Spatial and temporal control of cell aggregation efficiently directs human pluripotent stem cells towards neural commitment

Abstract

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work, we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation, after four days of culture, 3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm, which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up.

Early retinal differentiation of human pluripotent stem cells in microwell suspension cultures

“…This occurs at both 5000 and 10,000 cells/EB as does the persistence of a significant population of smaller EBs. Controlling the EB starting size and the implementation of mixed conditions was not sufficient to minimise the formation of a very wide range of aggregate sizes in agreement with previous studies using stirred tank bioreactors (Miranda et al 2015). Changing the EB size can affect O 2 diffusion, availability of soluble factors and the nutrient mass transfer rates.…”

Scaling up a chemically‐defined aggregate‐based suspension culture system for neural commitment of human pluripotent stem cells

“…We observed that using 55 rpm leads to a uniform aggregate population ( C v of 21.6%) averaging 140 ± 7 µm in diameter after 72 h of expansion, and similar results in terms of aggregate diameter were obtained by using a benchtop‐bioreactor hybrid with no impeller, which promotes agitation through gentle tube rotation . Moreover, this diameter was previously defined as optimal to initiate neural commitment of hiPSCs when culturing these cells as suspension aggregates and using a dual Smad inhibition protocol .…”

“…Although the majority of diameter values remain below 115 µm, 24 h after seeding, hiPSCs seeded at 2.0 × 10 6 cell/mL and agitated at 40 rpm already display aggregate diameters in the desirable range (139 ± 26 µm) after 24 h. However, this condition shows population heterogeneity, making it more difficult to attain a narrow distribution in aggregate sizes. By the 48 h of expansion, an increase in average diameter for every condition was verified, but only aggregates generated at 40 rpm were able to maintain the 135–145 µm range defined to be optimal to initiate the neural commitment.…”

“…Aggregates were seeded at densities ranging from 0.25 to 1.0 × 10 6 cells/ mL. At day 6 or 9 of neural commitment, hiPSC-derived NP cell aggregates were further differentiated according to the protocol previously described by Miranda et al [25].…”

Editorial: Stem Cell Engineering

“…They applied this differentiation platform to induced pluripotent stem cells (iPSCs) from Rett syndrome patients and found impaired neural commitment in the disease iPSCs. Miranda et al [9] used size-controlled aggregates of hPSCs to demonstrate that an optimal cell cluster size exists for differentiation to neural progenitors. This finding will lead to a guided development of an integrated hPSC expansion and neural differentiation process.…”