Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133

Blood ◽  
2004 ◽  
Vol 103 (6) ◽  
pp. 2055-2061 ◽  
Author(s):  
Sergey V. Shmelkov ◽  
Lin Jun ◽  
Ryan St Clair ◽  
Deirdre McGarrigle ◽  
Christopher A. Derderian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Surface Proteins ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Alternative Promoters ◽  
Tissue Specific

Abstract AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34+ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5′–untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5′-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissue-specific stem and progenitor cells.

Gastrointestinal Stem Cells. III. Emergent themes of liver stem cell biology: niche, quiescence, self-renewal, and plasticity

2006 ◽  
Vol 290 (2) ◽  
pp. G189-G193 ◽  
Author(s):  
Neil D. Theise
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Stem Cell Niche ◽  
Progenitor Cell ◽  
Stem Cell Biology ◽  
Cell Plasticity ◽  
Self Renewal ◽  
Cell Niche

This essay will address areas of liver stem/progenitor cell studies in which consensus has emerged and in which controversy still prevails over consensus, but it will also highlight important themes that inevitably should be a focus of liver stem/progenitor cell investigations in coming years. Thus concepts regarding cell plasticity, the existence of a physiological/anatomic stem cell niche, and whether intrahepatic liver stem/progenitor cells comprise true stem cells or progenitor cells (or both) will be approached in some detail.

Change in SP Distribution with Age Reflects Intrinsic Age-Based Changes in Stem Cell Biology: Implications for the Mechanism of Aging.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4209-4209
Author(s):  
Daniel J. Pearce ◽  
Catherine Simpson ◽  
Kirsty Allen ◽  
Ayad Eddaoudi ◽  
Derek Davies ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
The Absolute

Abstract It has been postulated that as we age, accumulated damage causes stem cells to die by apoptosis. This could lead to a diminished stem cell pool and consequently a reduced organ regeneration potential that contributes to somatic senescence. Hematopoietic stem cells have evolved many mechanisms to cope with their exposure to toxins during life. Cell surface transporters and anti-toxic enzymes are highly expressed in hematopoietic stem cells. If toxins do get the opportunity to damage the DNA of stem cells then the cell is more likely to die by apoptosis than attempt DNA repair and risk an error. Summarised below are our results from an investigation of the frequency, phenotype, cell cycle status and repopulation potential (in young recipients) of C57BL6 side population (SP) cells from mice with a range of ages. The absolute frequency of SP cells increases with age (Figure-A). The proportion of the lineage negative, Sca-1+, c-kit+ (KLS) cell population that is an SP stem cell increases from ~1% to over 30% during the murine lifetime (red bars in Figure-B). These SP cells from older mice have a reduced 4-month competitive repopulation potential when compared to SP cells from younger mice but contain a similarly low proportion of phenotypically-defined mature cells (blue bars in Figure-B) and have a similar cell cycle profile and progenitor cell output (2% of 3 x 96 well plates for each). SP cells from older mice contained a higher proportion of SP cells with the highest efflux ability (61 vs 414 days, p=<0.001, n=6) Engrafted cells derived from old SP cells 4 months previously still displayed an increased SP frequency when compared to engrafted cells derived from SP cells of young mice. Hence, more progenitors or committed cells have not gained the SP ability; rather this difference in SP distribution reflects an age-dependent change in hematopoietic stem cell biology that is independent of the microenvironment. Specifically, the proportion of stem and progenitor cells (KLS) that is a stem cell (SP fraction of KLS) increases with age. We hypothesize that this may be a progressive enrichment of primitive cells over time via selection. As we age, accumulative damage to hematopoietic stem and progenitor cells causes more cells to die by apoptosis. It may be that the stem/progenitor cells with the lowest hoechst efflux ability are most susceptible to damage and hence most likely to die by apoptosis. Since the HSCs with the highest efflux of hoechst are thought to be the most primitive, it may be that there is an enrichment of primitive cells. This could account for the increased SP proportion observed within KLS cells. As there may be cells with ABC/G2 activity that is undetectable via the SP technique, selection of cells with a higher pump activity could also explain the increased SP frequency we observed. This hypothetical mechanism would also be independent of microenvirinment. In summary, we surmise that HSCs have a mechanism that copes with cellular damage while compensating for the reduced cellular output of HSCs with age by increasing the absolute number of HSCs. Figure Figure

Identification of Novel Surface Markers and Targets In Neoplastic Stem Cells In AML and CML: a Flow-Gene-Flow Screen Approach.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1575-1575 ◽  
Author(s):  
Harald Herrmann ◽  
Sabine Cerny-Reiterer ◽  
Irina Sadovnik ◽  
Viviane Winter ◽  
Katharina Blatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Gene Chip ◽  
Cytokine Receptors ◽  
Stem Cell Markers ◽  
Surface Markers ◽  
Leukemic Stem Cells

