Parental Somatic Cells Research Articles

IPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors

BackgroundDozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming.MethodsWe established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells.ResultsHere we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells.ConclusionIn summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies.Trial registration Not applicable.

Targeted migration of pherophorin-S indicates extensive extracellular matrix dynamics in Volvox carteri.

Hydroxyproline-rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin-related genes in V.carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin-S is a soluble, non-cross-linked ECM protein. Using transformants expressing a YFP-tagged pherophorin-S we observed the synthesis and secretion of pherophorin-S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin-S, we conclude that pherophorin-S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.

Genomic Instability of iPSCs and Challenges in Their Clinical Applications.

Generation of human-induced pluripotent stem cells (iPSCs) from somatic cells has opened the possibility to design novel therapeutic approaches. In 2014, the first-in-human clinical trial of iPSC-based therapy was conducted. However, the transplantation for the second patient was discontinued at least in part due to genetic aberrations detected in iPSCs. Moreover, many studies have reported genetic aberrations in iPSCs with the rapid progress in genomic technologies. The presence of genomic instability raises serious safety concerns and can hamper the advancement of iPSC-based therapies. Here, we summarize our current knowledge on genomic instability of iPSCs and challenges in their clinical applications. In view of the recent expansion of stem cell therapies, it is crucial to gain deeper mechanistic insights into the genetic aberrations, ranging from chromosomal aberrations, copy number variations to point mutations. On the basis of their origin, these genetic aberrations in iPSCs can be classified as (i) preexisting mutations in parental somatic cells, (ii) reprogramming-induced mutations, and (iii) mutations that arise during in vitro culture. However, it is still unknown whether these genetic aberrations in iPSCs can be an actual risk factor for adverse effects. Intersection of the genomic data on iPSCs with the patients' clinical follow-up data will help to produce evidence-based criteria for clinical application. Furthermore, we discuss novel approaches to generate iPSCs with fewer genetic aberrations. Better understanding of iPSCs from both basic and clinical aspects will pave the way for iPSC-based therapies.

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells

SummaryWe show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes’ expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC.

DNA hypermethylation enhanced telomerase reverse transcriptase expression in human-induced pluripotent stem cells

During reprogramming into human induced pluripotent stem cells (iPSCs), several stem cell marker genes are induced, such as OCT-4, NANOG, SALL4, and TERT. OCT-4, NANOG, and SALL4 gene expression can be regulated by DNA methylation. Their promoters become hypomethylated in iPSCs during reprogramming, leading to their induced expression. However, epigenetic regulation of the TERT gene remains unclear. In this study, we focused on epigenetic regulation of the human TERT gene and identified a differentially methylated region (DMR) at a distal region in the TERT promoter between human iPSCs and their parental somatic cells. Interestingly, the TERT-DMR was highly methylated in iPSCs, but low-level methylation was observed in their parental somatic cells. Region-specific, methylated-promoter assays showed that the methylated TERT-DMR up-regulated the promoter activity in iPSCs. In addition, Lamin B1 accumulated at the TERT-DMR in iPSCs, but not in their parent somatic cells. These results suggested that the TERT transcription was enhanced by DNA methylation at the TERT-DMR via binding to nuclear lamina during reprogramming. Our findings shed light on a new functional aspect of DNA methylation in gene expression.

Environmental stresses induce transgenerationally inheritable survival advantages via germline-to-soma communication in Caenorhabditis elegans

Hormesis is a biological phenomenon, whereby exposure to low levels of toxic agents or conditions increases organismal viability. It thus represents a beneficial aspect of adaptive responses to harmful environmental stimuli. Here we show that hormesis effects induced in the parental generation can be passed on to the descendants in Caenorhabditis elegans. Animals subjected to various stressors during developmental stages exhibit increased resistance to oxidative stress and proteotoxicity. The increased resistance is transmitted to the subsequent generations grown under unstressed conditions through epigenetic alterations. Our analysis reveal that the insulin/insulin-like growth factor (IGF) signalling effector DAF-16/FOXO and the heat-shock factor HSF-1 in the parental somatic cells mediate the formation of epigenetic memory, which is maintained through the histone H3 lysine 4 trimethylase complex in the germline across generations. The elicitation of memory requires the transcription factor SKN-1/Nrf in somatic tissues. We propose that germ-to-soma communication across generations is an essential framework for the transgenerational inheritance of acquired traits, which provides the offspring with survival advantages to deal with environmental perturbation.

