Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

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

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 microM 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.

Genetic stability of human embryonic stem cells: A first-step toward the development of potential hESC-based systems for modeling childhood leukemia.

Human ESCs provide an opportunity for modeling human-specific strategies to study the earliest events leading to normal hematopoietic specification versus leukemic transformation. Of interest, are the human childhood acute leukemias harboring specific fusion oncogenes such as MLL-AF4, TEL-AML1 or BCR-ABL wherein clinically significant manifestations arise in utero. The mechanisms of transformation are not amenable to analysis with patient samples and, many mouse models for pediatric leukemias have fallen short in illuminating the human disease because they do not recapitulate key aspects of the actual disease, suggesting that the mouse models are missing essential components of oncogenesis present in the human embryo. Prior to using hESCs as a tentative system for modeling leukemia, robust studies aimed at demonstrating their genetic stability are required; otherwise, cooperating mutations already present could prime hESCs susceptible to transformation. We performed an extensive molecular cytogenetic and cellular in vitro and in vivo analysis which reveals an overall genomic stability of HS181 and HS293 hESCs maintained long-term by mechanical dissociation in human feeders. Importantly, we show for the first time that the genetically stable HS181 hESC line differentiates into CD45+ hematopoietic cells and clonogenic hematopoietic progenitors. This data should encourage stem cell researchers to implement robust cytogenetic tools when assessing hESC genetic stability, in order to detect tiny but relevant biological functional or structural chromosome abnormalities and, paves the way for generating fusion oncogene-expressing transgenic hESCs as a human-specific system for studying the early in utero events leading to normal hematopoietic specification versus childhood leukemic transformation.