Cell Expansion Process Research Articles

Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) can be differentiated to neural cells that model neurodegenerative diseases and be used in the screening of potential drugs to ameliorate the disease phenotype. Traditionally, NPCs are produced in 2D cultures, in low yields, using a laborious process that includes generation of embryonic bodies, plating, and colony selections. To simplify the process and generate large numbers of hiPSC-derived NPCs, we introduce a microcarrier (MC) system for the expansion of a hiPSC line and its subsequent differentiation to NPC, using iPS (IMR90) as a model cell line. In the expansion stage, a process of cell propagation in serum-free MC culture was developed first in static culture, which is then scaled up in stirred spinner flasks. A 7.7-fold expansion of iPS (IMR90) and cell yield of 1.3×10⁶ cells/mL in 7 days of static MC culture were achieved. These cells maintained expression of OCT 3/4 and TRA-1-60 and possessed a normal karyotype over 10 passages. A higher cell yield of 6.1×10⁶ cells/mL and 20-fold hiPSC expansion were attained using stirred spinner flasks (seeded from MC static cultures) and changing the medium-exchange regimen from once to twice a day. In the differentiation stage, NPCs were generated with 78%-85% efficiency from hiPSCs using a simple serum-free differentiation protocol. Finally, the integrated process of cell expansion and differentiation of hiPSCs into NPCs using an MC in spinner flasks yielded 333 NPCs per seeded hiPSC as compared to 53 in the classical 2D tissue culture protocol. Similar results were obtained with the HES-3 human embryonic stem cell line. These NPCs were further differentiated into βIII-tubulin⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes, showing that cells maintained their multilineage differentiation potential.

Effects of 3D microwell culture on growth kinetics and metabolism of human embryonic stem cells

Human embryonic stem cells (hESCs) hold potential in the field of tissue engineering, given their capacity for both limitless self-renewal and differentiation to any adult cell type. However, several limitations, including the ability to expand undifferentiated cells and efficiently direct differentiation at scales needed for commercial cell production, prevent realizing the potential of hESCs in tissue engineering. Numerous studies have illustrated that three-dimensional (3D) culture systems provide microenvironmental cues that affect hESC pluripotency and differentiation fates, but little is known about how 3D culture affects cell expansion. Here, we have used a 3D microwell array to model the differences in hESC growth kinetics and metabolism in two-dimensional (2D) versus 3D cultures. Our results demonstrated that 3D microwell culture reduced hESC size and proliferative capacity, and impacted cell cycle dynamics, lengthening the G1 phase and shortening the G2/M phase of the cell cycle. However, glucose and lactate metabolism were similar in 2D and 3D cultures. Elucidating the effects of 3D culture on growth and metabolism of hESCs may facilitate efforts for developing integrated, scalable cell expansion and differentiation processes with these cells.

Safety evaluation of stem cells used for clinical cell therapy in chronic liver diseases; with emphasize on biochemical markers

There are several issues to be considered to reduce the risk of rejection and minimize side effects associated with liver cell transplantation in chronic liver diseases. The source and the condition of stem cell proliferation and differentiation ex vivo and the transplantation protocols are important safety considerations for cell based therapy. The biochemical and molecular markers are important tools for safety evaluation of different processes of cell expansion and transplantation. Studies show that hepatocytes differentiated from adult and embryonic stem cells exhibit biochemical and metabolic properties resembling mature hepatocytes. Therefore these assays can help to assess the biological and metabolic performance of hepatocytes and progenitor stem cells. The assays also help in testing the contribution of transplanted hepatocytes in improving the repair and function of damaged liver in the recipient. Here we review the biochemical and metabolic markers, which are implicated in evaluation of safety issues of stem cells used for therapeutic purposes in chronic liver diseases and regeneration of damaged liver. We also highlight application of biochemical tests for assessment of liver cell transplantation.

Acute fasting decreases the expression of GLUT1 and glucose utilisation involved in mouse oocyte maturation and cumulus cell expansion

Acute fasting impairs meiotic resumption and glucose consumption in mouse cumulus cell and oocyte complexes (COCs). This study examines the effects of acute fasting on the regulation of glucose transporter 1 (GLUT1) expression and glucose consumption in oocyte maturation. Our results indicate that the restriction of glucose utilisation by 2-deoxyglucose (2-DG) mimicked the inhibitory effects of acute fasting on oocyte meiotic resumption and cumulus cell expansion, effects that were rescued by high glucose concentrations in the culture medium. GLUT1 protein levels were higher in cumulus cells compared with oocytes, and GLUT1 expression in COCs increased with FSH treatment in vitro. However, under acute fasting conditions, GLUT1 expression in COCs decreased and the response to FSH disappeared. Exposure to high glucose conditions (27.5mM and 55mM), significantly increased both glucose consumption and GLUT1 levels in COCs. Inhibition of GLUT1 function using an anti-GLUT1 antibody significantly inhibited FSH-induced oocyte meiotic resumption. Taken together, these results suggest that acute fasting decreases GLUT1 expression and glucose utilisation, inhibiting the processes of oocyte maturation and cumulus cell expansion.

Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes

Cotton is an important source of natural fibre used in the textile industry and the productivity of the crop is adversely affected by drought stress. High throughput transcriptomic analyses were used to identify genes involved in fibre development. However, not much information is available on cotton genome response in developing fibres under drought stress. In the present study a genome wide transcriptome analysis was carried out to identify differentially expressed genes at various stages of fibre growth under drought stress. Our study identified a number of genes differentially expressed during fibre elongation as compared to other stages. High level up-regulation of genes encoding for enzymes involved in pectin modification and cytoskeleton proteins was observed at fibre initiation stage. While a large number of genes encoding transcription factors (AP2-EREBP, WRKY, NAC and C2H2), osmoprotectants, ion transporters and heat shock proteins and pathways involved in hormone (ABA, ethylene and JA) biosynthesis and signal transduction were up-regulated and genes involved in phenylpropanoid and flavonoid biosynthesis, pentose and glucuronate interconversions and starch and sucrose metabolism pathways were down-regulated during fibre elongation. This study showed that drought has relatively less impact on fibre initiation but has profound effect on fibre elongation by down-regulating important genes involved in cell wall loosening and expansion process. The comprehensive transcriptome analysis under drought stress has provided valuable information on differentially expressed genes and pathways during fibre development that will be useful in developing drought tolerant cotton cultivars without compromising fibre quality.

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor

BackgroundThe creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings.ResultsWe performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs.ConclusionsBy varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

Redifferentiation of Dedifferentiated Chondrocytes by Adenoviral Vector-Mediated TGF-β3 and Collagen-1 Silencing shRNA in 3D Culture

Autologous chondrocytes remain one of the most preferable candidates among various therapeutic cell species because of their high efficacy, despite remarkable progress in discovery and development of therapeutic cells for cartilage regenerative medicine to date. However, the essential process of cell expansion via repeated monolayer sub-cultures inevitably induces chondrocytic dedifferentiation. In this study, we aimed to achieve and enhance redifferentiation of dedifferentiated chondrocytes with dual genes of transforming growth factor (TGF)-β3 and short hairpin RNA (shRNA) that restore chondrocytic phenotype and silence fibrous collagen type I (Col I), respectively. It was hypothesized that gene delivery of the two targets would promote chondrogenesis in chondrocytes, and meanwhile inhibit the expression of the undesired Col I. Three types of recombinant adenoviruses were constructed. Two of them were of single-function vectors with the ability to express either TGF-β3 (Ad-TGFβ3) or shRNA (specific for Col I, Ad-shRNA); the third type was of double-function vectors that encode both TGF-β3 and anti-Col I shRNA (Ad-double). We infected the dedifferentiated chondrocytes with Ad-double, or co-transduced them with Ad-TGFβ3 and Ad-shRNA at the same time (designated as Ad-combination). Data from real-time RT-PCR and histological staining suggested a restoration in the expression of cartilage-specific genes including aggrecan, type II collagen, and cartilage oligomeric matrix protein (COMP); while a significant down-regulation of Col I expression was observed in groups treated with Ad-double and Ad-combination compared to other control groups. These results demonstrated that, by genetic modification, dedifferentiated chondrocytes managed to redifferentiate back to chondrocytic phenotype, which may greatly facilitate cartilage regenerative medicine by providing sufficient number of competent therapeutic cells.

The specific overexpression of a cyclin‐dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin-dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over-expressing SlKRP1 under the control of a fruit-specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.

