title: 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.
authors.affiliation: Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, The Academic Center for Education, Culture and Research, Tehran, Iran.
authors.forename: Mehran Rezaei
authors.initials: MR
authors.lastname: Larijani
authors.forename: Ali
authors.initials: A
authors.lastname: Seifinejad
authors.forename: Behshad
authors.initials: B
authors.lastname: Pournasr
authors.forename: Vahid
authors.initials: V
authors.lastname: Hajihoseini
authors.forename: Seydeh-Nafiseh
authors.initials: SN
authors.lastname: Hassani
authors.forename: Mehdi
authors.initials: M
authors.lastname: Totonchi
authors.forename: Maryam
authors.initials: M
authors.lastname: Yousefi
authors.forename: Farnaz
authors.initials: F
authors.lastname: Shamsi
authors.forename: Ghasem Hosseini
authors.initials: GH
authors.lastname: Salekdeh
authors.forename: Hossein
authors.initials: H
authors.lastname: Baharvand
mesh_terms: D000943:Antigens, Differentiation / Q000378:metabolism; D017209:Apoptosis; D002449:Cell Aggregation; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D048430:Cell Shape; D002478:Cells, Cultured; D015925:Cryopreservation; D017077:Culture Media, Conditioned; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology / Q000502:physiology; D020869:Gene Expression Profiling; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D059785:Karyotype; D010446:Peptide Fragments / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors
chemical_list: D000943:Antigens, Differentiation; D017077:Culture Media, Conditioned; D010446:Peptide Fragments; C518603:protein kinase inhibitor peptide (5-24); D016222:Fibroblast Growth Factor 2; D054460:rho-Associated Kinases