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Improving the Therapeutic Relevance of Muscle Stem Cells

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Sustained Release of Bone Morphogenetic Protein 2 via Coacervate Improves the Osteogenic Potential of Muscle-Derived Stem Cells

From Stem Cells Translational Medicine

The group of Johnny Huard at the University of Pittsburgh, USA have previously isolated and characterized muscle-derived stem cells (MDSCs) (Gharaibeh et al) which have been shown by various groups by be able to undergo osteogenic differentiation given the correct stimuli.   They are therefore a potential alternative to bone marrow-derived mesenchymal stem cells for bone tissue engineering.   One of these stimuli is continued exposure to BMPs, hindered by the short half-lives in vivo (Jeon et al and Zhao et al) and the requirement of maintaining a localised concentration.   The group have also devised a delivery strategy;  a poly(ethylene argininylaspartate diglyceride)(PEAD)-heparin complex loaded with BMP2 which forms an emulsion-like aggregation of organic molecules separated from the aqueous phase, or a coacervate (Johnson and Wang), previously used to effectively deliver fibroblast growth factor-2 (FGF2) for therapeutic angiogenesis (Chu et al).   Now, in a report in Stem Cells TM, they report on the use of this system with BMP2 to stimulate osteogenesis in MDSCs in vitro and in vivo (Li and Johnson et al).

Loading of BMP2 into the coacervate was 98.2% efficient, and after 28 days incubation 25% of the BMP2 had been released indicating that the coacervate can efficiently control the release of incorporated BMP2. Using the stimulation of ALP expression in the myogenic cell line, C2C12, a common method of determining the activity of BMPs, it was demonstrated that coacervate-BMP2 induced more ALP than control (coacervate alone) and a similar concentration of free BMP2.   ALP is also a marker for osteogenic differentiation of MDSCs in response to BMP2 (>Lee et al ), and this system was next used to assess the effectiveness of coacervate-BMP2 on a cell monolayer and cells in a 3D fibrin gel, representing a potential scaffold for cell delivery to a bone defect.   In the monolayer system, 100ng of coacervate-BMP2, but not 100ng of free BMP2 stimulated ALP expression at 5 days, while multi-dose treatment of free BMP2 (300ng in total) to stimulate sustained release did allow for ALP expression, which was not significantly different to coacervate-BMP2. mRNA levels of Runx2 and collagen type I were also increased in coacervate-BMP2 and multi-dose free BMP2 treatment to a similar level suggesting that the coacervate system can mediate osteogenic differentiation at lower doses of BMP2. Analysis in the 3D fibrin gel culture system found that while free BMP2 (100ng) this time did allow for ALP expression, perhaps due to reduced growth factor degradation, coacervate-BMP2 stimulated expression significantly more.   Furthermore, due to the controlled release, BMP2 levels released from the coacervate, and therefore active, were estimated to be 90% less than free BMP2 at day 5.

Finally, in vivo analysis using a mouse ectopic bone formation model fount that MDSCs stimulated with coacervate-BMP2 displayed extensive bone formation at 2 and 4 weeks after implantation, while those treated with free BMP2 had minimal bone formation at 2 weeks but which did increase by week 4. Calcified osteoid matrix was obvious throughout the wound for the coacervate-BMP2 treated cells but was only found in the periphery of the wound for free BMP2 treated cells.   Furthermore within the coacervate treated wound, the interface between muscle and newly formed bone as detectable, with adjacent myofibers morphologically normal.

Overall, this study demonstrates the effectiveness of the polycation-heparin coacervate delivery system for the binding, protection and sustained release of BMP2, an important factor in many differentiation studies. While this study does underline the excellent therapeutic potential of this system in bone repair and highlights the potential of MDSCs, this strategy can surely be adapted for uses in many different differentiation strategies, and in the maintenance and production of pluripotent cell types. Furthermore, this could lead to an increased cost effectiveness of many protocols utilising expensive factors, as lower concentrations of coacervate-BMP2 were seen to function as well as concentrations of free BMP2 at 3 times the level. 

 

References

  • Chu H et al. (2011) Injectable fibroblast growth factor-2 coacervate for persistent angiogenesis. Proc Natl Acad Sci USA 108:13444–13449

  • Gharaibeh B et al. (2008) Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3:1501–1509

  • Jeon O et al. (2007) Enhancement of ectopic bone formation by bone morphogenetic protein-2 released from a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold. Biomaterials 28:2763–2771

  • Johnson NR, Wang Y (2013) Controlled delivery of heparin-binding EGF-like growth factor yields fast and comprehensive wound healing. J Control Release 166:124–129

  • Lee JY et al. (2000) Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 150:1085–1100

  • Zhao B et al. (2006) Heparin potentiates the in vivo ectopic bone formation induced by bone morphogenetic protein-2. J Biol Chem 281:23246–23253

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.