Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

Abstract

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology.

Optimization of agitation speed in spinner flask for microcarrier structural integrity and expansion of induced pluripotent stem cells

“…A, C, E EB from static culture, B, D, F EB from 25 RPM dynamic culture. Scale bar 200 lm have attempted the expansion iPSC as aggregates in spinner flask or stirred bioreactor and all three have used different agitation rates (Fluri et al 2012;Olmer et al 2012;Shafa et al 2012). The use of microcarrier for iPSC expansion has been reported by only two groups: Kehoe et al (2010) mentioned iPSC expansion in a review article, while recently, Bardy et al (2013) looked at the expansion of iPSC and its neural differentiation.…”

“…Extensive study in the field of large scale expansion of ES cells has given rise to novel and better systems for these cells but the field of dynamic culture of iPS cells has just started. Recently, efforts have also been made to expand iPSC in spinner flasks as aggregates (Fluri et al 2012;Olmer et al 2010Olmer et al , 2012Shafa et al 2012). However, expansion as aggregates is associated with some disadvantages.…”

Chemically controlled aggregation of pluripotent stem cells

“…Aggregates were dissociated and seeded into adherent conditions after three passages in suspension with DS. Directed differentiation into all three germ layers was performed as described previously for endoderm (Ungrin et al, 2012), mesoderm (Bauwens et al, 2011), and ectoderm (Fluri et al, 2012) lineages. G‐Band karyotyping was performed by WiCell (Madison, WI).…”

Scalable culture of human pluripotent stem cells in 3D

“…For example, because the materials facilitate the rapid growth of hPSCs, they might be coupled with reprogramming technologies to enhance the efficiency and yield of obtaining pluripotent cells from somatic sources in a manner similar to what has been reported for other suspension culture systems (15,16). Comparing the differentiation of various human induced PSC lines to neural (and other) lineages within the context of the 3D hydrogels might also be interesting in light of reports about the reduced efficiency and variability of differentiation of induced PSCs compared with human embryonic stem cells (17).…”