Single Progenitor Cell Research Articles

Reconstituting pancreas development from purified progenitor cells reveals genes essential for islet differentiation

Developmental biology is challenged to reveal the function of numerous candidate genes implicated by recent genome-scale studies as regulators of organ development and diseases. Recapitulating organogenesis from purified progenitor cells that can be genetically manipulated would provide powerful opportunities to dissect such gene functions. Here we describe systems for reconstructing pancreas development, including islet β-cell and α-cell differentiation, from single fetal progenitor cells. A strict requirement for native genetic regulators of in vivo pancreas development, such as Ngn3, Arx, and Pax4, revealed the authenticity of differentiation programs in vitro. Efficient genetic screens permitted by this system revealed that Prdm16 is required for pancreatic islet development in vivo. Discovering the function of genes regulating pancreas development with our system should enrich strategies for regenerating islets for treating diabetes mellitus.

Notch1 is required in newly postmitotic cells to inhibit the rod photoreceptor fate

Several models of cell fate determination can be invoked to explain how single retinal progenitor cells (RPCs) produce different cell types in a terminal division. To gain insight into this process, the effects of the removal of a cell fate regulator, Notch1, were studied in newly postmitotic cells using a conditional allele of Notch1 (N1-CKO) in mice. Almost all newly postmitotic N1-CKO cells became rod photoreceptors, whereas wild-type (WT) cells achieved a variety of fates. Single cell profiling of wild-type and N1-CKO retinal cells transitioning from progenitor to differentiated states revealed differential expression of inhibitor of DNA binding factors Id1 and Id3, as well as Notch-regulated ankyrin repeat protein (Nrarp). Misexpression of Id1 and Id3 was found to be sufficient to drive production of Müller glial cells and/or RPCs. Moreover, Id1 and Id3 were shown to partially rescue the production of bipolar and Müller glial cells in the absence of Notch1 in mitotic and newly postmitotic cells. Misexpression of Nrarp, a downstream target gene and inhibitor of the Notch signaling pathway, resulted in the overproduction of rod photoreceptors at the expense of Müller glial cells. These data demonstrate that cell fate decisions can be made in newly postmitotic retinal cells, and reveal some of the regulators downstream of Notch1 that influence the choice of rod and non-rod fates. Taken together, our results begin to address how different signals downstream from a common pathway lead to different fate outcomes.

Guest editorial: Pathophysiology and management of thrombocytopenia: possible clinical application of TPO receptor agonists

