A method for clonal analysis of epidermal growth factor-responsive neural progenitors

被引:27
作者
Engstrom, CM
Demers, D
Dooner, M
McAuliffe, C
Benoit, BO
Stencel, K
Joly, M
Hulspas, R
Reilly, JL
Savarese, T
Recht, LD
Ross, AH
Quesenberry, PJ [1 ]
机构
[1] Univ Massachusetts, Med Ctr, Ctr Canc, Dept Neurol, Worcester, MA 01605 USA
[2] Univ Massachusetts, Med Ctr, Ctr Canc, Dept Pharmacol & Mol Toxicol, Worcester, MA 01605 USA
[3] Roger Williams Gen Hosp, Res Dept, Providence, RI 02908 USA
关键词
neural progenitor cell; neural stem cell; epidermal growth factor; neurosphere differentiation; cell sorting;
D O I
10.1016/S0165-0270(02)00074-2
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Epidermal growth factor (EGF) responsive neural progenitors are defined by clonal growth from single cells. In previous studies we were unable to obtain clones at single cell densities using trypsinized cells and trituration alone always gave cellular aggregates. Here we report on single cell derived clones using a technique involving trituration of EGF responsive neurospheres, cell filtration, and single cell sorting using a MoFlo high speed fluorescence activated cell sorter. Single cell deposition was confirmed by labeling cells with Hoechst 33342 and Flow-check Fluorospheres, and visualization by fluorescence microscopy. The cells were deposited into liquid medium and grown from single cells in 10-20 ng/ml EGF for 12-14 days. This gave a cloning efficiency of 2.12%+/-0.37. New colonies occurred as late as day 18 post-sort. Tritiated thymidine suicide indicates that a percentage of these cells are cycling. Immunohistochemical analysis for oligodendrocytes, astroglia, and neuronal lineages performed on colonies at 10-14 and 21-28 days gave 39% uni-lineage, 36% bi-lineage, and 25% tri-lineage colonies. A total of five different types of progenitor cells were observed. In individual colonies, oligodendrons predominated with a lesser presence of astroglial or neuronal cell types. This approach establishes a reliable and reproducible method for single cell cloning of neurosphere cells. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:111 / 121
页数:11
相关论文
共 47 条
  • [1] AHMED S, 1995, J NEUROSCI, V15, P5765
  • [2] Arsenijevic Y, 1998, J NEUROSCI, V18, P2118
  • [3] Baas D, 1997, GLIA, V19, P324, DOI 10.1002/(SICI)1098-1136(199704)19:4<324::AID-GLIA5>3.0.CO
  • [4] 2-X
  • [5] BAYER SA, 1982, SCIENCE, V216, P890, DOI 10.1126/science.7079742
  • [6] Growth and fate of PSA-NCAM+ precursors of the postnatal brain
    Ben-Hur, T
    Rogister, B
    Murray, K
    Rougon, G
    Dubois-Dalcq, M
    [J]. JOURNAL OF NEUROSCIENCE, 1998, 18 (15) : 5777 - 5788
  • [7] Turning brain into blood: A hematopoietic fate adopted by adult neural stem cells in vivo
    Bjornson, CRR
    Rietze, RL
    Reynolds, BA
    Magli, MC
    Vescovi, AL
    [J]. SCIENCE, 1999, 283 (5401) : 534 - 537
  • [8] Enrichment of neurons and neural precursors from human embryonic stem cells
    Carpenter, MK
    Inokuma, MS
    Denham, J
    Mujtaba, T
    Chiu, CP
    Rao, MS
    [J]. EXPERIMENTAL NEUROLOGY, 2001, 172 (02) : 383 - 397
  • [9] Chiasson BJ, 1999, J NEUROSCI, V19, P4462
  • [10] Subventricular zone astrocytes are neural stem cells in the adult mammalian brain
    Doetsch, F
    Caillé, I
    Lim, DA
    García-Verdugo, JM
    Alvarez-Buylla, A
    [J]. CELL, 1999, 97 (06) : 703 - 716