Fgd5 identifies hematopoietic stem cells in the murine bone marrow

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence

Cancer Stem Cells and Side Population Cells in Breast Cancer and Metastasis

In breast cancer it is never the primary tumour that is fatal; instead it is the development of metastatic disease which is the major cause of cancer related mortality. There is accumulating evidence that suggests that Cancer Stem Cells (CSC) may play a role in breast cancer development and progression. Breast cancer stem cell populations, including side population cells (SP), have been shown to be primitive stem cell-like populations, being long-lived, self-renewing and highly proliferative. SP cells are identified using dual wavelength flow cytometry combined with Hoechst 33342 dye efflux, this ability is due to expression of one or more members of the ABC transporter family. They have increased resistance to chemotherapeutic agents and apoptotic stimuli and have increased migratory potential above that of the bulk tumour cells making them strong candidates for the metastatic spread of breast cancer. Treatment of nearly all cancers usually involves one first-line agent known to be a substrate of an ABC transporter thereby increasing the risk of developing drug resistant tumours. At present there is no marker available to identify SP cells using immunohistochemistry on breast cancer patient samples. If SP cells do play a role in breast cancer progression/Metastatic Breast Cancer (MBC), combining chemotherapy with ABC inhibitors may be able to destroy both the cells making up the bulk tumour and the cancer stem cell population thus preventing the risk of drug resistant disease, recurrence or metastasis

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Expression of Abcg2 in Murine Skeletal Muscle Cells Identifies Two Populations with Different Myogenic Potential.

Stem cells can be identified by a side population (SP) phenotype in a variety of adult and embryonic tissues. We have previously shown that expression of the Abcg2 serves as a prospective marker for isolating HSCs suggesting that Abcg2 expression may also serve as a marker for stem cell activity in other non-hematopoetic tissues. In particular, skeletal muscle SP cells have been shown to have stem cell activity in muscle reconstitution experiments and the SP population in skeletal muscle is significantly reduced in Abcg2 null mice. To investigate the possibility that Abcg2 can serve as a muscle stem cell marker, we used our mouse strain in which a GFP reporter gene was inserted into the Abcg2 locus. Skeletal muscle cells from adult Abcg2/GFP knock-in mice were isolated based on GFP expression and tested for stem cell activity. To exclude contamination by hematopoetic cells, all experiments were performed on cells gated for the CD45 −/Ter119− phenotype. Flow cytometric analysis showed that 11.6 ± 4.2 % of these muscle cells expressed the Abcg2/GFP allele. Since myogenic progenitor cells have the CD34+/ Sca-1−phenotype, GFP positive and negative cell populations were further analyzed for CD34 and Sca-1 expression. This analysis showed that 15.6 ± 5.3 % of Abcg2/GFP+ cells were CD34+/ Sca-1−. In contrast, 51.3 ±18.3 % of Abcg2/GFP− cells were CD34+/ Sca-1−. These results indicated that Abcg2/GFP− cell population may have a higher frequency of myogenic progenitor cells when compared to Abcg2/GFP+ cells. Analysis of skeletal muscle SP cells for GFP expression showed that 57.5±12 % of the SP and 10.8±0.9 % of non-SP or main population (MP) cells expressed the Abcg2/GFP allele. When SP and MP cell populations were analyzed for CD34 and Sca-1 expression, the highest percentage of CD34+/Sca-1− cells were found in MP/GFP− cell population (33.8±5.3%). Since 61.7 % of total cells were MP/GFP− cells, the greatest absolute number of cells with the myogenic phenotype were found to be located in MP/GFP− population. The growth characteristics and differentiation potential of Abcg2/GFP+ and Abcg2/GFP− cells were then assessed in a myogenic clonal culture assay. Sorted Abcg2/GFP+ and Abcg2/GFP− cells were plated in collagen-coated plates in proliferation medium. Both cell populations increased in number and formed large colonies after 7 days in culture. When these cells were then cultured in myogenic differentiation medium for 4 days, only GFP− cells differentiated into contracting myofibers. In contrast, GFP+ cells differentiated mostly into adherent fibroblast like cells. This data was further validated by DNA micro-arrays analysis of GFP+ and GFP− cell populations. We found that GFP− cells expressed skeletal muscle-specific genes such as MyoD, myf-5, myogenin and troponin whereas GFP+ population did not express any of these genes. Based on these data, we conclude that myogenic progenitor cells did not express the Abcg2/GFP allele. We are currently characterizing the Abcg2/GFP+ population for potential mesenchymal stem cell activity. Transplantation assays to determine myogenic activity of GFP+ and GFP− populations in vivo are in progress.</jats:p

Relationship between ABCG2 Expression and Side Population Stem Cell Phenotype in Murine Hematopoietic System Revealed by an Abcg2/GFP Knock-In Mouse Model.