Platelets are small, anucleated blood cells that exhibit a discoid shape when non-activated. Platelets play a crucial role in the formation of a pathologic thrombus as well as a normal hemostatic plug formation. Platelets express several adhesion receptors, such as GPIb–IX and GPIIb–IIIa, and circulate passively throughout the vascular tree covered by an intact monolayer of endothelial cells. In the initial step of hemostasis (and thrombus formation), platelets adhere to exposed subendothelial matrices at sites of vascular injury (or altered vascular surfaces). Following adhesion, they become activated, change shape, secrete granule contents, and aggregate to form a primary hemostatic plug and provide a catalytic surface to enhance blood coagulation. Platelets are released by megakaryocytes (MKs). Platelet counts are 130–320 9 10/lL in peripheral blood, and their average life span is 7–10 days. Red blood cell (RBC) counts in peripheral blood (4.4–5.6 9 10/lL) are approximately 20 times more than platelet counts, whereas their average life span is 120 days (approximately 12 times longer than that of platelets). Thus, the amounts of daily platelet production and RBC production in human body appear to be on the same order of magnitude. Essentially, only one mature blood cell is produced from a single progenitor cell within the bone marrow; e.g., one erythroblast differentiates to one mature erythrocyte. Bone marrow aspiration shows that erythroid progenitors are present at a concentration of approximately 100 9 10/lL. In sharp contrast, only 50–150 MKs/lL are present in the bone marrow, suggesting that roughly 1,000 platelets are generated from a single MK. For many years, the mechanism of thrombopoiesis remained largely obscure due to the paucity of MKs. The cloning and characterization of the primary regulator of thrombopoiesis, thrombopoietin (TPO), in 1994 greatly facilitated our understanding of platelet production. Thrombopoiesis is a complex process that induces the commitment of multipotent stem cells to the MK lineage, MK progenitor proliferation, and terminal differentiation, inducing platelet production [1]. TPO controls thrombopoiesis by affecting the proliferation and/or apoptosis of progenitor cells, as well as MKs ploidy. Moreover, TPO supports the proliferation of hematopoietic stem cells (HSCs) and progenitor cells. In vitro colony assays using bone marrow from TPO and/or c-Mpl mice show a reduction of CFU-mix, CFU-GM and BFU-E progenitors. Although RBC and white blood cell (WBC) counts in peripheral blood are normal in TPO and c-Mpl mice, despite the marked thrombocytopenia, and do not reflect the multilineage effect of TPO, TPO can shorten the duration of anemia and leukopenia in an experimental mouse model under myelosuppressive conditions [2]. In addition to TPO, MK-active chemokines, stromal-derived factor (SDF)-1, and a fibroblast growth factor (FGF-4), direct MK interaction with the bone marrow stroma and regulate TPO-independent cell maturation, and restore thrombopoiesis in TPO and c-Mpl mice [3]. A recent report of in vivo imaging of thrombopoiesis in mouse bone marrow revealed that MKs are enriched at bone marrow sinusoids and observed as sessile cells that extend dynamic proplatelet-like protrusions into microvessels, and that these cells routinely release heterogeneous particles, with properties resembling immature proplatelets, into BM microvessels [4]. In this issue of IJH, in light of the approval of TPO receptor agonists in the treatment of primary immune thrombocytopenia and significant advances in the field of Y. Tomiyama (&) Osaka University Hospital, Suita, Osaka, Japan e-mail: yoshi@hp-blood.med.osaka-u.ac.jp

Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis

Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors (TFs) in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterised by distinctive TF expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated TF pairings, including previously unrecognised relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease.

Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production

Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.

Evaluation of mechanical properties of fixed bone cells with sub-micrometer thickness by picosecond ultrasonics

The picosecond ultrasonics technique is used to investigate the viscoelastic properties of nucleus of fixed single osteoblast progenitor cells adhering on a titanium alloy substrate. A two-color probing picosecond ultrasonics and a fluorescence visualization setups were developed and combined to allow to distinguish subcomponents inside the cell under investigation. It opens the way for quantitative measurements of the viscoelastic properties of single cells and of their sub-micrometer thickness. It is shown that a blue probe, λ = 400 nm, is preferable to a red probe, λ = 800 nm, to perform these measurements with fixed sub-micrometer bone cells. 26 GHz acoustic frequencies are detected in cells as thin as 135 nm. A 1D analytical model of the acoustic generation and of the optical detection is used to describe the experimental results. The nucleus longitudinal elastic moduli (13–16 GPa) and dynamic viscosities (13–30 cP) are measured at high frequencies (GHz) from a time-frequency analysis of the experimental data of fixed single cells.

Genome-Wide Characterization of Human Hematopoietic Progenitor Cell Heterogeneity by Expression Profiling of Single Cells: A Pilot Study

