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. 2012;7(7):e41958.
doi: 10.1371/journal.pone.0041958. Epub 2012 Jul 30.

Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions

Stefan Frank  1 , Miao Zhang, Hans R Schöler, Boris Greber
Affiliations

Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions

Stefan Frank et al. PLoS One. 2012.

Abstract

Human pluripotent stem cells (hPSCs) are conventionally grown in a mouse feeder cell-dependent manner. Chemically defined culture conditions are, however, desirable not only for potential medically oriented applications but also for investigating mechanisms of self-renewal and differentiation. In light of the rather high complexity and cost of existing defined hPSC culture systems, we have systematically evaluated over 20 potential media ingredients. Only components that reproducibly gave beneficial effects were ultimately combined to yield a simple and cost-effective formulation termed FTDA. This xeno-free medium is based on mimicking self-renewal factor activities present in mouse embryonic fibroblast-conditioned medium, at minimal dosages. Additionally, small molecule inhibitors of BMP and WNT signaling served to specifically suppress typical types of spontaneous differentiation seen in hPSC cultures. FTDA medium was suitable for the generation of human induced pluripotent stem cells and enabled robust long-term maintenance of diverse hPSC lines including hard-to-grow ones. Comparisons with existing defined media suggested reduced spontaneous differentiation rates in FTDA. Our results imply that using supportive factors at minimal concentrations may still promote robust self-renewal and preserve pluripotency of hPSCs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Dorsomorphin prevents spontaneous TE-like differentiation in chemically defined hESC medium.
(A) Colonies in N2B27+ FGF2 fail to expand well after several passages (line HuES6). (B) Typical spontaneous differentiation formed in centers of colonies. Representative differentiated and undifferentiated parts were collected for further analysis (right panels). (C) Real-time RT-PCR analysis of samples in (B). (D) Immunocytochemistry confirms that inner differentiated cells stain positive for HAND1. (E) Dorsomorphin addition to N2B27 completely prevents TE-like differentiation in routine culture (line H7). (F) Titration of optimal dorsomorphin dosage. At 50 nM, both TE-like and neural differentiation rates were minimal.
Figure 2
Figure 2. FGF2, TGFβ1, and Activin A cooperatively sustain self-renewal in hESCs.
(A) Colonies in N2B27+ FGF2 with or without DM typically exhibit weak NANOG staining in their centres. (B) RT-qPCR analysis showing that FGF2 and TGFβ1 sustain NANOG in a cooperative or synergistic manner (passage 1: n = 2, passage 3: n = 3, all in presence of DM). (C) Typical morphology at passage 2 showing that FGF2 or TGFβ1 alone fail to robustly sustain self-renewal. When applied in combination, however, larger colonies with reduced rates of differentiation are obtained. (D) Titration of FGF2 and TGFβ1 dosage using the NANOG expression level as a readout, in presence of DM. Arrows mark concentrations chosen for further investigation. (E) RT-qPCR analysis showing that additional supplementation with Activin A further enhances the expression of self-renewal genes in hESCs (left chart, n = 5). Note that a dosage of 2.5 to 5 ng/ml was sufficient to achieve near-saturating expression levels. In contrast, the induction of mesendodermal differentiation genes at this concentration of Activin A was still moderate (right chart, n = 5, in presence of FGF2, TGFβ1, and DM over three passages). (F) Analysis of the mesendodermal gene induction signature by Activin A (n = 3, in presence of FGF2, TGFβ1, and DM). Arrows mark the supplementation with Activin A. Note that the Activin-induced upregulation of mesendodermal markers EOMES, GSC, and MIXL1 is fully reversible. Data in this figure: Line HuES6.
Figure 3
Figure 3. Optimization of accessory media ingredients.
(A) HSA supplementation caused superior colony expansion compared to BSA. (B) 0.1% HSA was sufficient for obtaining saturated expression levels of OCT4, NANOG, and SOX2. (C) HSA supplementation promoted improved colony expansion as compared to albumin-free conditions. (D) Addition of defined lipids enabled improved colony expansion with most lines (NCL3 hESCs, n = 2). (E) Optimized FTDA medium enabled robust maintenance of arbitrarily chosen hES and hiPS cell lines over multiple passages, with minimal spontaneous differentiation. Top: Representative phase contrast morphology. Bottom: Stereo microscopic view. (F) Microarray cluster analysis of several hPSC lines cultured in MEF-CM and FTDA as well as human fibroblasts demonstrated clustering of individual lines independent of the medium used. (G) Global gene expression comparison of HuES6 hESCs cultivated in MEF-CM and FTDA showed highly similar expression patterns for most genes, including pluripotency markers.
Figure 4
Figure 4. Validation of optimized FTDA medium.
(A) hESCs grown in FTDA medium for more than 20 passages exhibited robust expression of self-renewal markers (top row), stable karyotype, and rapid growth rates (bottom left). Bottom right: FTDA allowed clonal expansion from single cells replated in the presence of Y27632 as well as effective formation of embryoid bodies. (B) hES cells grown in FTDA for multiple passages could be induced to form early neuroectodermal, mesodermal, or endodermal precursors, using the same basal medium but different growth factors (top row). Using directed or spontaneous differentiation protocols, cells gave rise to terminally differentiated cell types of all three germ layers (middle row). Bottom: H&E stained teratoma sections formed by HuES6 cells grown for more than 20 passages in the defined medium.
Figure 5
Figure 5. Direct reprogramming in FTDA.
(A) FTDA allowed derivation of hiPS colonies after viral transduction of adult dermal fibroblasts with OCT4, SOX2, KLF4 and c-MYC. (B) The number of hiPS colonies increased over time, reaching an average of 24 per 12,500 seeded cells (0.2%). (C) Emerging hiPS colonies could be picked and expanded in FTDA and homogenously stained positive for pluripotency markers OCT4 and NANOG. (D) Spontaneous in-vitro differentiation of hiPSCs derived under FTDA conditions yielded cell types representative of all 3 germ layers.
Figure 6
Figure 6. Media comparison and model of FTDA mechanism.
(A) hESC line NCL3 and (B) iPS line 1 were cultured for 3 passages in FTDA, in MEF-CM, and in proprietary media. All media sustained pluripotency, but differentiation tended to be increased in MEF-CM and some proprietary media, compared to FTDA. Top: Stereo microscopic view. Bottom: Gene expression analysis relative to FTDA (n = 3). (C) Model of our understanding of the pluripotency-sustaining mechanism of FTDA. FGF2, TGFβ1 and Activin A cooperatively support self-renewal, whereas DM inhibits spontaneous extraembryonic differentiation. IWP-2 may additionally be used if cells tend to spontaneously differentiate into mesendoderm.
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