IngentaConnect A culture system using human foreskin fibroblasts as feeder cells...

Authors: Outi Hovatta¹; Milla Mikkola¹; Karin Gertow²; Anne-Marie Strömberg¹; José Inzunza¹; Julius Hreinsson¹; Björn Rozell²; Elisabeth Blennow³; Michael Andäng¹; Lars Ährlund-Richter²

Source: Human Reproduction, Volume 18, Number 7, July 2003 , pp. 1404-1409(6)

Publisher: Oxford University Press

Abstract:

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24–36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.

Keywords: blastocyst/culture/feeder cells/fibroblasts/human

Document Type: Research article

DOI: 10.1093/humrep/deg290

Affiliations: 1: Departments of Clinical Science, 2: Clinical Research Centre, Karolinska Institutet, Huddinge University Hospital, S-141 86 Stockholm, 3: Centre for Molecular Medicine, Karolinska Institutet, S-171 77, Stockholm, Sweden

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A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells