| 1. |
|
|
| 2. |
- Baudet, Aurelie, et al.
(författare)
-
RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion.
- 2012
-
Ingår i: Blood. - : American Society of Hematology. - 1528-0020 .- 0006-4971. ; 119:26, s. 6255-6258
-
Tidskriftsartikel (refereegranskat)abstract
- We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells, using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacological inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit.
|
|
| 3. |
- Baudet, Aurélie, et al.
(författare)
-
Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis
- 2022
-
Ingår i: Bio-protocol. - 2331-8325. ; 12:6
-
Tidskriftsartikel (refereegranskat)abstract
- Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner.
|
|
| 4. |
- Galeev, Roman, et al.
(författare)
-
Forward RNAi screens in human hematopoietic stem cells
- 2017
-
Ingår i: Methods in Molecular Biology. - New York, NY : Springer New York. - 1064-3745. ; 1622, s. 29-50
-
Bokkapitel (refereegranskat)abstract
- Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNA interference (RNAi)-based screens combined with next-generation sequencing (NGS) in primary human hematopoietic stem and progenitor cells (HSPCs). These cells are suitable targets for complex, selection-based screens using pooled lentiviral short hairpin RNA (shRNA) libraries. The screening approach presented in this chapter is a promising tool to dissect regulatory mechanisms in hematopoietic stem cells (HSCs) and somatic stem cells in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.
|
|
| 5. |
- Galeev, Roman, et al.
(författare)
-
Genome-wide RNAi Screen Identifies Cohesin Genes as Modifiers of Renewal and Differentiation in Human HSCs
- 2016
-
Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 14:12, s. 2988-3000
-
Tidskriftsartikel (refereegranskat)abstract
- To gain insights into the regulatory mechanisms of hematopoietic stem cells (HSCs), we employed a genome-wide RNAi screen in human cord-blood derived cells and identified candidate genes whose knockdown maintained the HSC phenotype during culture. A striking finding was the identification of members of the cohesin complex (STAG2, RAD21, STAG1, and SMC3) among the top 20 genes from the screen. Upon individual validation of these cohesin genes, we found that their knockdown led to an immediate expansion of cells with an HSC phenotype in vitro. A similar expansion was observed in vivo following transplantation to immunodeficient mice. Transcriptome analysis of cohesin-deficient CD34(+) cells showed an upregulation of HSC-specific genes, demonstrating an immediate shift toward a more stem-cell-like gene expression signature upon cohesin deficiency. Our findings implicate cohesin as a major regulator of HSCs and illustrate the power of global RNAi screens to identify modifiers of cell fate.
|
|
| 6. |
- Hultmark, Simon, et al.
(författare)
-
Combinatorial molecule screening identifies a novel diterpene and the BET inhibitor CPI-203 as differentiation inducers of primary acute myeloid leukemia cells
- 2021
-
Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 1592-8721 .- 0390-6078. ; 106:10
-
Tidskriftsartikel (refereegranskat)abstract
- Combination treatment has proven effective for patients with acute promyelocytic leukemia, exemplifying the importance of therapy targeting multiple components of oncogenic regulation for a successful outcome. However, recent studies have shown that the mutational complexity of acute myeloid leukemia (AML) precludes the translation of molecular targeting into clinical success. Here as a complement to genetic profiling, we used unbiased, combinatorial in vitro drug screening to identify pathways that drive AML and to develop personalized combinatorial treatments. First, we screened 513 natural compounds on primary AML cells and identified a novel diterpene (H4) that preferentially induced differentiation of FLT3 wild-type AMLs, while FLT3-ITD/mutations conferred resistance. The responding samples to H4, displayed increased expression of myeloid markers, a clear decrease in the nuclear-cytoplasmic ratio and the potential of re-activation of the monocytic transcriptional program reducing leukemia propagation in vivo. By combinatorial screening using H4 and molecules with defined targets, we demonstrated that H4 induces differentiation by the activation of protein kinase C (PKC) signaling pathway, and in line with this, activates PKC phosphorylation and translocation of PKC to the cell membrane. Furthermore, the combinatorial screening identified a bromo- and extra-terminal domain (BET) inhibitor that could further improve H4-dependent leukemic differentiation in FLT3 wild-type monocytic AML. Taken together, this illustrates the value of an unbiased and multiplex screening platform for developing combinatorial therapeutic approaches for AML.
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
- Karlsson, Christine, et al.
(författare)
-
Forward RNAi screens in human stem cells.
- 2010
-
Ingår i: Methods in Molecular Biology. - Totowa, NJ : Humana Press. - 1940-6029. ; 650, s. 29-43
-
Tidskriftsartikel (refereegranskat)abstract
- Identifying the genes and pathways that regulate self-renewal and differentiation in somatic stem cells is a central goal in stem cell and cancer biology. Here, we describe a method for RNAi-based screens in primary human hematopoietic stem and progenitor cells. These cells are suitable targets for complex, selection-based screens using pooled lentiviral shRNA libraries. The screening approach is a promising new tool to dissect regulatory mechanisms in hematopoietic and somatic stem cells, in general, and may be particularly useful to identify gene targets and modifiers that can be further exploited in strategies for ex vivo stem cell expansion.
|
|
