High quality clinical grade human embryonic stem cell lines derived from fresh discarded embryos

Abstract

Background: Human embryonic stem cells (hESCs) hold tremendous promise for cell replacement therapies for a range of degenerative diseases. In order to provide cost-effective treatments affordable by public health systems, HLA-matched allogeneic tissue banks of the highest quality clinical-grade hESCs will be required. However only a small number of existing hESC lines are suitable for clinical use; they are limited by moral and ethical concerns and none of them apply Good Manufacturing Practice (GMP) standards to the earliest and critical stages of gamete and embryo procurement. We thus aimed to derive new clinical grade hESC lines of highest quality from fresh surplus GMP grade human embryos.

Methods: A comprehensive screen was performed for suitable combinations of culture media with supporting feeder cells or feeder-free matrix, at different stages, to support expansion of the inner cell mass and to establish new hESC lines.

Results: We developed a novel two-step and sequential media system of clinical-grade hESC derivation and successfully generated seven new hESC lines of widely varying HLA type, carefully screened for genetic health, from human embryos donated under the highest ethical and moral standards under an integrated GMP system which extends from hESC banking all the way back to gamete and embryo procurement.

Conclusions: The present study, for the first time, reports the successful derivation of highest-quality clinical-grade hESC lines from fresh poor-quality surplus human embryos generated in a GMP-grade IVF laboratory. The availability of hESC lines of this status represents an important step towards more widespread application of regenerative medicine therapies.

Keywords: Embryo; Good Manufacturing Practice; Human embryonic stem cells; Pluripotency.

Fig. 1

a Derivation of human embryonic stem cell lines MAN lines 10–16 from surplus fresh IVF embryos. The derivation of human embryonic stem cell line MAN13 from (a) surplus fresh IVF embryos donated on day 4 post-fertilisation, which gave rise to (b) a blastocyst on day 6 of grade BL3Dc, with no discernible inner cell mass (two different focal planes through the blastocyst, bi and bii). Following plating onto hDF feeder cells, an outgrowth was established (c) and passaged (passage 1, P1) (d) in order to establish MAN13 at P5 – P12 (e- g), which could be maintained on a different line of hDF feeder cells (f). b The derivation of human embryonic stem cell lines MAN10–12 and MAN 14–16 on hDF feeder cells. Arrows indicate blastocysts used in derivation

Fig. 2

Immunostaining of MAN lines 10–16 for pluripotency markers. At approximately p10 after 5 days of culture on human dermal fibroblasts (hDFs), positive pluripotency markers (green) were assessed including OCT4, Nanog, SOX2, SSEA4 and TRA160. The differentiation marker SSEA1 was used as a negative control with DAPI (blue) as a counterstain. Isotype controls were mouse IgG, mouse IgM and rabbit IgG. Scale bars represent 100 μM

Fig. 3

Immunostaining of in vitro differentiated cells from MAN lines 10–16. hESCs were subjected to in vitro differentiation via embryoid body formation and each line analysed by immunocytochemistry for two markers of each germ cell layer (green) with DAPI (blue) as a counterstain. Ectoderm markers: β-tubulin III and neurofilament; mesoderm markers alpha-smooth muscle actin (alpha-SMA) and vimentin; endoderm markers: GATA6 and FOXA2. Scale bars represent 100uM

Fig. 4

Histological sections of teratomas derived from MAN lines 13–16. The teratomas were formed by subcutaneous injection of MAN13–16 cells under the kidney capsule of a SCID mouse. Stained sections show hematoxylin and eosin staining (A-L) of structures representative of the three germ layers: endoderm (A, D, G, J), mesoderm (B, E, H, K) and ectoderm (C, F, I, L) for MAN13 (A-C), MAN14 (D-F), MAN15 (G-I) and MAN16 (J-L). A, Kidney from endoderm; B, smooth muscle from mesoderm; C, retinal pigment epithelium from ectoderm; D, oesophagus from endoderm; E, artery from mesoderm; F, primitive neuroepithelium from ectoderm; G, intestine from endoderm; H, skeletal muscle from mesoderm; I, neural epithelium from ectoderm; J, sebaceous gland from endoderm; K, smooth muscle from mesoderm; L, skin tissue from ectoderm. a-d Alcian blue-fast red staining showing cartilage (mesoderm) in (a) MAN13, (b) MAN14, (c) MAN15 and (d) MAN16. Scale bars represent 100uM

Fig. 5

Copy number variants of unknown clinical significance. Data for individual microarray probes are represented by dots and plotted on a log₂ scale of the ratio of hESC DNA/reference DNA. Sub-images are not to scale. Aberrations detected by Cytosure™ Interpret software are visible as shaded segments and the average log₂ ratio of these segments is indicated by thick solid lines. a Approximately 3.1–3.2 Mb loss of 10q21.1 seen in MAN10 (b) approximately 87–94 kb gain of Xp22.33 seen in MAN11 (c) approximately 94–189 kb gain of Xp11.21 seen in MAN13 (d) gain of 20q11.21 seen in MAN13 (blue data approximately 1.1–1.4 Mb ) and MAN14 (green data approximately 1.01–1.1 Mb) (e) approximately 187–277 kb loss of 2p15 seen in MAN15 (f) approximately 346–465 kb gain of 6q26 seen in MAN15