Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors

Abstract

The promise of pluripotent stem cells as a research and therapeutic tool is partly undermined by the technical challenges of generating and maintaining these cells in culture. Human embryonic stem cells (hESCs) are exquisitely sensitive to culture conditions, and require constant signaling by growth factors and cell-cell and cell-matrix interactions to prevent apoptosis, senescence, and differentiation. Previous work from our laboratory demonstrated that overexpression of the prosurvival gene BCL2 in mouse embryonic stem cells overrode the requirement of serum factors and feeder cells to maintain mESCs in culture. To determine whether this prosurvival gene could similarly protect hESCs, we generated hESC lines that constitutively or inducibly express BCL2. We find that BCL2 overexpression significantly decreases dissociation-induced apoptosis, resulting in enhanced colony formation from sorted single cells, and enhanced embryoid body formation. In addition, BCL2-hESCs exhibit normal growth in the absence of serum, but require basic fibroblast growth factor to remain undifferentiated. Furthermore, they maintain their pluripotency markers, form teratomas in vivo, and differentiate into all three germ layers. Our data suggest that the BCL2 signaling pathway plays an important role in inhibiting hESC apoptosis, such that its overexpression in hESCs offers both a survival benefit in conditions of stress by resisting apoptosis and obviates the requirement for serum or a feeder layer for maintenance.

Fig. 1.

Improvement in colony and EB formation with BCL2-hESCs. (A–D) Colony formation from FACS-sorted wild-type (red line), GFP- (green line), and BCL2- (blue line) hESCs when plated onto matrigel (A and B) or MEFs (C and D) in the absence (A and C) or presence (B and D) of ROCK inhibitor. Colony formation efficiency was plotted versus cells plated per well. Cell counts were capped at 50 colonies per well. (E and F) EBs were generated from RFP-hESCs (E) and BCL2-hESCs (F) after single-cell dissociation. Shown are brightfield (Left) and RFP/GFP fluorescence (Right) of hESC clusters in microwells. (G) Chimeric EBs generated from BCL2-hESCs and RFP-hESCs, mixed at multiple ratios. Plotted is the percentage of BCL2-hESCs within the EBs at 0, 1, and 4 d after generation, determined by FACS. (H) Absolute number of live hESC-derived cells per EB of GFP-hESCs (orange), constitutively expressed BCL2-hESCs (red), and inducible BCL2-hESCs in the absence (green) or presence (blue) of DOX. Two-thousand hESCs were seeded per well (40 wells per line) at day 0, then pooled at day 1. Half the EBs were harvested and counted at day 1, and the other half at day 4. Error bars are SD (n = 3) of three independent experiments.

Fig. 2.

Enhanced survival of BCL2-hESCs in serum-free media. (A) Growth kinetics of hESCs at different KOSR concentrations. Wild-type and BCL2-hESCs were plated on Matrigel and cultured in media with KOSR concentrations of 20, 1, and 0%, and cells were counted daily. Error bars are SD (n = 3). (B) Representative BCL2-hESC colonies grown in media with 20% KOSR (Left) and in 0% KOSR (Right). (C) Representative colony of BCL2-hESCs grown in media lacking both KOSR and bFGF. The same colony is shown at day 3 (Left) and day 7 (Right) after plating. (D) Alkaline phosphatase activity of BCL2-hESCs (Left) and wild-type hESCs (Right) cultured for two passages under different concentrations of KOSR and bFGF. (E) Cell-cycle analysis of GFP- (Upper Left plot and Lower graph, white bars) or BCL2-hESCs (Upper Right plot and Lower graph, black bars) cultured in complete hESC media. EdU was added 2 h before analysis. Shown are the percentages of cells at each cell cycle stage.

Fig. 3.

Human ESCs overexpressing BCL2 maintain their pluripotency markers when cultured for short-term (3 passages) or long-term (12 passages) in serum-free media. (A) Immunofluorescence staining of BCL2-hESCs after three passages in minimal media containing no KOSR with anti-Oct3/4, anti-Nanog, anti-SSEA4, and anti-Sox 17. Compared with control GFP-hESCs, BCL2 expressing hESCs had normal expression of Oct3/4, Nanog, and SSEA4. Absence of Sox 17 indicates lack of differentiation in the BCL2-hESC colonies. (B) Representative colonies of parental H9 and BCL2 expressing hESCs cultured on MEFs for 67 d (12 passages) in complete media, in minimal media lacking serum, or in minimal media for 61 d, then passaged into complete media for additional 6 d. Unmanipulated H9 cells in minimal media did not survive beyond three passages, and therefore were not analyzed. (C) Intracellular FACS profile of Oct3/4 (Left) and Nanog (Right) expression of hESC cultures at day 67. H9 and BCL2 isotype controls are shown in red and green lines, respectively. For ease of comparison, the crest of Oct3/4 and Nanog expression curves for the H9 culture in complete media are indicated by the dotted line.

Fig. 4.

Apoptosis analysis of BCL2-hESCs in minimal media. (A) Experimental design. Control and BCL2 hESCs were passaged from feeder plates onto Matrigel-coated wells in complete media. At 48 (day 2) or 24 (day 3) h before analysis, wells were changed into minimal (0% KOSR, 1 ng/mL bFGF) media for the 48 and 24 h time points, respectively. At day 4, all floating and attached cells were harvested and analyzed. (B) Photographs of hESC colonies taken at day 4 from control (H9, Upper) and BCL2 (Lower) wells in complete media (Left), or in minimal media 24 h (Center) or 48 h (Right) before analysis. (C) Apoptosis analysis of control and BCL2 hESCs, using Annexin V (y axis) and 7-AAD (x axis). Percentages of live cells (Annexin V−, 7-AAD−), dying cells (Annexin V+, 7AAD−), and dead cells (Annexin V+, 7AAD+) are shown. (D) Average percent of live (white), dying (gray), and dead (cells) from control and BCL2 hESCs in complete and minimal media. Error bars are SD (n = 3) and P values are calculated by the t test. (E) Average absolute number of live cells from control (white) and BCL2 (black) hESCs. Error bars are SD (n = 3) and P values are calculated by the t test. (F) Analysis of apoptosis of H9- and BCL2-hESCs treated with the caspase inhibitor ZVAD-FMK in minimal media. H9- and BCL2-hESCs were plated as described in A, but some wells were pretreated for 1 h with 25 μM ZVAD-FMK before changing to minimal media, then retreated with an additional 25 μM ZVAD-FMK after each media change. Human ESCs were harvested 48 h after changing to minimal media, and analyzed for Annexin V binding (y axis) and 7-AAD incorporation (x axis). Values shown are the percent of live, dying, and dead cells.