Molecular characterization and transcriptome profiling of expansin genes isolated from Calotropis procera fibers

The Calotropis procera seed fibers provide an excellent model system to study the genes involved in fiber elongation, fineness and strength. Expansins constitute one of the important gene families involved in plant cell expansion and other cell wall modification processes. Four homologs of Expansin A gene i.e. CpEXPA 1, CpEXPA 2, CpEXPA 3 and CpEXPA 4 were isolated from the cDNA library obtained from fast growing Calotropis procera fibers. These homologs represented typical Expansin A family. Each of them had two conserved domains including GH45 like domain and the putative polysaccharide binding domain. The deduced amino acid sequences of the homologs indicated three conserved motifs: i) eight cysteine residues at N-terminus, ii) four tryptophan residues at C-terminus and iii) a Histidine-Phenylalanine-Aspartate motif in the center of the sequence. The presence of N-terminal signal peptide consisting of hydrophobic amino acids and a transmembrane region in all these expansin isoforms suggests their cotranslational insertion into the endoplasmic reticulum and then transportation to the cell wall by secretory pathway. The relative quantification of the four expansins in root, stem, fiber and leave tissues indicated that the transcripts of CpEXPA1 , CpEXPA2, CpEXPA3 and CpEXPA4 are variably transcribed in these tissues. The lowest transcription of all the four Expansin A isoforms was observed in elongating roots indicating that root tissue might be having specific expansins other than those confined to air grown organs.

Scalable expansion of multipotent adult progenitor cells as three‐dimensional cell aggregates

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 10⁶/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells.

Angiogenic and Osteogenic Potential of Bone Repair Cells for Craniofacial Regeneration

There has been increased interest in the therapeutic potential of bone marrow derived cells for tissue engineering applications. Bone repair cells (BRCs) represent a unique cell population generated via an ex vivo, closed-system, automated cell expansion process, to drive the propagation of highly osteogenic and angiogenic cells for bone engineering applications. The aims of this study were (1) to evaluate the in vitro osteogenic and angiogenic potential of BRCs, and (2) to evaluate the bone and vascular regenerative potential of BRCs in a craniofacial clinical application. BRCs were produced from bone marrow aspirates and their phenotypes and multipotent potential characterized. Flow cytometry demonstrated that BRCs were enriched for mesenchymal and vascular phenotypes. Alkaline phosphatase and von Kossa staining were performed to assess osteogenic differentiation, and reverse transcriptase-polymerase chain reaction was used to determine the expression levels of bone specific factors. Angiogenic differentiation was determined through in vitro formation of tube-like structures and fluorescent labeling of endothelial cells. Finally, 6 weeks after BRC transplantation into a human jawbone defect, a biopsy of the regenerated site revealed highly vascularized, mineralized bone tissue formation. Taken together, these data provide evidence for the multilineage and clinical potential of BRCs for craniofacial regeneration.

Bioprocessing of Human Glioblastoma Brain Cancer Tissue

Solid cancer tumors are thought to arise from aberrant stem cell populations, called cancer stem cells (CSCs). Hence, the development of effective cancer therapies may rely on developing methods that specifically target these cells. However, the scarcity of CSCs in vivo represents a major impediment to such research, as there is an insufficient supply for basic biochemical and genetic analyses. It is therefore necessary to develop methods to expand reproducibly CSC tissue in vitro in a controlled environment. To date, we have developed bioreactor protocols for the suspension culture of an aggressive and deadly type of brain cancer called glioblastoma multiforme (GBM). Human GBM-derived cells achieved a maximum cell density of 2.4 x 10(6) cells/mL after 24 days under high shear conditions in batch culture conditions. In comparison, fed-batch cultures achieved 4.5 x 10(6) cells/mL after 32 days. Characterization of bioreactor-expanded cells using both flow cytometry and a differentiation assay indicated that bioreactor-generated human GBM-derived cells have similar characteristics to the initial cell population and achieve >90% CD133 expression. Additionally, genomic characterization indicated that a very small number of key genes were differentially expressed in the bioreactor-expanded GBM-derived cells, thereby conserving the basic nature of the brain cancer tissue in the cell expansion process.

Differential and functional interactions emphasize the multiple roles of polyamines in plants

Biogenic amines putrescine, spermidine and spermine are ubiquitous in nature and have interested researchers because they are essential for cell division and viability, and due to a large body of their pharmacological effects on growth and development in most living cells. The genes and enzymes involved in their biosynthetic pathways are now established and characterized. In recent years, molecular aspects of polyamine action have also begun to emerge. Our model is the ripening tomato fruit in which processes of cell division, cell expansion and cell growth have ceased, and yet the cells are responsive at biochemical and molecular levels to genetically manipulated concentrations of putrescine (Put), spermidine (Spd) and spermine (Spm). Thus, transcriptome, limited protein profiling, and metabolome studies of transgenic tomato fruit have yielded significant new information on cellular processes impacted by polyamine manipulation. We have used these datasets to determine the linear correlation coefficients between the endogenous levels of Put, Spd and Spm with several parameters. Results of our analysis presented here show that effects of the diamine Put generally contrast those with polyamines Spd and Spm, emphasizing that individual biogenic amines should be considered to have defined action in plant biology and that they differentially affect growth and development. A multiple function model of polyamine action is discussed to explain the role of polyamines in most organisms, in general, and ripening fruit, in particular.