Nucleosome organizations in induced pluripotent stem cells reprogrammed from somatic cells belonging to three different germ layers.

BackgroundNucleosome organization determines the chromatin state, which in turn controls gene expression or silencing. Nucleosome remodeling occurs during somatic cell reprogramming, but it is still unclear to what degree the re-established nucleosome organization of induced pluripotent stem cells (iPSCs) resembles embryonic stem cells (ESCs), and whether the iPSCs inherit some residual gene expression from the parental fibroblast cells.ResultsWe generated genome-wide nucleosome maps in mouse ESCs and in iPSCs reprogrammed from somatic cells belonging to three different germ layers using a secondary reprogramming system. Pairwise comparisons showed that the nucleosome organizations in the iPSCs, regardless of the iPSCs’ tissue of origin, were nearly identical to the ESCs, but distinct from mouse embryonic fibroblasts (MEF). There is a canonical nucleosome arrangement of -1, nucleosome depletion region, +1, +2, +3, and so on nucleosomes around the transcription start sites of active genes whereas only a nucleosome occupies silent transcriptional units. Transcription factor binding sites possessed characteristic nucleosomal architecture, such that their access was governed by the rotational and translational settings of the nucleosome. Interestingly, the tissue-specific genes were highly expressed only in the parental somatic cells of the corresponding iPS cell line before reprogramming, but had a similar expression level in all the resultant iPSCs and ESCs.ConclusionsThe re-established nucleosome landscape during nuclear reprogramming provides a conserved setting for accessibility of DNA sequences in mouse pluripotent stem cells. No persistent residual expression program or nucleosome positioning of the parental somatic cells that reflected their tissue of origin was passed on to the resulting mouse iPSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0109-x) contains supplementary material, which is available to authorized users.

Stem cells: The quest for the perfect reprogrammed cell.

There are two methods for reprogramming mature cells to pluripotent stem cells, which can give rise to all cells of the body. The first direct comparison of the methods reveals that both can cause subtle molecular defects. See Article p.177 This study compares the distinct genetic, epigenetic and transcriptional signatures of human pluripotent stem cells produced by somatic cell nuclear transfer (SCNT) with induced pluripotent stem (iPS) cells produced by transcription-factor-mediated reprogramming. Both cell types were produced from the same pool of somatic donor cells to ensure a genetic match. iPS cells and SCNT-derived embryonic stem cells contained comparable de novo copy number variations. The abnormalities previously reported — residual DNA methylation patterns typical of parental somatic cells — were observed as anticipated in the iPS cells, but not in the SCNT-derived cells. This suggests that human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and therefore might be more appropriate than iPS cells for use in cell replacement therapies.

Abnormalities in human pluripotent cells due to reprogramming mechanisms.

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the 'gold standard', they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies.

Induced Pluripotent Stem Cells from Human Placental Chorion for Perinatal Tissue Engineering Applications

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly, we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4, SOX2, KLF4, and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology, immunocytochemistry, and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives, including neural stem cells, beating cardiomyocytes, and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications.

Modeling Genomic Imprinting Disorders Using Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology has allowed for the invaluable modeling of many genetic disorders including disorders associated with genomic imprinting. Genomic imprinting involves differential DNA and histone methylation and results in allele-specific gene expression. Most of the epigenetic marks in somatic cells are erased and reestablished during the process of reprogramming into iPSCs. Therefore, in generating models of disorders associated with genomic imprinting, it is important to verify that the imprinting status and allele-specific gene expression patterns of the parental somatic cells are maintained in their derivative iPSCs. Here, we describe three techniques: DNA methylation analysis, allele-specific PCR, and RNA FISH, which we use to analyze genomic imprinting in iPSC models of neurogenetic disorders involving copy number variations of the chromosome 15q11-q13 region.

