Pax3-induced expansion enables the genetic correction of dystrophic satellite cells

Abstract

BackgroundSatellite cells (SCs) are indispensable for muscle regeneration and repair; however, due to low frequency in primary muscle and loss of engraftment potential after ex vivo expansion, their use in cell therapy is currently unfeasible. To date, an alternative to this limitation has been the transplantation of SC-derived myogenic progenitor cells (MPCs), although these do not hold the same attractive properties of stem cells, such as self-renewal and long-term regenerative potential.MethodsWe develop a method to expand wild-type and dystrophic fresh isolated satellite cells using transient expression of Pax3. This approach can be combined with genetic correction of dystrophic satellite cells and utilized to promote muscle regeneration when transplanted into dystrophic mice.ResultsHere, we show that SCs from wild-type and dystrophic mice can be expanded in culture through transient expression of Pax3, and these expanded activated SCs can regenerate the muscle. We test this approach in a gene therapy model by correcting dystrophic SCs from a mouse lacking dystrophin using a Sleeping Beauty transposon carrying the human μDYSTROPHIN gene. Transplantation of these expanded corrected cells into immune-deficient, dystrophin-deficient mice generated large numbers of dystrophin-expressing myofibers and improved contractile strength. Importantly, in vitro expanded SCs engrafted the SC compartment and could regenerate muscle after secondary injury.ConclusionThese results demonstrate that Pax3 is able to promote the ex vivo expansion of SCs while maintaining their stem cell regenerative properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0061-7) contains supplementary material, which is available to authorized users.

Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells

“…We have found that the three factors HEYL, KLF4, and MYOD, without exogenous PAX3 also induced PAX3-GFP cells from mesodermal cells; however, PAX7 , CALCR , or NAP1L5 transcripts in GFP + cells without exogenous PAX3 were all decreased and showed less myogenic differentiation in vitro . Furthermore, additional expression of PAX3/PAX7 is probably important for the long-term maintenance of MuSCs (Buckingham and Relaix, 2015), and the activation of transient PAX3 promotes ex vivo expansion of mouse PAX7-positive MuSCs (Filareto et al., 2015). Manipulation of culture medium by itself, without the introduction of expression vectors, is being used to promote the formation of myogenic cells for cell therapy (Chal et al., 2015), although the addition of the four factors that we describe increased the production of MuSCs and also conferred more adult MuSC-like properties as discussed.…”

Making muscle: skeletal myogenesisin vivoandin vitro

“…Both Pax3-and Pax7-reprogrammed mouse progenitors were shown to engraft when transplanted in a mouse model. Pax3-or Pax7-reprogrammed hPSCs can generate myogenic progenitors that are CD29 (ITGβ1) + /CD44 + / CD56 + and CXCR4 − /CD106 (VCAM1) − and which can engraft into mouse muscle (Darabi et al, 2012;Filareto et al, 2015) (Box 1). Overexpression of Mesp1 has also been used to produce putative cardio-pharyngeal progenitors with the capacity to differentiate into both cardiac and cranial paraxial mesoderms (Chan et al, 2013;Lescroart et al, 2014).…”

Muscle injury, impaired muscle function and insulin resistance in Chromogranin A-knockout mice

“…5A), but no change in satellite marker Myf5 (Relaix et al . 2006, Young & Wagers 2010, Filareto et al . 2015).…”

“…It is possible that although the increased expression of MyoD1 and Pax3 may drive the proliferation and differentiation of progenitor cells (Relaix et al . 2006, Young & Wagers 2010, Filareto et al . 2015), increased expression of Ifnγ may inhibit myogenin activity and prevent differentiation (Londhe & Davie 2011).…”