Protocol

Basic Cell Culture Protocols

Volume 946 of the series Methods in Molecular Biology pp 257-266

Date:

Long-Term Culture-Initiating Cell Assay for Mouse Cells

  • Stefan WoehrerAffiliated withTerry Fox Laboratory, BC Cancer AgencyMedical University of Vienna
  • , Cindy L. MillerAffiliated withSTEMCELL Technologies Email author 
  • , Connie J. EavesAffiliated withTerry Fox Laboratory, British Columbia Cancer Agency

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Abstract

The long-term culture-initiating cell (LTC-IC) assay is a well-established in vitro assay used to enumerate primitive mouse hematopoietic stem cells (HSCs) and relies on the two cardinal functions of HSCs: ability to self-renew and differentiation capacity. LTC-ICs present in minimally processed and purified cell suspensions and cocultured on a supportive feeder layer are detected by their sustained ability to produce hematopoietic progenitors (colony forming cells) after ≥ 4 weeks in culture. Refinements including the use of a defined stromal cell line, and extending the in vitro culture to 6 weeks allow detection of LTC-IC at similar frequencies to transplantable HSCs quantified using in vivo assays.

Key words

Hematopoietic stem cells Stromal cells Stem cell niche LTC-IC Long-term culture-initiating cell