Abstract
BackgroundMuscle growth and repair is accomplished by the satellite cell pool, a self-renewing population of myogenic progenitors. Functional heterogeneity within the satellite cell compartment and changes in potential with experimental intervention can be revealed by in vitro colony-forming cell (CFC) assays, however large numbers of colonies need to be assayed to give meaningful data, and manually quantifying nuclei and scoring markers of differentiation is experimentally limiting.MethodsWe present G-Tool, a multiplatform (Java) open-source algorithm that analyzes an ensemble of fluorescent micrographs of satellite cell-derived colonies to provide quantitative and statistically meaningful metrics of myogenic potential, including proliferation capacity and propensity to differentiate.ResultsWe demonstrate the utility of G-Tool in two applications: first, we quantify the response of satellite cells to oxygen concentration. Compared to 3% oxygen which approximates tissue levels, we find that 21% oxygen, the ambient level, markedly limits the proliferative potential of transit amplifying progeny but at the same time inhibits the rate of terminal myogenic differentiation. We also test whether satellite cells from different muscles have intrinsic differences that can be read out in vitro. Compared to masseter, dorsi, forelimb and hindlimb muscles, we find that the diaphragm satellite cells have significantly increased proliferative potential and a reduced propensity to spontaneously differentiate. These features may be related to the unique always-active status of the diaphragm.ConclusionsG-Tool facilitates consistent and reproducible CFC analysis between experiments and individuals. It is released under an open-source license that enables further development by interested members of the community.
Highlights
Muscle growth and repair is accomplished by the satellite cell pool, a self-renewing population of myogenic progenitors
We describe here G-Tool, an open source Java algorithm with a simple graphical user interface (GUI), that allows the evaluation of an ensemble of colony photographs and provides quantitative metrics of proliferation and differentiation potential for each colony-forming cell (CFC) and statistical summaries allowing different experimental groups to be compared for differences in CFC activity
The satellite cell CFC assay Within adult muscle, satellite cells are marked by expression of the transcription factor Pax7 [11]
Summary
Muscle growth and repair is accomplished by the satellite cell pool, a self-renewing population of myogenic progenitors. Likewise, understanding how cells respond to ex vivo culture, and developing optimized conditions for expansion of myogenic progenitors, could have a significant impact if this knowledge can be applied to myogenic cell transplantation. Skeletal muscle clearly have the intrinsic potential to self-renew and generate satellite cell progeny in vivo [3], so it remains possible that appropriate culture conditions may enable expansion of repopulating activity, and thereby enable cell therapies. The colony-forming cell (CFC) assay has had a tremendous impact in the field of hematopoiesis, both on our understanding of the hematopoietic stem cell (HSC), and of its transit amplifying progeny. Gleaning information about the state of the cell or its response to the culture condition requires analysis of the cellular composition of the colony, for example determining how many total cells the colony has (a measure of proliferative potential of the CFC), and the ratio of undifferentiated to terminally differentiated cells (a measure of propensity to differentiate). An automated way of processing this information would make the CFC assay much more accessible to the analysis of skeletal muscle progenitors
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