Embryonic stem cell derived macrophages as a model for studying liver fibrosis and a potential source of cells for therapy
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Date
08/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Haideri, Sharmin Shabbir
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Abstract
The difference between the number of patients needing transplantation for chronic
liver disease and the number of organ donors is growing, drawing attention to the
urgent requirement for novel therapies. Chronic liver injury is commonly caused by
viral hepatitis, alcohol consumption, obesity and metabolic disorders. Prolonged liver
injury leads to fibrosis, hepatic scarring and eventually cirrhosis. This project is based
on previous studies demonstrating the therapeutic effects of bone marrow-derived
macrophages (BMDM) in a murine model of liver fibrosis. BMDM facilitated fibrosis
regression and improved liver regeneration. Pro-resolution macrophages exhibited
increased expression of MMPs, growth factors and phagocytosis-related genes.
However, macrophages derived from bone marrow are inherently heterogeneous
and difficult to genetically manipulate. To overcome this limitation, our laboratory
has established a protocol whereby pure populations of macrophages can be
produced in significant numbers from murine embryonic stem cells (ESC) in vitro,
providing an essentially limitless source of macrophages. The first goal of this project
was to compare macrophages derived from ESCs (ESDM) with classical BMDM. ESDM
displayed characteristic macrophage morphology, could be activated and responded
to different cytokines in vitro, and were functionally phagocytic. However, they
displayed some differences in their gene expression profile, and were found to be
less phagocytic than BMDM. We then assessed whether ESDM could be used in the
treatment of a murine model of hepatic injury induced by carbon tetrachloride
administration. ESDM therapy helped in the regression of liver fibrosis, down-regulated
the number of fibrogenic myofibroblasts, and activated liver progenitor
cells. However, a higher number of ESDM compared to BMDMs were required to
exert that effect. To assess whether ESDM may be similar to yolk sac derived tissue-resident
macrophages, rather than monocyte-derived, we compared their behaviour
in a Kupffer cell repopulation assay. Macrophages were depleted using liposomal
clodronate treatment then animals were transplanted with either ESDM or BMDM.
We demonstrated that ESDM were more efficient than BMDM at repopulating the
Kupffer cell compartment and reversing the effects of liposomal clodronate
treatment in mice. It is well known that macrophages are very difficult to genetically
modify. So our strategy was to genetically modify ESC and then differentiate them to
macrophages that carry the modification. By genetically modifying ESCs, we
attempted to produce pro-fibrolytic ESDM that over-express MMP12 which is a
member of the matrix metalloproteinase family of genes that mainly degrades
elastin, an extracellular matrix component. We initially employed a Tet-On 3G
expression system to create an ESC line where MMP12 could be expressed in an
inducible manner in differentiated macrophages. However, although this inducible
strategy functioned in undifferentiated ESCs we could not induce the expression of
MMP12 in differentiated macrophages. In an attempt to overcome possible gene-silencing
issues, we designed and constructed an expression strategy such that
Mmp12 was expressed specifically in macrophages. The ESC line was built such that
Mmp12 expression would be driven by the promoter of macrophage colony
stimulating factor-1 receptor gene (Csf-1r or c-fms). Using the CRISPR/Cas9 strategy,
we successfully targeted the Mmp12 cDNA to the Csf-1r locus but ESDM that were
differentiated from targeted ESC lines did not express Mmp12. Thus, despite having
adopted two independent strategies, we have failed to generate genetically modified
macrophages. As a first step to translate the therapeutic effects of macrophages into
the clinical setting, we optimized a feeder- and serum-free protocol to efficiently
generate macrophages from human induced pluripotent stem cells.