Project Summary/ Abstract
Human induced pluripotent stem cells (hiPSCs) are among the top candidates for cell therapy due to their
pluripotency and isogenic source. In case of skeletal muscle disorders, stem cell-based therapies can replace
defective or damaged muscle tissue with healthy muscle stem cells and progenitors. Therefore, hiPSCs have
been the focus of recent research for derivation of skeletal muscle progenitor cells (MPCs). However, cell
therapies are generally limited by poor cell survival due to lack of oxygen, nutrients and local trophic factors.
Meanwhile, endothelial cells (ECs) have a key role in regulation of muscle stem cells through secretion of trophic
factors controlling their activation, function and maturation. In addition, ECs directly contribute in angiogenesis
and formation of new vessels, improving local circulation. Indeed, coupled activation of MPCs and ECs supports
optimal muscle regeneration and plasticity after injuries or increased physiological demand. Therefore, ECs can
be considered as a potential adjunctive cell therapy to improve MPC survival and engraftment outcome in vivo.
Although the interaction between ECs and MPCs have been studied using primary cell lines and mouse models,
their possible cross-talk, underlying molecular mechanisms and their combined in vivo engraftment efficiency
has not been evaluated in a hiPSC-derived model system. Therefore, current application aims to: A) study the
effect of hiPSC-ECs on in vitro activity and function of their myogenic counterparts (hiPSC-MPCs) and to identify
and validate underlying mechanisms, and B) to evaluate the therapeutic efficiency of a combined hiPSC-EC+
MPC therapy in dystrophic or injury mouse models. In Aim1, hiPSC-ECs and MPCs will be grown using a
transwell co-culture system to allow paracrine interaction of the cells. The paracrine effect of ECs on MPCs will
be evaluated on cell proliferation, migration and differentiation using gene expression, transwell migration assay
and immunostaining methods. In addition, time-course RNA-Seq and secretome proteomics will be performed
to identify differentially expressed genes and proteins, such as ligands and receptor pairs, growth factors and
pathways. Top candidates will be validated by proteomics and over-expression/inhibition studies to validate their
role in the predicted cellular function. In Aim2, hiPSC-ECs and MPCs will be injected into muscle of dystrophic
or injury mouse models using different cell ratios and their in vivo survival and engraftment will be evaluated by
live cell bioluminescence, as well as histologic evaluation for donor cell engraftment into myofibers, muscle stem
cell and vessel compartment. Data will be quantified for engraftment and vascularization among different
experimental conditions to determine the efficiency and appropriate cell ratio of combined hiPSC-EC + MPC
therapy in the studied models. Completion of these studies will elucidate the role and underlying mechanisms of
hiPSC-EC/MPC interaction, as well as defining their combined in vivo efficiency to improve donor cell survival,
vascularization and engraftment in muscle disorders as a proof of principle study. Outcome of this study will
likely be able to move the field toward generation of multi-cellular hiPSC models for in vitro and in vivo studies.
