# Regenerating Vascularized and Innervated Skeletal Muscle to Treat VML Defects

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2024 · $411,500

## Abstract

Skeletal muscle makes up almost half of the human lean body mass and approximately 40% of all traumatic
injuries involve skeletal muscle damage. This results in a global economic burden of roughly $6 billion. While
skeletal muscle possesses an intrinsic self-regeneration capacity, in clinical scenarios of volumetric muscle
loss (VML) where the muscle's natural repair mechanisms are overwhelmed, regeneration fails. Tissue
engineering strategies using human skeletal muscle stem or progenitor cells combined with novel biomaterials
have unprecedented potential to provide effective therapies. In this study, we propose to harness the myogenic
potential and regenerative capacity of sorted skeletal muscle stem/progenitor reporter cells (PAX7::GFP+)
derived from human pluripotent stem cells (hPSCs). Specifically, we hypothesize that PAX7::GFP+ myogenic
progenitors grown on electrospun fibrin microfiber bundles will proliferate, upregulate their expression of
myogenic genes and form aligned, multi-nucleated myotubes assembled into 3D muscle grafts. These
engineered grafts will be used to regenerate skeletal muscle tissue and restore normal function following VML.
We further hypothesize that the use of agrin in combination with insulin-like growth factor-1 (IGF-1) will
promote the formation of more densely packed PAX7::GFP+ derived myotubes in the engineered muscle
grafts and enable the formation of mature neuromuscular junctions (NMJs) in the regenerating skeletal muscle.
We will test these hypotheses in three Specific Aims. In Sp. Aim 1, we will engineer uniform, densely seeded
skeletal muscle grafts by (i) electrospinning PAX7::GFP+ cell aggregates into the fibrin microfiber bundles and
(ii) coating the microfiber bundles with PAX7::GFP+ cell-seeded bulk fibrin. We will stimulate their maturation
into contractile 3D skeletal muscle tissues using biophysical stimulation. We will quantitatively evaluate cell
morphology, proliferation, multi-nucleation, and myogenic differentiation and utilize single-cell RNA-sequencing
to compare the cellular heterogeneity and myogenic gene expression profiles with that of native muscle cells.
In Sp. Aim 2, we will evaluate the potential of soluble and tethered agrin/IGF-1 individually and in combination
to enhance the proliferation and myogenesis of PAX7::GFP+ cells. We will also characterize the effects of
tethering these molecules on the physicochemical and pro-myogenic properties of the modified scaffolds. In
Sp. Aim 3, we will implant PAX7::GFP+ derived muscle grafts engineered with and without soluble or tethered
agrin/IGF-1 into small incisions into the tibialis anterior (TA) muscle of immunodeficient mice to assess cell
survival, integration, and regenerative potential. We will use these data to optimize the engineered skeletal
muscle grafts that we will implant into VML defects to quantitatively assess muscle regeneration, vascular and
neural infiltration, the formation of mature neuromuscular junctions, and funct...

## Key facts

- **NIH application ID:** 10868617
- **Project number:** 5R01AR077581-05
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Warren L Grayson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $411,500
- **Award type:** 5
- **Project period:** 2020-08-05 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10868617

## Citation

> US National Institutes of Health, RePORTER application 10868617, Regenerating Vascularized and Innervated Skeletal Muscle to Treat VML Defects (5R01AR077581-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10868617. Licensed CC0.

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