AbstractAbstract 1231 Background:Current methods for defining and isolating human hematopoietic stem and progenitor cells using surface markers enrich for unique functional properties of these populations. However, significant functional heterogeneity in these compartments remains with important implications for understanding normal and altered hematopoiesis. Using flow sorting to enrich >10,000 cells as progenitor subpopulations, we previously characterized the gene expression signature of normal human HSC (Majetiet al 2009 PNAS 106(9):3396–3401). We hypothesized that interrogation of the transcriptomes of single cells from this compartment could resolve remaining heterogeneity and help identify and better define features of progenitor cells and hematopoietic stem cells (HSCs). Methods:Using normal human bone marrow aspirates and a FACS Aria II instrument equipped with a specialized single-cell sorting apparatus, we sorted cells enriched for HSCs based on expression of Lin-CD34+CD38-CD90+CD45RA− into 1-cell, 10-cell, 100-cell, and 40000-cell (bulk) representations. We used at least 5 replicates per group and verified single cell deposition by direct visualization. We amplified cDNA from these corresponding inputs using an exponential whole transcriptome amplification (WTA) scheme (Miltenyi SuperAmp), and evaluated gene expression profiles by two microarray platforms (Agilent/GE Healthcare 60K, and Affymetrix U133 plus 2.0), and by RNA-Seq (Illumina). We used gene expression correlation between replicates within and between microarrays as means of assessing methodological reproducibility and estimating population heterogeneity. Results:Whole transcriptome amplification yielded cDNA ranging from 0.2–1 kb for 10 and 100 cells, with significantly lower size distribution of amplified cDNA observed for single cells. Gene expression profiles had significantly better replicate reproducibility and array coverage with the Agilent microarray platform when compared with the Affymetrix U133 Plus 2.0 platform (gene coverage of 84 % for 100 cells, 73 % for 10 cells and 50% for 1 cell for Agilent vs 24 % for 100 cells, 11 % for 10 cells and 5.7% for 1 cell for Affymetrix). RNA-Seq profiling of the same populations is ongoing with major technical optimizations focused on reducing amplification of non-human templates while maintaining library complexity and representation. Using biological replicates for each input size, we observed high inter-replicate correlation levels for expression profiles obtained for bulk sorted HSCs from 8 healthy donors (∼40000-cells, average r=0.97) and for 100-cell and 10-cell inputs from a single donor (r=0.96–0.99, respectively). While intra-array concordance of replicate measurements (n=14642) was high (r>0.91) within each of 5 single cells from a single donor, comparison of 5-single cells from the same donor identified significant heterogeneity, when compared to the 10-cell and 100-cell sub-clusters (Figure 1). Individual genes characteristically expressed by these heterogeneous single cell populations are currently being investigated by FACS and Fluidigm arrays. A larger experiment characterizing 192 single progenitor cells, employing Agilent microarrays and RNA-Seq is currently in progress. Conclusions:Single cell transcriptome profiling is feasible, with best performance on 60-mer microarrays. Single cell transcriptomes exhibit lower, but reasonable levels of reproducibility (r>0.7) and precision as compared with higher cell numbers. Gene expression profiles of single cells capture gene expression heterogeneity in HSCs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini

The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.

Similarity of Visual Selectivity among Clonally Related Neurons in Visual Cortex

Neurons in rodent visual cortex are organized in a salt-and-pepper fashion for orientation selectivity, but it is still unknown how this functional architecture develops. A recent study reported that the progeny of single cortical progenitor cells are preferentially connected in the postnatal cortex. If these neurons acquire similar selectivity through their connections, a salt-and-pepper organization may be generated, because neurons derived from different progenitors are intermingled in rodents. Here we investigated whether clonally related cells have similar preferred orientation by using a transgenic mouse, which labels all the progeny of single cortical progenitor cells. We found that preferred orientations of clonally related cells are similar to each other, suggesting that cell lineage is involved in the development of response selectivity of neurons in the cortex. However, not all clonally related cells share response selectivity, suggesting that cell lineage is not the only determinant of response selectivity.

Tbx6 is a determinant of cardiac and neural cell fate decisions in multipotent P19CL6 cells

Multipotent P19CL6 cells differentiate into cardiac myocytes or neural lineages when stimulated with dimethyl sulfoxide (DMSO) or retinoic acid (RA), respectively. Expression of the transcription factor Tbx6 was found to increase during cardiac myocyte differentiation and to decrease during neural differentiation. Overexpression of Tbx6 was not sufficient to drive P19CL6 cells to a cardiac myocyte fate or to accelerate DMSO-induced differentiation. In contrast, knockdown of Tbx6 dramatically inhibited DMSO-induced differentiation of P19CL6 cells to cardiac myocytes, as evidenced by the loss of striated muscle-specific markers and spontaneous beating. Tbx6 knockdown was also accompanied by almost complete loss of Nkx2.5, a transcription factor involved in the specification of the cardiac myocyte lineage, indicating that Nkx2.5 is downstream of Tbx6. In distinction to its positive role in cardiac myocyte differentiation, Tbx6 knockdown augmented RA-induced differentiation of P19CL6 cells to both neurons and glia, and accelerated the rate of neurite formation. Conversely, Tbx6 overexpression attenuated differentiation to neural lineages. Thus, in the P19CL6 model, Tbx6 is required for cardiac myocyte differentiation and represses neural differentiation. We propose a model in which Tbx6 is a part of a molecular switch that modulates divergent differentiation programs within a single progenitor cell.