Global Indiscriminate Methylation in Cell-Specific Gene Promoters following Reprogramming into Human Induced Pluripotent Stem Cells

SummaryMolecular reprogramming of somatic cells into human induced pluripotent stem cells (iPSCs) is accompanied by extensive changes in gene expression patterns and epigenetic marks. To better understand the link between gene expression and DNA methylation, we have profiled human somatic cells from different embryonic cell types (endoderm, mesoderm, and parthenogenetic germ cells) and the iPSCs generated from them. We show that reprogramming is accompanied by extensive DNA methylation in CpG-poor promoters, sparing CpG-rich promoters. Intriguingly, methylation in CpG-poor promoters occurred not only in downregulated genes, but also in genes that are not expressed in the parental somatic cells or their respective iPSCs. These genes are predominantly tissue-specific genes of other cell types from different lineages. Our results suggest a role of DNA methylation in the silencing of the somatic cell identity by global nonspecific methylation of tissue-specific genes from all lineages, regardless of their expression in the parental somatic cells.

Comprehensive Comparison Of Gene Expression, Genomic DNA Methylation, and In Vitro Hematopoietic Differentiation Among Many Human iPS and ES Cell Lines

ObjectiveHematopoietic differentiation from human induced pluripotent stem (iPS)/embryonic stem (ES) cell attracts much attention due to its huge potential for regenerative medicine. As indicated by some earlier papers, there is large variation in differentiation potential among pluripotent stem cell (PSC) lines, and this is one of major concerns in clinical application of PSCs. If it becomes possible to predict which PSC line has high differentiation potential without real differentiation experiment, it would greatly contribute to clinical application of PSCs. Although some papers reported about presence of epigenetic memories of parental somatic cells in iPS cells, the amount of the influence on differentiation potential remains to be known. Furthermore, especially in studies using human PSCs, genetic difference among individual donors of iPS/ES cells seems to be large, thus the study using many PSC lines from many donors is warranted. To address these issues, we planned to collect data of many iPS/ES cell lines on genome-wide gene expression and genomic DNA methylation, and differentiation potentials of individual lines, and identify the factors which affected difference in differentiation potential among PSC lines. The final goal of this study is to create data base about gene expression and DNA methylation profile and differentiation potentials of many PSC lines. We believe that this dataset will allow us to predict differentiation potentials of individual PSC lines, and accelerate clinical application of PSC lines in hematology field. MethodWe utilized 39 iPS/ES lines (iPS 35 lines, ES 4 lines) in this study. The iPS cell lines were derived from dermal fibroblast (n = 16), cord blood (n = 3), peripheral blood (n = 10), keratinocyte (n = 3), and dental pulp cell (n = 3), and were generated by retrovirus vector (n = 9), episomal vector (n = 25), and sendai virus vector (n = 1). The iPS cells were derived from 15 donors, and the ES cells were derived from 4 donors. We assessed hematopoietic differentiation potential by investigating hematopoietic differentiation efficiency for the first 15 days from start of differentiation, and colony forming potential of hematopoietic precursor cells (CD34+CD38-CD43+lineage marker- population) generated from PSC lines using semi-solid methylcellulose based-media. In addition, we collected genome-wide mRNA expression and DNA methylation profile of PSC lines, parental lines of iPS cells, hematopoietic precursor cells generated from PSCs by using mRNA microarray, genomic methylation beads array, and next generation sequencers, and analyzed correlation of these data with differentiation potentials of individual PSC lines. ResultWe have found that there is large variation in hematopoietic differentiation efficiency and colony forming ability as reported previously. Genome-wide investigation of gene expression and genomic DNA methylation revealed that expression of some genes or some factors were significantly correlated with hematopoietic differentiation efficiency or colony forming ability of hematopoietic precursor cells. Importantly, the factors affecting differentiation efficiency for first 15 days and those affecting colony-forming ability were absolutely different. More importantly, by combining several factors discovered in this analysis, we can predict hematopoietic differentiation potential of individual iPS/ES cell lines regardless of what parental cell lines iPS cells are derived or whether it is an iPS cell or ES cell. ConclusionFrom genome-wide analysis of gene expression and genomic DNA methylation, and hematopoietic differentiation experiments, we discovered the factors that were associated with difference in differentiation potential among PSC lines. Now, we are focusing on investigating molecular mechanisms by which the discovered factors are responsible for the difference in hematopoietic differentiation potentials among PSC lines. We believe that our findings will contribute not only to clinical application of hematopoietic cells generated from human PSCs, but also to further understanding of human developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Morphological Differences of Primary Cilia Between Human Induced Pluripotent Stem Cells and Their Parental Somatic Cells

