Cell Expansion Process Research Articles

CM from intact hAM: an easily obtained product with relevant implications for translation in regenerative medicine

BackgroundIt is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs.MethodsAmniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM.ResultsWe demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC.ConclusionsThe results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreover, the possibility of having a lyophilized secretome that maintains its original properties would allow for a ready-to-use product with easier handling, shipping and storage. The use of a lyophilized product will also facilitate clinicians by permitting customized reconstitution volumes and methods according to the most suitable formula required by the clinical application.

Formins: the key regulators of plant cell morphology and development

Formins are widely distributed in eukaryotes such as fungi, plants and animals. They play crucial roles in regulating the polymerization of actin, coordinating the synergistic interactions between actin and microtubules, and determining cell growth and morphology. Unlike formins from fungi and animals, plant formins have been evolved into two plant-specific types. Generally, type Ⅱ formins are believed to regulate the polarized growth of cells, and type Ⅰ formins may regulate the cell expansion and division processes. Recent studies on the function of plant formins suggest it is inappropriate to classify the function of formins purely based on their structures. This review summarizes the domain organization of formins and their corresponding functions, as well as the underpinning mechanisms. Furthermore, the unsolved or unexplored issues along with future perspectives on plant formins are proposed and discussed.

Reactive Oxygen Species Link Gene Regulatory Networks During Arabidopsis Root Development.

Plant development under altered nutritional status and environmental conditions and during attack from invaders is highly regulated by plant hormones at the molecular level by various signaling pathways. Previously, reactive oxygen species (ROS) were believed to be harmful as they cause oxidative damage to cells; however, in the last decade, the essential role of ROS as signaling molecules regulating plant growth has been revealed. Plant roots accumulate relatively high levels of ROS, and thus, maintaining ROS homeostasis, which has been shown to regulate the balance between cell proliferation and differentiation at the root tip, is important for proper root growth. However, when the balance is disturbed, plants are unable to respond to the changes in the surrounding conditions and cannot grow and survive. Moreover, ROS control cell expansion and cell differentiation processes such as root hair formation and lateral root development. In these processes, the transcription factor-mediated gene expression network is important downstream of ROS. Although ROS can independently regulate root growth to some extent, a complex crosstalk occurs between ROS and other signaling molecules. Hormone signals are known to regulate root growth, and ROS are thought to merge with these signals. In fact, the crosstalk between ROS and these hormones has been elucidated, and the central transcription factors that act as a hub between these signals have been identified. In addition, ROS are known to act as important signaling factors in plant immune responses; however, how they also regulate plant growth is not clear. Recent studies have strongly indicated that ROS link these two events. In this review, we describe and discuss the role of ROS signaling in root development, with a particular focus on transcriptional regulation. We also summarize the crosstalk with other signals and discuss the importance of ROS as signaling molecules for plant root development.

Micro computed tomography with and without contrast enhancement for the characterization of microcarriers in dry and wet state

In the field of regenerative medicine, microcarriers are used as support matrix for the growth of adherent cells. They are increasingly recognised as promising biomaterials for large scale, cost-effective cell expansion bioreactor processes. However, their individual morphologies can be highly heterogeneous which increases bioprocesses’ variability. Additionally, only limited information is available on the microcarriers’ 3D morphology and how it affects cell proliferation. Most imaging modalities do not provide sufficient 3D information or have a too limited field of view to appropriately study the 3D morphology. While microfocus X-ray computed tomography (microCT) could be appropriate, many microcarriers are hydrated before in-vitro use. This wet state makes them swell, changing considerably their morphology and making them indistinguishable from the culture solution in regular microCT images due to their physical density close to water. The use of contrast-enhanced microCT (CE-CT) has been recently reported for 3D imaging of soft materials. In this study, we selected a range of commercially available microcarrier types and used a combination of microCT and CE-CT for full 3D morphological characterization of large numbers of microcarriers, both in their dry and wet state. With in-house developed image processing and analysis tools, morphometrics of individual microcarriers were collected. Also, the morphology in wet state was assessed and related to accessible attachment surface area as a function of cell size. The morphological information on all microcarriers was collected in a publicly available database. This work provides a quantitative basis for optimization and modelling of microcarrier based cell expansion processes.

Cell signaling regulation in salivary gland development.