Experimental study of cell pattern formation induced by internal heat sources in a horizontal fluid layer

This study determines the flow structure in a convection cell with an internally heated layer by PIV to elucidate the convection cell transition mechanisms. The vertical velocity component is determined and the cell behaviour with respect to Rayleigh number is investigated quantitatively. Cell expansion process is described as a consequence of development of the descending flow at the centre of cells. The results suggest that a spoke-like structure is stable in this system in ideal conditions and a double-cell structure is formed when there are restrictions on the system, i.e. finite lateral boundaries.

Automating the Expansion Process of Human Skeletal Muscle Myoblasts with Suppression of Myotube Formation

An intelligent culture system accompanied by automated operations (liquid transfer and cell passage) was newly developed to perform serial cultures of human skeletal muscle myoblasts. To realize a desired performance, a laminin-coated surface was applied to myoblast expansion in a culture flask. It was found that the laminin coating enhanced the overall growth ability attributable not to shortening of the doubling time but to prevention of differentiation toward myotube formation, compared with that on a conventional plain surface. In addition, the effects of seeding density and confluence degree on the growth were investigated quantitatively in terms of cell attachment and division as well as proliferative cell population in the culture on the laminin-coated surface. With increasing in seeding density, the number of proliferative cells decreased at the end of culture accompanied by an increase in the confluence degree, which caused poor attachment of the passaged cells on the surface in the subsequent culture. The quantitative analyses of these cell behaviors helped us determine the appropriate seeding density and attainable confluence degree during one passage, which were 1.0 x 10(3) cells/cm(2) and 0.5 as the initial and boundary conditions, respectively. An automated culture system that could manage two serial cultures by monitoring the confluence degree was constructed. The automated operation with the intelligent determination of the time for passage was successfully performed without serious loss of growth activity, compared with manual operation using conventional flasks. These results indicated that the monitoring of confluence degree is effective to perform the culture passage of myoblasts, being contributable to automating the cell expansion process.

Omega 3 fatty acids reduce myeloid progenitor cell frequency in the bone marrow of mice and promote progenitor cell differentiation.

BackgroundOmega 3 fatty acids have been found to inhibit proliferation, induce apoptosis, and promote differentiation in various cell types. The processes of cell survival, expansion, and differentiation are of key importance in the regulation of hematopoiesis. We investigated the role of omega 3 fatty acids in controlling the frequency of various myeloid progenitor cells in the bone marrow of mice. Increased progenitor cell frequency and blocked differentiation are characteristics of hematopoietic disorders of the myeloid lineage, such as myeloproliferative diseases and myeloid leukemias.ResultsWe found that increasing the proportion of omega 3 fatty acids relative to the proportion of omega 6 fatty acids in the diet caused increased differentiation and reduced the frequency of myeloid progenitor cells in the bone marrow of mice. Furthermore, this had no adverse effect on peripheral white blood cell counts.ConclusionOur results indicate that omega 3 fatty acids impact hematopoietic differentiation by reducing myeloid progenitor cell frequency in the bone marrow and promoting progenitor cell differentiation. Further exploration of this discovery could lead to the use of omega 3 fatty acids as a therapeutic option for patients that have various disorders of hematopoiesis.

Signal transducer and activator of transcription 3—A key molecular switch for human mesenchymal stem cell proliferation