Abstract Abstract 1575 The concept of leukemic stem cells (LSC) is increasingly employed to explain the biology of various myeloid neoplasms and to screen for pivotal targets, with the hope to improve drug therapy through elimination of disease-initiating cells. Although the stem cell hypothesis may apply to all neoplasms, leukemia-initiating cells have so far only been characterized in some detail in myeloid leukemias. In an attempt to identify novel cell surface markers and targets on leukemic stem cells (LSC) in acute (AML) and chronic myeloid leukemia (CML), we examined CD34+/CD38- and CD34+/CD38+ populations of leukemic cells in a cohort of patients with AML (n=55) and CML (n=20). In a first step, cell surface antigen profiles were determined by multicolor flow cytometry. In this screen, we were able to show that CD34+/CD38- LSC in AML and CML consistently express certain cytokine receptors, including G-CSFR (CD114), SCFR/KIT (CD117), and IL-3RA (CD123). The low affinity IL-2R (CD25) was detectable on CD34+/CD38- stem cells in patients with CML, and in a subset of AML patients. Other cytokine receptors (R) such as FLT3, IGF-1R, endoglin (CD105), GM-CSFRA (CD116), and OSMR were expressed variably on CD34+/CD38- progenitor cells, whereas the EPOR was not detectable on LSC. We were also able to detect several established therapeutic targets on LSC, including CD33 and CD44. Whereas CD44 was consistently expressed on all LSC in all donors, CD33 was found to be expressed variably on subpopulations of LSC in AML and CML, depending on the phase and type of disease. By using cytokine ligands (G-CSF, IL-3, SCF, EPO) and targeted drugs, we were also able to confirm that identified cytokine receptors and targets were functionally active molecules and potentially relevant targets. In a next step, highly enriched (purity >98%) sorted CD34+/CD38- cells, CD34+/CD38+ cells, and CD34- cells were collected in patients with AML and CML, and in 3 cord blood samples as controls. Purified cells were subjected to gene chip analyses, qPCR, and functional analyses. The identity of leukemic progenitors was confirmed by FISH, and expression of markers and targets in CML stem cells and AML stem cells was confirmed by qPCR. In gene chip analyses, we screened for novel LSC markers and targets. Candidate genes were selected based and their specific expression in progenitor cell fractions and surface membrane location, which was confirmed by antibody staining experiments. Novel stem cell markers identified so far include ROBO4, NPDC-1, and CXCR7. The previously described surface markers MDR-1 and CLL-1 were also identified by flow cytometry, but were also found to be expressed on more mature hematopoietic cells. By contrast, ROBO4 was found to be expressed preferentially on CD34+/CD38- stem cells, but less abundantly on CD34+/CD38+ progenitor cells in CML. Interestingly, whereas ROBO4 was expressed on CD34+/CD38- stem cells in most patients with CML, ROBO4 expression on leukemic stem cells was only found in a subset of AML patients. By contrast, CD34+/CD38- stem cells in AML frequently expressed CLL-1 and NPDC-1 on their surface. In conclusion, we have identified novel markers and targets in CD34+/CD38- progenitor cells in AML and CML. These markers may be useful for the identification and isolation of leukemic stem cells in AML and CML, and for the validation of drug effects on these cells. Disclosures: De Angelis: Biopharm R&D, GSK: Employment. Holmes:Biopharm R&D, GSK: Employment. Valent:Domantis: Research Funding.

scholarly journals ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Mohd Farid Abdul Halim ◽  
Jonathan D. Stoltzfus ◽  
Stefan Schulze ◽  
Micheal Hippler ◽  
Mechthild Pohlschroder
Keyword(s):  
Cell Surface ◽  
In Silico ◽  
Cell Biology ◽  
Surface Proteins ◽  
Signal Peptidase ◽  
Secreted Proteins ◽  
Dependent Manner ◽  
Content Type ◽  
C Terminus ◽  
Signal Peptidase I

ABSTRACT Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of Haloferax volcanii, previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, in silico analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved “twin” arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry. IMPORTANCE The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon H. volcanii, through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for in silico subcellular localization predictions of archaeal and bacterial proteins.

Abstract 402: Plasminogen Inhibits Sdf-1-induced Cxcr4 Internalization In Bone Marrow-derived Mononuclear Cells

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Hongyu Han ◽  
Lindi Zhang ◽  
Yi Fan ◽  
Yanqing Gong
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Surface ◽  
Mononuclear Cells ◽  
Cxcr4 Expression ◽  
Stem Cell Mobilization ◽  
Immunofluorescent Staining ◽  
Dependent Manner ◽  
Cell Mobilization

