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

ABSTRACT: 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.

SUBMITTER: Vieyra DS

PROVIDER: S-EPMC3136721 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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

Vieyra Diego S DS Rosen Allison A Goodell Margaret A MA

Stem cells and development 20091001 8

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 un ...[more]

PMID: 19113897

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Project description:BackgroundAutologous chondrocyte implantation is an established technique for the repair of degenerated articular cartilage. Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 (Ho) fluorescence dye, has attracted attention as a method of stem cell isolation. Although SP cells from synovial tissue were expected to be an excellent source for this tissue engineering, their precise character in the synovial tissue has not been determined.MethodsSynovial tissues from bovine metacarpophalangeal joints were used as a stem cell source. For efficient collection of stem cells, we first prepared a preculture before sorting in medium containing FBS at variable concentrations for 4 days. Using a cell sorter and the Ho-dye, a poorly stained population enriched with stem cells was then isolated. To determine the characteristics of the stem cells, specific marker genes such as CD34, Flk-1, c-Kit, Abcg-2 were identified by real-time PCR. Sorted SP cells were cultured in a stem cell medium supplemented with bFGF, SCF and fibronectin, and evaluated for their differentiation potentials into chondrocytes, osteocytes and myocytes.ResultsSP cells of synovium tissue were increased from 2% of the total cell population to approximately 10% of the total cells by preculture in the 1%FBS contained medium. Sorted SP cells expressed CD34, Flk-1, c-Kit, Abcg-2 and Mdr-1 -all are important marker genes for stem cell characteristics. The SP cells could be further expanded ex vivo while maintaining stem cell potentials such as marker gene expression, Ho-dye efflux potential and multiple differentiation potentials into chondrocyte, osteocyte and myocyte.ConclusionIn the present study, we demonstrated that the cells with outstanding stem cell properties were efficiently collected as a SP fraction from bovine synovial membrane. Furthermore, we have described an efficient isolation method and the culture conditions for ex vivo expansion that maintains their important characteristics. Our results suggest that the SP cells of synovium tissue might be important candidates as sources for cell transplantation.

Project description:Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

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

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

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