Human bone marrow mesenchymal stem cells are multipotent cells with enormous potential for cellular therapies. Identifying those mediators that induce human bone marrow mesenchymal stem cell proliferation and elucidating the signaling networks involved will encourage clinical efforts exploiting such cells. Here, we demonstrate that platelet-derived growth factor-BB and basic fibroblast growth factor induce human bone marrow mesenchymal stem cell proliferation. Platelet-derived growth factor-BB induced human bone marrow mesenchymal stem cell proliferation via complete activation of the Janus-activated kinase-signal transducers and activators of transcription cascade, inducing signal transducer and activator of transcription 3 tyrosine and serine phosphorylation as well as Janus-activated kinase 2 tyrosine phosphorylation. Janus-activated kinase 2 was required for signal transducer and activator of transcription 3 tyrosine phosphorylation, whereas the extracellular signal-regulated kinase 1/2 mediated signal transducer and activator of transcription 3 serine phosphorylation in response to platelet-derived growth factor-BB. Furthermore, platelet-derived growth factor-BB was shown to promote nuclear translocation of signal transducer and activator of transcription 3. By contrast, basic fibroblast growth factor-stimulated human bone marrow mesenchymal stem cell proliferation was mediated via the extracellular signal-regulated kinase 1/2 pathway without involvement of the Janus-activated kinase-signal transducers and activators of transcription cascade. Importantly, platelet-derived growth factor-BB and basic fibroblast growth factor induced human bone marrow mesenchymal stem cell proliferation without affecting their osteogenic differentiation potential. Together, our study highlights the role of several growth factors in human bone marrow mesenchymal stem cell proliferation and the signaling pathways involved in the process. This information is crucial for achieving a better control over the human bone marrow mesenchymal stem cell expansion process.

Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses

Current understanding of the integration of cell division and expansion in the development of plant lateral organs such as leaves is limited. Cell number is established during a mitotic phase, and subsequent growth into a mature organ relies primarily on cell expansion accompanied by endocycles. Here we show that the three Arabidopsis cyclin D3 (CYCD3) genes are expressed in overlapping but distinct patterns in developing lateral organs and the shoot meristem. Triple loss-of-function mutants show that CYCD3 function is essential neither for the mitotic cell cycle nor for morphogenesis. Rather, analysis of mutant and reciprocal overexpression phenotypes shows that CYCD3 function contributes to the control of cell number in developing leaves by regulating the duration of the mitotic phase and timing of the transition to endocycles. Petals, which normally do not endoreduplicate, respond to loss of CYCD3 function with larger cells that initiate endocycles. The phytohormone cytokinin regulates cell division in the shoot meristem and developing leaves and induces CYCD3 expression. Loss of CYCD3 impairs shoot meristem function and leads to reduced cytokinin responses, including the inability to initiate shoots on callus, without affecting endogenous cytokinin levels. We conclude that CYCD3 activity is important for determining cell number in developing lateral organs and the relative contribution of the alternative processes of cell production and cell expansion to overall organ growth, as well as mediating cytokinin effects in apical growth and development.

Genetic Relationship Between Angustifolia3 and Extra-Small Sisters Highlights Novel Mechanisms Controlling Leaf Size

Coordination between cell proliferation and cell expansion is pivotal in leaf size determination. A group of mutants that are impaired in cell proliferation such as the angustifolia3 (an3) has provided a clue to understanding how these cellular processes are coordinated. In these mutants, impaired cell proliferation is accompanied by enhanced cell enlargement. We propose to call this phenomenon “compensated cell enlargement.” Previously, we isolated 10 extra-small sisters (xs) mutants that are specifically impaired in post-mitotic cell expansion and found that several xs mutations are able to suppress compensated cell enlargement in an3. Thus, the enhanced cell expansion observed in an3 results from the hyperactivation of post-mitotic cell expansion involving specific members of the XS gene family. These results suggested that cell proliferation process(es) and post-mitotic cell expansion process(es) are somehow linked in an as yet unknown fashion in leaf primordia. In this addendum, we propose possible models for the linking mechanisms that coordinate AN3-dependent cell proliferation and XS-dependent cell expansion in leaf development.

The cell cycle‐associated protein kinase WEE1 regulates cell size in relation to endoreduplication in developing tomato fruit

Tomato fruit size results from the combination of cell number and cell size which are respectively determined by cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of pericarp and locular tissues is characterized by the concomitant arrest of mitotic activity, inhibition of cyclin-dependent kinase (CDK) activity, and numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. To decipher the molecular basis of the endoreduplication-associated cell growth in fruit, we investigated the putative involvement of the WEE1 kinase (Solly;WEE1). We here report a functional analysis of Solly;WEE1 in tomato. Impairing the expression of Solly;WEE1 in transgenic tomato plants resulted in a reduction of plant size and fruit size. In the most altered phenotypes, fruits displayed a reduced number of seeds without embryo development. The reduction of plant-, fruit- and seed size originated from a reduction in cell size which could be correlated with a decrease of the DNA ploidy levels. At the molecular level downregulating Solly;WEE1 in planta resulted in the increase of CDKA activity levels originating from a decrease of the amount of Y15-phosphorylated CDKA, thus indicating a release of the negative regulation on CDK activity exerted by WEE1. Our data indicated that Solly;WEE1 participates in the control of cell size and/or the onset of the endoreduplication process putatively driving cell expansion.