MicroRNAs 221 and 222 Inhibit Normal Erythropoiesis and Erythroleukemic Cell Growth Via Kit Receptor Downmodulation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 830-830 ◽  
Author(s):  
Nadia Felli ◽  
Laura Fontana ◽  
Elvira Pelosi ◽  
Rosanna Botta ◽  
Desirée Bonci ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
Cell Activity ◽  
Regulate Gene Expression ◽  
Functional Studies ◽  
Kit Receptor ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Erythroleukemic Cell ◽  
Erythroleukemic Cell Line

Abstract MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression primarily through translational repression. In unilineage erythropoietic (E) culture of cord blood (CB) CD34+ progenitor cells, the level of miR 221 and 222 is gradually and sharply downmodulated. Hypothetically, this decline could promote erythropoiesis by unblocking expression of key functional proteins. Our studies indicate that miR 221 and 222 target the Kit receptor: specifically, (a) the luciferase targeting assay showed that miR 221 and 222 directly interact with the 3′UTR of Kit mRNA; (b) in E culture the miR 221 and 222 level is inversely related to Kit protein expression, whereas the abundance of Kit mRNA is relatively stable. Functional studies show that treatment of CD34+ cells with miR 221 and 222, via oligonucleotide transfection or lentiviral vector infection, causes impaired proliferation and accelerated differentiation of E cells, coupled with downmodulation of Kit protein: this phenomenon, observed in E culture releasing endogenous Kit ligand (KL), is magnified in E culture supplemented with KL. Furthermore, transplantation experiments into NOD-SCID mice reveal that miR 221 or 222 treatment of CD34+ cells impairs their engraftment capacity and stem cell activity. Finally, miR 221 and 222 gene transfer impairs proliferation of the TF1 erythroleukemic cell line, expressing the Kit receptor. Altogether, our studies indicate that in human erythropoiesis the decline of miR 221 and 222 unblocks Kit protein production at translational level, thus leading to expansion of early E cells. Furthermore, overexpression of miR 221 and 222 inhibits proliferation of Kit+ erythroleukemic cells, suggesting a potential role of these microRNAs in cancer therapy.

Clinical-Scale Cultures of Cord Blood CD34+Cells to Amplify Committed Progenitors and Maintain Stem Cell Activity

2011 ◽  
Vol 20 (9) ◽  
pp. 1453-1464 ◽  
Author(s):  
Zoran Ivanovic ◽  
Pascale Duchez ◽  
Jean Chevaleyre ◽  
Marija Vlaski ◽  
Xavier Lafarge ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Cell Activity ◽  
Clinical Scale ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cord Blood Cd34

Inhibition Of CXCR2 As a Therapeutic Strategy In AML and MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 484-484 ◽  
Author(s):  
Carolina Schinke ◽  
Orsolya Giricz ◽  
Shanisha A. K. Gordon ◽  
Laura Barreyro ◽  
Tushar D. Bhagat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Leukemic Cell ◽  
Leukemia Stem Cells ◽  
P Value ◽  
Healthy Controls ◽  
Functional Studies ◽  
Cxcr2 Expression ◽  
Cd34 Cells

Abstract Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in individual patients of MDS and AML (N=16) and identified candidate genes that are consistently dysregulated at multiple immature stem and progenitor cell stages. Interleukin 8 (IL8), was one of the most consistently overexpressed genes in MDS/AML Hematolpoetic Stem Cells (HSCs) and progenitors when compared to healthy control HSCs and progenitors. IL8 is a pro-inflammatory chemokine, which is able to activate multiple intracellular signaling pathways after binding to its surface receptor CXCR2. Even though increased IL8-CXCR2 signaling has been shown to promote angiogenesis, metastasis and chemotherapy resistance in many solid tumors, its role in AML and MDS is not well elucidated. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of CXCR2 in MDS when compared to healthy controls (FDR<0.1). Most importantly, analysis of The Cancer Genome Atlas (TCGA) AML (n=200) dataset showed that CXCR2 expression was predictive of significantly adverse prognosis (log rank P value=0.0182; median survival of 245 days in cxcr2 high vs 607 days in cxcr2 low) in patients, further pointing to a critical role of IL8-CXCR2 signaling in AML/MDS. Next, we studied the functional role of IL8 and CXCR2 in AML. A panel of leukemic cell lines (THP-1, U937, KG-1, MOLM13, HL-60, K532) were screened for CXCR2 expression and revealed significantly higher expression when compared to healthy CD34+ control cells. SB-332235, a specific inhibitor of CXCR2 was used for functional studies. CXCR2 inhibition led to significant, (p<0.05) reduction in proliferation in all 6 cell lines tested and an effect was seen as early as 24 hrs of exposure. CXCR2 inhibition was found to lead to G0/G1 cell cycle arrest and trigged apoptosis in THP-1 and U937 cells (p-value 0.004 and 0.02 respectively). Incubation of primary AML/MDS bone marrow samples with SB-332235 similarly lead to significantly reduced proliferation at 24hrs, when compared to healthy CD34+ cells. Selective, and highly significant inhibition of leukemic cell growth was also seen in colony assays from primary MDS/AML samples (mean leukemic colonies in AML/MDS= 73 vs 313 in controls, P < 0.001). Interestingly, inhibition of CXCR2 in primary AML marrow samples led to induction of apoptosis in immature CD34+/CD38- cells when compared to healthy controls. Lastly, xenografting studies with THP-1 leukemic cells revealed that CXCR2 inhibitor treatment led to decreased leukemic burden and organ infiltration when compared to placebo controls in vivo. In summary we have found significantly increased expression of IL8 and its receptor CXCR2 in sorted HSCs and progenitors from AML and MDS patients. High CXCR2 expression was a marker of adverse prognosis in a large cohort of AML patients. Most importantly, in vitro and in vivo functional studies showed that CXCR2 is a potential therapeutic target in AML/MDS and is able to selectively target immature, LSC-enriched cell fractions in AML. Disclosures: No relevant conflicts of interest to declare.

