Retracted: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension

Abstract

Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.

A Universal and Robust Integrated Platform for the Scalable Production of Human Cardiomyocytes From Pluripotent Stem Cells

“…hPSCs were expanded using a previously described suspension culture method [27,32]. In brief, to initiate suspension cultures, 2 3 10 5 viable cells per milliliter were transferred to nonadhesive bacterial plates (60 mm; catalog no.…”

Cleavage of E-Cadherin and β-Catenin by Calpain Affects Wnt Signaling and Spheroid Formation in Suspension Cultures of Human Pluripotent Stem Cells

“…Published suspension culture approaches differ in several aspects such as cell dissociation and inoculation protocols, feeding strategies, and culture media composition. However, the most commonly used culture media comprise mTeSR TM 1 (5,9,12) or mouse embryonic fibroblast-conditioned medium (MEF-CM) (6,10) and usually include supplementation of the Rho-associated coiled-coil kinase inhibitor Y27632 (RI), which supports the survival of hPSCs after their dissociation into single cells (13).…”

Small-Scale Bioreactors for the Culture of Embryonic Stem Cells

“…However, Singh et al noted cell line differences in the maintenance of pluripotency within the suspension bioreactors; pluripotency markers remained high for the hES2 and hES3 lines, but Oct4, Tra-160 and SSEA4 down regulated for the ESI049 hESC line (Singh et al 2010). Other research groups have reported successful expansion of hESCs in suspension culture where the suspension culture in this case is not a suspension bioreactor but rather a shake flask (Amit et al 2010), low-attachment well plate (Olmer et al 2010) or in some cases, not described (Rezaei Larijani et al 2011;Steiner et al 2010). The data obtained from these researchers provides valuable information about the hESCs ability to differentiate in suspension where hESCs were differentiated to neural cells in suspension (Steiner et al 2010) and to neural, cardiomyocytes and hepatocytes in suspension (Rezaei Larijani et al 2011).…”

“…Other research groups have reported successful expansion of hESCs in suspension culture where the suspension culture in this case is not a suspension bioreactor but rather a shake flask (Amit et al 2010), low-attachment well plate (Olmer et al 2010) or in some cases, not described (Rezaei Larijani et al 2011;Steiner et al 2010). The data obtained from these researchers provides valuable information about the hESCs ability to differentiate in suspension where hESCs were differentiated to neural cells in suspension (Steiner et al 2010) and to neural, cardiomyocytes and hepatocytes in suspension (Rezaei Larijani et al 2011). Additionally, the ability of hESCs to maintain stable karyotypes and maintain pluripotency in suspension was established (Amit et al 2010;Olmer et al 2010;Rezaei Larijani et al 2011;Steiner et al 2010).…”

“…The data obtained from these researchers provides valuable information about the hESCs ability to differentiate in suspension where hESCs were differentiated to neural cells in suspension (Steiner et al 2010) and to neural, cardiomyocytes and hepatocytes in suspension (Rezaei Larijani et al 2011). Additionally, the ability of hESCs to maintain stable karyotypes and maintain pluripotency in suspension was established (Amit et al 2010;Olmer et al 2010;Rezaei Larijani et al 2011;Steiner et al 2010). However, these cell culture strategies are not amenable to substantial scale-up for clinical purposes due to heterogeneity and lack of control over bioprocess parameters.…”