Biomimetic Self-Assembled Scaffolds Enhance Muscle Stem Cell Transplantation

Muscle stem cells (also known as satellite cells) are essential for endogenous skeletal muscle regeneration but their utility in cell transplantation therapy is currently limited by their inefficient survival, self-renewal, and differentiation after injection into muscle tissue. To improve the contributions of MuSCs post-transplant, we developed biomimetic scaffolds based on synthetic peptide amphiphiles (PAs) that self-assemble to encapsulate cells and growth factors within a muscle-like unidirectionally-ordered environment of long and aligned nanofibers directly as they are injected into damaged muscle tissue.

We generated PAs consisting of an aliphatic (C₁₆) palmitoyl tail and hydrophilic nine amino acid cap (e.g. V₃A₃E₃, in various rearrangements) using a peptide synthesizer. We annealed PAs into randomly-ordered, long-axis nanofibers, which externally expose the amino acid cap and internally aggregate the hydrophobic tail, by entropic mixing. We extruded annealed PAs (aPAs) into physiological calcium concentrations in culture medium to fabricate a stable, noodle-like aPA scaffolds with highly-ordered nanofibers in parallel to the direction of extrusion. We measured the storage modulus of aPA scaffolds using shear rheometry and observed shear storage moduli (G’) spanning three orders of magnitude, from G’ = 3-20 kPa, encompassing a range of physiological muscle stiffness.

We evaluated myogenic progenitor cell viability, proliferation and differentiation after encapsulation in assembled aPA scaffolds in vitro. We observed that the aPA scaffold stiffness determined the macroscopic degree of muscle progenitor cell alignment with the ordered nanofiber template. The aPA scaffolds supported primary myoblast survival and proliferation in vitro, and, when tuning their stiffness to G’ = 9 kPa, optimal myoblast differentiation into mature myosin heavy chain-positive myofibers.

Upon injection into hindlimb muscles, we observed longitudinal co-alignment of scaffold nanofibers with recipient muscle myofibers to achieve desired templating of injected cells into differentiation tracks in parallel with the native architecture. aPA scaffold degradation in vivo was assayed using a Gd(III)-doped PA molecules and MRI imaging every five days. These scaffolds displayed characteristic degradation rates in vivo matching the time course of tissue regeneration (t_1/2 = ~14 days).

We transplanted FACS-isolated α7-integrin⁺ CD34⁺ muscle stem cells from GFP/Luciferase transgenic mice mixed with a 1wt%/vol aPA solution and then immediately injected into hindlimb muscles of pre-irradiated immunodeficient NOD/scid mice. When transplanted within aPA scaffolds, we observed profound improvements in the frequency of MuSC engraftment, as measured by bioluminescence imaging longitudinally up to one-month post-transplant, compared to MuSCs in control solutions. We also observed enhanced donor cell-derived myofiber contributions, as assayed by anti-GFP immunohistochemistry, a standard measure of muscle repair.

This work establishes a new stem cell encapsulation technology to aid muscle cell therapies. Importantly, this scaffold system self-assembles without the need for in vivo polymerization or cross-linking and has emergent nanofiber alignment to template muscle cell maturation. Simple rearrangement of PA amino acid sequence controls scaffold stiffness and can be varied to optimize muscle stem cell function in vitro and in vivo.