Characteristics Of Pluripotent Cells Research Articles

Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells.

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.

The Characteristics of Human iPS Cells and siRNA Transfection Under Hypoxia.

The characteristics of pluripotent cells have great potential for basic and clinical research and application. We describe the effect of normoxia or hypoxia regarding the proliferation and pluripotency of human iPS cells using colony number counting and real-time polymerase chain reaction (PCR). In addition, the function of hypoxia-inducible factors (HIFs) in human iPS cells under hypoxic conditions is evaluated in relation to the expression of pluripotency markers by siRNA and real-time PCR. Furthermore, we introduce the change of HIF-2α expression when signal transducer and activator of transcription 3 (STAT3) is suppressed by its inhibitor, Stattic or S31 201, using RT-PCR.

Establishment and identification of congenital clubfoot induced pluripotent stem cells

Objective To establish specific induced pluripotent stem cells (iPSCs)fromthe patient with congenitalclubfoot by taking exfoliated cells in urine as source. Methods 180 ml of urine from clubfoot patient were collected, and the exfoliated cells in urine were isolated and cultured by centrifugation. Then, the cells were reprogrammed with virus system and identified by pluripotency gene detection, alkaline phosphatase (ALP) staining, karyotype identification, Fluorescein activated cell sorter (FACS) surface markers detection, embryoid body (EB) suspension ball assay and methylation assay. Results The expression of pluripotentgenes between obtained cellsand human embryonic stem cells H9 was similar, ALP staining showed strong positive. The cells possessed normal karyotype and 84.1% positive rate of pluripotency marker Tra-1-60, they could form a large number of EB suspension balls and almost lost methylation of the promoter regionsin Nanog homeobox (NANOG) and organic cation/carnitine transporter4 (Oct4) genes. Therefore, werevealed that the obtained cells owned all of the characteristics of pluripotent cells, and wecan identified them as iPSCs. Conclusion iPSCs were successfully constructed in this study, which provided cell materials for the clarification of evolution characteristics of congenital clubfootdisease. Key words: Congenital clubfoot; Induced pluripotent stem cells; Urine-derivedcells; Reprogramming

Functional evaluation of ES-somatic cell hybrids in vitro and in vivo.

Embryonic stem cells (ESCs) have previously been reported to reprogram somatic cells following fusion. The resulting ES-somatic cell hybrids have been shown to adopt the transcriptional profile of ESCs, suggesting that the pluripotent program is dominant. ES-somatic cell hybrids have most characteristics of pluripotent cells in vitro; however, it remains unclear whether the somatic genome is an active partner in the hybrid cells or simply retained predominately as silent cargo. Furthermore, the functional properties of ES-somatic cell hybrids in vivo have been limited to studies on their contribution to teratomas and developing embryos/chimeras. The extent of their pluripotency remains largely unclear. Here we determined that the somatic genome is actively transcribed by generating ES-somatic cell hybrids using Rag2-deficient ESCs fused to autologous wild-type somatic cells. Rag2 expression was detected during in vitro differentiation, suggesting that the somatic genome follows the correct temporal cues during differentiation. Furthermore, ES-somatic cell hybrids maintain their tetraploid state following 4 weeks of differentiation in vivo and are immune tolerated when transferred into matched individuals. The ES-somatic cell hybrids can efficiently differentiate into hematopoietic precursors in both myeloid and lymphoid lineages in vitro, suggesting that the somatic genome is actively transcribed following cell fusion based reprogramming. However, the ES-somatic cell hybrids showed an altered hematopoietic potential following in vitro differentiation and were unable to show hematopoietic engraftment in a mouse model.

281 USE OF SMALL MOLECULES ENHANCES REPROGRAMMING SUCCESS IN BOVINE DERMAL FIBROBLASTS

Reprogramming of differentiated cells to induced pluripotency holds tremendous potential for livestock reproduction. Here we report use of small molecules to support the generation of stem cell-like colonies from bovine dermal fibroblasts that show characteristics of pluripotent cells. Reprogramming of bovine somatic cells was undertaken by introducing canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc through a lentiviral drug-inducible polycistronic vector. Several small molecules were tested to support reprogramming. A combination of 3 small molecules (sodium butyrate, PD0325901, and SB431542; NaB-PD-SB) in iPS medium [Minimum Essential Medium Alpha, 20% fetal bovine serum, 1× insulin-transferrin-selenium (ITS), 2 mM Glutamax, 100 µM NEAA, 50 U mol–1 penicillin, 50 mg mL–1 streptomycin, 0.1 mM β-mercaptoethanol, 4 ng mL–1 human leukemia inhibitory factor, and 10 ng mL–1 basic fibroblast growth factor] was found to accelerate the kinetics of the reprogramming process. Colonies appeared at 12 days of culture compared with 21 days in iPS medium alone (P < 0.01, n = 5). Colonies in NaB-PD-SB iPS medium had consistent morphology, with small cells in compact dome formations with well-defined colony borders. Cells were not passaged but were either harvested for mRNA extraction or differentiated into embryoid bodies. Gene expression of the reprogramming factors in stem cell-like colonies was confirmed by qRT-PCR, and further gene expression analysis revealed activation of Nanog and ALPL mRNA expression (P < 0.01, n = 2). Embryoid bodies were analysed for gene expression by quantitative RT-PCR. Silencing of the transgene was not observed in embryoid bodies despite withdrawal of the inducer doxycycline. Expression of the endoderm marker FOXA2, ectoderm markers NES and TUBB3, and mesoderm marker DES was strongly increased in embryoid bodies compared with non-reprogrammed bovine fibroblasts (P < 0.01, n = 2), confirming expression of marker genes for the 3 germ layers.

