| 1. |
- Guibentif, Carolina, et al.
(författare)
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Single-Cell Analysis Identifies Distinct Stages of Human Endothelial-to-Hematopoietic Transition
- 2017
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 19:1, s. 10-19
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Tidskriftsartikel (refereegranskat)abstract
- During development, hematopoietic cells originate from endothelium in a process known as endothelial-to-hematopoietic transition (EHT). To study human EHT, we coupled flow cytometry and single-cell transcriptional analyses of human pluripotent stem cell-derived CD34(+) cells. The resulting transcriptional hierarchy showed a continuum of endothelial and hematopoietic signatures. At the interface of these two signatures, a unique group of cells displayed both an endothelial signature and high levels of key hematopoietic stem cell-associated genes. This interphase group was validated via sort and subculture as an immediate precursor to hematopoietic cells. Differential expression analyses further divided this population into subgroups, which, upon subculture, showed distinct hematopoietic lineage differentiation potentials. We therefore propose that immediate precursors to hematopoietic cells already have their hematopoietic lineage restrictions defined prior to complete downregulation of the endothelial signature. These findings increase our understanding of the processes of de novo hematopoietic cell generation in the human developmental context.
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| 2. |
- Rönn, Roger E., et al.
(författare)
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Reactive Oxygen Species Impair the Function of CD90+ Hematopoietic Progenitors Generated from Human Pluripotent Stem Cells
- 2017
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 35:1, s. 197-206
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Tidskriftsartikel (refereegranskat)abstract
- Cell stressors, such as elevated levels of reactive oxygen species (ROS), adversely affect hematopoietic stem cell (HSC) reconstituting ability. However, the effects of ROS have not been evaluated in the context of hematopoietic development from human pluripotent stem cells (hPSCs). Using our previously described in vitro system for efficient derivation of hematopoietic cells from hPSCs, we show that the vast majority of generated hematopoietic cells display supraphysiological levels of ROS compared to fresh cord blood cells. Elevated ROS resulted in DNA damage of the CD34+ hematopoietic fraction and, following functional assays, reduced colony formation and impaired proliferative capacity. Interestingly, all the proliferative potential of the most primitive hematopoietic cells was limited to a small fraction with low ROS levels. We show that elevation of ROS in hPSC-derived hematopoietic cells is contributed by multiple distinct cellular processes. Furthermore, by targeting these molecular processes with 4 unique factors, we could reduce ROS levels significantly, yielding a 22-fold increase in the most primitive CD90+ CD34+ hematopoietic cells with robust growth capacity. We demonstrate that the ROS reducing factors specifically reduced ROS in more primitive hematopoietic fractions, in contrast to endothelial cells that maintained low ROS levels in the cultures. We conclude that high levels of ROS in in vitro differentiation systems of hPSCs is a major determinant in the lack of ability to generate hematopoietic cells with similar proliferation/differentiation potential to in vivo hematopoietic progenitors, and suggest that elevated ROS is a significant barrier to generating hPSC-derived repopulating HSCs. Stem Cells 2017;35:197–206.
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| 3. |
- Rönn, Roger, et al.
(författare)
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Retinoic Acid regulates hematopoietic development from human pluripotent stem cells.
- 2015
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 4:2, s. 269-281
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Tidskriftsartikel (refereegranskat)abstract
- The functions of retinoic acid (RA), a potent morphogen with crucial roles in embryogenesis including developmental hematopoiesis, have not been thoroughly investigated in the human setting. Using an in vitro model of human hematopoietic development, we evaluated the effects of RA signaling on the development of blood and on generated hematopoietic progenitors. Decreased RA signaling increases the generation of cells with a hematopoietic stem cell (HSC)-like phenotype, capable of differentiation into myeloid and lymphoid lineages, through two separate mechanisms: by increasing the commitment of pluripotent stem cells toward the hematopoietic lineage during the developmental process and by decreasing the differentiation of generated blood progenitors. Our results demonstrate that controlled low-level RA signaling is a requirement in human blood development, and we propose a new interpretation of RA as a regulatory factor, where appropriate control of RA signaling enables increased generation of hematopoietic progenitor cells from pluripotent stem cells in vitro.
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| 4. |
- Saxena, Shobhit, et al.
(författare)
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Cyclic AMP Signaling through Epac Axis Modulates Human Hemogenic Endothelium and Enhances Hematopoietic Cell Generation
- 2016
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Ingår i: Stem Cell Reports. - : Elsevier BV. - 2213-6711. ; 6:5, s. 692-703
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoietic cells emerge from hemogenic endothelium in the developing embryo. Mechanisms behind human hematopoietic stem and progenitor cell development remain unclear. Using a human pluripotent stem cell differentiation model, we report that cyclic AMP (cAMP) induction dramatically increases HSC-like cell frequencies. We show that hematopoietic cell generation requires cAMP signaling through the Exchange proteins activated by cAMP (cAMP-Epac) axis; Epac signaling inhibition decreased both hemogenic and non-hemogenic endothelium, and abrogated hematopoietic cell generation. Furthermore, in hematopoietic progenitor and stem-like cells, cAMP induction mitigated oxidative stress, created a redox-state balance, and enhanced C-X-C chemokine receptor type 4 (CXCR4) expression, benefiting the maintenance of these primitive cells. Collectively, our study provides insights and mechanistic details on the previously unrecognized role of cAMP signaling in regulating human hematopoietic development. These findings advance the mechanistic understanding of hematopoietic development toward the development of transplantable human hematopoietic cells for therapeutic needs.
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