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals Role of Microparticles as Messengers Enhancing Stem Cell Activity After Genetic Engineering

2012 ◽  
Vol 111 (3) ◽  
pp. 265-267 ◽  
Author(s):  
Elisa A. Liehn ◽  
Octavian Bucur ◽  
Christian Weber
Keyword(s):  
Stem Cell ◽  
Genetic Engineering ◽  
Cell Activity ◽  
Stem Cell Activity

scholarly journals The Role of Smoothened in Cancer

2020 ◽  
Vol 21 (18) ◽  
pp. 6863 ◽  
Author(s):  
Kuo-Shyang Jeng ◽  
I-Shyan Sheen ◽  
Chuen-Miin Leu ◽  
Ping-Hui Tseng ◽  
Chiung-Fang Chang
Keyword(s):  
Stem Cells ◽  
Signaling Pathway ◽  
Cell Activity ◽  
Therapeutic Approaches ◽  
Colon Cancers ◽  
Stem Cell Activity ◽  
Different Types ◽  
Hh Signaling ◽  
Novel Therapeutic

Smoothened (SMO) belongs to the Hedgehog (HH) signaling pathway, which regulates cell growth, migration, invasion and stem cells in cancer. The HH signaling pathway includes both canonical and noncanonical pathways. The canonical HH pathway functions through major HH molecules such as HH ligands, PTCH, SMO and GLI, whereas the noncanonical HH pathway involves the activation of SMO or GLI through other pathways. The role of SMO has been discussed in different types of cancer, including breast, liver, pancreatic and colon cancers. SMO expression correlates with tumor size, invasiveness, metastasis and recurrence. In addition, SMO inhibitors can suppress cancer formation, reduce the proliferation of cancer cells, trigger apoptosis and suppress cancer stem cell activity. A better understanding of the role of SMO in cancer could contribute to the development of novel therapeutic approaches.

GATA-1 Overexpression Does Not Restore Deficient Erythropoiesis in Myelodysplastic Syndromes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3430-3430
Author(s):  
Alexandra Rideau ◽  
Stephane Durual ◽  
Maciej Wiznerowics ◽  
Sylvie Ruault ◽  
Vincent Piguet ◽  
...  
Keyword(s):  
Myelodysplastic Syndromes ◽  
Culture System ◽  
Cell Cycle Control ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Differentiation Markers ◽  
Cd34 Cells ◽  
Erythroleukemic Cell

