Microsphere size effects on embryoid body incorporation and embryonic stem cell differentiation

Abstract

Differentiation of pluripotent embryonic stem cells (ESCs) in vitro via multicellular spheroids called embryoid bodies (EBs) is commonly performed to model aspects of early mammalian development and initiate differentiation of cells for regenerative medicine technologies. However, the three-dimensional nature of EBs poses unique challenges for directed ESC differentiation, including limited diffusion into EBs of morphogenic molecules capable of specifying cell fate. Degradable polymer microspheres incorporated within EBs can present morphogenic molecules to ESCs in a spatiotemporally controlled manner to more efficiently direct differentiation. In this study, the effect of microsphere size on incorporation into EBs and ESC differentiation in response to microsphere- mediated morphogen delivery were assessed. PLGA microspheres with mean diameters of 1, 3, or 11 microm were fabricated and mixed with ESCs during EB formation. Smaller microspheres were incorporated more efficiently throughout EBs than larger microspheres, and regardless of size, retained for at least 10 days of differentiation. Retinoic acid release from incorporated microspheres induced EB cavitation in a size-dependent manner, with smaller microspheres triggering accelerated and more complete cavitation than larger particles. These results demonstrate that engineering the size of microsphere delivery vehicles incorporated within stem cell environments can be used to modulate the course of differentiation.

Incorporation of Retinoic Acid Releasing Microspheres into Pluripotent Stem Cell Aggregates for Inducing Neuronal Differentiation

“…Carpenedo et al showed that microsphere diameter affects incorporation efficiency within mESC aggregates with smaller diameter microspheres (1 µm) being more readily incorporated within mESC aggregates than larger ones (3 µm). 10 Additionally, the study revealed that incorporated microspheres ranged in size from 2 to 5 µm while larger diameter microspheres (11 µm) were excluded. The microspheres used by When hiPSCs were cultured with 0.5 mg of microspheres, these instabilities were not observed.…”

“…Other groups have shown that microspheres manufactured from variety of biodegradable polymers can be used to deliver morphogens throughout mESC aggregates. [7][8][9][10] However, the effects of incorporating drug-releasing microspheres into hiPSC aggregates have not been investigated. Moreover, such a strategy has not been used to generate neural tissue by combining hiPSC aggregates with morphogen releasing microspheres.…”

Pluripotent stem cells to hepatocytes, the journey so far

“…Pluripotent stem cells can be aggregated to form embryoid bodies and can be differentiated by exposure to specific cytokine and growth factor cocktails (40). However, all the existing embryoid body-based protocols suffer from low differentiation efficiency and spontaneous differentiation which gives rise to unwanted cell lineages (41)(42)(43). As monolayer culture can be adjusted to avoid most of these issues, it has been utilised widely and is capable of producing relatively pure populations of cells with hepatic function.…”

Generating Cartilage Repair from Pluripotent Stem Cells

“…The three-dimentional (3D) structure of the EB is similar to the early postimplantation embryo, and the cells in EBs are able to differentiate into the three germ layers in serum containing medium. 69,70 EBs have been used as a model to study embryo development and to validate ESCs differentiation. [70][71][72][73][74][75][76][77][78][79] Since it was thought that key signaling occurs within the 3D EB, inducing more efficient early differentiation than culture in two dimensions, EBs have also been used as a starting point for targeting differentiation to particular cell phenotypes.…”