Chemically defined conditions for human iPSC derivation and culture

Abstract

We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.

Fig. 1. Albumin is not required for human ES cell culture

(a) The plots show cloning efficiency (day 5) of H1 ES cells plated at clonal density (500 cells/well) with ROCK inhibitor on a Matrigel-coated surface in TeSR media made with different commercial batches of BSA. (*p < 0.05, n = 3, relative to BSA batch 1, the only batch supporting long-term self-renewal). (b) Micrographs show colonies after 72 hours of incubation in TeSR with different batches of BSA. Scale bar = 70 µm. (c) FACS analysis of OCT4 expression of cells depicted in (b). Green peak: OCT4 staining; unshaded peak: mouse IgG control. (d) The plots show survival of dissociated H1 ES cells 24 hours after plating into simplified medium (TeSR core) with or without BSA (Supplementary Table 1, Supplementary Fig. 1A). The “Survival Index” represents the number of surviving cells divided by the number of input cells (*p < 0.05, n = 3). (e) Plots show cell survival of dissociated cells 24 hours after plating into TeSR-based medium with the indicated combinations of BSA and β-mecaptoethanol (BME). (The control medium is TeSR core, Supplementary Table 1). (*p < 0.05, n = 3). (f) In the cell culture described in (e), cell proliferation was measured 120 hours after plating (*p < 0.05, n = 3). The “Proliferation Index” represents the cell number at a specific time point divided by the number of input cells at time 0.

Fig. 2. Essential media components for human ES cell survival and proliferation

(a) The plots show survival of dissociated H1 ES cells 24 hours after plating into the indicated media (Supplementary Table 1) on Matrigel-coated plates. (*p < 0.05, n = 3, relative to survival in TeSR) (b) The plots show proliferation of cells from (a) 96 hours after plating and culture in the same media with daily media change.(*p<0.05, n=3, relative to proliferation in TeSR) (c) The plots show survival (blue) and proliferation (red) of human ES cells dissociated and plated in the indicated media, 22 h and 129 h after plating, respectively. (*p < 0.05, n = 3). (d) The plots show cell numbers over time of H1 cells maintained in defined media (DMEM/F12, NaHCO₃, Insulin, FGF2 and LAA) for multiple passages with or without selenium. 150,000 starting cells were seeded in each passage on day 0, 4 and 7. (e) FACS analysis of OCT4 expression in H1 cells grown in the indicated media for 4 passages. Green peak, OCT4 staining; unshaded peak: mouse IgG control. (f) The plots show cloning efficiency of human foreskin iPS cells grown in the indicated defined media, with ROCK inhibitor HA100, in hypoxic conditions (*p < 0.05, n = 3). Similar results were obtained for human ES and other iPS cell lines. (g) The plots show cloning efficiency of ES cells in the indicated media under hypoxic (red) and normoxic (blue) conditions. (*p < 0.05, n = 3). Similar results were obtained from iPS cell lines. (h) The plots show fold expansion of ES (H1) and iPS cells cultured under hypoxic conditions in the indicated media. 200,000 cells were plated at each passage.

Fig. 3. Vitronectin coated surfaces support human ES and iPS cells cultured in E8 medium

(a) The schematics show the four vitronectin variants (VTN-WT, VTN-C, VTN-N, VTC-NC) that were expressed and purified as coating materials for ES cells. SMB (Somatomedin B domain) and V10 are functional domains of wild type vitronectin; RGD, integrin-binding site. (b) The plots show cell survival on the four vitronectin variants in E8 media after 24 hours. The survival index was normalized to cell survival on Matrigel (*p < 0.05, n = 3). (c) The plots show cell survival after 24 hours on VTN-NC (right) and matrigel (left) in E8(TGFβ) media after passaging with EDTA (red) and TrypLE (blue) (*p < 0.05, n = 3). (d, e) The plots show cell survival after 24 hours (d) or cloning efficiency (e) on VTN-NC (red) and matrigel (blue) surfaces in the presence of the indicated small chemical inhibitors (*p < 0.05, n = 3).

Fig. 4. Reprogramming fibroblast cells in fully defined condition

(a) Schematic of procedure to derive integration-free iPS cells from fibroblast cells in fully defined conditions. (b) The micrographs show a typical iPS colony 25 days after reprogramming (left, scale bar = 200 µm.) and prior to picking, and after first passage (right, scale bar = 25 µm.). This particular iPS clone was maintained in E8 (NODAL) on Matrigel. (c) FACS analysis of OCT4 and SSEA4 of a typical iPS cell line derived from foreskin fibroblasts and maintained in E8 for 20 passages. Greeⁿ peak: OCT4 staining; unshaded peak: mouse IgG control. (d – g) Human foreskin fibroblasts were reprogrammed in the indicated media (see Methods for details). The plots show reprogramming efficiency scored after 30 days. In (d), sodium butyrate (100 µM) was added to both conditions to improve efficiency. (In (d, f, g), *p < 0.05, n = 3, experiments were repeated twice (d, f) and five times (g), with similar results. In (e), due to the inconsistency of reprogramming efficiency in TeSR, four independent experiments were each repeated three times, *p < 0.05, n = 12).

Fig. 5. Derivation of human iPS cells directly from biopsy samples in chemically defined conditions

(a, b) The plots show growth (fold change in cell number) of adult fibroblast cells (a) plated onto the indicated plate coating materials in E8-based fibroblast media (see Methods) and counted after 4 days (*p < 0.05, n = 3), or (b) cultured in the indicated media on vitronectin (see Methods) and counted 96 hours after plating (*p < 0.05, n = 3). (c) The micrographs show three fibroblast cell lines derived from skin biopsies in defined fibroblast medium on vitronectin-coated plates. Scale bar = 100 µm. (d) The micrographs show representative iPS colonies obtained by reprogramming fibroblasts from (c) according to the procedure in Figure 4a, in the presence of butyrate. Colonies are shown after multiple passages in E8 (TGFβ). Scale bar = 100 µm. (e and f) FACS analysis of pluripotency markers OCT4 and SSEA4 in iPS cells after 10 passages.