Islet1-expressing cardiac progenitor cells: a comparison across species

Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-012-0400-1) contains supplementary material, which is available to authorized users.

Abstract 5363: Aggregation rather than monoclonal expansion explains ovarian cancer spheroid formation

Abstract Purpose: It is thought that a subpopulation of ovarian cancer cells possess stem cell like properties and are responsible for both the emergence of cancer and for subsequent chemoresistance and recurrence. A common approach used to enrich for cancer stems cells in vitro involves culture in selective media on low attachment plates resulting in non-attached multicellular spheres. Spheroids are present in ascites fluid of women with ovarian cancer, and may contain stem cells responsible for metastasis and recurrence. Our objective was to address whether spheroids are truly monoclonal in origin and arise from expansion of a single common progenitor cell. Procedures: We cultivated 5 ovarian cancer cell lines (DOV13, HEYC2, OVCAR2, OVCAR3, and PEO4) to confluence in monolayer cultures. The cell lines were chosen for their ability to form spheroids as well as variation in expression of the stem cell marker CD133 (OVCAR2, OVCAR3 CD133+, others negative). The cells were trypsinized to form single cell suspensions and plated at densities ranging from 100 to 5000 cells per plate in stem cell-selective media in ultra-low attachment culture dishes. A Zeiss Axio Observer microscope was used for time-lapse photography of the plated cells at 5-minute intervals over a 24-hour period. In addition, single cell suspensions were subjected to flow activated cell sorting to plate one cell in each well of 96-well Costar ultra-low cluster plates to monitor clonal formation of spheroids. Results: Cells were dissociated and plated under stem cell-selective conditions and 288 images of each cell line were recorded over 24 hours. The cells were found to begin to aggregate within two to four hours, regardless of cell numbers plated. By 24 hours post-plating, there was pronounced aggregation in 5/5 (100%) cell lines with formation of tightly compacted spheroid structures in 4/5 (80%) cell lines. Single cells in the 96-well plates showed no evidence of spheroid formation over a 14-day observation period. Conclusion: We observed spheroid formation in both CD133 positive and negative ovarian cancer cell lines. Formation occured due to cellular aggregation rather than clonal expansion from a single progenitor with stem cell properties. Although cellular aggregation may be relevant to ovarian cancer biology, these data suggest that spheroid formation should be viewed with caution if used as a proxy for monoclonal expansion of ovarian cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5363. doi:1538-7445.AM2012-5363

Ectopic transgenic expression of NKX2.2 induces differentiation of adult pancreatic progenitors and mediates islet regeneration

To pursue islet regeneration in situ in adult pancreas with a diabetic animal model, we used ultrasound targeted microbubble destruction (UTMD) to deliver islet transcription factor genes to the pancreas of STZ-treated rats, specifically using a piggyBac transposon gene delivery system for long-term transgene expression of Nkx2.2 in STZ rat pancreas. Our results show that Nkx2.2 gene induced robust proliferation and differentiation of adult pancreatic progenitors. Our high-resolution confocal images precisely displayed how one single pancreatic progenitor cell differentiated into islets-like clusters and, further, into mature islets with normal morphology in situ in postnatal pancreas. Nkx2.2 targeted to the pancreas by UTMD induces pancreatic progenitor cell proliferation and differentiation with subsequent islet regeneration and cure of STZ-induced diabetes for three months.

