Nanofiber-Based in Vitro System for High Myogenic Differentiation of Human Embryonic Stem Cells

Myogenic progenitor cells derived from human embryonic stem cells (hESCs) can provide unlimited sources of cells in muscle regeneration but their clinical uses are largely hindered by the lack of efficient methods to induce differentiation of stem cells into myogenic cells. We present a novel approach to effectively enhance myogenic differentiation of human embryonic stem cells using aligned chitosan-polycaprolactone (C-PCL) nanofibers constructed to resemble the microenvironment of the native muscle extracellular matrix (ECM) in concert with Wnt3a protein. The myogenic differentiation was assessed by cell morphology, gene activities, and protein expression. hESCs grown on C-PCL uniaxially aligned nanofibers in media containing Wnt3a displayed an elongated morphology uniformly aligned in the direction of fiber orientation, with increased expressions of marker genes and proteins associated with myogenic differentiation as compared to control substrates. The combination of Wnt3a signaling and aligned C-PCL nanofibers resulted in high percentages of myogenic-protein expressing cells over total treated hESCs (83% My5, 91% Myf6, 83% myogenin, and 63% MHC) after 2 days of cell culture. Significantly, this unprecedented high-level and fast myogenic differentiation of hESC was demonstrated in a culture medium containing no feeder cells. This study suggests that chitosan-based aligned nanofibers combined with Wnt3a can potentially act as a model system for embryonic myogenesis and muscle regeneration

Chitosan-Based Thermoreversible Hydrogel as an <i>in Vitro</i> Tumor Microenvironment for Testing Breast Cancer Therapies

Breast cancer is a major health problem for women worldwide. Although <i>in vitro</i> culture of established breast cancer cell lines is the most widely used model for preclinical assessment, it poorly represents the behavior of breast cancers <i>in vivo</i>. Acceleration of the development of effective therapeutic strategies requires a cost-efficient <i>in vitro</i> model that can more accurately resemble the <i>in vivo</i> tumor microenvironment. Here, we report the use of a thermoreversible poly­(ethylene glycol)-<i>g</i>-chitosan hydrogel (PCgel) as an <i>in vitro</i> breast cancer model. We hypothesized that PCgel could provide a tumor microenvironment that promotes cultured cancer cells to a more malignant phenotype with drug and immune resistance. Traditional tissue culture plates and Matrigel were applied as controls in our studies. <i>In vitro</i> cellular proliferation and morphology, the secretion of angiogenesis-related growth factors and cytokines, and drug and immune resistance were assessed. Our results show that PCgel cultures promoted tumor aggregate formation, increased secretion of various angiogenesis- and metastasis-related growth factors and cytokines, and increased tumor cell resistance to chemotherapeutic drugs and immunotherapeutic T cells. This PCgel platform may offer a valuable strategy to bridge the gap between standard <i>in vitro</i> and costly animal studies for a wide variety of experimental designs