title: Generation of human secondary cardiospheres as a potent cell processing strategy for cell-based cardiac repair.
abstract: Cell therapy is a promising approach for repairing damaged heart. However, there are large rooms to be improved in therapeutic efficacy. We cultured a small quantity (5-10 mg) of heart biopsy tissues from 16 patients who received heart transplantation. We produced primary and secondary cardiospheres (CSs) using repeated three-dimensional culture strategy and characterized the cells. Approximately 5000 secondary CSs were acquired after 45 days. Genetic analysis confirmed that the progenitor cells in the secondary CSs originated from the innate heart, but not from extra-cardiac organs. The expressions of Oct4 and Nanog were significantly induced in secondary CSs compared with adherent cells derived from primary CSs. Those expressions in secondary CSs were higher in a cytokine-deprived medium than in a cytokine-supplemented one, suggesting that formation of the three-dimensional structure was important to enhance stemness whereas supplementation with various cytokines was not essential. Signal blocking experiments showed that the ERK and VEGF pathways are indispensable for sphere formation. To optimize cell processing, we compared four different methods of generating spheres. Method based on the hanging-drop or AggreWell™ was superior to that based on the poly-d-lysine-coated dish or Petri dish with respect to homogeneity of the product, cellular potency and overall simplicity of the process. When transplanted into the ischemic myocardium of immunocompromised mice, human secondary CSs differentiated into cardiomyocytes and endothelial cells. These results demonstrate that generation of secondary CSs from a small quantity of adult human cardiac tissue is a feasible and effective cell processing strategy to improve the therapeutic efficacy of cell therapy.
authors.affiliation: Cardiovascular Center & Department of Internal Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul, Republic of Korea.
authors.forename: Hyun-Jai
authors.initials: HJ
authors.lastname: Cho
authors.forename: Ho-Jae
authors.initials: HJ
authors.lastname: Lee
authors.forename: Yeon-Ju
authors.initials: YJ
authors.lastname: Chung
authors.forename: Ju-Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Hyun-Ju
authors.initials: HJ
authors.lastname: Cho
authors.forename: Han-Mo
authors.initials: HM
authors.lastname: Yang
authors.forename: Yoo-Wook
authors.initials: YW
authors.lastname: Kwon
authors.forename: Hae-Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Byung-Hee
authors.initials: BH
authors.lastname: Oh
authors.forename: Young-Bae
authors.initials: YB
authors.lastname: Park
authors.forename: Hyo-Soo
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D002454:Cell Differentiation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D005260:Female; D016027:Heart Transplantation; D006801:Humans; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008875:Middle Aged; D009203:Myocardial Infarction / Q000601:surgery; D009206:Myocardium / Q000166:cytology* / Q000473:pathology; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D046509:Tissue Culture Techniques / Q000379:methods*; D023822:Tissue Engineering / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D055815:Young Adult
chemical_list: D042461:Vascular Endothelial Growth Factor A