The Temporal Sequence of the Mammalian Neocortical Neurogenetic Program Drives Mediolateral Pattern in the Chick Pallium

The six-layered neocortex permits complex information processing in all mammalian species. Because its homologous region (the pallium) in nonmammalian amniotes has a different architecture, the ability of neocortical progenitors to generate an orderly sequence of distinct cell types was thought to have arisen in the mammalian lineage. This study, however, shows that layer-specific neuron subtypes do exist in the chick pallium. Deep- and upper-layer neurons are not layered but are segregated in distinct mediolateral domains in vivo. Surprisingly, cultured chick neural progenitors produce multiple layer-specific neuronal subtypes in the same chronological sequence as seen in mammals. These results suggest that the temporal sequence of the neocortical neurogenetic program was already inherent in the last common ancestor of mammals and birds and that mammals use this conserved program to generate a uniformly layered neocortex, whereas birds impose spatial constraints on the sequence to pattern the pallium.

A new approach to manipulate the fate of single neural stem cells in tissue

A challenge in the field of neural stem cell biology is the mechanistic dissection of single stem cell behavior in tissue. Although such behavior can be tracked by sophisticated imaging techniques, current methods of genetic manipulation do not allow researchers to change the level of a defined gene product on a truly acute time scale and are limited to very few genes at a time. To overcome these limitations, we established microinjection of neuroepithelial/radial glial cells (apical progenitors) in organotypic slice culture of embryonic mouse brain. Microinjected apical progenitors showed cell cycle parameters that were indistinguishable to apical progenitors in utero, underwent self-renewing divisions and generated neurons. Microinjection of single genes, recombinant proteins or complex mixtures of RNA was found to elicit acute and defined changes in apical progenitor behavior and progeny fate. Thus, apical progenitor microinjection provides a new approach to acutely manipulating single neural stem and progenitor cells in tissue.

P1-04-02: In Vitro Qualification and Quantification of Murine Mammary Stem/Progenitor Cells.

Abstract Increasing evidence suggest that tumors with a cell origin of more basal or stem-like sources are often highly metastatic and associated with poor prognosis. Identification of the cell of origin thus has important implications for development of new preventive and therapeutic approaches. To identifying the cells of origin of various breast cancers, understanding the normal cellular hierarchy within the breast tissue is an important prerequisite. However, current understanding of normal mammary stem/progenitor cell is limited due to the lack of a robust in vitro assay. The newly developed cell surface markers (CD24+CD49fhi) can only be used for low percent of enrichment. The only gold standard assay for functional evaluation of stem cell property is the in vivo transplantation of cleared mammary fat pad assay, yet this assay is expensive, time consuming, and requires highly trained skills, and thus cannot be used routinely for experiments. The goal of our study is thus to develop a robust in vitro assay to qualify and quantify murine mammary stem/progenitor cells. Our rationale lies on our recent findings of in vitro study of different fractions of murine stem/progenitor cells. When we use isolated primary epithelial cells for in vitro mammosphere formation, we found that stem cell enriched fraction and progenitor cell enriched fraction both formed very small mammospheres (≤ 50 μm) and these spheres appear to be of clonal origin (one cell gives rise to one sphere). When we plated these small spheres into 3D extracellular matrix for colony formation, however, they gave rise to two distinct structures: stem-enriched fraction generated predominately solid structure while progenitor-enriched fraction is dominated by hollow structures. Previous studies have linked the solid structure to the basal/myoepithelial lineage and the hollow structure to the luminal lineage. We thus suspected that the small mammospheres derived from the stem or progenitor enriched fractions could be originated from single stem or progenitor cell, and the number of spheres could indicate the number of original stem/progenitor cells within these enriched fractions. Subsequent in vivo transplantation with single sphere or single solid structure cultured in 3D extracellular matrix derived from green fluorescent protein transgenic mice proved that single sphere or 3D solid structure can repopulate the gland-free mammary fat pad. To conclude, the in vitro mammosphere formation in combination with subsequent differentiation in 3D extracellular matrix can be used as a robust in vitro assay for qualification and quantification of murine mammary stem/progenitor cells. This in vitro assay will greatly facilitate our understanding of genes regulate stem cell self-renewal, proliferation, differentiation as well as mechanisms keeping them at quiescent state within the niche. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-02.

