corpus and cauda epididymis. In conclusion, treatment with Fulvestrant induced regional-specific changes in hormonal and steroid receptor content, and affected expression of proteins important for epithelial organization and absorption/secretion. The mechanisms of oestrogen action may differ among epididymal regions, which may contribute to determine region-specific sperm functions.Andrology 04/2014; 2(4):559-571. DOI:10.1111/j.2047-2927.2014.00219.x · 2.30 Impact Factor
[Show abstract] [Hide abstract]
- "As already mentioned in the introduction CD133 alone or in a combination with other markers is actually used for the isolation of normal stem cells from several tissues such as bone marrow  , brain  , kidney  prostate , liver , pancreas  , sarcoma   and skin . "
ABSTRACT: CD133 (promini-1) is a member of the transmembrane glycoprotein family, was initially described as a specific marker to select human hematopoietic progenitor cells. Then, it was recognised as important marker to identify and isolate the specific cell subpopulation termed "cancer stem cells". Many studies showed that CD133(+) cells have stemness properties such as self-renewal, differentiation ability, high proliferation and they are able also to form tumours in xenografts. Moreover it has been demonstrated that CD133(+) cells more resistant to radiation and standard chemotherapy than CD133(-) cells. Although this, others investigations demonstrated that also CD133(-) cells can show the same characteristics of those positive for CD133(+). Hence, some inconsistencies among published data on CD133 function can be ascribed to different causes questioning the main role as specific marker of cancer stem cells. In fact, many authors indicate that CD133 is expressed both in differentiated and undifferentiated cells, and CD133(-) cancer cells can also initiate tumours. Indeed, it is still a matter of debate whether CD133(+) cells truly represent the ultimate tumourigenic population. However, the belief that CD133 may act as a universal marker of CSCs has been met with a high degree of controversy in the research community. In this review there is an attempt to highlight: i) the role and function of CD133, with an overview on the current stage of knowledge about this molecule, ii) the difficulty often encountered in its identification iii) the utility of CD133 expression as a prognostic marker.American Journal of Translational Research 10/2013; 5(6):563-581. · 3.40 Impact Factor
[Show abstract] [Hide abstract]
- "About ten years ago murine ductal cells cultured in vitro were reported to form 3D clusters that differentiate to functional islet cells, which are able to respond to a glucose challenge and to reverse diabetes in mice . An interesting strategy for the prospective isolation of putative progenitors from an enriched ductal cell population is also being pursued by Taniguchi and colleagues [61, 62]. The approach combines immunohistochemical analysis of mouse pancreas to define new phenotypic markers and flow cytometry cell sorting to isolate clonal cell populations that are able to differentiate toward the endocrine lineage in vitro or in vivo. "
ABSTRACT: β-cell replacement by allogeneic islet transplantation is a promising approach for patients with type 1 diabetes, but the shortage of organ donors requires new sources of β cells. Islet regeneration in vivo and generation of β-cells ex vivo followed by transplantation represent attractive therapeutic alternatives to restore the β-cell mass. In this paper, we discuss different postnatal cell types that have been envisaged as potential sources for future β-cell replacement therapy. The ultimate goal being translation to the clinic, a particular attention is given to the discrepancies between findings from studies performed in rodents (both ex vivo on primary cells and in vivo on animal models), when compared with clinical data and studies performed on human cells.Journal of Transplantation 10/2011; 2011:892453. DOI:10.1155/2011/892453