Human satellite cells have regenerative capacity and are genetically manipulable
Andreas Marg, … , Zsuzsanna Izsvák, Simone Spuler
Andreas Marg, … , Zsuzsanna Izsvák, Simone Spuler
Published August 26, 2014
Citation Information: J Clin Invest. 2014;124(10):4257-4265. https://doi.org/10.1172/JCI63992.
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Technical Advance Genetics Muscle biology Stem cells Therapeutics

Human satellite cells have regenerative capacity and are genetically manipulable

Abstract

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.

Authors

Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler

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Figure 3

Cell colonies originating from HMFF cultures contain satellite cells and retain normal proliferative and fusion capacity.

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Cell colonies originating from HMFF cultures contain satell...
(A) Four HMFFs after 14-day tissue culture on Matrigel (green: desmin, blue: Hoechst). Large colonies of desmin+ cells are found as well as spontaneously fused multinucleated myotubes (top row). Scale bar: 200 μm (first and second image); 50 μm (third image). Approximately 50% of cells are PAX7+ satellite cells after 19 days in culture (red: PAX7, green: desmin, blue: Hoechst) (middle row). Scale bar: 20 μm. At 19 days of tissue culture, BrdU staining (red: BrdU, green: desmin, blue: Hoechst) confirms that myogenic cells are proliferating (bottom row). Scale bar: 20 μm. The asterisk indicates a dividing PAX7+ cell. (B) Sleeping Beauty transposon–mediated gene transfer of a Venus transgene (pT2-caggs-Venus). Venus expressed in satellite cells (top image). Venus+ cells form colonies outside the HMFF (middle and bottom images). Scale bar: 200 μm. The double asterisks indicate HMFF. (C) Cell colonies from HMFF cultures after 35 days of hypothermic treatment in serum-reduced medium (OptiMEM) and subsequent culture in SMGM at 37°C for 32 days (left). Solution A did not permit cells to survive hypothermic treatment (right). Scale bar: 200 μm. The double asterisks indicate HMFF. (D) Myogenic cells derived from HMFFs after 35 days of hypothermic treatment followed by 27 days (BrdU, PAX7, MYF5) and 48 days (MYOD, desmin) in SMGM at 37°C. Desmin stain demonstrates a pure myogenic population without contaminating fibroblasts. PAX7-desmin double stain after HMFFs were placed in 15 μM DAPT during hypothermic treatment (21 days). PAX7+ cells were abundant after 35 days at 37°C. Scale bar: 50 μm.