Reestablishment of the inactive X chromosome to the ground state through cell fusion-induced reprogramming.

The restricted gene expression pattern of a differentiated cell can be reversed by fusion of the somatic cell with a more developmentally potent cell type, such as an embryonic stem (ES) cell. During this reprogramming process, somatic cells obtain most of the characteristics of pluripotent cells. Reactivation of an inactive X chromosome (Xi) is an important epigenetic marker confirming the pluripotent reprogramming of somatic cells. Female somatic cells contain one active X chromosome (Xa) and one Xi, and following the fusion of these cells with male ES cells, the Xi becomes activated, resulting in XaXaXaY fusion hybrid cells. To monitor Xi reactivation, transgenic female neural stem cells (fNSCs) carrying a green fluorescent protein (GFP) reporter gene expressed on the Xa (X-GFP), but not on the Xi, were used for reprogramming. XaXi(GFP) NSCs, whose GFP reporter was silenced, were fused with HM1 ES cells (XY) to induce pluripotent reprogramming. The Xi(GFP) of NSCs were found to be activated on day 4 post-fusion, indicating reactivation of the Xi. Hybrid cells showed pluripotent cell-specific characteristics cells including inactivation of the NSC marker Nestin, DNA demethylation of Oct4, DNA methylation of Nestin, and reactivation of the Xi. Following differentiation of the (GFP-positive) hybrid cells through embryoid body formation, the proportion of GFP-negative cells was found to be approximately 26%, indicating that there was random inactivation of one of the three Xas. Here, we showed that the Xi of somatic cells is reprogrammed to the Xa state and that cellular differentiation occurs randomly, i.e., regardless of the Xa or Xi state, indicating that the memory of the Xi of somatic cells has been erased and reset to the ground state (i.e., inner cell mass-like state), indicating that random X-chromosome inactivation occurs upon differentiation.

Research status ofpluripotent stem cells from different origins

：Pluripotent stem cellsare type of cells that can differentiate into cells of ectoderm,mesoderm and endodermlayers.Cells isolated from developing embryos such as embryonic stem cells,embryonic germcells,as well as cells derived from adult tissues and bone marrow and testis,have beenproved to be pluripotent.Recent studies demonstrated that differentiated cells from adultcould be reprogrammed to become pluripotent by genetic manipulation.The characteristics ofpluripotent cells from different origins are summarized in this review,and the possiblerelationship between those populations is discussed. Key words: Pluripotency; Embryonic stem cell; Embryonic germ cell; Inducedpluripotent stem cell

Reprogramming somatic gene activity by fusion with pluripotent cells

Fertilized eggs and early blastomeres, that have the potential to develop to fetuses when placed into a uterus, are totipotent. Those cells in the embryo, that can give rise to all cell types of an organism, but not to an organism itself, are pluripotent. Embryonic stem (ES), embryonic carcinoma (EC), and embryonic germ (EG) cells are powerful in vitro artifacts derived from different embryonic stages and are pluripotent. Totipotent and pluripotent cells have the potential to greatly benefit biological research and medicine. One powerful feature is that the genetic program of somatic cells can be converted into that of totipotent or pluripotent cells, as shown by nuclear transfer or cell fusion experiments. During reprogramming by cell fusion various features of pluripotent cells are acquired. These include the typical morphology of the respective pluripotent fusion partner, a specific epigenetic state, a specific gene profile, inactivation of tissue-specific genes expressed in the somatic fusion partner, and the developmental as well as differentiation potential of pluripotent cells. In this review, we will discuss what is known about the reprogramming process mediated by cell fusion and the potential use of fusion-induced reprogramming for therapeutic applications.

When microspores decide to become embryos — cellular and molecular changesThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

Cultured microspores can be induced to develop into fully functional haploid embryos instead of mature pollen. The ability of these cells to change their development in response to environmental stimuli is an exceptional example of totipotency in plants. Discovering the triggers of embryo development in microspore cultures could lead to a greater understanding of the early stages of embryogenesis in plants and might be used to increase the range of crop plants to which microspore culture can be applied for the production of double haploid homozygous lines. The information might also help to define the general characteristics of pluripotent cells in any organism. In this review, the changes that occur in cellular organization and gene expression in early-stage microspore cultures of several species are discussed. Responding cells in these cultures enlarge, their nuclei are repositioned to their cell centres, and their cytoplasms become filled with fragmented vacuoles. We used flow cytometry to track cellular changes in canola ( Brassica napus L.) microspore cultures as well as microarray analyses and real-time PCR to compare gene expression in embryogenic and nonembryogenic cells. A model for embryogenic cell activation in plants that involves alkalinization, Ca2+ signaling, and changes in GTPase activity that lead to significant changes in gene expression is discussed.