Identification and characterization of side population cells in embryonic stem cell cultures.

Marker and functional heterogeneity has been described for embryonic stem cells (ESCs). This property has been correlated with the presence of ESC subpopulations resembling pluripotent cell lineages of the embryo. The ability to efflux Hoechst (Ho) displayed by side population (SP) cells has proven valuable as a marker to identify multipotent stem cells from a variety of tissues. Here we report that cultures from different ESC lines consistently show an SP population that displays antigens of undifferentiated ESCs, distinct drug efflux properties, and an expression pattern of ABC transporters, inner cell mass (ICM), and epiblast genes, which distinguish it from the non-SP ESC fraction. This SP population contains pluripotent cells that differentiate into ectoderm, mesoderm, and endoderm in embryoid body and teratoma assays. Further, purified SP cells efficiently integrate into developing morulae and contribute to ICM. Under standard ESC culture conditions, SP and non-SP populations display ability to convert into each other; however, an equilibrium establishes between these fractions. Using protocols customized for SP ESCs, we report that cells with similar efflux properties can be identified in the ICM of peri-implanted blastocysts. Our results indicate that ESCs display heterogeneity for the SP marker, and the SP population of these cultures contains cells that phenotypically and functionally resemble efflux-active ICM cells of the peri-implanted embryo. Our observations suggest an involvement of the SP phenotype in ESC maintenance and early embryo development, and support the idea that ESCs are composed of distinct phenotypic and functional pluripotent subpopulations in dynamic equilibrium.

Pluripotentiality and conditional transgene regulation in human embryonic stem cells expressing insulated tetracycline-ON transactivator.

Conditional manipulation of gene expression by using tetracycline (TET)-ON based approaches has proven invaluable to study fundamental aspects of biology; however, the functionality of these systems in human embryonic stem cells (hESC) has not been established. Given the sensitivity of these cells to both genetic manipulation and variations of culture conditions, constitutive expression of TET transactivators might not only be toxic for hESC but might also impair their ability to self-renew or differentiate into multiple tissues. Therefore, the effect of these transactivators on the biology and pluripotentiality of hESC must first be evaluated before broad use of TET-ON methodologies is applied in these cells. Improved insulated lentivectors that display stable transgene expression and minimal insertional transactivation have been described for hESC. By using insulated lentivectors that allow simultaneous expression of TET components and fluorescent reporters, here we demonstrate that hESC constitutively expressing the TET-ON transactivator rtTA2SM2 can be derived and expanded in culture while retaining inducible transgene expression and pluripotentiality, including marker expression, a normal karyotype, and the ability to generate multiple tissues of different germ layer origin in teratomas. We also show that these cells retain the ability to control the expression of a stable integrated transgene in a doxycycline-dependent manner, which demonstrates that an insulated TET-ON lentiviral system is functional in hESC. Together, our results indicate that improved TET regulators like rtTA2SM2 in combination with insulated lentiviral-based systems offer alternative strategies for conditional gene expression in hESC. Disclosure of potential conflicts of interest is found at the end of this article.

Plasticity and tissue regenerative potential of bone marrow-derived cells.

Diverse in vivo studies have suggested that adult stem cells might have the ability to differentiate into cell types other than those of the tissues in which they reside or derive during embryonic development. This idea of stem cell "plasticity" has led investigators to hypothesize that, similar to embryonic stem cells, adult stem cells might have unlimited tissue regenerative potential in vivo, and therefore, broad and novel therapeutic applications. Since the beginning of these observations, our group has critically examined these exciting possibilities for mouse bone marrow-derived cells by taking advantage of well-characterized models of tissue regeneration, Cre/lox technology, and novel stem cell isolation protocols. Our experimental evidence does not support plasticity of hematopoietic stem cells as a frequent physiological event, but rather indicates that cell fusion could account for reported cases of hematopoietic stem cell plasticity or "transdifferentiation" in vivo. Our studies highlight the need for meticulous technical controls during the isolation, transplantation, tracking, and analysis of bone marrow-derived cells during in vivo studies on plasticity. Further studies will be necessary to better define experimental conditions and criteria to unequivocally prove or reject plasticity in vivo. In this review, we focus on results from several studies from our laboratory, and discuss their conclusions and implications.