scholarly journals Conditional Activation of Bmi1 Expression Regulates Self-renewal, Apoptosis, and Differentiation of Neural Stem/Progenitor Cells In Vitro and In Vivo

Stem Cells ◽  
2011 ◽  
Vol 29 (4) ◽  
pp. 700-712 ◽  
Author(s):  
Gokhan Yadirgi ◽  
Veronica Leinster ◽  
Serena Acquati ◽  
Heeta Bhagat ◽  
Olga Shakhova ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Self Renewal ◽  
Bmi1 Expression ◽  
Neural Stem Progenitor Cells

Oncogenic Wilms Tumor 1 (WT1) Mutation Augments Hematopoietic Progenitor Cell Clonogenicity and Promotes Expansion Of The Long-Term Hematopoietic Stem Cell (LT-HSC) Compartment: Implications For WT1-Mediated Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1269-1269
Author(s):  
Colleen E. Annesley ◽  
Rachel E. Rau ◽  
Daniel Magoon ◽  
David Loeb ◽  
Patrick Brown
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Wilms Tumor ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Exon 9

Abstract Background The WT1 gene encodes for a zinc finger-containing transcription factor involved in differentiation, cell cycle regulation and apoptosis. WT1 expression is developmentally regulated and tissue-specific, with expression maintained in the kidney and in CD34+ hematopoietic progenitor cells. Inactivating mutations of this tumor suppressor gene are well-described in sporadic Wilms tumor and as germline mutations in Wilms tumor predisposition syndromes. WT1 mutations have been reported in approximately 10% of both adult and pediatric patients with cytogenetically-normal acute myeloid leukemia (CN-AML), and have been associated with treatment failure and a poor prognosis. These reported mutations consist of insertions, deletions or point mutations. Many are frameshift mutations in exon 7, can occur as biallelic double mutations, and result in truncated proteins which may alter DNA-binding ability. Missense mutations in exon 9 have also been identified, and reports suggest that these may act in a dominant-negative manner, resulting in a loss of function. Despite these observations, the functional contribution of WT1 mutations to leukemogenesis is still largely undetermined. Methods/Results We obtained a novel knock-in WT1 mutant mouse model, which is heterozygous for the missense mutation R394W in exon 9, and homologous to exon 9 mutations seen in human AML. We hypothesized that WT1 mutations may have an aberrant effect on hematopoiesis, and specifically, could alter progenitor cell differentiation or proliferation. To investigate this, we collected lineage-negative bone marrow (lin- BM) cells from two-month old WT1 mutant (WT1mut) and wild-type (wt) mice. We performed methylcellulose colony-forming assays, serially replating cells every 10-12 days. Strikingly, WT1mut progenitor cells showed higher in vitro colony-forming capacity and an increased ability to serially replate, suggesting aberrantly enhanced self-renewal capability. Furthermore, WT1mut colonies from secondary and tertiary passages were larger and more cohesive than wild-type colonies, demonstrating increased proliferation and morphology consistent with blast colony-forming units (CFU-blast). Flow cytometric analysis of these WT1mut cells at tertiary replating revealed an immature, largely c-Kit+ population. Next, in order to study the effects of WT1mut on HSCs in vivo, we performed serial competitive transplantation of HSC-enriched, lineage-depleted BM into lethally irradiated mice. At 14 weeks post-transplant, the donor bone marrow cells were harvested and analyzed by flow cytometry. We observed a significant expansion of the LT-HSC compartment in the WT1mut mice compared to wild-type mice. These data provide new insight into the biology and functional role of WT1 mutations in the aberrant regulation of hematopoietic stem and progenitor cell expansion. Conclusion Oncogenic WT1 mutations confer enhanced proliferation and renewal of myeloid progenitor cells in vitro and expansion of LT-HSCs in vivo. Our findings suggest that WT1 mutations enhance stem cell self-renewal, potentially priming these cells for leukemic transformation upon acquisition of cooperative events. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Visualization of individual cell division history in complex tissues

2020 ◽  
Author(s):  
Annina Denoth-Lippuner ◽  
Baptiste N. Jaeger ◽  
Tong Liang ◽  
Stefanie E. Chie ◽  
Lars N. Royall ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Progenitor Cells ◽  
Individual Cell ◽  
Cell Divisions ◽  
Mouse Tissues ◽  
Single Cell Rna Sequencing ◽  
Neural Stem Progenitor Cells

SummaryThe division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. We developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analysing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT allows for novel insights into stem cell behaviour. Further, we used single cell RNA-sequencing (scRNA-seq) of iCOUNT-labelled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a novel tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair.HighlightsiCOUNT reports previous cell divisions in mouse and human cells in vitroiCOUNT detects cell division biographies in complex mouse tissues in vivoiCOUNT allows for the analysis of human neural stem/progenitor cells in human forebrain organoidsSingle cell RNA-sequencing of iCOUNT cells derived from the mouse developing cortex and human forebrain organoids identifies molecular consequences of previous rounds of cell divisionsGraphical abstract

Human hematopoietic stem/progenitor cells overexpressing Smad4 exhibit impaired reconstitution potential in vivo

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4343-4351 ◽  
Author(s):  
Emma Rörby ◽  
Matilda Nifelt Hägerström ◽  
Ulrika Blank ◽  
Göran Karlsson ◽  
Stefan Karlsson
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Cell System ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Smad Pathway ◽  
Smad Signaling ◽  
Smad Signaling Pathway

Abstract Hematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-β superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-β in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-β, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.

Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2411-2411
Author(s):  
Hein Schepers ◽  
Patrick M. Korthuis ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Ectopic Expression ◽  
Fold Increase ◽  
Negative Regulator ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells. We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells. To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice. Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures: No relevant conflicts of interest to declare.

Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 248-248
Author(s):  
Xiao Yan ◽  
Heather A. Himburg ◽  
Phuong L. Doan ◽  
Mamle Quarmyne ◽  
Nelson J. Chao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Growth Factor Receptor ◽  
Self Renewal ◽  
Donor Cells ◽  
Growth Factor Receptor Bound

Abstract The mechanisms which regulate HSC regeneration following stress or injury remain poorly understood. Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. Following 550cGy TBI, BM KSL cells were depleted by 7 days post-TBI, whereas KSL cell recovery was evident at day+14. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells. We identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed in regenerating BM KSL cells compared to non-irradiated KSL cells (3.3 fold, p<0.0001). Grb10 is a member of the family of imprinted genes which are predominately expressed in numerous stem cell populations, including embryonic stem cells, skin and muscle stem cells. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) in detected in 7 day culture (p=0.03 and p=0.002, respectively). Furthermore, mice which were competitively transplanted with Grb10-deficient KSL cells had 10-fold decreased donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed > 10-fold deficit in long-term repopulating capacity in Grb10 deficient KSL cells compared to Grb10 expressing KSL cells (p=0.006 for %CD45.1+ donor cells in secondary mice). In order to examine the effect of Grb10-deficiency on HSC fate and hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8-week old Grb10m/+ (1 mutant allele, 1 wild type allele) were found to have 10-fold decreased Grb10 expression in BM lin- cells and had normal range complete blood counts. However, BM CFCs were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006) and competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (1/14, 7% vs. 5/14, 38% of mice with > 0.1% donor CD45.2+ cells). These results suggest that Grb10 regulates HSC self-renewal in vitro and in vivo. Mechanistically, Grb10m/+ mice displayed no alterations in the cell cycle status or frequency of apoptotic cells within BM HSCs compared to Grb10+/+ mice. However, single cytokine functional screening suggested that Grb10 regulates SCF-mediated proliferation of HSCs. Grb10m/+ BM KSL cells generated significantly less CFCs in culture in response to SCF treatment compared to Grb10+/+ KSL cells (p=0.008). Commensurate with this, SCF-mediated activation of mTOR was significantly increased in Grb10m/+ KSL cells compared to that observed in Grb10+/+ KSL cells (p=0.006). These data suggest that cytokine-mediated induction of mTOR signaling, which has been shown to deplete functional HSCs, is antagonized by Grb10, and that Grb10 is necessary to block cytokine-mediated HSC differentiation in vitro and in vivo. Grb10 represents a novel regulator of HSC fate determination and a new mechanistic target to facilitate HSC self-renewal. Studies are underway to determine whether Grb10 is also necessary for HSC regeneration after TBI. Disclosures No relevant conflicts of interest to declare.

Macrophage migration inhibitory factor (MIF) promotes cell survival and proliferation of neural stem/progenitor cells

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Cell Survival ◽  
Progenitor Cells ◽  
Migration Inhibitory Factor ◽  
Macrophage Migration Inhibitory Factor ◽  
Inhibitory Factor ◽  
Macrophage Migration ◽  
Ganglionic Eminence ◽  
Neural Stem Progenitor Cells

In a previous study, we showed that murine dendritic cells (DCs) can increase the number of neural stem/progenitor cells (NSPCs) in vitroand in vivo. In the present study, we identified macrophage migration inhibitory factor (MIF) as a novel factor that can support theproliferation and/or survival of NSPCs in vitro. MIF is secreted by DCs and NSPCs, and its function in the normal brain remains largelyunknown. It was previously shown that in macrophages, MIF binds to a CD74–CD44 complex. In the present study, we observed theexpression of MIF receptors in mouse ganglionic-eminence-derived neurospheres using flow cytometry in vitro. We also found CD74expression in the ganglionic eminence of E14 mouse brains, suggesting that MIF plays a physiological role in vivo. MIF increased thenumber of primary and secondary neurospheres. By contrast, retrovirally expressed MIF shRNA and MIF inhibitor (ISO-1) suppressedprimary and secondary neurosphere formation, as well as cell proliferation. In the neurospheres, MIF knockdown by shRNA increasedcaspase 3/7 activity, and MIF increased the phosphorylation of Akt, Erk, AMPK and Stat3 (Ser727), as well as expression of Hes3 and Egfr,the products of which are known to support cell survival, proliferation and/or maintenance of NSPCs. MIF also acted as a chemoattractantfor NSPCs. These results show that MIF can induce NSPC proliferation and maintenance by multiple signaling pathways actingsynergistically, and it may be a potential therapeutic factor, capable of activating NSPC, for the treatment of degenerative brain disorders.

