Establishing and optimizing feeder cell-free culture methods for human embryonic stem cells
OJALA, MARISA (2009)
2009
Biokemia - Biochemistry
Lääketieteellinen tiedekunta - Faculty of Medicine
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Hyväksymispäivämäärä
2009-09-21
Julkaisun pysyvä osoite on
https://urn.fi/urn:nbn:fi:uta-1-20064
https://urn.fi/urn:nbn:fi:uta-1-20064
Tiivistelmä
Background and Aims: Human embryonic stem cells (hESCs) are pluripotent cells and thus provide a promising cell source for clinical applications of regenerative medicine. Currently hESCs are cultured on fibroblast feeder cell layers, which provide necessary cell-cell interactions for the attachment and soluble factors enabling the undifferentiated growth of hESCs. However, culturing of feeder cells is expensive and laborious. In addition, xeno-products, used in feeder cell and hESC cultures could transmit animal pathogens to hESCs, and cause rejections when transplanted to patients. Therefore there is a need to develop xeno- and feeder cell-free culturing methods for hESCs. The first aim of this research project was to set up and compare two commercial xeno-products containing feeder cell-free culturing methods for hESCs. The second aim was to optimize a novel, defined, serum- and xeno-free Reges medium, developed in Regea, into feeder cell-free conditions.
Methods: Regea 06/015 hESC line was cultured in mTeSRTM1 medium on MatrigelTM attachment matrix and in STEMPRO® medium on CELLStartTM attachment matrix. Human ESCs were characterized by the expression of typical genes and proteins for undifferentiated and differentiated hESCs. Gene expressions were analyzed by quantitative real time PCR and protein expressions by immunocytochemistry and fluorescence-activated cell sorter (FACS). The pluripotency of hESCs was studied by the expression of genes from different germ layers in embryoid bodies. Human ESCs were also karyotyped. Total 15 different combinations of Reges media were tested in Reges optimization experiments.
Results: Both of the commercial feeder cell-free methods and none of the tested Reges media compositions supported the undifferentiated growth of hESCs.
Conclusions: Although both of the commercial feeder cell-free methods supported the undifferentiated growth of hESCs, they both contained xeno-products and thus are not optimal methods for culturing hESCs. However, a functional feeder cell-free method could ease the workload related to the preparation of feeder cells. Despite none of the Reges media supported the undifferentiated growth of hESCs, the results provided valuable information and showed a direction to further studies.
Methods: Regea 06/015 hESC line was cultured in mTeSRTM1 medium on MatrigelTM attachment matrix and in STEMPRO® medium on CELLStartTM attachment matrix. Human ESCs were characterized by the expression of typical genes and proteins for undifferentiated and differentiated hESCs. Gene expressions were analyzed by quantitative real time PCR and protein expressions by immunocytochemistry and fluorescence-activated cell sorter (FACS). The pluripotency of hESCs was studied by the expression of genes from different germ layers in embryoid bodies. Human ESCs were also karyotyped. Total 15 different combinations of Reges media were tested in Reges optimization experiments.
Results: Both of the commercial feeder cell-free methods and none of the tested Reges media compositions supported the undifferentiated growth of hESCs.
Conclusions: Although both of the commercial feeder cell-free methods supported the undifferentiated growth of hESCs, they both contained xeno-products and thus are not optimal methods for culturing hESCs. However, a functional feeder cell-free method could ease the workload related to the preparation of feeder cells. Despite none of the Reges media supported the undifferentiated growth of hESCs, the results provided valuable information and showed a direction to further studies.