Utilizing Humanized Mice to Study the Human Immune Response to Pro-Regenerative Biomaterials

Introduction: The immune response
plays an integral role in tissue repair and regulation of endogenous stem cells
to regenerate damaged tissue.� Effectiveness and safety of biomaterial
therapies that stimulate repair are dependent on our understanding of their
interaction with the human immune response.� However, standard preclinical animal
models provide limited representation of the human immune response and its
numerous unique characteristics. �We studied the use of a humanized mouse model,
which generates multi-lineages of human immune cells, for evaluating the human
immune response to xenogeneic and allogeneic decellularized extracellular
matrix (ECM) based biomaterials.

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Methods: NOD/SCID/γ immunodeficient
mice were implanted with human liver, thymus and CD34+ fetal liver cells to
generate humanized mice.� Humanized and Balb/c mice were subcutaneously
injected with myocardial ECM hydrogels derived from decellularized xenogeneic porcine
and allogeneic human tissue, and a nondecellularized pro-inflammatory control.
Injections isolated at 7 days were assessed for immune cell infiltration by
immunohistochemistry and polarization by PCR (n = 8-12 injections).� Immune
cell infiltration was assessed overall and 200 �m into the core of the injection.�
T-cells (CD3, CD4, CD8) and macrophages (F4/80, iNOS, CD206) were stained for
specific phenotypic profiles.� Pro-remodeling versus pro-inflammatory T-helper
(Th-) cell (Th2/Th1) and macrophage (M2/M1) polarization was assessed by gene
expression ratio of GATA3/TBX21 and ARG1/NOS2, respectively, with CRTH2/CCR5
uniquely assessed for human Th-cells.� Results are reported as mean�SEM with
significance considered at p < 0.05.

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Results: Although overall CD3⁺CD4⁺
Th-cell response to decellularized materials was not significantly different, significantly
greater Th-cell infiltration into the material core was observed for porcine
compared to human myocardial hydrogels (53.6�9.9 versus 14.3�6.9 cells/mm²)
in humanized mice with no significant difference in Balb/c infiltration (97.1�29.8
versus 92.1�10.4 cells/mm²).� Significantly fewer CD3⁺CD8⁺
cytotoxic T-cells and pro-inflammatory F4/80⁺iNOS⁺CD206^-
macrophages were found for the whole material in porcine (4.5�1.0, 44.5�8.7 cells/mm²) and human (17.6�8.9, 31.8�11.5
cells/mm²) compared to nondecellularized myocardial hydrogels (233.5�78.0,
129.2�.7 cells/mm²) in humanized mice with similar results observed
in Balb/c mice.� Numbers of pro-remodeling F4/80⁺iNOS^-CD206⁺
and triple stained F4/80⁺iNOS⁺CD206⁺ macrophages
were not significantly different for all materials in both mouse models, however,
ratios of pro-remodeling F4/80⁺iNOS^-CD206⁺ versus
pro-inflammatory F4/80⁺iNOS⁺CD206^- macrophages
suggested M2 trends for decellularized materials compared to nondecellularized.
�Gene expression ratio analysis (GATA3/TBX21, CRTH2/CCR5, ARG1/NOS2) demonstrated
significant Th2 and trending M2 profiles for porcine (2.95�0.2, 2.76�0.3, 1.7�0.4)
compared to nondecellularized (1.0�0.2, 1.0�0.6, 1.0�0.2) with Th2 trends for human
myocardial hydrogels (1.36�0.2, 1.65�0.1, 0.84�0.2) in humanized mice.� In
comparison, Balb/c results (GATA3/TBX21, ARG1/NOS2) gave trending Th2 and
significant M2 profiles for porcine (3.4�2.2, 3.3�1.3), and trends for human (2.1�0.5,
1.7�0.2) compared to nondecellularized myocardial hydrogels (1.0�0.4, 1.0�0.3).���

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Conclusions: Humanized mice uniquely
responded to porcine myocardial hydrogels with greater Th-cell infiltration
based on immunohistochemistry and a stronger Th2 and M2 profile based on gene
expression ratios compared to human and nondecellularized materials, suggesting
a greater pro-remodeling response to the xenogeneic material.� These results
demonstrate the use of this model for assessing the degree and polarization of
the human immune response to biomaterial therapies, and could be used to
further model endogenous regeneration.