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Single-Cell PCR Methods for Studying Stem Cells and Progenitors

  • Jane E. Aubin
  • Fina Liu
  • G. Antonio Candeliere
Part of the Methods in Molecular Biology™ book series (MIMB, volume 185)

Abstract

Knowledge of the molecular and cellular events characterizing osteoblast development is growing as new markers, including important classes of regulatory molecules such as transcription factors (e.g., Cbfa-1 [1]), are elucidated. Nevertheless, a paucity of definitive and specific markers, especially for the more primitive progenitors and the hemopoietic lineages (2). One useful model, however, has been culture of mixed populations of freshly isolated cells derived from a variety of bones (e.g., 21-d fetal rat calvaria [RC]) or bone marrow stroma under conditions that favor osteoblast development (2). For example, when such heterogeneous primary cultures are grown long-term (approx 3 wk) in medium supplemented with ascorbic acid and α-glycerophosphate, a low frequency (about 0.00001–1% of unfractionated freshly isolated populations) of osteoprogenitor cells present divide and differentiate to form 3-dimensional mineralized bone nodules (3,4). These infrequent cells comprise the colony forming units or colony forming cells-osteoprogenitor (CFU-Os or CFC-Os, respectively) in populations from the whole tissue and appear analogous to the nonstem cell CFU/CFCs in lineages such as the hemopoietic. Notably, the frequency of such cells can be determined by limiting dilution, and they appear to have limited capacity for self-renewal (3,4). On the other hand, morphological, immunohistochemical, and molecular analyses have confirmed that differentiation of CFU-Os and formation of bone nodules reproducibly recapitulates a proliferation-differentiation sequence from an early precursor cell to a mature osteoblast (2).

Keywords

Polymerase Chain Reaction Terminal Deoxynucleotidyl Transferase Laser Capture Microdissection Polyester Cloth Bone Marrow Stroma 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Jane E. Aubin
    • 1
  • Fina Liu
    • 2
  • G. Antonio Candeliere
    • 1
  1. 1.Department of Anatomy and Cell BiologyUniversity of TorontoTorontoCanada
  2. 2.INSERMHõpïtal Edouard HerriotLyonFrance

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