Genetic programming of human iPSC-derived macrophages provides a tool to study the Erythroblastic Island niche in vitro
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Date
25/11/2019Author
Lopez Yrigoyen, Martha Beatriz
Metadata
Abstract
Macrophages have been attracting much attention as they are present in many
tissues and organs, and are involved in homeostatic tissue function and disease.
Studies involving human macrophages have been hampered due to the ethical
constraints and technical difficulties related to their isolation/derivation for in vitro
expansion, and to the difficulty of genetically modifying them to interrogate the role
of specific factors in macrophage behaviour. Thus, an off-the-shelf source of
macrophages that is amenable to the vast arsenal of genetic manipulation
techniques, such as induced pluripotent stem cells (iPSCs), represents a valuable
tool in the macrophage field.
As a proof of principle, we used a human iPSC-line that was targeted in the safe
harbour AAVS1 locus to express the fluorescent protein ZsGreen constitutively
(SFCi55-ZsG). We demonstrate efficient production of terminally differentiated
macrophages from the SFCi55-ZsG iPSC-line that fluoresce green. Macrophages
derived from this targeted cell line are indistinguishable from those generated from
their parental line in terms of gene expression, cell surface marker expression and
phagocytic ability. Furthermore, genetically modified macrophages could be
activated/polarised to an M (LPS+ IFN-ϒ), M (IL10) or M (IL4) phenotype and
retained their plasticity-related abilities, as they were able to switch from one
activated state to another. These results showed that targeting of iPSCs via the
AAVS1 locus allows for the production of fully functional genetically engineered
macrophages that could be used for in vivo tracking of macrophages in disease.
Furthermore, this strategy provides a platform for the introduction of genes/factors
that are predicted to modulate and/or stabilise macrophage phenotype and function
in diverse biological settings.
We then used the AAVS1 targeting strategy to programme iPSC-derived
macrophages into a phenotype comparable to macrophages associated with the
erythroid island. Human red blood cell (RBC) precursors proliferate and mature
within an erythroblastic island (EI) niche consisting of a central macrophage
surrounded by 5-30 erythroblasts. Emulating this EI niche in vitro could provide a
tool to study the molecular mechanisms involved in terminal erythropoiesis and
might ultimately overcome the limitations associated with the production of RBCs in
vitro. The transcription factor KLF1 has been reported to play an important role in
murine EI-like macrophages, and we noted the deficient expression of KLF1 in
macrophages derived from iPSCs in vitro. We, therefore, hypothesised that
enforced expression of KLF1 might programme these macrophages to a more EIlike
phenotype. Indeed, activation of KLF1 in iPSC-derived macrophages (iPSC-DM)
increased the expression of some EI-associated genes and cell surface markers;
and enhanced their phagocytic activity.
We established a co-culture system with iPSC-DMs and umbilical cord bloodderived
CD34+ haematopoietic progenitor cells, as well as iPSC-derived
haematopoietic cells and demonstrated that co-culture with macrophages increased
the production of mature and enucleated erythroid cells and this was further
enhanced when KLF1 was activated. The effect of KLF1 activation was partially
retained when contact with macrophages was inhibited, suggesting that a paracrine
effect (secreted factors encoded by KLF1 targets genes) is associated with its
mechanisms of action.
RNA sequencing of KLF1 activated iPSC-DMs revealed potential cell surface
proteins/receptors and secreted factors that might be involved in the enhanced
proliferation, enucleation and maturation of erythroid cells. We identified three
potential mediators: ANGPTL7, IL33 and SERPINB2 because when added together
in erythroid cultures, there was a significant increase in erythroid cell maturation and
enucleation in the absence of macrophages. Pilot studies revealed that secreted
factors NRG1, IGFBP6, CCL13 and TNFSF10 could also play a role in the later
stages of the erythroid differentiation protocol.
To our knowledge, this is the first time that macrophage phenotype and function
have been manipulated by transcription factor programming. In this particular
scenario, our novel co-culture system with programmed macrophages provides a
model to study the EI niche and terminal erythropoiesis in vitro, and brings us a step
closer to the ultimate goal of replacing blood transfusion with a manufactured
product.