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International Journal of Bioprinting Bioprinted stem cell niche regenerates muscles

2.5. Myogenesis and proliferation assays 2.10. Statistical analysis
For myogenic differentiation assay, WT MPCs were Image analysis was performed with ImageJ software
seeded as ~90% confluence in the DMEM containing (version 1.32j; National Institutes of Health, Bethesda, MD,
2% horse serum (HS) and allowed for differentiation USA). Data from at least six samples from each subject
for 96 h before being fixed and imaged to compare the were pooled for statistical analysis. Results are given as
number of myotubes formed by WT MPCs in different the mean ± standard deviation (SD). Statistical differences
groups. For proliferation assay, WT MPCs were seeded between groups in the functional assays were determined
as 2000 cells/cm in the DMEM containing 20% FBS and by two-way repeated analysis of variance (ANOVA). For
2
1% CEE and allowed for proliferation for 72 h before other experiments, statistical differences were determined
being observed to compare the number of WT MPCs in by Student’s t-test (2 categories) or one-way ANOVA (>2
different groups. categories).

2.6. In vitro migration assay
DLL1(mouse):Fc (human)(rec) was bioprinted onto 3. Results
DermaMatrix construct (4 × 4 × 1 mm in dimensions). 3.1. Skeletal muscle cells in mdx mice developed
Cell seeding was performed by injecting of 0.2 × 10 lower Notch activity, and Notch reactivation in
5
green fluorescent protein (GFP)-labeled MPCs into dystrophic muscle promoted stem cell proliferation
both the DLL1-bioprinted DermaMatrix constructs and Notch activity in skeletal muscle declines with age in
control DermaMatrix constructs (IgG Fc) and cultured both mice and humans, and this decline is responsible for
in medium. Three days after cell seeding, DermaMatrix the depletion of functional muscle stem cells [11-13] . When
constructs were placed in the Matrigel-coated upper compared to WT mice (6-month-old), we observed a
chamber of an electrode-impedance-based invasion assay down-regulation in the expression of key Notch signaling
system (xCELLigence) . The Matrigel layer was made of factors (i.e., Notch1, Hes1, Jagged1 and DLL1/ Delta-like
[39]
Matrigel dissolved in medium (1 mg/mL). The numbers protein-1) in the skeletal muscle of mdx mice (Figure 1A).
of cells migrating out of the DermaMatrix constructs and To determine the effects of reactivating Notch signaling in
through the 3D Matrigel layer were monitored. dystrophic muscle, we then chose a recombinant protein
of Notch ligand DLL1 [DLL1 (mouse): Fc (human) (rec),
2.7. Bioprinting of Notch ligand on coverslip Adipogen] as a Notch activator to be tested in our system.
DLL1(mouse): Fc(human) (rec.) was bioprinted onto DLL1 (mouse): Fc (human) (rec) was injected into the limb
fibrin/protein G-coated coverslips, as a circular dot muscles (gastrocnemius) of mdx mice, and the number of
with a diameter of 1.5 mm. WT MPCs (0.5 × 10 /cm ) Pax7-positive cells was found to be obviously increased 3
2
4
were then seeded on the slides and cultured for 4 days. days after injection when compared to the control mice
Immunostaining with Myosin Heavy Chain antibody injected with inactive IgG (Figure 1B and C). Therefore,
(MHC-MF20) were performed to track the myogenic this result indicates that the reactivation of Notch signaling
differentiation potential of MPC.
in dystrophic muscle is effective in promoting self-renewal
2.8. Implantation of bioprinted constructs of muscle stem cells. However, the effect of this local
The DermaMatrix construct (4 × 4 × 1 mm in dimensions) injection of soluble protein could be temporary, and we
seeded with MPCs (4 × 10 ) was implanted into the expect that the delivery of bioprinted Notch ligands on
4
gastrocnemius (GM) muscle of mdx/scid mice (6-month- some type of biocompatible carrier could generate a much
old). At 10 days after implantation, cell engraftment was longer effect in promoting stem cell proliferation.
compared between DermaMatrix constructs with or
without bioprinted DLL1. 3.2. Treatment of muscle progenitor cells with DLL1
recombinant protein in vitro effectively promoted
2.9. Histology and immunohistochemistry cell proliferation capacity and inhibited myogenic
GM muscles of mdx/scid mice implanted with MPC- differentiation capacity.
seeded DLL1-DermaMatrix constructs were harvested 10 We then tested the way of DLL1-Fc in affecting the function
days after implantation. Snap-frozen skeletal muscles were of muscle stem cells. MPCs were isolated from 8-week-
cryo-sectioned at a thickness of 10 microns and processed old WT mice (C57BL/6J, Jackson Lab) using a modified
for histological analysis. Muscle stem cells seeded in preplate technique . MPCs were then treated with DLL1
[29]
DermaMatrix constructs were identified and tracked by (mouse): Fc (human) (rec) (200 ng/mL) for 3 days for either
the expression of GFP. Muscle regeneration involving the the proliferation assay or the myogenic differentiation
direct fusion of implanted WT-MPCs was tracked and assay. Results showed that the proliferation potential of
confirmed by immunostaining of dystrophin. the MPCs was increased and the myogenic potential was

Volume 9 Issue 3 (2023) 302 https://doi.org/10.18063/ijb.711