The effect of vitronectin on the differentiation of embryonic stem cells in a 3D culture system

Abstract

While stem cell niches in vivo are complex three-dimensional (3D) microenvironments, the relationship between the dimensionality of the niche to its function is unknown. We have created a 3D microenvironment through electrospinning to study the impact of geometry and different extracellular proteins on the development of cardiac progenitor cells (Flk-1(+)) from resident stem cells and their differentiation into functional cardiovascular cells. We have investigated the effect of collagen IV, fibronectin, laminin and vitronectin on the adhesion and proliferation of murine ES cells as well as the effects of these proteins on the number of Flk-1(+) cells cultured in 2D conditions compared to 3D system in a feeder free condition. We found that the number of Flk-1(+) cells was significantly higher in 3D scaffolds coated with laminin or vitronectin compared to colIV-coated scaffolds. Our results show the importance of defined culture systems in vitro for studying the guided differentiation of pluripotent embryonic stem cells in the field of cardiovascular tissue engineering and regenerative medicine.

Hyaluronan-Based Three-Dimensional Microenvironment Potently Induces Cardiovascular Progenitor Cell Populations

“…Using a live/dead cytotoxicity assay, we can see in Figure 1 that the environment of the hydrogel supports mES cell growth. We did observe a slower proliferation rate (data not shown) of cells encapsulated within the hydrogel as compared to cells maintained on gelatin-(for control) and collagen IV-coated dishes in 2D culture conditions, yet this was similar to the proliferation rates we observed previously when cells were cultured on nanofibrous scaffolds [2,4]. Additionally, we observed different morphologies of cells in 2D culture versus 3D culture.…”

“…However, the role of the ECM within the cardiovascular progenitor cell niche is still relatively unknown. Recently, it has been shown that the addition of ECM proteins to an in vitro culture contributes to the induction of a CPC population [2,4]. Here, we demonstrate that the addition of a 3D element further enhances that induction.…”

“…We have previously shown that collagen IV in 2D culture conditions induces a greater CPC population and that the addition of an electrospun scaffold further enhances induction of a Flk-1 + CPC population [2,4]. While electrospun scaffolds can be classified as a 3D microenvironment, it can take mES cells up to seven days to migrate to the interior of the scaffold (data not shown).…”

“…Additionally, incorporating a modified 2D substrate, a nanofibrous scaffold, into the culture system further induces a CPC population [4]. In order to further examine the full effects of a 3D microenvironment, we will develop a 3D in vitro culture system using a HA-based hydrogel.…”

Introduction to Stem Cells and Regenerative Medicine

“…A feeder layer of embryonic fibroblast cells (MEFCs) or medium that contains the anti-differentiation cytokine leukemia inhibitory factor (LIF) are used. Withdrawal of LIF from the medium or removal of the ESCs from the feeder layer results in the formation of ‘embryoid bodies’, in which all 3 germ layers (endoderm, mesoderm, and ectoderm) are present [56,57,58,59,60]. …”