scholarly journals Direct Stimulation of Adult Neural Stem/Progenitor Cells In Vitro and Neurogenesis In Vivo by Salvianolic Acid B

PLoS ONE ◽  
2012 ◽  
Vol 7 (4) ◽  
pp. e35636 ◽  
Author(s):  
Pengwei Zhuang ◽  
Yanjun Zhang ◽  
Guangzhi Cui ◽  
Yuhong Bian ◽  
Mixia Zhang ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Salvianolic Acid B ◽  
Direct Stimulation ◽  
Salvianolic Acid ◽  
Neural Stem Progenitor Cells ◽  
Stimulation Of

scholarly journals Structurally Distinct LewisX Glycans Distinguish Subpopulations of Neural Stem/Progenitor Cells

2011 ◽  
Vol 286 (18) ◽  
pp. 16321-16331 ◽  
Author(s):  
Eva Hennen ◽  
Tim Czopka ◽  
Andreas Faissner
Keyword(s):  
Progenitor Cells ◽  
Carbohydrate Binding ◽  
Neural Cells ◽  
Stem Cell Markers ◽  
Binding Assays ◽  
Embryonic Mouse ◽  
Isolation And Characterization ◽  
Neural Stem Progenitor Cells

There is increasing evidence that the stem and progenitor cell population that builds the central nervous system is very heterogeneous. Stem cell markers with the potential to divide this cell pool into subpopulations with distinct characteristics are sparse. We were looking for new cell type-specific antigens to further subdivide the progenitor pool. Here, we introduce the novel monoclonal antibody clone 5750. We show that it specifically labels cell surfaces of neural stem and progenitor cells. When 5750-expressing cells were isolated by fluorescence-activated cell sorting from embryonic mouse brains, the sorted population showed increased neurosphere forming capacity and multipotency. Neurospheres generated from 5750-positive cells could self-renew and remained multipotent even after prolonged passaging. Carbohydrate binding assays revealed that the 5750 antibody specifically binds to LewisX-related carbohydrates. Interestingly, we found that the LewisX epitope recognized by clone 5750 differs from those detected by other anti-LewisX antibody clones like 487LeX, SSEA-1LeX, and MMALeX. Our data further reveal that individual anti-LewisX clones can be successfully used to label and deplete different subpopulations of neural cells in vivo and in vitro. In conclusion, we present a new tool for the isolation and characterization of neural subpopulations and provide insights into the complexity of cell surface glycosylation.

163. POTENTIAL MARKERS FOR THE PROSPECTIVE ISOLATION OF HUMAN ENDOMETRIAL EPITHELIAL STEM/PROGENITOR CELLS

2010 ◽  
Vol 22 (9) ◽  
pp. 81
Author(s):  
C. E. Gargett ◽  
C. S. C. Tan ◽  
H. Buhring
Keyword(s):  
Progenitor Cells ◽  
Hormone Replacement ◽  
Proliferative Potential ◽  
Cell Marker ◽  
Self Renewal ◽  
Epithelial Cell Marker ◽  
Post Menopausal ◽  
Prospective Isolation

The human endometrium regenerates each month following menstruation, parturition and in post-menopausal women taking hormone replacement therapy. Adult stem/progenitor cells discovered residing in human and mouse endometrium may be responsible for this regenerative capacity. However, assays used to identify these stem/progenitor cells are retrospective. The aim of this study is to identify surface markers for the prospective isolation of human endometrial epithelial progenitor cells using a panel of 22 antibodies. Flow cytometry and was used in the initial screen and immunohistochemistry was used to reveal the location of marker expression. Multi-colour FACS protocols were developed with promising markers in conjunction with EpCAM (epithelial cell marker) and to exclude endothelial (CD31+), leukocytes (CD45+) and stromal (CD90+) cells. Sorted subpopulations were assessed for clonogenicity and self-renewal activity using in vitro cloning assays. Six antibodies were short-listed. 2D1D12 enriched for progenitor cells that formed epithelial clones in culture (n = 2). The 2D1D12+EpCAM+ fraction produced very few colony-forming units (CFU). 2D1D12–EpCAM+ fraction gave rise to small CFU. However the 2D1D12+EpCAM– population showed the greatest progenitor activity, producing many large CFU that could be serially cloned twice, indicating self renewal activity. This preliminary data suggests that 2D1D12+EpCAM–CD90–CD31–CD45– population may enrich for human endometrial epithelial stem/progenitor cells. Importantly, large CFU have previously been reported to exhibit stem cell properties of self-renewal, differentiation and high proliferative potential (1). Future studies will focus on xenografting this population to assess tissue reconstitution ability in vivo. The identification of endometrial epithelial stem/progenitor cell marker(s) will enable their prospective isolation for further characterisation and will assist in the investigation of their potential role in endometrial proliferative disorders such as endometriosis and endometrial cancer. (1) Gargett CE et al (2009) Biol Reprod 80: 1136–45.

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