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

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

The Plasticity of Adult Stem CellThe Plasticity of Adult Stem Cell

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

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

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

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

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

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.

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

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

Table 1. Engraftment of rat Table 1. Engraftment of rat GFPGFP++ cells in chick embryoscells in chick embryos

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.

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.

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.

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.

Thank you!Thank you!