Research Topics

  • Human embryonic stem cell (hESC) culture methods

Human embryonic stem cells are typically cultured over cell-based layers, originally mouse embryonic fibroblasts, which support indefinite pluripotential stem cell growth. Which factors are responsible for stem cell maintenance are uncharacterized at present, and it is hypothesized that embryonic origin is important for pluripotential stem cell growth. In fact, reported experiments showed that human cell lines of embryonic origin were more efficient as feeders as those isolated from neonatal or adult tissues.

Apart from the feeder layer, culture media for human stem cells has to contain certain factors which are needed for undifferentiated cell growth: the basic fibroblast growth factor (bFGF) is one of them, and the use of a serum replacement instead of complete serum (FBS) is important to maintain stemness in cell cultures.

This research line aims at developing defined and optimal conditions for the development of human embryonic stem cell lines devoid of animal-origin products. Feeder cell monolayers are substituted by proteic coatings, either synthetic, recombinant or derived from extracellular matrix, and culture media will contain only human or recombinant-origin factors.

This research line develops different methodologies for the derivation of hESC lines, based in mechanical or enzymatic splitting of the inner cell mass outgrowths, and using entire blastocysts as well as single cell derived from pre-blastocysts as initial material. The establishment of new hESC lines from blastocysts posses additional difficulties to stem cell culture, which is intrinsically laborious and tricky. Inner cell masses isolation requires high technical skills, mostly when there are no current standard and optimized protocols for the establishment of indefinite pluripotential human embryonic stem cell cultures.

This line implements workflow conditions to produce differentiated stem cells amenable to being used for future transplantation therapy. That is, to comply with GMP (good manufacturing practices) regulations, which refer to normalized procedures and their quality control processes needed to guarantee efficacy and safety in products of therapeutic use. Similar to any other pharmaceutical product, cell-based ones for human use have to be manufactured in compliance with GMP regulations and be subjected to strict and rigorous quality control policies. Pre-establishing standard operation procedures (SOPs) and quality control checkpoints, as long as contingency plans and troubleshooting measurements in case of failures occur during the overall process.

This project aims at derivation of new hESC lines from genetically abnormal embryos, either chromosomally abnormal or punctual mutation carriers. The new lines are intended to serve as models of corresponding pathologies, as a powerful tool for the study of their molecular mechanisms and the discovering of novel therapies as result of cell-based and cell-free high throughput screenings.

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