Rat adult stem cells (marrow stromal cells) engraft and 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

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

The Plasticity of Adult Stem Cell

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

Outline of this Study rat GFP + MSC collections and primary cultures rat GFP + MSC collections and primary cultures MSCs transplantation into organgenesis-stage chick embyros Histological Analysis Real-time PCR Assays Karyotyping 4 days postinjection

Methods Primary Marrow Stromal Cell Cultures Primary 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

Methods Microsurgery Microsurgery 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

Methods Immunocytochemistry Immunocytochemistry Preparation of the embryo sections Embryos harvest Flash frozen FixRinse Cut into 20 μm section Mount every 9th and 10th section Incubation of the slides with antibodies Anti-α-heavy chain myosin Anti-cardiotin Alexa-594-tagged secondary anti mouse antibody

Methods Real-Time PCR Analysis for the Rat Y chromosome 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.

Methods Fluorescence-Activated Cell Sorting (FACS) and Karyotyping 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

Results Fifteen of 65 live embryos tested were shown to contain the donor cells 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 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 0.03–0.8% chimerism The extent of chimerism varied widely, but 5 of the 15 embryos showed 0.03–0.8% chimerism

Table 1. Engraftment of rat GFP + cells in chick embryos

Conclusion The rat GFP + cells expanded, but not as rapidly as the endogenous chick cells. 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 rat GFP + cells had engrafted and expanded without any major genomic alterations. The possibility of transient reductive division exists. The possibility of transient reductive division exists.

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

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

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

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