Page 44 - 2018_09-Mondo

Haematologica 2018 Volume 103(9):1444-1450
Ferrata Storti Foundation
Hematopoiesis
Transient inhibition of NF-κB signaling enhances ex vivo propagation of human hematopoietic stem cells
Mehrnaz Safaee Talkhoncheh, Agatheeswaran Subramaniam,
Mattias Magnusson, Praveen Kumar, Jonas Larsson and Aurélie Baudet
Division of Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Sweden
ABSTRACT
Despite extensive studies, defining culture conditions in which hematopoietic stem cells can be expanded ex vivo has been chal- lenging. Here we show that chemical inhibition of the NF-κB sig- naling pathway leads to a significant improvement of hematopoietic stem cell function from ex vivo cultured human umbilical cord blood derived CD34+ cells. We found a distinct peak of activation of the NF-κB pathway shortly after cells were put in culture, and consequently inhibi- tion of the pathway was both necessary and sufficient during the first 24 hours of culture where it reduced the levels of several pro-inflammatory cytokines. Taken together, NF-κB pathway inhibition facilitates propaga- tion of hematopoietic stem cells in culture and may complement other strategies for hematopoietic stem cell expansion by relieving stress sig- nals that are induced as an immediate response to culture initiation.
Introduction
Umbilical cord blood (CB) has emerged as a promising source of hematopoietic stem and progenitor cells (HSPCs) for transplantation. Unfortunately, the use of CB grafts is mainly restricted to pediatric transplantation as the number of HSPCs per unit is usually too low to allow the infusion of the minimal cell dose required for successful transplantation in adults.1-3 Potentially, the ex vivo expansion of CB- derived HSPCs prior to transplantation could extend the use of CB transplantation to adult patients.4 Successful HSPC expansion would further facilitate the develop- ment of more advanced cell therapies for hematologic diseases, including gene ther- apy applications.5
Hematopoietic stem cell self-renewal is regulated by a combination of positive- negative feedback signaling.6 An incomplete understanding of this complex regula- tory mechanism and how it would fit in a culture system has limited successful HSC ex vivo expansion. Despite the well-studied role of positive signals such as growth factors on HSC self-renewal, several studies highlighted the importance of inhibito- ry signals in restricting HSC self-renewal and function ex vivo. It has been suggested that, when under culture, HSCs undergo an immediate and transient “culture shock” that compromises their maintenance and function. This is due to upregulation of negative regulators of HSC expansion such as tumor necrosis factor (TNF signaling) and members of the aryl hydrocarbon receptor (AhR) signaling pathway.7,8 Furthermore, the secretion of inhibitory signaling factors, mainly from more differ- entiated cells, constitutes a major restriction to all HSC culture systems.8 To further explore this notion, our laboratory has performed high-throughput forward RNAi screens and identified negative regulators of ex vivo expansion of human HSPCs, including the cohesin family of genes, and p38 (MAPK14).9,10 Through these studies, we also identified shRNAs which display a remarkable ability to expand phenotyp- ically-defined HSPCs in culture but whose gene target has not been validated. We reasoned that, although it would be very difficult to identify the gene targets responsible for the phenotype, the molecular characterization of cells targeted by such off-target shRNAs could provide general clues about the context under which HSPCs can be propagated ex vivo. Here, we report that the expression signature of cells showing enhanced expansion from one specific off-target shRNA (sh758)
Correspondence:
jonas.larsson@med.lu.se
Received: January 12, 2018. Accepted: May 17, 2018. Pre-published: June 7, 2018.
doi:10.3324/haematol.2018.188466
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/9/xxx
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