Unique Differentiation Profile of Mouse Embryonic Stem Cells in Rotary and Stirred Tank Bioreactors

Abstract

Embryonic stem (ES)-cell-derived lineage-specific stem cells, for example, hematopoietic stem cells, could provide a potentially unlimited source for transplantable cells, especially for cell-based therapies. However, reproducible methods must be developed to maximize and scale-up ES cell differentiation to produce clinically relevant numbers of therapeutic cells. Bioreactor-based dynamic culture conditions are amenable to large-scale cell production, but few studies have evaluated how various bioreactor types and culture parameters influence ES cell differentiation, especially hematopoiesis. Our results indicate that cell seeding density and bioreactor speed significantly affect embryoid body formation and subsequent generation of hematopoietic stem and progenitor cells in both stirred tank (spinner flask) and rotary microgravity (SyntheconÔ) type bioreactors. In general, high percentages of hematopoietic stem and progenitor cells were generated in both bioreactors, especially at high cell densities. In addition, Synthecon bioreactors produced more sca-1 þ progenitors and spinner flasks generated more c-Kit þ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that on day 7 of differentiation, embryoid bodies from both bioreactors consisted of all three germ layers of embryonic development. However, unique gene expression profiles were observed in the two bioreactors; for example, expression of specific hematopoietic genes were significantly more upregulated in the Synthecon cultures than in spinner flasks. We conclude that bioreactor type and culture parameters can be used to control ES cell differentiation, enhance unique progenitor cell populations, and provide means for large-scale production of transplantable therapeutic cells.

Differential Expression of Extracellular Matrix and Growth Factors by Embryoid Bodies in Hydrodynamic and Static Cultures

“…have previously been demonstrated to influence ESC differentiation compared with static cultures by modulating global phenotypic gene expression profiles, specific cell lineages, and endogenous signaling pathways. [10][11][12]25 Rotary orbital culture was previously found to support increased cardiomyogenic differentiation, 10 which is consistent here with increased expression of several genes (Pdgfa, Tgfb2, Vegfc, Col5a1, Col6a1, Lama2, Lama3, Lamc1, and Vcan) involved in cardiac development relative to static culture (Supplementary Fig. S3).…”

“…8,9 Hydrodynamic culture systems for PSCs are increasingly being used for scale-up purposes and have demonstrated influence over the fate of differentiating PSCs. [10][11][12][13] The use of continuous microfluidic perfusion devices, for example, can alter extracellular matrix (ECM) remodeling processes and cause mouse ESCs to spontaneously differentiate. 14 Furthermore, the expression profile of extracellular molecules has been shown to be dynamically modulated during ESC differentiation in a rotary orbital culture system.…”

Stem cell bioprocess engineering towards cGMP production and clinical applications

“…Moreover, 3-D stem cell aggregates have been recently shown to increase the therapeutic potential and differentiation efficiency of stem cells through the sustainment of endogenous signaling (Sart et al 2013c, d;Fridley et al 2010). In addition, to meet the requirement for high dose cell therapy (e.g.…”

Dendritic cells derived from pluripotent stem cells: Potential of large scale production

“…Bioreactors not only allow the scale up of the process, but may also regulate the differentiation pathway due to the unique hydrodynamic environment, especially the presence of shear stress [21,22] . Several types of bioreactors have been used for hPSC expansion and differentiation, including spinner flasks, rotating wall vessels, and perfusion bioreactors [23][24][25] . These bioreactors provide a closed and homogenous culture environment along with the capability for online monitoring and control of culture parameters that fulfill regulatory requirements [26] .…”