Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells

Galat, Yekaterina; Elcheva, Irina; Dambaeva, Svetlana; Katukurundage, Dimantha; Beaman, Kenneth D.; Iannaccone, Philip M.; Galat, Vasiliy

doi:10.1016/j.exphem.2018.05.007

Cited by 15 publications

(8 citation statements)

References 53 publications

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“…Regarding isoDSV-iPSCs, this cell line was established as a result of the spontaneous loss of an extra copy of Chromosome 21 in DSV-iPSCs. These three iPS cell lines were differentiated into endothelial cells (SR2-iECs, DSV-iECs, isoDSV-iECs), which were previously characterized in [24,31,32].…”

Section: Characterization and Bioinformatic Functional Assessment Of mentioning

confidence: 99%

Down syndrome iPSC model: endothelial perspective on tumor development

et al. 2020

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Trisomy 21 (T21), known as Down syndrome (DS), is a widely studied chromosomal abnormality. Previous studies have shown that DS individuals have a unique cancer profile. While exhibiting low solid tumor prevalence, DS patients are at risk for hematologic cancers, such as acute megakaryocytic leukemia and acute lymphoblastic leukemia. We speculated that endothelial cells are active players in this clinical background. To this end, we hypothesized that impaired DS endothelial development and functionality, impacted by genome-wide T21 alterations, potentially results in a suboptimal endothelial microenvironment with the capability to prevent solid tumor growth. To test this hypothesis, we assessed molecular and phenotypic differences of endothelial cells differentiated from Down syndrome and euploid iPS cells. Microarray, RNA-Seq, and bioinformatic analyses revealed that most significantly expressed genes belong to angiogenic, cytoskeletal rearrangement, extracellular matrix remodeling, and inflammatory pathways. Interestingly, the majority of these genes are not located on Chromosome 21. To substantiate these findings, we carried out functional assays. The obtained phenotypic results correlated with the molecular data and showed that Down syndrome endothelial cells exhibit decreased proliferation, reduced migration, and a weak TNF-α inflammatory response. Based on this data, we provide a set of genes potentially associated with Down syndrome's elevated leukemic incidence and its unfavorable solid tumor microenvironment-highlighting the potential use of these genes as therapeutic targets in translational cancer research.

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Section: Characterization and Bioinformatic Functional Assessment Of mentioning

confidence: 99%

Down syndrome iPSC model: endothelial perspective on tumor development

et al. 2020

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“…In particular, Wnt activates mesendodermal genes during colony maturation towards the pluripotent state. Furthermore, in many studies [ 17 , 41 , 49 ] exogenous Wnt/catenin was used to stimulate the production of hematopoietic populations from hPSCs. The comparative cytometry assays revealed that β-catenin was abundantly expressed by the 3D/EB and was almost non-expressed by the 2D/monolayer cells ( Figure 4 A) ( Figure S6 ).…”

Section: Resultsmentioning

confidence: 99%

Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems

Mora-Roldan

Ramírez-Ramírez

Pelayo

et al. 2021

Cells

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Background. In vitro methods for hematopoietic differentiation of human pluripotent stem cells (hPSC) are a matter of priority for the in-depth research into the mechanisms of early embryogenesis. So-far, published results regarding the generation of hematopoietic cells come from studies using either 2D or 3D culture formats, hence, it is difficult to discern their particular contribution to the development of the concept of a unique in vitro model in close resemblance to in vivo hematopoiesis. Aim of the study. To assess using the same culture conditions and the same time course, the potential of each of these two formats to support differentiation of human pluripotent stem cells to primitive hematopoiesis without exogenous activation of Wnt signaling. Methods. We used in parallel 2D and 3D formats, the same culture environment and assay methods (flow cytometry, IF, qPCR) to investigate stages of commitment and specification of mesodermal, and hemogenic endothelial cells to CD34 hematopoietic cells and evaluated their clonogenic capacity in a CFU system. Results. We show an adequate formation of mesoderm, an efficient commitment to hemogenic endothelium, a higher number of CD34 hematopoietic cells, and colony-forming capacity potential only in the 3D format-supported differentiation. Conclusions. This study shows that the 3D but not the 2D format ensures the induction and realization by endogenous mechanisms of human pluripotent stem cells’ intrinsic differentiation program to primitive hematopoietic cells. We propose that the 3D format provides an adequate level of upregulation of the endogenous Wnt/β-catenin signaling.

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“…At later stages, formation of endothelium was significantly inhibited. Furthermore, expression of RUNX1, an endothelial marker with elevated expression levels in emergent hematopoietic progenitor cells 48 and during definitive hematopoiesis 49,50 was significantly downregulated in GLI‐edited clones.…”

Section: Discussionmentioning

confidence: 99%

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment

Galat

Perepitchka

et al. 2021

Stem Cells

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GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

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Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells

Cited by 15 publications

References 53 publications

Down syndrome iPSC model: endothelial perspective on tumor development

Down syndrome iPSC model: endothelial perspective on tumor development

Assessment of the Hematopoietic Differentiation Potential of Human Pluripotent Stem Cells in 2D and 3D Culture Systems

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment

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