A UTF1-based selection system for stable homogeneously pluripotent human embryonic stem cell cultures

Abstract

Undifferentiated transcription factor 1 (UTF1) was identified first in mouse embryonic stem cells and is also expressed in human embryonic and adult stem cells. UTF1 transcription ceases at the onset of differentiation, which clearly distinguishes it from less sensitive pluripotency markers, such as Oct4 or Nanog. We present here two transgenic hESC lines, named ZUN. Each line harbors one copy of the UTF1 promoter/enhancer driving a resistance gene and yielded highly homogeneous cultures under selection pressure, with a larger proportion of Oct4 and Sox2 positive cells. While ZUN cultures, like parental HUES8 cultures, retained the capacity to differentiate into tissues of all three germ layers using a SICD mouse teratoma model, they surprisingly exhibited an increased refractoriness to various differentiation cues in vitro. Together with its small size of only 2.4 kb for the entire cassette, these features render our selection system a powerful novel tool for many stem cell applications and human somatic cell reprogramming strategies.

Figure 1.

UTF1 expression in hESCs. (A) qRT-PCR Expression analysis of RA- (for 12 days) and DMSO-induced (for 7 days) differentiation in hESCs, normalized to that of respective undifferentiated hESC lines, with two biological replicates for each sample. (B) Schematic drawing of vectors. Full-length UTF1 was cloned into phagemid pTZ-18R yielding pTZ-UTF1. Its coding region was subsequently replaced with enhanced green florescent protein (EGFP) or neomycin (Neo), yielding pTZ-UTF1-EGFP and pTZ-UTF1-Neo, respectively. Red text indicates alterations in pTZ-UTF1-EGFP. (C) Flow cytometry of UTF1-driven EGFP expression in various cell lines following transient transfection of pTZ-UTF1-EGFP. Transfection efficiencies were normalized to those determined in parallel using pCMV-EGFP and are the average of three biological replicates. See Supplementary Figure S7 for representative dot plots.

Figure 2.

Characterization of ZUN hESC lines. (A) Expression analysis of ZUN hESCs. qRT-PCR comparison of pluripotency markers across the three hESC cultures normalized to HUES8 samples, performed with biological triplicates. (B) Oct4 and Sox2 expression analysis of ZUN hESCs. Percentage of hESCs expressing Oct4 and Sox2. ZUN cultures consist of significantly more Oct4- and Sox2-expressing cells compared to HUES8, based on the two-tailed paired Student's t-test (P-values < 0.05). (C) Surface marker analysis of ZUN hESCs. Percentage of positively stained hESCs for TRA-1-60 and TRA-1-81. Both ZUN lines consist of significantly more positively stained cells compared to parental HUES8, based on the two-tailed paired Student's t-test (P-values < 0.05). (D) qRT-PCR analysis of removal and restoration of G418 selection pressure on ZUN1 and ZUN2 cultures. ZUN lines were cultured in the presence of G418 for 90 days (+G418); G418 removed for 30 (−G418) or 60 (−/−G418) days; G418 removed for 30 days and re-introduced for 30 days (−/+G418). Expression levels were normalized to +G418 values and presented as an average of three experimental repeats.

Figure 3.

Differentiation of ZUN hESC lines on Matrigel and Gelatin. (A) Expression analysis of spontaneous differentiation using pluripotency markers. The qRT-PCR of hESCs without MEFs plated on gelatin or matrigel for 12 days was performed with biological triplicates, and presented as expression levels relative to that of respective undifferentiated cell lines. (B) AP staining of hESCs on gelatin and matrigel. Representative phase contrast photomicrographs comparing HUES8 and ZUN cell morphologies on the 12th day and visualization of the ablative effects after 2 days of exposure to G418. Scale bar: 55 µm.

Figure 4.

Differentiation of ZUN hESCs induced by RA and DMSO. (A) Expression analysis of induced differentiation using pluripotency markers. qRT-PCR of hESCs on MEFs induced to differentiate via RA (for 12 days) or DMSO (for 7 days) in differentiation medium. (B) Transcript analysis of induced differentiation using differentiation markers. (C) TRA-1-60 and TRA-1-81 surface marker immunostaining for induced differentiation, comparing HUES8 with ZUN cultures. The bottom row shows a control panel of undifferentiated HUES8 cells stained with secondary antibody only, TRA-1-60 or TRA-1-81 antibodies (left to right). Scale bar: 30 μm. (D) Transcript analysis after EB formation. qRT-PCR of EBs from hESCs induced to differentiate for 21 days in suspension cultures. The qRT-PCR experiments were performed with biological duplicates, and presented as expression levels relative to that of respective undifferentiated cell lines.

Figure 5.

Teratoma formation SCID mice studies. The hESC lines ZUN1 (A), ZUN2 (B) and the parental line HUES8 (C) were injected into the hind leg of male SCID mice, and the teratoma formation was followed by a simple grading system (see Materials and Methods section). When teratomas were full developed (1–2 g tissue weight, 6–8 weeks), they were harvested and processed by routine histological procedures, followed by heamatoxilin/eosin staining. (D) Detailed histology of ectodermal (epithelium, neuroectoderm), mesodermal (bone in red, cartilage in blue) and endodermal (glandular structures) structures are shown for each hESC line.