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Hum. Reprod. Advance Access published online on April 28, 2009
Human Reproduction, doi:10.1093/humrep/dep107
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mesenchymal stem cells facilitate the derivation of human embryonic stem cells from cryopreserved poor-quality embryos

J.L. Cortes1, L. Sanchez1, G. Ligero1, I. Gutierrez-Aranda1, P. Catalina1, C. Elosua1, P.E. Leone1, R. Montes1, C. Bueno1, V. Ramos-Mejía1, I. Maleno2, J.L. García-Pérez1 and P. Menendez1,3

1 Andalusian Stem Cell Bank (BACM), Centro de Investigación Biomédica, CSJA-UGR, Parque Tecnológico de la Salud, Avda del Conocimiento s/n, Granada, Spain 2 Servicio de Análisis Clínicos, Hospital Virgen de las Nieves, Granada, Spain

3 Correspondence address. Tel: +34-958-894672; Fax: +34-958-894652; E-mail: pablo.menendez{at}juntadeandalucia.es

BACKGROUND: Human embryonic stem cells (hESCs) have opened up a new area of research in biomedicine. The efficiency of hESC derivation from frozen poor-quality embryos is low and normally achieved by plating embryos on mouse or human foreskin feeders (HFFs). We attempted to optimize embryo survival and hESC derivation.

METHODS: Three conditions were tested on frozen poor-quality embryos: (i) embryo treatment with the Rho-associated kinase (ROCK) inhibitor, Y-27632; (ii) use of human mesenchymal stem cells (hMSCs) as feeders; and (iii) laser drilling (LD) for inner cell mass (ICM) isolation. Two hundred and nineteen thawed embryos were randomly treated with (n = 110) or without (n = 109) 10 µM Y-27632. Surviving embryos that developed to blastocyst stage (n = 50) were randomly co-cultured on HFFs (n = 21) or hMSCs (n = 29). ICM isolation was either by whole-blastocyst culture (WBC) or WBC plus LD.

RESULTS: Embryo survival was 52% higher with Y-27632. hMSCs appeared to facilitate ICM outgrowth and hESC derivation: three hESC lines were derived on hMSCs (10.3% efficiency) whereas no hESC line was derived on HFFs. ROCK inhibition and ICM isolation method did not affect hESC efficiency. The lines derived on hMSCs (AND-1, -2, -3) were characterized and showed typical hESC morphology, euploidy, surface marker and transcription factor expression and multilineage developmental potential. The hESC lines have been stable for over 38 passages on hMSCs.

CONCLUSION: Our data suggest that Y-27632 increases post-thaw embryo survival and that hMSCs may facilitate the efficiency of hESC derivation from frozen poor-quality embryos.

Key words: human embryonic stem cell derivation/human mesenchymal stem cells/ROCK inhibitor/Y-27632/laser drilling

Submitted on February 2, 2009; resubmitted on March 24, 2009; accepted on March 30, 2009.


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