Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells  Michaela Kunova, Kamil Matulka, Livia Eiselleova, Petra Trckova, Ales Hampl, Petr Dvorak  Reproductive BioMedicine Online  Volume 21, Issue 5, Pages 676-686 (November 2010) DOI: 10.1016/j.rbmo.2010.06.027 Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Morphology of human embryonic stem cells (ESC) growing on different feeder layers in knockout serum replacement (KSR)- and VegetaCell-supplemented medium. (A) Control human ESC maintained on mouse embryonic fibroblast feeder cells in medium with KSR formed rounded, compact colonies. (B) Many human ESC colonies grown on human foreskin fibroblasts (HFF) formed spindle-shaped colonies. (C) In addition, some human ESC colonies grown on HFF showed a peripheral layer of morphologically distinct cells (arrows). (D) The vast majority of human ESC colonies maintained on HFF in medium with VegetaCell showed rounded colonies with the occasional appearance of peripheral regions of more flattened cells (arrows). Bars=200μm. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Morphology of human embryonic stem cells (ESC) growing without feeder cells on Matrigel in mouse embryonic fibroblast (MEF)-conditioned and/or VegetaCell-supplemented medium. (A) Control human ESC maintained at low density on Matrigel in MEF-conditioned medium with knockout serum replacement (KSR) displayed rounded compact colonies. (B) When MEF-conditioned medium with KSR was replaced by medium with VegetaCell and humanized reagents, only a minority of human ESC attached and formed colonies consisting of several tens of cells that underwent progressive cell death and detached after 2–3 days in culture. (C) Increasing the density of human ESC dissociated into a single-cell suspension and grown in medium with VegetaCell resulted in a monolayer with cell morphology that was identical to that of (D) human ESC grown in MEF-conditioned medium with KSR. Insets show similar positive alkaline phosphatase activity (red) which indicated the undifferentiated status of human ESC. Bars=200μm. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Expression of undifferentiated stem cell markers in human embryonic stem cells (ESC) maintained in different culture systems. (A) Colonies growing on human foreskin fibroblasts (HFF) in medium with VegetaCell (VC) strongly expressed Oct4, NANOG, SSEA3, SSEA4, TRA-1-60 and TRA-1-81. (B) Feeder-free monolayer human ESC in medium with VegetaCell highly expressed Oct4, NANOG and SSEA3. (C) Semiquantitative reverse-transcriptase PCR shows comparable expression of POU5F1, NANOG and SOX2 among all culture conditions tested. (D) Alkaline phosphatase was highly active in human ESC growing in medium with VegetaCell both as colonies on HFF (left panel) or as a monolayer on Matrigel (right panel). (E) Flow cytometry analysis displayed high positivity for pluripotency markers SSEA3 and TRA-1-81 in human ESC cultured under all conditions tested. (F) Quantitative real-time PCR revealed differences in the expression of NANOG, TDGF1, GABRB3, DNMT3B, GDF3, POU5F1, LEFTB and GAL between standard human ESC culture on mouse embryonic fibroblast (MEF) feeder cells in knockout serum replacement (KSR) medium and monolayer culture in VegetaCell medium. The control was a sample without cDNA. FITC=fluorescein isothiocyanate; MEF-CM=MEF-conditioned medium; ML=monolayer culture. Bars=100μm. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Growth characteristics of human embryonic stem cells (ESC) maintained in different culture systems. (A) Metabolic activity of human ESC determined by a WST-1-based proliferation assay showed that human ESC on mouse embryonic fibroblast (MEF) feeder cells in medium with knockout serum replacement (KSR) grew better than human ESC on human foreskin fibroblasts (HFF) in medium with KSR, which grew better than human ESC on HFF in medium with VegetaCell. (B) Cell counts were used to establish the growth curve of monolayer human ESC. Doubling time determined at time period between 72 and 120h after plating was comparable for both culture media tested. (C) A 5-ethynyl-2′-deoxyuridine incorporation assay showed longer cell cycles in human ESC maintained on HFF and Matrigel compared with standard culture on MEF feeder cells. This prolongation was even more pronounced in VegetaCell medium. (D) TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay revealed that culture of human ESC on HFF significantly increased the number of apoptotic cells compared with culture on MEF feeder cells, while monolayer culture on Matrigel did not. Importantly, VegetaCell medium reduced the level of cell death in both culture systems. A two-tailed Student’s t-test was used to determine the significance level. ∗P=0.05; ∗∗P=0.01; ∗∗∗P=0.001. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Differentiation of human embryonic stem cells (ESC) maintained in medium with VegetaCell. (A) Human ESC maintained in standard culture conditions with mouse embryonic fibroblasts and knockout serum replacement (MEF/KSR) and human ESC growing on Matrigel as a monolayer in medium with VegetaCell for 10 passages (MG/VC) developed similar embryoid bodies after 20 days of culture in nonadherent conditions (see insets). In addition, the 5+10 day differentiation protocol produced similarly massive outgrowth of differentiated cells from human ESC maintained under both culture conditions. (B) Differentiated cells generated from two different culture settings expressed gene markers of all three germ layers indicating similar differentiation potential. Undifferentiated human ESC are shown as a reference. (C) Human ESC maintained in medium with VegetaCell showed a capacity to differentiate into neural precursor cells (left upper panel), which could further form neuron-like cells (right upper panel) strongly positive for β-tubulin III (lower panels; arrow). (D) Neurodifferentiation protocol employed in human ESC expanded in medium with VegetaCell (MG/VC) generated neural cells that showed lower expression of NOG, NCAM, PAX6 and SOX2 compared with cells generated from human ESC expanded under standard conditions (MEF/KSR). Undifferentiated human ESC are shown as a reference. Control, sample without cDNA. Bars=100μm. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions

Supplementary Figure 1 Karyotype analysis of human embryonic stem cells (ESC) maintained in medium with VegetaCell. Human ESC maintained in medium with VegetaCell as (A) colonies on human foreskin fibroblast-derived feeder layers for 10 passages and (B) a monolayer on Matrigel for 15 passages showed a normal 46,XX karyotype. Reproductive BioMedicine Online 2010 21, 676-686DOI: (10.1016/j.rbmo.2010.06.027) Copyright © 2010 Reproductive Healthcare Ltd. Terms and Conditions