Basic fibroblast growth factor support of human embryonic stem cell self-renewal

Abstract

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic fibroblast growth factor (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. It has recently been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Herein we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100, and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture, the cells formed teratomas when injected into severe combined immunodeficient beige mice and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells and suggest that fibroblasts and fibro-blast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold.

Figure 1

Morphology of human ES cells grown in high FGF2 concentrations. 5× phase contrast images show representative morphology of H14 cells maintained for 5 days in A) CM, B) UM, C) UM 40, or D) UM 100. Arrows identify centralized regions of differentiation within UM 40 colonies. Abbreviations: CM, conditioned medium plus 4 ng/ml FGF2; UM, unconditioned medium plus 4 ng/ml FGF2; UM 40, unconditioned medium plus 40 ng/ml FGF2; UM 100, unconditioned medium plus 100 ng/ml FGF2.

Figure 2

FGF2 dose response for human ES cell self-renewal. Growth curve analysis of H9 cells cultured in CM, UM 4, UM 24, UM 40, UM 80, UM 100, and UM 250 for three passages. 5 × 10⁵ cells from CM-cultured H9 cells were plated on Day 0 of passage 1. Cell numbers were counted from triplicate wells on Days 3, 5, and 7 of each passage. Initial plating density and sampling times were repeated, when possible, for 3 passages. Average cell numbers (×10⁵ cells) with standard deviations are listed below the graph for each condition and time point. For conditions that allowed cell survival through three passages, cells were analyzed on Day 7 of passage 3 by FACS for multiple human embryonic stem cell markers (right hand box). Abbreviations: CM, conditioned medium plus 4 ng/ml FGF2; UM 4, unconditioned medium plus 4 ng/ml FGF2; UM 24, unconditioned medium plus 24 ng/ml FGF2; UM 40, unconditioned medium plus 40 ng/ml FGF2; UM 80, unconditioned medium plus 80 ng/ml FGF2; UM 100, unconditioned medium plus 100 ng/ml FGF2; UM 250, unconditioned medium plus 250 ng/ml FGF2; OCT4, octamer-binding transcription factor-3/4; SSEA4, stage-specific embryonic antigen −4; Tra1–60, tumor rejection antigen 1–60.

Figure 3

Human ES cell marker expression in UM100 cultured cells. A. 5× Phase contrast and immunofluorescence for OCT4 on H1 cells (i, iv: 24 passages in UM100), H9 cells (ii, v: 10 passages in UM100), and H1 CM-cultured controls (iii, vi: 23 passages). B. RT-PCR analysis for molecular markers expressed in H1 cells cultured in CM (17 passages) or UM100 (19 passages). C. Western blot analysis for OCT4 and NANOG of H1 cells cultured in CM (24 passages) or UM100 (26 passages). F9 cell lysate (Santa Cruz Biotechnology, Inc.) served as an OCT4-positive control. D. FACS analysis of Oct4 (i – iii), SSEA4 (iv – vi), and Tra1–60 (vii – ix) expression by H1 cells (passage 30) cultured 7 days in CM (i, iv, vii), UM 100 (ii, v, viii), or UM 4/RA (iii, vi, ix). Abbreviations: CM, conditioned medium plus 4 ng/ml FGF2; UM 100, unconditioned medium plus 100 ng/ml FGF2; M, markers; OCT4, octamer-binding transcription factor-3/4; Rex1, Reduced expression-1; hTERT, human telomerase reverse transcriptase; F9, mouse embryonal carcinoma cells; SSEA4, stage-specific embryonic antigen −4; Tra1–60, tumor rejection antigen 1–60; UM 4/RA, unconditioned media plus 4 ng/ml FGF2 and 10µM retinoic acid.

Figure 3

Human ES cell marker expression in UM100 cultured cells. A. 5× Phase contrast and immunofluorescence for OCT4 on H1 cells (i, iv: 24 passages in UM100), H9 cells (ii, v: 10 passages in UM100), and H1 CM-cultured controls (iii, vi: 23 passages). B. RT-PCR analysis for molecular markers expressed in H1 cells cultured in CM (17 passages) or UM100 (19 passages). C. Western blot analysis for OCT4 and NANOG of H1 cells cultured in CM (24 passages) or UM100 (26 passages). F9 cell lysate (Santa Cruz Biotechnology, Inc.) served as an OCT4-positive control. D. FACS analysis of Oct4 (i – iii), SSEA4 (iv – vi), and Tra1–60 (vii – ix) expression by H1 cells (passage 30) cultured 7 days in CM (i, iv, vii), UM 100 (ii, v, viii), or UM 4/RA (iii, vi, ix). Abbreviations: CM, conditioned medium plus 4 ng/ml FGF2; UM 100, unconditioned medium plus 100 ng/ml FGF2; M, markers; OCT4, octamer-binding transcription factor-3/4; Rex1, Reduced expression-1; hTERT, human telomerase reverse transcriptase; F9, mouse embryonal carcinoma cells; SSEA4, stage-specific embryonic antigen −4; Tra1–60, tumor rejection antigen 1–60; UM 4/RA, unconditioned media plus 4 ng/ml FGF2 and 10µM retinoic acid.

Figure 4

Teratoma Formation. UM 100-cultured H1 cells form teratomas generating structures representative of all developmental lineages. A. Neural rosette. B. Cartilage. C. Epithelium.

Figure 5

FGF2 Stability. Different starting concentrations of FGF2 added to conditioned or unconditioned medium were analyzed for remaining FGF2 concentrations after overnight incubation at 4° C (Blue Bars) or on H1 cells at 37° C (Red Bars). Samples were subjected to ELISA-based assays for final FGF2 concentrations. Values with standard deviations are listed below bars in ng/ml. Abbreviations: CM 4, conditioned medium plus 4 ng/ml FGF2; UM 4, unconditioned medium plus 4 ng/ml FGF2; UM 24, unconditioned medium plus 24 ng/ml FGF2; UM 40, unconditioned medium plus 40 ng/ml FGF2; UM 80, unconditioned medium plus 80 ng/ml FGF2; UM 100, unconditioned medium plus 100 ng/ml FGF2; UM 250, unconditioned medium plus 250 ng/ml FGF2.