Rat%20adult%20stem%20cells%20(marrow%20stromal%20cells)%20engraft%20and%20differentiate%20in%20chick%20embryos%20without%20evidence%20of%20cell%20fusion - PowerPoint PPT Presentation

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Rat%20adult%20stem%20cells%20(marrow%20stromal%20cells)%20engraft%20and%20differentiate%20in%20chick%20embryos%20without%20evidence%20of%20cell%20fusion

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The Plasticity of Adult Stem Cell. Marrow Stromal Cells ... or fusion are generally biased to detect either one process or the other. ... – PowerPoint PPT presentation

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Title: Rat%20adult%20stem%20cells%20(marrow%20stromal%20cells)%20engraft%20and%20differentiate%20in%20chick%20embryos%20without%20evidence%20of%20cell%20fusion


1
Rat adult stem cells (marrow stromal cells)
engraftand differentiate in chick embryos
without evidence of cell fusion
  • Radhika R. Pochampally, Brian T. Neville, Emily
    J. Schwarz, Marilyn M. Li, and Darwin J.
    Prockop
  • PNAS June 22, 2004 vol. 101 no. 25

2
Introduction
  • Identification of Stem cells
  • Classification and origin of Stem Cells
  • According to the developmental stage
  • According to the differentiation potential

3
The Plasticity of Adult Stem Cell
4
Marrow Stromal Cells
  • Mesenchymal Stem Cell(MSC), pluripotent stem
    cells
  • Migration to a variety of tissues----natural
    repair system
  • Differentiation in coculture with lung epithelial
    cells----1/4 nuclear fusion

5
Outline of this Study
  • rat GFP MSC collections and primary
    cultures
  • MSCs transplantation into organgenesis-stage
    chick embyros

  • Histological Analysis Real-time PCR Assays
    Karyotyping

4 days postinjection
6
Methods
  • Primary Marrow Stromal Cell Cultures

Kill the transgenetic GFPrats, male
Take out femurs and tibias
Flush out and filter bone marrow
Culture and passage the MSCs for 2-5 times
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Methods
  • Microsurgery

Incubate chick embryos at 39C for 40 to 48 h
Remove 2 or 3 recently formed somitis on the right
Inject the rat GFP MSC suspension into the
somitie-removal space
Harvest the embryos 4 days postinjection
Control an equal volume of PBS
9
Methods
  • Immunocytochemistry

Preparation of the embryo sections
Embryos harvest
Fix
Rinse
Flash frozen
Cut into 20 µm section
Mount every 9th and 10th section
Incubation of the slides with antibodies
Anti-a-heavy chain myosin
Anti-cardiotin
Alexa-594-tagged secondary anti mouse antibody
10
Methods
  • Real-Time PCR Analysis for the Rat Y chromosome

Extract and purify DNA from both frozen sections
and whole embryos
Real-time PCR assays for rat Y chrommosome
Control pure uninjected chicken embryo DNA and
purified DNA from rat MSCs.
11
Methods
  • Fluorescence-Activated Cell Sorting (FACS) and
    Karyotyping

Disperse the embryos injected with GFP MSC
Sort the GFP cells by a flow cytometer
Culture the sorted cells
G bands staining
12
Results
  • Fifteen of 65 live embryos tested were shown to
    contain the donor cells
  • a 100- to 160-fold increase in size of embryos /
    1.5- to 33-fold increase in male rat chromosomes
  • The extent of chimerism varied widely, but 5 of
    the 15 embryos showed 0.030.8 chimerism

13
Table 1. Engraftment of rat GFP cells in chick
embryos
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Conclusion
  • The rat GFP cells expanded, but not as rapidly
    as the endogenous chick cells.
  • The rat GFP cells had engrafted and expanded
    without any major genomic alterations.
  • The possibility of transient reductive division
    exists.

20
Discussion
  • Cell fusion is a normal event during the
    development of several tissues.

21
Discussion
  • The experimental strategies used to follow
    differentiation or fusion are generally biased to
    detect either one process or the other.

22
Discussion
  • Detection of transdifferentiation and fusion in
    vivo of the adult stem cells depends highly on
    many factors.

23
Thank you!
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