Activation of Endogenous Transcription Factors Expands Arterial Endothelial Cells and Enhances Hematopoietic Progenitor Production from Human Pluripotent Stem Cells

מושגי ליבה

Activation of nine endogenous transcription factors, including RUNX1T1, in human pluripotent stem cells leads to an expansion of arterial endothelial cells and increased production of functional hematopoietic progenitors, mediated in part by the paracrine factor IGFBP2.

תקציר

The researchers compared the transcriptomes of endothelial and hematopoietic cells derived in vitro from human induced pluripotent stem cells (iPSCs) to those derived in vivo from the human aorta-gonad-mesonephros (AGM) region. This analysis identified nine transcription factors (RUNX1T1, NR4A1, GATA2, SMAD7, ZNF124, SOX6, ZNF33A, NFAT5, TFDP2) that were expressed at lower levels in the in vitro-derived cells compared to their in vivo counterparts.
The researchers then developed a novel doxycycline-inducible CRISPR activation (iCRISPRa) system to induce the expression of these nine transcription factors in human iPSCs. Single-cell RNA sequencing analysis revealed that activation of these factors led to an expansion of an arterial-like endothelial cell population, which was associated with increased production of functional hematopoietic progenitors.
Further investigation showed that the increased hematopoietic progenitor potential was mediated in part by the paracrine factor IGFBP2, which was highly upregulated in the expanded arterial endothelial cell population upon transcription factor activation. IGFBP2 was found to induce a metabolic shift in the endothelial cells, reducing glycolysis and increasing mitochondrial respiration, which is known to be important for the endothelial-to-hematopoietic transition.
These findings provide new insights into the molecular regulation of hematopoietic development and demonstrate the utility of the iCRISPRa system for studying dynamic processes controlling cell fate decisions in human pluripotent stem cell models.

סטטיסטיקה

The expression level of the nine target transcription factors (RUNX1T1, NR4A1, GATA2, SMAD7, ZNF124, SOX6, ZNF33A, NFAT5, TFDP2) was significantly higher in the arterial hemogenic endothelial cells derived from the human AGM region compared to the in vitro-derived endothelial and hematopoietic cells.
Activation of the nine target genes in human iPSCs led to a 3.33 ± 0.73 fold increase in the proportion of CD34+DLL4+ arterial-like cells.
The number of hematopoietic colony-forming units (CFU-E and CFU-GM) was significantly increased upon activation of the target genes in human iPSCs.
Addition of recombinant IGFBP2 protein to the in vitro differentiation culture resulted in a significant increase in the total number of hematopoietic CFU colonies.

ציטוטים

"Activation of target transcription factors using our novel CRISPR strategy results in transcriptional remodelling and a steer in cell identity that we detected as a functional difference in the hematopoietic progenitor profile."
"IGFBP2 induces the upregulation of RUNX1 and OxPhos genes and the metabolic remodelling of endothelial cells."

תובנות מפתח מזוקקות מ:

A novel human pluripotent stem cell-based gene activation system identifies IGFBP2 as a mediator in the production of hematopoietic progenitors in vitro

by Petazzi,P., ... ב- www.biorxiv.org 01-14-2024

https://www.biorxiv.org/content/10.1101/2024.01.14.575573v1

שאלות מעמיקות

What other signaling pathways or transcriptional regulators might interact with RUNX1T1 to control IGFBP2 expression and hematopoietic progenitor development?

RUNX1T1, as a transcriptional regulator, may interact with various signaling pathways and transcription factors to control IGFBP2 expression and hematopoietic progenitor development. One potential interaction could be with the Wnt signaling pathway, known to play a crucial role in hematopoietic development. Activation of Wnt signaling has been linked to the regulation of hematopoietic stem cell self-renewal and differentiation. Additionally, transcription factors such as GATA2, which was identified as a target gene in this study, may also interact with RUNX1T1 to regulate IGFBP2 expression. GATA2 is known to be involved in hematopoietic stem cell development and maintenance. Furthermore, SMAD7, another target gene identified, may interact with RUNX1T1 to modulate the TGF-beta signaling pathway, which has been implicated in hematopoietic development.

How do the in vitro-derived arterial endothelial cells differ functionally from their in vivo counterparts, beyond their hematopoietic potential?

In addition to their hematopoietic potential, in vitro-derived arterial endothelial cells may differ functionally from their in vivo counterparts in several ways. One significant difference could be in their response to environmental cues and signaling molecules. In vivo, arterial endothelial cells interact with a complex microenvironment that includes neighboring cells, extracellular matrix components, and circulating factors, which may influence their behavior and function. In contrast, in vitro-derived arterial endothelial cells may lack some of these interactions, leading to differences in their functional properties.
Another difference could be in their maturation and specialization. In vivo, arterial endothelial cells undergo a process of maturation and specialization to perform specific functions within the vascular system. This process may be more dynamic and regulated in vivo compared to in vitro conditions, leading to differences in the functional characteristics of the cells.
Additionally, the in vitro-derived arterial endothelial cells may lack the full spectrum of cellular interactions and signaling cascades present in the in vivo environment, which could impact their functional properties, such as response to stimuli, gene expression profiles, and overall behavior.

Could the iCRISPRa approach be applied to other developmental processes to better recapitulate in vivo cellular differentiation and maturation in vitro?

Yes, the iCRISPRa approach could be applied to other developmental processes to better recapitulate in vivo cellular differentiation and maturation in vitro. By using this inducible CRISPR-mediated activation strategy, researchers can precisely control the expression of key transcription factors and signaling molecules involved in various developmental pathways. This approach allows for the manipulation of gene expression levels in a temporal and spatial manner, mimicking the dynamic changes that occur during development.
For example, the iCRISPRa approach could be used to study the development of other cell lineages, such as neuronal cells, muscle cells, or epithelial cells, by activating specific transcription factors and signaling pathways known to be critical for their differentiation and maturation. By modulating the expression of key regulators, researchers can guide the cells towards a desired fate and study the molecular mechanisms underlying their development.
Overall, the iCRISPRa approach provides a powerful tool for studying dynamic processes controlling developmental events and can be applied to a wide range of systems to recapitulate abnormal phenotypes characterized by ectopic activation of specific endogenous gene expression. This approach holds great potential for advancing our understanding of developmental biology and disease modeling in vitro.