Human induced pluripotent stem cell (hiPSC) reprogramming possesses enormous potential in stem cell research and disease modeling. Chemical and mechanical signaling has been implicated in the maintenance of pluripotency of hiPSCs, as well as their differentiation pathways toward various lineages. Primary cilia have been shown to play a critical role in mechanochemical signaling across a wide spectrum of cell types. The functions of primary cilia in hiPSCs and their characteristic changes during the reprogramming process remain largely vague. This work focused on understanding how reprogramming affects the mechanical characteristics of primary cilia. Using immunofluorescence imaging assays, we validated the presence of primary cilia on reprogrammed cells. These reprogrammed cells had high expression levels of pluripotency markers, Nanog and Cripto, shown by quantitative polymerase chain reaction assays. We also found high expression of hedgehog signaling proteins Patched1 (Ptch1), Smoothened (Smo), Gli1, and Gli2 in reprogrammed cells. Stimulation of the hedgehog pathway resulted in the concerted movement of Ptch1 out of the cilia and Smo into the cilia, implying that the cilia on iPSCs contain functioning hedgehog machinery. The mean length of primary cilia in reprogrammed cells was shorter than those of parental human fibroblasts. Morphometric analyses revealed that reprogramming resulted in an increase in the curvature of primary cilia from ∼0.015 to 0.064 μm(-1), indicating an underlying approximately fourfold decrease in their rigidity, and a decrease in length of primary cilia from ∼2.38 to ∼1.45 μm. Furthermore, reprogramming resulted in fewer primary cilia displaying kinked geometries.

Generation of porcine induced pluripotent stem cells and evaluation of their major histocompatibility complex protein expression in vitro

Induced pluripotent stem cells (iPSCs) are thought to be highly beneficial in the field of regenerative medicine and are believed to overcome immunogenic barriers to cell transplantation. However, issues remain regarding their safety and efficiency for medical use. Furthermore, some recent reports have suggested that iPSCs could be targeted by the autologous immune system. To promote practical applications of iPSCs, in depth research using appropriate animal models is needed and porcine species appear to provide an ideal model. Recent studies have focused on the generation of porcine iPSC cells, but no investigations of their immunological properties have been conducted to date. In the present study, we generated putative iPSCs from porcine somatic cells and measured major histocompatibility complex (MHC) expression on the iPSCs and their derivatives. Compact colonies that expressed pluripotent markers appeared 11 days after viral infection. Embryonic bodies (EB) were produced and differentiated into three germ layers in vitro. Karyotyping and swine leukocyte antigen (SLA) typing showed that the iPSCs were identical to parental somatic cells. Porcine iPSCs expressed only low levels of MHC class I and moderately increased levels on their differentiated derivatives, whereas MHC class II was rarely expressed. In the presence of interferon-gamma (IFN-γ), the expression of MHC class I was elevated on differentiated iPSCs, and gradually decreased after withdrawal of the cytokine. Our data suggest that porcine iPSCs could be useful for preclinical studies of the efficiency and viability of iPSCs, and for devising strategies to rescue transplanted cells from the autologous immune system.

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

A Gender-Specific Retinoblastoma-Related Protein inVolvox carteriImplies a Role for the Retinoblastoma Protein Family in Sexual Development

Here, we describe the cloning and characterization of RETINOBLASTOMA-RELATED PROTEIN1 (RBR1) from the green alga Volvox carteri. RBR1 expression increases substantially during embryogenesis and in response to the sex-inducer glycoprotein, but it decreases significantly under heat stress. While RBR1 is expressed in gonidia (asexual reproductive cells) and embryos, the largest proportion of RBR1 mRNA is found in parental somatic cells. The presence of 4 splice variants and 15 potential cyclin-dependent kinase phosphorylation sites suggests that RBR1 is subject to control at the posttranscriptional and posttranslational levels. Surprisingly, RBR1 is a gender-specific gene, mapping exclusively to the female mating-type locus. A procedure for stable nuclear transformation of males was established to generate RBR1-expressing males. These transformants exhibit enlarged reproductive cells, altered growth characteristics, and a prolonged embryogenesis. The results suggest that a functionally related analog of RBR1 exists in males. The reason for the divergent evolution of RBRs in females and males appears to be based on sexual development: males and females respond to the same sex-inducer with different cleavage programs and substantial differences in cellular differentiation. Thus, the gender-specific presence of RBR1 provides evidence for an additional, novel role for retinoblastoma family proteins in sexual development.

