Supplementary Material for: Silk-based matrices and c-Kit positive cardiac progenitor cells for a cellularized silk fibroin scaffold: study of an in vivo model

The production of a cellularized silk fibroin scaffold is very difficult because it is actually impossible to differentiate cells into a well-organized cardiac tissue. Without vascularization, not only do cell masses fail to grow, but they may also exhibit an area of necrosis, indicating a lack of oxygen and nutrients. In the present study, we used the so-called tyrosine protein kinase kit (c-kit)-positive cardiac progenitor cells (CPCs) to generate cardiac cellularized silk fibroin scaffolds, multipotent cells isolated from the adult heart to date that can show some degree of differentiation toward the cardiac phenotype. To test their ability to differentiate into the cardiac phenotype in vivo as well, CPC and collagen organoid-like masses were implanted into nude mice and their behavior observed. Since the three-dimensional structure of cardiac tissue can be preserved by scaffolds, we prepared in parallel different silk fibroin scaffolds with three different geometries and tested their behavior in three different models of immunosuppressed animals. Unfortunately, CPC cellularized silk fibroin scaffolds cannot be used in vivo. CPCs implanted alone or in collagen type I gel were destroyed by CD3+ lymphocytes’ aggregates, whereas the porous and partially oriented scaffolds elicited a consistent foreign body response characterized by giant cells. Only the electrospun meshes were resistant to the foreign body reaction. In conclusion, c-kit-positive CPCs, although expressing a good level of cardiac differentiation markers in vitro with or without fibroin meshes, are not suitable for an in vivo model of cardiac organoids because they are degraded by a T-cell-mediated immune response. Even scaffolds, which may preserve the survival of these cells in vivo, also induced a host response. However, among the tested scaffolds, the electrospun meshes (F-scaffold) induced a lower response compared to all the other tested structures.

细胞化丝素蛋白支架（cellularized silk fibroin scaffold）的制备极具挑战性，因实际上无法将细胞诱导分化为结构规整的心肌组织。若无血管化（vascularization）构建，细胞团不仅无法存活增殖，还可能出现坏死区域，提示其缺乏氧气与营养供给。
本研究中，我们采用所谓的酪氨酸蛋白激酶Kit（c-kit）阳性心脏祖细胞（cardiac progenitor cells, CPCs）来制备心肌细胞化丝素蛋白支架；这类多能细胞可从成年心脏中分离获得，是目前已证实可向心肌表型（cardiac phenotype）实现一定程度分化的细胞群。为验证其体内（in vivo）向心肌表型分化的潜能，我们将CPCs与类胶原类器官（collagen organoid-like）团块植入裸鼠体内，并对其存活与行为变化进行观测。
鉴于支架可维持心肌组织的三维结构，我们同步制备了三种不同几何构型的丝素蛋白支架，并在三种不同的免疫抑制动物模型中测试其性能表现。遗憾的是，负载CPCs的细胞化丝素蛋白支架无法应用于体内（in vivo）研究：单独植入或植入I型胶原（collagen type I）凝胶中的CPCs会被CD3+淋巴细胞（CD3+ lymphocytes）聚集物破坏；而多孔且部分取向的支架则会引发持续的异物反应（foreign body response），其特征为出现巨细胞（giant cells）。仅静电纺丝网状支架（electrospun meshes）可抵御该异物反应。
综上，尽管无论是否搭载丝素蛋白网状支架，c-kit阳性CPCs在体外（in vitro）均能表达较高水平的心肌分化标志物，但这类细胞并不适用于心肌类器官的体内（in vivo）模型，因其会被T细胞介导的免疫反应所降解。即便支架可在体内（in vivo）维持这类细胞的存活，其本身也会引发宿主免疫反应。不过在本次测试的所有支架中，静电纺丝网状支架（electrospun meshes, F-支架（F-scaffold））引发的免疫反应相较于其余受试结构更低。