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Adult hematopoietic progenitors are multipotent in chimeric mice

2012-07-02 15:24:21

Comptes Rendus Biologies; 2012 Jul; 335(7):454-62


Bernard Pessac, Vamshi K. Nimmagadda, Tapas Makar, Paul S. Fishman, Christopher T. Bever, Jr., and David Trisler



Introduction



The search for pluripotent stem cells capable of restoring damaged functions of altered organs is the Holy Grail of tissue and organ engineering in mammals. Early investigations had shown that differentiation gene products are present in tissues other that their main site of expression. Indeed, we have reported that
bone stem marrow cells, as well as differentiated lineages to which they give rise, express the myelin basic protein gene that codes for a brain structural protein. In parallel, it has been claimed that cells derived from bone marrow can give rise to various cell types including neural cells, pancreatic cells or cardiac muscle cells. Taken together, these data suggested that bone marrow cells, or a subset of them, might be multipotent.



Another line of research has been to engineer pluripotent cell lines: as a first step, embryonic stem cells (ESCs) were derived from the inner cell mass of
early embryo blastocysts, cells that are able to form each of the three embryonic germ layers ectoderm, mesoderm and endoderm. But, because of practical issues ESCs are problematic for human cell replacement therapy. Then an important step was the creation of a mouse clone from olfactory neurons showing that a differentiated cell could be reprogrammed back to an embryonic stem cell like stage. The more recent breakthrough has been the induction of pluripotent stem cells (iPSCs) by the genetic manipulation of normal adult somatic cells such as fibroblasts. However, induction of pluripotency may evoke genetic changes leading
eventually to tumorigenicity. This lead us to investigate the existence of natural adult stem cells whose pluripotency could lead to or take part in the generation of a new organ(s).



Indeed, we had previously reported that a subpopulation of mouse bone marrow hematopoietic progenitors with the CD34+ antigen naturally expresses an array of
ESC genes including Oct-4, Rex-1, Sox-2, Klf-4 and LIN-28, genes that have been initially used to induce iPSCs. We had also found that this same subset of adult stem cells expresses ectodermal neural lineage genes, mesodermal cardiac lineage genes as well as endodermal pancreatic and intestinal genes. Taken together, the fact that these bone marrow cells express both embryonic stem cell genes and lineage genes for each of the three embryonic germ layers suggests that they may be
pluripotent. Here we illustrate that when these adult bone marrow CD34+ cells are transplanted into mouse blastocysts, they give rise to organs of adult mice such as ectodermal derived brains, mesodermal cardiac cells, bone and bone marrow cells and endodermal pancreas and intestinal cells, indicating the pluripotency of these CD34+ bone marrow.



Results



Generations of Chimeric Mice



Because a subset of adult CD34+ bone marrow stem cells expresses both embryonic stem cell genes and lineage genes for each of the three embryonic germ cell layers, pluripotency of these cells was suggested. We ascertained their pluripotency by transplanting the adult CD34+ cells into mouse blastocysts. Adult CD34+ stem cells from a male C57Bl/2J transgenic mouse that expressed β-galactosidase, B6.129S7-Gt(ROSA)26Sor/J were cultured as previously reported. When these ROSA CD34+ cells were treated with X-gal they stained blue, whereas wildtype CD34+ cells do not stain with X-gal (Fig. 2). These male ROSA CD34+ cells were transplanted into wildtype C57Bl/6J blastocysts that do not express β-galactosidase. As a control, wildtype CD34+ cells that do not have the β-galactosidase transgene were transplanted into separate wildtype mouse blastocysts. Eight to fifteen adult ROSA or wildtype CD34+ cells were transplanted into each E 3.5 stage blastocyst. At E 3.5, each blastocyst is composed of 64 cells including both the trophoblasts that make up a single cell layer thick wall of the blastocyst that go on to become all of the extra-embryonic structures, e.g., placenta, umbilical cord, amnion and chorion, as well as the inner cell mass of embryonic stem cells that forms the embryo itself. After transplantation of the ROSA CD34+ cells, staining of the chimeric blastocyst with X-gal revealed the blue-stained ROSA CD34+ cells within the blastocoel of the unstained wildtype blastocysts (Fig. 2). The chimeric blastocysts then were implanted into pseudo-pregnant mice. The chimeric embryos were allowed to develop normally through birth to weaning. All chimeric mice were robust and behaved normally.



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