Germ cell specification in Volvox carteri.

Volvox carteri illustrates with diagrammatic clarity Weismann's concept of an immortal germline that produces a mortal soma that will carry it for a time, but then perish. Each V. carteri adult consists of about 16 asexual reproductive cells (gonidia) in the interior of a sphere that consists at its surface of about 2000 biflagellate somatic cells. When mature, each gonidium divides to form a juvenile with this same cellular composition. Half-way through their maturation, juveniles hatch out of the parenteral spheroid, whereupon parental somatic cells undergo programmed death while juvenile gonidia prepare for a new round of reproduction. The first visible step in V. carteri germ-soma differentiation is asymmetric cleavage, which sets apart large gonidial initials from small somatic initials. Experimental analysis indicates that it is a difference in size, not any difference in cytoplasmic quality, that determines whether a cell will become germinal or somatic. Mutational and molecular studies lead to the following model for the genetic control of the germ-soma dichotomy: first, the gls locus acts to cause asymmetric division; then large cells activate a set of lag loci that suppress expression of somatic genes, while small cells activate the regA locus that suppresses gonidial genes.

Advantage or Disadvantage: Is Asexual Reproduction Beneficial to Survival of the Tunicate, Polyandrocarpa misakiensis

It has been believed that clonal propagation by asexual reproduction has serious disadvantages for long-term survival, because asexual reproduction seems not to remove harmful mutations, it seems not to give rise to genetic variations upon which evolution depends and it seems not to reset cell aging. In this article, we re-consider those arguments, by reviewing asexual reproduction of the tunicate, Polyandrocarpa misakiensis. Tracer experiments of bud formation and growth using morphological and chimeric phenotypes showed that the parental epithelial tissues surrounding the bud primordium do not enter the growing bud. It is possible, therefore, to assume that budding involves the purge of a large number of parental somatic cells and tissues. Unlike sexuals, asexuals do not carry out meiotic recombination nor gene shuffling that are two major sources of genetic variation, but we can show that in P. misakiensis at least two genes have significant redundancy and genetic variation even in a clonal colony. Telomerase expressed in germlines is thought to reset the molecular clock executed by telomere shortening. In our Polyandrocarpa cDNA projects, four out of about 2,000 cDNAs examined were matched with retroviral reverse transcriptase that is the catalytic subunit of telomerase, suggesting that telomerase might work in asexual reproduction. In P. misakiensis, dedifferentiation system is used to make new asexual generations. TC14 lectin plays an important role in the maintenance of multipotent but differentiated state of the formative tissue. It is antagonized by tunicate serine protease (TRAMP) that has striking mitogenic and dedifferentiation-inducing activities on the multipotent cells. This system would serve to delay aging of somatic cells. In conclusion, empirical arguments that asexual reproduction is disadvantageous to long-term life do not appear to be tenable to budding of P. misakiensis.

Early and late gene expression programs in developing somatic cells of Volvox carteri

In situ hybridization was used to resolve details of the temporal and spatial aspects of expression of a number of cell-type-specific genes of Volvox carteri. In confirmation of earlier results obtained by Northern-blot analysis, this study revealed that accumulation of transcripts of somatic genes was largely restricted to the somatic-cell lineage. In extension of the previous study, two distinct gene expression programs during somatic-cell development were more fully defined: “early” somatic genes were expressed in the somatic-cell lineage even before visible differentiation began, whereas “late” somatic genes were not expressed until after somatic cells had been visibly differentiated for more than a day and had already reached the presenescent-adult stage of development. We postulate that products of the early somatic genes may be involved in generalized somatic functions that are required throughout somatic-cell development, whereas the late somatic gene products may be involved in more specialized processes that characterize mature, parental somatic cells. The most significant finding of the present study is that transcripts of early somatic genes are significantly more abundant in presumptive somatic cells than in presumptive gonidia during embryonic stages, while the two cell types are linked by a network of cytoplasmic bridges. The fact that the two cell lineages can establish and maintain different transcript populations despite the extensive cytoplasmic continuity that exists between them is somewhat surprising and must be taken into account in future attempts to elucidate the way in which dichotomous differentiation is initiated in the organism.