FANCA knockout in human embryonic stem cells causes severe growth disadvantage

Abstract

Fanconi anemia (FA) is an autosomal recessive disease characterized by congenital abnormalities and progressive bone marrow failure (BMF) in the first decades of life, which develops into hematologic malignancies in 33% of cases. Although FA knockout mouse models have been established, they do not display spontaneous BMF and therefore do not fully mimic the hematopoietic condition seen in FA patients. As there is mounting evidence that the hematopoietic impairment starts already before birth, a human pluripotent stem cell model would constitute a more appropriate system to investigate the mechanism underlying BMF in FA and its developmental basis. Human pluripotent stem cells such as human embryonic stem cells (hESC) offer an unlimited source of cells with the capacity of differentiating into all cell types of the body. When supplied with the right combination of cytokines and growth factors, these cells can be fated into hematopoietic cells during in vitro hematopoietic differentiation, a process known to closely mimic the ontogeny of the hematopoietic system as it occurs in vivo. Modeling FA in human pluripotent stem cells would thus allow us to assess the hematopoietic differentiation potential of FA-deficient hESC and try to unravel at which point in the development of the blood system the FA deficiency causes problems.
To generate a human pluripotent stem cell model for FA, we have created a knockout of FANCA in hESC using zinc finger nuclease (ZFN) technology. We found that whereas cells with one disrupted allele retained an unaltered proliferation potential, disruption of both alleles caused a severe growth disadvantage. As a result, heterogeneous cultures arose due to the presence of cells still carrying an unaffected FANCA allele, quickly outgrowing the knockout cells. When pure cultures of FANCA knockout hESC were pursued either through selection or single cell cloning, this rapidly resulted in growth arrest and such cultures could not be maintained. Although the inherent growth defect hampers the establishment of a human pluripotent FA disease model, our findings highlight the importance of a functional FA pathway at the pluripotent stem cell stage.
In parallel, we have tested several protocols for their ability to induce a robust and efficient in vitro hematopoietic differentiation from hESC. Out of three different hematopoietic differentiation systems (stromal co-­culture system, spin embryoid body system, monolayer culture system) the serum-­free, feeder-­free monolayer culture system showed the most consistent results in our hands and we have used this system to conduct a kinase inhibitor (KI) screen to identify compounds that can improve the in vitro hematopoietic differentiation from hESC. Despite some degree of variation inherent to biological processes such as differentiation, we were able to identify two KIs that led to increased hematopoietic marker expression and the presence of large numbers of cells with a hematopoietic progenitor appearance. Both KIs inhibited TGF? receptors (ALK1 and ALK5) and we saw the most prominent induction in cells expressing hematopoietic marker such as CD43 and CD45 when adding the KIs early in the differentiation process (between day 4-­6). As such, our findings confirmed recent reports on the role of TGFbeta during in vitro hematopoietic differentiation from hESC.

Date of Award	14 Oct 2014
Original language	English
Supervisor	Karen Sermon (Jury) & C. Verfaillie (Promotor)