Derivation of human embryonic stem cell lines from single blastomeres of low-quality embryos by direct plating

Abstract

Purpose: To explore a simple method of establishing pluripotent human embryonic stem cell (hESC) lines from single blastomeres of low-quality (LQ) embryos.

Methods: Blastomeres were isolated from normally fertilized, day-3 pre-implantation LQ embryos by dissolving of the zona pellucida and were then plated directly onto inactivated human foreskin fibroblasts. The subsequent culture was identical to that used to derive a hESC line from the inner cell mass of a blastocyst. The established hESC lines were passaged and characterized.

Results: Two hESC lines were produced by culturing the blastomeres individually in a hESC culture system (hESC-CS). Both of the hESC lines maintained a normal 46-chromosome XY karyotype, expressed stemness markers, and showed a pluripotent phenotype, including the ability to differentiate into all three germ layers in vitro and in vivo.

Conclusions: The blastomeres of LQ embryos have a developmental capacity that necessitates prolonged culture. Plating of blastomeres from LQ embryos directly into the hESC-CS is a feasible method for deriving hESC lines.

Fig. 1

Morphology of the typical LQ embryos. LQ embryos were at 4-cell (a, 400 ×), 5-cell (b, 400 ×) and 6-cell (c, 400 ×) stages

Fig. 2

Derivation of the first hESC line (S1) from a single blastomere of a 5-cell stage LQ embryo (50 % fragmentation, uneven symmetry, age 30 years). a The single blastomere developed into a small morula (3–4 cells) on day-2 (D2) after plating (100 ×). b On D4, the blastomere-derived morula implanted into HFFs (100 ×). c On D7, the blastomere-derived morula produced a trophectoderm (TE)-like outgrowth without showing any sign of cavitation (100 ×). d By D9, hESC-like cells were visible in the core of the outgrowth (50 ×). e These cells developed into a hESC-like clone and TE-like cells vanished in the following days (50 ×). f The clone was mechanically passaged on D12, and a typical hESC clone was observed at 7 days after the first passage (50 ×)

Fig. 3

Derivation of the second hESC line (S2) from a single blastomere of a 5-cell stage LQ embryo (40 % fragmentation, uneven symmetry, age 28 years). a Similar to the first derivation, the single blastomere split into two cells on day-1 (D1) after plating (100 ×). b On D3, it developed into a compacted morula (100 ×). c On D8, flattened TE-like cells were visible, but they did not survive in further culture (100 ×). d–e By D12-14, a hESC-like clone was produced (50 ×). f The clone was mechanically passaged on D14, and a typical hESC clone was observed at 6 days after the first passage (50 ×)

Fig. 4

G-banding results of S1 and S2 cells. All cells of S1 (a) and S2 (b) cell lines displayed a normal male karyotype (46, XY)

Fig. 5

Pluripotency marker expression of S1 and S2 cells as detected by immunocytochemistry. S1: SSEA-3 (a), SSEA-4 (b), TRA-1-60 (c) and TRA-1-81 (d). S2: SSEA-3 (e), SSEA-4 (f), TRA-1-60 (g) and TRA-1-81 (h)

Fig. 6

Gene expression of pluripotency markers in S1 and S2 cells as detected by RT-PCR. Both S1 (a) and S2 (b) cell lines expressed stemness markers: NANOG, POU5F1, REX-1, SOX-2, LIN 28, NPM1 and GDF3 (lanes 1–7). β-actin was used as a reference marker (lane 8). No primer and no template PCR control was negative (lane 9)

Fig. 7

In vitro pluripotency of S1 and S2 cells as confirmed by RT-PCR. Embryoid bodies obtained from S1 (a) and S2 (b) cells showed the presence of cells from all three germ layers: ectoderm (lane 2: SOX1), mesoderm (lane 3: ACTC1) and endoderm (lane 4: AFP). β-actin was used as a reference marker (lane 5). No primer and no template PCR control was negative (lane 6)

Fig. 8

In vivo pluripotency of S1 and S2 cells as shown by immunohistochemistry. Teratomas obtained from S1 (a, 200 ×) and S2 (b, 200 ×) cells showed the presence of cells from all three germ layers: ectoderm (a1 and b1: squamous epithelium), mesoderm (a2 and b2: cartilage) and endoderm (a3 and b3: glandular epithelium)