A system for the surveillance of stem cell fate and function

Cell based therapies represent a promising area of research in regenerative medicine. However, the mechanism by which transplanted cells contribute to bone healing remains unclear. The authors utilized a transgenic mouse strain expressing both the topaz variant of green fluorescent protein under the control of the collagen type I alpha 1 promoter/enhancer sequence (Col1a1 GFP ) and membrane-bound tomato red constitutively in all cells types (R26 mTmG ) to decipher how both transplanted and endogenous cells mediate bone healing [1] . Calvarial healing was assessed using both parietal and frontal defects and showed that frontal osteoblasts express Col1a1 to a significantly greater degree than parietal osteoblasts. Col1a1 GFP ; R26 mTmG mice were also used to observe the behavior of adipose-derived stromal cells (ASCs), bone marrow-derived mesenchymal stem cells (BM-MSCs), and osteoblasts following transplantation into critical-sized calvarial defects. ASCs significantly increased the rate of bone healing and exhibited both increased survival and Col1a1 expression when compared to BM-MSCs and osteoblasts. These results support the Col1a1 GFP ; R26 mTmG system as a promising technology for the evaluation of stem cell populations in cell-based therapeutics for the purposes of bone healing.

Stem cell therapies are typically directed at healing diseased and wounded tissues.Inflammatory signals are widespread and some studies have suggested that stem cells do not persist for long in these environments post-transplantation [2,3] .Two general models exist: 1) transplanted cells survive for prolonged periods of time and contribute directly to repair or 2) transplanted cells rapidly undergo programmed cell death following transplantation and thus primarily exert a paracrine effect by releasing cytokines and signaling molecules into the transplantation microenvironment.The fact remains that transplantation of many types of stem cells leads to enhanced repair and regeneration across a wide spectrum of tissue types and disease states.Understanding the precise mechanisms by which stem cells contribute to enhanced bone healing holds promise for improving upon the efficacy of cell-based therapies.For this reason, the utility of a system that provides a reliable readout of cellular functionality and survival post-transplantation is apparent.The Col1a1 GFP ; R26 mTmG reporter system enables both histologic and FACS-based readouts of collagen expression and cell viability in situ.Mostly importantly, the system may be applied to analyze

RESEARCH HIGHLIGHT
any cell expressing collagen type 1a1 in the context of tissue repair.
The study revealed that the survival of a given transplanted cell type positively correlated with increased rates of calvarial healing.ASCs showed the highest overall rates of calvarial healing, as well as 72-hour and two week post-transplant survival.BM-MSCs and osteoblasts showed lower rates of calvarial healing and decreased survival post-transplant at both 72 hours and two weeks.However, osteoblasts expressed Col1a1 to a significantly greater degree than the other cell types at 72 hours and two weeks post-transplant.This raises the possibility that if osteoblast survival was increased through genetic manipulation or pre-treatment with a small molecule, the benefits of their apparently superior functionality for bone production could be harnessed for cell-based therapies.
In addition to studying cells in the context of transplantation, the Col1a1 GFP ; R26 mTmG reporter system was also successfully utilized to investigate the behavior of endogenous cells during normal physiologic bone healing processes.Existing literature suggests that neural crest-derived frontal bone osteoblasts possess enhanced endogenous activation of pro-osteogenic signaling pathways relative to parietal bone osteoblasts [4][5][6][7][8] .Using the Col1a1 GFP ; R26 mTmG system, we determined that temporospatial differences in collagen expression exist between frontal and parietal bone during bone healing.Frontal-derived osteoblasts exhibit increased expression of Col1a1 in comparison to parietal-derived osteoblasts.
The identification of markers that correlate with a functional property (i.e.collagen production) has shown promise for enhancing cell-based therapeutics.The markers CD90 and CD105 have been shown to select for adipose-derived stromal cells with enhanced osteogenic activity [9,10] .An important future use for the Col1a1 GFP ; R26 mTmG system would involve the rapid screening and identification of other surface markers that correlate with Col1a1 expression by flow cytometry analysis.In this manner, subpopulations with increased Col1a1, and thus enhanced functional capacity in the context of bone healing, could be easily identified.
In conclusion, understanding how transplanted cells contribute to tissue healing and repair is a fundamental next step towards improving cell-based therapies [11] .There is evidence that certain stem cell types would benefit considerably from modifications that would increase cell survival after transplantation.For example, Hyun et al.
overexpressed Bcl-2 in adipose-derived stromal cells and demonstrated increased tissue regeneration through enhanced survival [12] .Other stem cell types tolerate transplantation and survive for longer periods of time but may lack the capacity to efficiently contribute to repair.For cell types such as this, enhanced repair through targeted genome editing may represent the best path to improve therapeutic efficacy.Selection of the optimal strategy for any given stem cell type first depends upon a detailed understanding of both its survival and functionality following transplantation.