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S-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

ABSTRACT: Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a subpopulation of the Lin- CD24+ Sca-1+ CD49f+ cells and are capable of self-renewal together with enhanced growth potential in sphere-forming assay in vitro, a phenotype consistent with that of a PSC population. Transplantation assays of prostate GFP+ cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables PSCs in situ identification and isolation via a single consistent parameter. Transcriptional profiling of these GFP+ neonatal stem cells showed an increased expression of several components of the Wnt signaling pathway. It also identified stem cell regulators with potential applications for further analyses of normal and cancer stem cells.

SUBMITTER: Brocqueville G

PROVIDER: S-EPMC5045392 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

Brocqueville Guillaume G Chmelar Renee S RS Bauderlique-Le Roy Hélène H Deruy Emeric E Tian Lu L Vessella Robert L RL Greenberg Norman M NM Rohrschneider Larry R LR Bourette Roland P RP

Oncotarget 20160501 20

Isolation of prostate stem cells (PSCs) is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. Here we show that cells identified by GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate GFP+ cells are a s ...[more]

PMID: 27081082

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Project description:Isolation of prostate stem cells is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. We showed that cells identified by s-SHIP/GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate s-SHIP/GFP-positive cells represent a subpopulation of the lineage-negative / CD24-positive / Sca-1-positive / CD49f-positive (LSC) cells and are capable of selfârenewal together with enhanced growth potential in sphereâforming assay in vitro, a phenotype consistent with that of a prostate stem cell population. Transplantation assays of these prostate GFP-positive cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables prostate stem cells in situ identification and isolation via a single consistent parameter. Since the GFP-positive cell population is a small subset of basal LSC cells and is most responsible for stem-like activity, we performed transcriptional profiling of GFP-negative LSC and GFP-positive LSC cells to distinguish a basal cell profile from a tissue stem cell profile. Prostate tissue was collected from 6-day-old male mice, minced into small fragment, digested with 200 U/ml collagenase IAâS (Sigma; C5894) in Dulbeccoâs modified Eagles medium (DME, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) (DME-10% FBS) at 37Â°C for 60 min with gentle agitation. The digested cells were filtered through a 40-Î¼m cell strainer (BD Biosciences) washed, and resuspended in DME-10% FBS., filtered and labeled with antibodies against lineage (Ter119, CD31, CD45), CD49f and Sca-1 cell surface markers (Affymetrix ebiosciences). Labeled cells were analyzed and the GFP-negative LSC and GFP-positive-LSC populations were sorted by FACS. For each cell population, 3 independent samples were collected and analysed.

Project description:BackgroundThe stem cell characteristic makes basal cells desirable for ex vivo modeling of airway diseases. However, to date, approaches allowing them extensively in vitro serial expansion and maintaining bona fide stem cell property are still awaiting to be established. This study aims to develop a feeder-free culture system of mouse airway basal stem cells (ABSCs) that sustain their stem cell potential in vitro, providing an experimental basis for further in-depth research and mechanism exploration.MethodsWe used ROCK inhibitor Y-27632-containing 3T3-CM, MEF-CM, and RbEF-CM to determine the proper feeder-free culture system that could maintain in vitro stem cell morphology of mouse ABSCs. Immunocytofluorescence was used to identify the basal cell markers of obtained cells. Serial propagation was carried out to observe whether the stem cell morphology and basal cell markers could be preserved in this cultivation system. Next, we examined the in vitro expansion and self-renewal ability by evaluating population doubling time and colony-forming efficiency. Moreover, the differentiation potential was detected by an in vitro differentiation culture and a 3D tracheosphere assay.ResultsWhen the mouse ABSCs were cultured using 3T3-CM containing ROCK inhibitor Y-27632 in combination with Matrigel-coated culture dishes, they could stably expand and maintain stem cell-like clones. We confirmed that the obtained clones comprised p63/Krt5 double-positive ABSCs. In continuous passage and maintenance culture, we found that it could be subculture to at least 15 passages in vitro, stably maintaining its stem cell morphology, basal cell markers, and in vitro expansion and self-renewal capabilities. Meanwhile, through in vitro differentiation culture and 3D tracheosphere culture, we found that in addition to maintaining self-renewal, mouse ABSCs could differentiate into other airway epithelial cells such as acetylated tubulin (Act-Tub) + ciliated and MUC5AC + mucus-secreting cells. However, they failed to differentiate into alveoli epithelial cells, including alveolar type I and alveolar type II.ConclusionWe established an in vitro feeder-free culture system that allows mouse ABSCs to maintain their stem cell characteristics, including self-renewal and airway epithelium differentiation potential, while keeping up in vitro expansion stability.

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S-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

Publications

s-SHIP expression identifies a subset of murine basal prostate cells as neonatal stem cells.

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