rat adult stem cells (marrow stromal cells) engraft and differentiate in chick embryos without...
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Rat adult stem cells (marrow stromal cells) engraftRat adult stem cells (marrow stromal cells) engraftand differentiate in chick embryos without evidencand differentiate in chick embryos without evidenc
e of cell fusione of cell fusion
Radhika R. Pochampally*, Brian T. Neville*, Emily J. ScRadhika R. Pochampally*, Brian T. Neville*, Emily J. Schwarz*, Marilyn M. Li†, and Darwin J. Prockop*‡hwarz*, Marilyn M. Li†, and Darwin J. Prockop*‡
PNAS PNAS June 22, 2004 June 22, 2004 vol. 101 no. 25 vol. 101 no. 25
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IntroductionIntroduction
• Identification of Stem cellsIdentification of Stem cells
• Classification and origin of Stem CellsClassification and origin of Stem Cells According to the developmental stageAccording to the developmental stage
According to the differentiation potential According to the differentiation potential
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The Plasticity of Adult Stem CellThe Plasticity of Adult Stem Cell
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Marrow Stromal CellsMarrow Stromal Cells
• Mesenchymal Stem Cell(MSC), pluripotent stem cellMesenchymal Stem Cell(MSC), pluripotent stem cellss
• Migration to a variety of tissues----natural repair systMigration to a variety of tissues----natural repair systemem
• Differentiation in coculture with lung epithelial cellDifferentiation in coculture with lung epithelial cells----1/4 nuclear fusions----1/4 nuclear fusion
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Outline of this StudyOutline of this Study rat GFPrat GFP++ MSC collections and primary cultures MSC collections and primary cultures
MSCs transplantation into organgenesis-stage chick embyrosMSCs transplantation into organgenesis-stage chick embyros
Histological Analysis Real-time PCR Assays KaryotypingHistological Analysis Real-time PCR Assays Karyotyping
4 days postinjection
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MethodsMethods
Primary Marrow Stromal Cell CulturesPrimary Marrow Stromal Cell Cultures
Kill the transgenetic GFP+rats, 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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MethodsMethods• MicrosurgeryMicrosurgery
Incubate chick embryos at 39°C 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
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MethodsMethods• ImmunocytochemistryImmunocytochemistry
Preparation of the embryo sections
Embryos harvest
Flash frozen
FixRinse
Cut into 20 μm sectionMount every 9th and 10th section
Incubation of the slides with antibodies
Anti-α-heavy chain myosinAnti-cardiotin
Alexa-594-tagged secondary anti mouse antibody
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MethodsMethods• Real-Time PCR Analysis for the Rat Y Real-Time PCR Analysis for the Rat Y
chromosomechromosomeExtract 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.
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MethodsMethods
• Fluorescence-Activated Cell Sorting (FACS) and Fluorescence-Activated Cell Sorting (FACS) and KaryotypingKaryotyping
Disperse the embryos injected with GFP+ MSC
Sort the GFP+ cells by a flow cytometer
Culture the sorted cells
G bands staining
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ResultsResults
• Fifteen of 65 live embryos tested were shown to contFifteen of 65 live embryos tested were shown to contain the donor cells ain the donor cells
• a 100- to 160-fold increase in size of embryos / 1.5- ta 100- to 160-fold increase in size of embryos / 1.5- to 33-fold increase in male rat chromosomeso 33-fold increase in male rat chromosomes
• The extent of chimerism varied widely, but 5 of the 1The extent of chimerism varied widely, but 5 of the 15 embryos showed 0.03–0.8% chimerism5 embryos showed 0.03–0.8% chimerism
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Table 1. Engraftment of rat Table 1. Engraftment of rat GFPGFP++ cells in chick embryoscells in chick embryos
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ConclusionConclusion
• The rat GFPThe rat GFP++ cells expanded, but not as rapidly as the cells expanded, but not as rapidly as the endogenous chick cells.endogenous chick cells.
• The rat GFPThe rat GFP++ cells had engrafted and expanded cells had engrafted and expanded without any major genomic alterations.without any major genomic alterations.
• The possibility of transient reductive division exists.The possibility of transient reductive division exists.
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DiscussionDiscussion
• Cell fusion is a normal event during the development Cell fusion is a normal event during the development of several tissues. of several tissues.
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DiscussionDiscussion
• The experimental strategies used to follow differentiaThe experimental strategies used to follow differentiation or fusion are generally biased to detect either one tion or fusion are generally biased to detect either one process or the other.process or the other.
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DiscussionDiscussion
• Detection of transdifferentiation and fusion Detection of transdifferentiation and fusion in vivoin vivo of of the adult stem cells depends highly on many factors. the adult stem cells depends highly on many factors.
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Thank you!Thank you!