In multiple pathological settings including stokes and myocardial infarction (MI), stem cells are mobilized from the bone marrow to injury site to promote tissue repair. However, naturally recruited stem cells are not sufficient for heart function recovery. How to enhance stem cell mobilization remains challenge but may lead to develop novel strategy for stem cell therapies. Our previous studies have shown that Plasminogen (Plg) enhances stem cell mobilization and contributes to cardiac repair after MI. Mechanism studies reveal that Plg regulates CXCR4 expression, the receptor of SDF-1, a major chemoattractant for stem cells, during stem cell mobilization. Using bone marrow mononuclear cells, our data show that Plg is able to increase CXCR4 expression in a dose-dependent manner. Moreover, Plg decreases CXCR4 internalization induced by SDF-1. SDF-1 (50nM) induces a rapid internalization of CXCR4 on BMNC indicated as much lower CXCR4 expression compared with control (0.9 ± 0.5% vs 10.5 ± 2.6%) in cell surface. However, Plg treatment (100nM) decreases CXCR4 internalization, indicated as significantly higher expression (5.5 ± 0.9%) of CXCR4 on the cell surface. Immunofluorescent staining results show that CXCR4 translocates into cytoplasma after SDF-1 treatment. Plg rescued the CXCR4 translocation induced by SDF-1 and CXCR4 is mainly expressed on the cell surface as shown in the figure, confirming that Plg may prolong CXCR4 expression through inhibiting CXCR4 internalization by SDF-1. These data has indicated a novel mechanism for Plg-regulated CXCR4 expression and may contribute stem cell mobilization from bone marrow to the circulation during MI injury.

scholarly journals Isolation of a multipotent mesenchymal stem cell-like population from human adrenal cortex

2018 ◽  
Vol 7 (5) ◽  
pp. 617-629 ◽  
Author(s):  
Earn H Gan ◽  
Wendy Robson ◽  
Peter Murphy ◽  
Robert Pickard ◽  
Simon Pearce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Cell Surface ◽  
Adrenal Cortex ◽  
Cell Biology ◽  
Growth Promotion ◽  
Pluripotency Markers

Background The highly plastic nature of adrenal cortex suggests the presence of adrenocortical stem cells (ACSC), but the exact in vivo identity of ACSC remains elusive. A few studies have demonstrated the differentiation of adipose or bone marrow-derived mesenchymal stem cells (MSC) into steroid-producing cells. We therefore investigated the isolation of multipotent MSC from human adrenal cortex. Methods Human adrenals were obtained as discarded surgical material. Single-cell suspensions from human adrenal cortex (n = 3) were cultured onto either complete growth medium (CM) or MSC growth promotion medium (MGPM) in hypoxic condition. Following ex vivo expansion, their multilineage differentiation capacity was evaluated. Phenotype markers were analysed by immunocytochemistry and flow cytometry for cell-surface antigens associated with bone marrow MSCs and adrenocortical-specific phenotype. Expression of mRNAs for pluripotency markers was assessed by q-PCR. Results The formation of colony-forming unit fibroblasts comprising adherent cells with fibroblast-like morphology were observed from the monolayer cell culture, in both CM and MGPM. Cells derived from MGPM revealed differentiation towards osteogenic and adipogenic cell lineages. These cells expressed cell-surface MSC markers (CD44, CD90, CD105 and CD166) but did not express the haematopoietic, lymphocytic or HLA-DR markers. Flow cytometry demonstrated significantly higher expression of GLI1 in cell population harvested from MGPM, which were highly proliferative. They also exhibited increased expression of the pluripotency markers. Conclusion Our study demonstrates that human adrenal cortex harbours a mesenchymal stem cell-like population. Understanding the cell biology of adrenal cortex- derived MSCs will inform regenerative medicine approaches in autoimmune Addison’s disease.

scholarly journals Nuclear Export of Smads by RanBP3L Regulates Bone Morphogenetic Protein Signaling and Mesenchymal Stem Cell Differentiation

2015 ◽  
Vol 35 (10) ◽  
pp. 1700-1711 ◽  
Author(s):  
Fenfang Chen ◽  
Xia Lin ◽  
Pinglong Xu ◽  
Zhengmao Zhang ◽  
Yanzhen Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Mesenchymal Stem Cell ◽  
Nuclear Export ◽  
Stem Cell Differentiation ◽  
Bmp Signaling ◽  
Dependent Manner ◽  
Mesenchymal Stem Cell Differentiation

Bone morphogenetic proteins (BMPs) play vital roles in regulating stem cell maintenance and differentiation. BMPs can induce osteogenesis and inhibit myogenesis of mesenchymal stem cells. Canonical BMP signaling is stringently controlled through reversible phosphorylation and nucleocytoplasmic shuttling of Smad1, Smad5, and Smad8 (Smad1/5/8). However, how the nuclear export of Smad1/5/8 is regulated remains unclear. Here we report that the Ran-binding protein RanBP3L acts as a nuclear export factor for Smad1/5/8. RanBP3L directly recognizes dephosphorylated Smad1/5/8 and mediates their nuclear export in a Ran-dependent manner. Increased expression of RanBP3L blocks BMP-induced osteogenesis of mouse bone marrow-derived mesenchymal stem cells and promotes myogenic induction of C2C12 mouse myoblasts, whereas depletion of RanBP3L expression enhances BMP-dependent stem cell differentiation activity and transcriptional responses. In conclusion, our results demonstrate that RanBP3L, as a nuclear exporter for BMP-specific Smads, plays a critical role in terminating BMP signaling and regulating mesenchymal stem cell differentiation.

scholarly journals A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takamasa Hirai ◽  
Ken Kono ◽  
Rumi Sawada ◽  
Takuya Kuroda ◽  
Satoshi Yasuda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Neural Progenitor Cells ◽  
Neural Progenitor ◽  
Sensitive Detection ◽  
Quality And Safety

AbstractHighly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

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