Abstract Stem cells from a variety of tissues could be identified by a side population (SP) phenotype. We have identified an association between expression of the ABCG2 transporter and the SP phenotype, although the exact relationship between these two cellular characteristics is still not well understood. To further explore the relationship between Abcg2 expression and SP phenotype, we have generated a Abcg2/GFP reporter mouse model in which an IRES-GFP expression cassette was inserted down-stream of the stop codon of Abcg2 gene by homologous recombination, so that the expression of GFP is under the control of Abcg2 locus but endogenous Abcg2 expression was unperturbed. Immunohistochemistry of tissue sections using an anti-GFP antibody confirmed expression of GFP in tissues that normally express Abcg2, such as renal proximal tubules and intestinal epithelium. Flow cytometry analysis showed that about 10% of total bone marrow mononuclear cells expressed the Abcg2/GFP allele. Within this total GFP+ population, greater than 99% of the cells fall outside the SP region. Staining with lineage specific antibodies showed that 77% of the non-SP/GFP+ cells are Ter119+ erythroid cells. In contrast, only 0.5% of the total GFP+ population falls within the SP region. Approximately 90% of these bone marrow SP cells expressed the Abcg2/GFP allele. Transplantation studies with bone marrow cells that were depleted of lineage positive cells were then performed. All 5 mice transplanted with 100 Lin−, SP+, GFP+ cells were reconstituted in both myeloid and lymphoid lineages. In contrast, no repopulating activity was detected with up to 10,000 Lin−, non-SP, GFP− cells. When Lin−, non-SP, GFP+ cells were transplanted, only 2 of 5 animals were reconstituted with 5000 cells, and no repopulation was seen with lower cell doses. These results show a complex relationship between Abcg2 expression and the SP phenotype. Virtually all SP cells express the Abcg2/GFP allele, and these cells are highly enriched for repopulating activity in transplant assays. However, the majority of bone marrow cells that expressed the Abcg2/GFP allele were Ter119+ erythroid cells that do not bear the SP phenotype. In addition, there are non-SP, GFP+ cells that lack expression of Ter119 and other mature lineage markers. These Lin−, non-SP, GFP+ cells have relatively lower repopulating activity compared to SP cells.</jats:p

ABCG2 Expression as Prospective Marker for Hematopoeitic Stem Cell Purification.

Abstract ABCG2 (BCRP/MXR/ABCP) is an ABC transporter that effluxes Hoechst 33342 and is required for the side population (SP) phenotype of hematopoietic stem cells (HSCs). Although SP cells are enriched for HSC activity, they are not a pure stem cell population and contain a significant number of differentiated and committed cells. We hypothesized that expression of ABCG2 would provide a more specific marker for HSCs, given our prior results showing that other ABC transporters were expressed in committed SP cells. To test this hypothesis, we have generated a mouse strain in which an IRES-GFP element was inserted immediately after the stop codon of Abcg2, so that GFP and the endogenous Abcg2 gene were co-expressed from the same transcriptional regulatory elements. Immunohistochemical analysis using an anti-GFP antibody showed that Abcg2/GFP allele was strongly expressed in proximal tubule cells of kidney, in the epithelial cells of small intestine, and in vascular endothelium, recapitulating the normal expression pattern of Abcg2. Using flow cytometry to analyze GFP expression in bone marrow cells, we found that 48% of SP cells expressed GFP. In the distal tip of the SP region, previously shown to have the highest concentration of HSCs, 93% of cells expressed GFP. In gated Lin- GFP+ cells within the whole bone marrow population, 55 % of the cells expressed c-Kit and Sca1, a previously identified phenotype for HSCs. We sorted Lin- cells into GFP+ and GFP− subpopulations and determined repopulation frequencies in a limiting dilution assay. Fifteen percent of recipient mice were reconstituted with 10 Lin- GFP+ cells, and 50% mice with 60 Lin- GFP+ cells. In groups transplanted with 125 to 1000 Lin- GFP+ cells, 100% of the mice were reconstituted. In contrast, no reconstitution was seen in any mice transplanted with 60,000 Lin− GFP− cells, indicating that most if not all HSCs expressed the Abcg2/GFP allele. Secondary transplant experiments and individual lineage analysis of peripheral blood cells confirmed that the Lin- GFP+ cells were long-term HSCs. These results demonstrate that expression of Abcg2 in Lin- cells defines a highly enriched population of HSCs, and is a more specific HSC marker than the SP phenotype. We are now increasing the stringency of the GFP sorting gate and transplanting mice with single cells to determine if a pure HSC population can be isolated with this simple 2 marker system.</jats:p

Transcriptome changes during intestinal cell differentiation

AbstractThe expression of 18 149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transcriptome was observed during the differentiation of the Caco-2 cells. 8762 of the 18 149 genes analysed were expressed above background level in the undifferentiated Caco-2 cells, whereas only 5767 genes were expressed above background in differentiated Caco-2 cells. This pattern of expression was caused by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial cells by performing reverse transcriptase–polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one – SART3 – was identified as a marker for human colonic crypts