Abstract Introduction: Transcription factor GATA-1 is essential for erythroid and megakaryocytic maturation. A role of GATA-1 in cell cycle control is suggested by the fact that GATA-1 mutations are associated with hematopoietic precursor proliferation and leukemogenesis and that defective GATA-1 expression is observed in in vitro cultures of erythroid myelodysplastic precursors. In order to study more in detail a potential role of GATA-1 dysregulation in myelodysplastic syndromes (MDS), we constructed lentiviral vectors with the aim to overexpress GATA-1 protein or to inhibit its production in erythroid progenitors. Methods and Results: Using RNA interference technology we tested how GATA-1 inhibition interfered with erythroid differentiation. We selected one GATA-1 specific siRNA, which abolished expression of GATA-1 protein in K562 and HEL erythroleukemic cell lines, as verified by Western blot. Interestingly, we observed in parallel to the disappearance of GATA-1 protein, decreased proliferation rates (170x for K562 and 30x for HEL after 17 days of culture) and increased apoptosis. Normal CD34+ cells cultured in our culture system and transduced with the siRNA vector were practically blocked in their erythroid differentiation: 14 % glyco+/CD36- mature erythroid cells versus 81 % in untransduced and 80 % in cultures transduced with control lentivector (obtained after 14 days of culture). Differentiation into myeloid cells was not affected. To overexpress GATA-1 we cloned the wild-type as well as a mutated, caspase-resistant, form of GATA-1 in a pWPIR-ires-GFP bicistronic lentivector. Functionality of both lentivectors was validated in HeLa cells. For the study of GATA-1 in primary human hematopoietic cells we used an in vitro culture system in which CD34+ progenitors differentiate into mature red blood cells in the presence of erythropoietin, IL-3, and SCF. Transduction of CD34+ cells with lentivectors led to increase of GATA-1 mRNA (400-fold) measured by Realtime RT-PCR and to detection of protein. No difference was observed in cell numbers, expression of erythroid differentiation markers and survival between cells transduced with control vector and with pWPIR-GATA-1-ires-GFP. CD34+ cells from 3 patients with low-risk MDS in this culture system proliferated less (15x ± 13 amplification after 14 days of culture versus 72x ± 35 for normal precursors) differentiated less, and became apoptotic earlier than normal cells. However, overexpression of GATA-1 did not restore proliferation rate, nor did it lead to increased erythroid differentiation, or increase in survival. Conclusion: GATA-1 overexpression was not able to overcome defective erythroid differentiation of myelodysplastic progenitors, nor did it increase differentiation of normal erythroid progenitors. On the other hand, GATA-1 inhibition in normal erythroid precursors led to blockage of erythroid differentiation. We therefore assume that either factors upstream of GATA-1 or additional, GATA-1 independent factors, are responsible for the myelodysplastic phenotype.

A Forward RNAi Screen Identifies p38 MAP Kinase As a Druggable Target for Expansion of Human Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2352-2352
Author(s):  
Aurelie Baudet ◽  
Jonas Larsson
Keyword(s):  
Stem Cell ◽  
Next Generation Sequencing ◽  
Ex Vivo ◽  
Cell Activity ◽  
P38 Map Kinase ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Generation Sequencing

Abstract Abstract 2352 While the mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood, intense efforts are being devoted to develop conditions that would enable ex vivo amplification of transplantable stem cells. We have developed a screening strategy to assess modulators of human HSCPs using pooled lentiviral shRNA libraries transduced into cord blood-derived stem/progenitor cells. We use the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs leading to prolonged maintenance or expansion of undifferentiated HSPCs. This approach enables complex, pooled screens in large numbers of cells. We further take advantage of next generation sequencing to track shRNA-transduced cells with high fidelity, allowing thousands of perturbations to be tested in parallel in a single pool of cells. Here we used a library of 2500 shRNAs targeting around 800 genes, mainly kinases and phosphatases, which include large numbers of “druggable” genes. The shRNAs composing the library were monitored by next generation sequencing in cord blood CD34+ cells sampled one day after transduction and following 20 days of culture, to determine their relative change in distribution during the culture period. The sequencing of all integrated proviruses containing shRNAs generated over 3 million sequences per sample. Analysis of the shRNA distribution before and after culture in 3 replicate screens revealed a dramatic enrichment of 3 independent shRNAs targeting MAPK14 (p38α). We could confirm that inhibition of MAPK14, mediated by RNA interference, leads to a proliferation advantage of CD34+ cells in culture, identifying p38 as a possible target for ex vivo stem cell expansion. We next used the chemical inhibitor SB203580 to inhibit p38 without genetic perturbation and in a non-permanent fashion. Culture of CD34+ cells under optimized conditions for expansion (serum-free medium supplemented with SCF, TPO and FLT3) with or without SB203580 showed a 3-fold increase of the stem cell enriched CD34+CD90+ cell population during 5 days of culture in SB203580 treated cells compared to control cells. Furthermore, when transplanted to immune-deficient NSG mice, SB203580 treated cells showed a dramatic increase in repopulating activity, as evidenced by the percentage of human engraftment 10 weeks after transplantation (SB203580: 30±6.4% vs control: 7.5±3.6%, p< 0,001). Thus, under otherwise optimized culture conditions for stem cell expansion, the addition of the p38 inhibitor leads to a significant increase in stem cell activity. To understand the basis for the increase in stem cell activity, we assayed SB203580 treated cells with respect to cell cycling and survival rate, but found a similar cell division history (shown by cytoplasmic dye dilution assays) and similar levels of apoptotic cells (shown by Annexin V staining) compared to control cells. Interestingly, however, when the cells were assayed for reactive oxygen species (ROS), we detected significantly reduced levels of ROS in SB203580 treated cells, implicating modulation of ROS as a possible mechanism behind the enhanced stem cell output. Taken together, using a functional forward genetic screen, we have been able to identify p38 MAP kinase as a highly promising target to enhance hematopoietic stem cell activity in ex vivo expansion settings. These results further support the feasibility of pooled RNAi screens in conjunction with next generation sequencing to identify genes and pathways that regulate primary human stem cell populations. Disclosures: No relevant conflicts of interest to declare.

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