Fate Restriction and Multipotency in Retinal Stem Cells

Stem cells have the capacity to both self-renew and generate postmitotic cells. Long-term tracking of individual clones in their natural environment constitutes the ultimate way to validate postembryonic stem cells. We identify retinal stem cells (RSCs) using the spatiotemporal organization of the fish retina and follow the complete offspring of a single cell during the postnatal life. RSCs generate two tissues of the adult fish retina, the neural retina (NR) and the retinal-pigmented epithelium (RPE). Despite their common embryonic origin and tight coordination during continuous organ growth, we prove that NR and RPE are maintained by dedicated RSCs that contribute in a fate-restricted manner to either one or the other tissue. We show that in the NR, RSCs are multipotent and generate all neuron types and glia. The clonal origin of these different cell types from a multipotent NSC has far-reaching implications for cell type and tissue homeostasis.

Abstract IA12: Induction of pluripotency by defined factors

Abstract Induced pluripotent stem (iPS) cells were originally generated from mouse and human fibroblasts by the retroviral introduction of Oct3/4, Sox2, c-Myc, and Klf4. iPS cells are similar to embryonic stem (ES) cells in morphology, proliferation, gene expression, and most importantly, pluripotency. Patient-specific iPS cells provide us with unprecedented opportunities in disease research, drug screening, toxicology, and cell transplantation therapies. Banks of iPS cells from individuals with HLA homozygous alleles may also provide another unprecedented source for regenerative medicine. iPS cells have many variations in comparison to ES cells. In addition to fibroblasts, iPS cells can be generated from various somatic cells, such as hepatic cells, gastric epithelial cells, keratinocytes, neural cells, peripheral blood cells, and cord blood cells. In addition to retroviral transduction, iPS cells can be integration-freely generated by adenoviruses, sendaiviruses, plasmids, transposons, recombinant proteins, and synthetic mRNA. Several chemicals and inhibitors have been shown to enhance iPS cell generation. Some of the original four factors can either be omitted or replaced by other genes or chemicals. Finally, each induction experiment can result in 100 or more independent iPS cell clones. These various iPS cells may vary significantly in regard to each cell's peculiar characteristics, such as the efficacy of in vitro directed differentiation and a propensity to produce tumors. In fact, the origins of iPS cells have a profound effect on their tumorigenicity. It is therefore necessary to determine the best origins, the best induction protocols, and the best methods to evaluate iPS cell clones and subclones for each application of iPS cells. Here, significance of epigenome analyses, including methylome, histone modifications, and imprinting increases as the integration-free method is becoming practical. It is also important to note that an iPS cell clone can be heterogeneous despite the fact that all the cells within the clone are derived from a single progenitor cell. This is because the process requires multiple cell divisions which cannot be completed by the four exogenous factors alone. Additional factors including p53 and Rb pathways are considered to play various roles in achieving full reprogramming. Obtaining a better understanding of the reprogramming mechanisms will therefore facilitate the generation of more uniform iPS cells in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr IA12.

Cytohistological Studies During Cotton Somatic Embryogenesis With Brassinosteroid Application

The histology and chromosome alterations of somatic embryo initiation and development in cotton ( Gossypium hirsutum L. cv. Coker) was examined during in vitro exogenous brassinosteroid (BR) application. Calli developed from hypocotyl explants were subjected to 0.5 μM BR. After 10 weeks of calli cultures with somatic embryos at different developmental stages were fixed for histological examinations. The histological examination showed that two types of embryogenic cells induced; epidermal or subepidermal. The embryogenic mass and somatic embryos were mostly derived from several morphologically competent cells from subepidermal cases; however single progenitor cells for epidermal situations. Chromosome behaviour of the cells surrounding embryogenic cells showed abnormal divisions and higher level of polyploidy on BR treated calli when compared to control group.

Clonal analysis reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo

Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to functional nlacZ after a rare intragenic recombination event, is targeted to the alpha(c)-actin gene, expressed in all developing skeletal and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract myocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk, with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate with genetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart, reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.