Mass Transfer Limitations in Embryoid Bodies during Human Embryonic Stem Cell Differentiation

Abstract

Due to their ability to differentiate into cell types from all the three germ layers and their potential unlimited capacity for expansion, embryonic stem cells have tremendous potential to treat diseases and injuries. Spontaneous differentiation of human embryonic stem cells (hESCs) is influenced by the size of the differentiating embryoid bodies (EBs). To further understand the dynamics between nutrient mass transfer, EB size, and stem cell differentiation, a transient mass diffusion model of a single hESC EB was constructed. The results revealed that the oxygen concentration at the centers of large EBs (400-µm radius) was 50% lower when compared to that in smaller EBs (200-µm radius). In addition, the concentration profile of cytokines within an EB depended strongly on their depletion rate, with higher depletion rates resulting in cytokine concentrations that varied significantly throughout the EB. A comparison of the results of our model with published experimental data reveals a close correlation between the fraction of cells that differentiate to a given lineage and the fraction of cells exposed to different oxygen or cytokine concentrations. This, along with other data from the literature, suggests that diffusive mass transfer influences the differentiation of hESCs within EBs by controlling the spatial distribution of soluble factors. This has important implications for research involving the differentiation of embryonic stem cells in EBs, as well as for bioprocess design and the development of robust differentiation protocols where mass transfer could be altered to control the cell differentiation trajectory.

Three-Dimensional Aggregates of Mesenchymal Stem Cells: Cellular Mechanisms, Biological Properties, and Applications

“…The multicellular aggregate structure creates a heterogeneous microenvironment with size-dependent gradients of nutrients, oxygen, and cytokines. 107 Among these factors, oxygen availability has been frequently suggested as a primary factor that influences MSC fate. G-MSC and A-MSC aggregate formation has been reported to induce a mild hypoxic environment, increasing the expression of hypoxiainducible factor (HIF)-1a and HIF-2a.…”

“…38 However, whether oxygen gradient in the aggregates is solely responsible or even sufficient to induce the observed alternation in MSC behaviors and secretome requires in-depth experimental and modeling analysis. 107 Although oxygen tension is a primary factor that activates HIF transcription, glycolytic metabolites such as pyruvate and lactate can also activate and stabilize HIF under normoxic condition. 108,109 Therefore, the molecular profiles of the 3D MSC aggregates remain to be fully characterized.…”

Glucose diffusion in tissue engineering membranes and scaffolds

“…This was attributed to the higher dynamic viscosity of CCM compared to water as well as the multi-component interactions present in CCM, although the latter is believed not to be as significant as the former. While many authors assumed the diffusivity in cell culture media to be equal to that in water (Li 1982, Abdullah and Das 2007, Clark et al 2011, Van Winkle et al 2012, Suhaimi et al (2015a) highlighted the significant differences between the diffusivities in both media. Table 8 summarises some examples of diffusing solutes and the corresponding diffusivity values that have been reviewed in this section.…”

Engineering Strategies for the Formation of Embryoid Bodies from Human Pluripotent Stem Cells

“…Although EB permits the generation of cells arising from all three primary germ layers, the differentiation outcomes are highly dependent upon the endogenous parameters of EBs, including the media composition [14], the cell numbers, the size, and the morphology of EBs [9,15]. For example, EB viability and the yield in terminal differentiation vary in a size-dependent manner [16]. While too small EBs did not survive well during the differentiation procedures, too large EBs underwent core necrosis [16].…”

“…For example, EB viability and the yield in terminal differentiation vary in a size-dependent manner [16]. While too small EBs did not survive well during the differentiation procedures, too large EBs underwent core necrosis [16]. A wide distribution in the EB size introduces a source of variability in their downstream differentiation [17], which depends on the immediate microenvironment perceived by individual cells in the EBs, that is, the position of cells relative to others in the EBs.…”