The impact of manual processing on the expansion and directed differentiation of embryonic stem cells

Abstract

Embryonic stem cells (ESC) have the developmental potential to form every adult cell type, even after prolonged culture. Reproducibly culturing pluripotent populations and directing differentiation has proven technically challenging yet will underpin the provision of stem cells for both screening and therapeutic applications. This study investigated whether the variations inherent in manual handling procedures cause inconsistent proliferation and phenotypic variability. Two mouse ESC green fluorescent protein (GFP) reporter cells lines, Oct4-GiP and 46C, were used to assess Oct4 expression during expansion and Sox1 expression during directed neuroectoderm differentiation. High inoculation cell densities (ICD) had a negative impact on Oct4-GFP expression. Similarly, increasing ICD caused a drop in Sox1-GFP expression in differentiating cultures. The expansion process had an optimum ICD of 31,800 cells cm(-2) whilst the highest yield of Sox1-GFP positive cells were found at an ICD of 16,400 cells cm(-2). These results implicate variable cell density as a major cause of interindividual variability. Passaging exposes cells to dynamic and repeated changes in their micro-environment. This was associated with a rapid drop in temperature and rise in pH. Extended exposure of 1, 2 and 3 h to ambient conditions resulted in the inhibition of ESC proliferation and Oct4-GFP expression. Dissociation subjects cells to fluid flow and centrifugal forces. Repeated exposure to fluid flow in capillaries prior to cultivation reduced the proliferative capacity of undifferentiated ESCs and caused a significant drop in differentiated neuroectoderm yield. Excessive centrifugal forces up to 1,000g caused shifts in phenotype and proliferation during expansion and differentiation. These studies highlight the need for automated cultivation systems which reproducibly control cell density, fluid flow, centrifugal forces, pH and temperature for the dissociation and inoculation of ESC processes.

Influence of Initial Seeding Density on Gene Expression during Neuronal Priming

“…This trend was repeated for otx2 at 10 k/cm 2 where higher expression was noted compared to the other ICSDs ( Figure 4C), possibly indicating more rapid differentiation towards mature neuronal states at lower ICSD. This supports previous observations in mouse ESCs, where increased neurectoderm differentiation was noted at low ICSD [7]. Expression of neuronal genes by hiPSCs was not as prominent as those observed for hESCs, with relative expression of the majority of genes screened never showing greater than 1×10 2 ΔΔCq increase.…”

“…Multiple bioprocess parameters can impact on the responses of human cells during processing [7]. ICSD and degree of confluence at the point of initiation of differentiation protocols should also be considered.…”

“…In the case of pluripotent cells, passaging hESC colonies at lower density of 1:8 resulted in a high degree of spontaneous differentiation versus those passaged at high density of 1:3 [6]. Using mouse 46C ESCs, Veraitch et al demonstrated that lower seeding density specifically enhanced neuroectoderm differentiation, identified by enhanced Sox1 expression [7].…”

Precisely delivered nanomechanical forces induce blebbing in undifferentiated mouse embryonic stem cells

“…OCT4-GiP and the parental line E14tg2a (kindly donated by Stem Cell Sciences Ltd, Cambridge, UK) cells were routinely passaged following a previously published protocol. 9 Briefly, cells were cultured in Glasgow Minimum Essential Media (Sigma Aldrich, Dorset, UK), 10% fetal calf serum (FCS; Invitrogen, Paisley, UK), 1% MEM nonessential amino acids (Gibco, Paisley, UK), 1% 2-β mercaptoethanol (Gibco), and 1% glutamate-pyruvate (Invitrogen, Carlsbad, CA), with or without leukemia inhibitory factor (LIF; Millipore, Watford, UK) (1 µL/mL). Cells were routinely passaged every 2 days in Iwaki flasks (SLS, Nottingham, UK) coated with 0.1% gelatine (Sigma).…”

Advances in Stem Cell Biology – an ASGBI International Conference held at Durham University

“…Chris Mason (London, UK) started the session and gave a broad overview of the requirements for translational research, including the need for consistency of materials, understanding of bioprocessing, scale-up procedures, reproducible procedures and cost effectiveness. Some of these issues are highlighted practically in a recently published article on the culture of embryonic stem cells (Veraitch et al 2008). He demonstrated how automation and the recruitment of robots could be employed to address some of these issues, such as the reproducible expansion of cultured human embryonic stem cells.…”