94. Modulation of Postnatal Muscle-Derived Stem Cell Fate Using siRNA
Authors: Jonathan B. Pollett, Karin A. Corsi, Jessica Tebbets, Bridget M. Deasy, Johnny Huard
Journal: Molecular Therapy (2006)

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Primary somatic stem cell isolations tend to be heterogeneous by nature. We have shown that postnatal muscle-derived stem cells (MDSCs) isolated from a mouse muscle biopsy can undergo multilineage differentiation, and that the cells generated within each given isolation are heterogeneous. From a single muscle biopsy we isolate various MDSC subpopulations. Although all of the subpopulations are capable of multilineage differentiation, not all have the same propensity to differentiate toward various lineages. When we compared the regenerative index of these cells to assess their myogenic potential (RI; number of dystrophin[+] fibers in an MDX mouse per 105 donor cells), we found RI values that ranged from 14|[ndash]|741 in 11 subpopulations isolated from the same biopsy. When we assessed their osteogenic capacity by examining their alkaline phosphatase (ALP) expression in response to BMP4, we observed values ranging from 84.4|[ndash]|507 nmol p-nitrophenyl phosphate/mg protein. Comparison of osteogenic potential with ability to engraft in dystrophic muscle revealed one population (mMDSC) that had the highest RI and the lowest ALP activity. As this population had a strong predilection for myogenic differentiation, we wanted to test if we could use siRNA to modify the cells' ability to differentiate. Specifically, we targeted MyoD1, a master regulatory gene for myogenesis, and Smad6, a member of the MAD-related family of molecules that specifically inhibits BMP-induced signaling. We hypothesized that a loss-of-function approach would enable us to modulate the myogenic priming of our cells and potentially increase their osteogenic potential. Using the RNAi-Ready pSIREN-RetroQ vector from Clontech, we generated siRNA retroviruses to murine MyoD1 (siMD) and Smad6 (siS6). mMDSCs transduced with siMD had impaired myotube formation when placed in fusion medium (DMEM with 2% horse serum). Furthermore, when subjected to myogenic conditions, these cells detached from the dish and formed non-adherent colonies. In contrast, control cells underwent myotube formation. Examination of the osteogenic potential of siMDtransduced cells revealed that inhibition of MyoD increased the percentage of ALP[+] cells from 8% to 40% 4 days after BMP4 stimulation. Hence modulation of MyoD levels enabled us to decrease the myogenic potential of mMDSCs and increase their osteogenic potential.

Though very myogenic, mMDSCs respond poorly to BMP4 stimulation. We believe that this is partially due to increased levels of Smad6. To counteract the effects of Smad6, we infected mMDSCs with siS6. Similar to siMD, siS6 increased the percentage of ALP[+] cells significantly but, unlike siMD, did not impair their ability to form myotubes. In contrast to siMD, siS6 increased the percentage of ALP[+] cells as soon as 24 hours after BMP4 stimulation. mMDSCs transduced to express BMP4 and implanted into mouse skeletal muscle always fail to form bone. However, when we transduced these cells with siS6 before implantation, 60% of host mice developed radiologically detectable bone within 3 weeks. Hence, we believe that targeting specific genetic factors in a loss-of-function approach can enable modulation of the differentiation capacity of MDSCs.