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review focuses on research conducted on mammalian salivary gland development, particularly on the differentiation of acinar, ductal, and myoepithelial cells. We discuss recent studies that provide conceptual advances in the understanding of the molecular mechanisms of salivary gland development. In addition, we describe the organogenesis of submandibular glands (SMGs), model systems used for the study of SMG development, and the key signaling pathways as well as cellular processes involved in salivary gland development. The findings from the recent studies elucidating the identity of stem/progenitor cells in the SMGs, and the process by which they are directed along a series of cell fate decisions to form functional glands, are also discussed. Advances in genetic tools and tissue engineering strategies will significantly increase our knowledge about the mechanisms by which signaling pathways and cells establish tissue architecture and function during salivary gland development, which may also be conserved in the growth and development of other organ systems. An increased knowledge of organ development mechanisms will have profound implications in the design of therapies for the regrowth or repair of injured tissues. In addition, understanding how the processes of cell survival, expansion, specification, movement, and communication with neighboring cells are regulated under physiological and pathological conditions is critical to the development of future treatments.

Inoculation of maize seeds with Pseudomonas putida leads to enhanced seedling growth in combination with modified regulation of miRNAs and antioxidant enzymes

Bacteria that positively interact with plant roots are defined as plant growth promoting rhizobacteria (PGPR). Although the positive effect of PGPR on plant growth has been widely studied, their impact on genetic regulation during plant growth processes remains largely unknown. Therefore, this study aimed to gain a deeper understanding of the regulatory role of miRNA and redox enzymes in response to PGPR at the maize seedling stage in leaf growth zone which consists of the meristem, elongation and mature zones. For this purpose, growth of the third leaf was monitored in response to Pseudomonas putida (P. putida) KT2440 at phenotypic, physiological, cellular, kinematic, and transcriptional levels. This application resulted in an increase of 15% in shoot length, 56% in both shoot fresh/dry weight, 10% in chlorophyll amount, 8% in mature cell length, 15% in leaf elongation rate, and 7% in cell production; meanwhile final leaf length was unchanged, while leaf area and leaf width decreased by 22% and 16%, respectively. Ascorbate peroxidase and glutathione reductase activity increased throughout the leaf growth zone indicating a possible role in PGPR-plant interaction during transition between cell division, expansion and differentiation processes. The expression analysis of cell cycle check point marker genes revealed that CycA2_1 was mainly responsible for promoting cell proliferation in meristem. miR160, miR169 and miR408 were differentially expressed in the meristem, indicating their indirect regulatory roles in the cell division response to PGPR. In addition, miR160, miR319 and miR396 were downregulated in the elongation zone, which draws attention to their possible role in regulating cell elongation processes. In summary, cell cycle, redox and miRNA regulation in maize seedling growth zones in response to P. putida were investigated for the first time in this study.

T Cell Receptor Engineered Lymphocytes for Cancer Therapy.

T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.

Developing adoptive T cell therapy for Polyomavirus-associated diseases using HLA-defined peptide platform

Background & Aim Polyomavirus infection is a growing threat among immunocompromised patients. Reconstitution of T cell immunity has been shown to control the infection. Hence, the adoptive transfer of polyomavirus-specific T cells is a promising approach to revive T cell immunity and tackle viral reactivation and disease progression. We aimed at mapping T cell epitopes across multiple viral antigens of BKV and JCV with broad HLA coverage. Methods, Results & Conclusion Methods Proteome wide analysis of T cell responses against BKV and JCV antigens was carried out in a cohort of healthy volunteers and transplant recipients to identify immunodominant T cell epitopes. This epitope screening was conducted using overlapping peptides from VP1, VP2, VP3, LTA and STA antigens of BKV and JCV. HLA restriction and fine mapping of these epitopes was confirmed using peptide loaded HLA-matched and mismatched targets. Finally a novel pepmix based on these epitopes was used for optimization of GMP-compliant ex vivo expansion of BKV and JCV-specific T cell therapy. Results We have mapped 57 novel epitopes from BKV and JCV restricted to HLA class I and class II alleles. Many of these epitopes show cross protective potential where BKV-specific T cells could detect JCV peptide and vice versa. Molecular analysis of T cell directed against polyomaviruses showed a unique transcriptional signature when compared to T cell specific for other persistent viruses. Using this knowledge we have developed a rapid in vitro T cell expansion process for manufacturing of adoptive T cell therapy which can be used for the treatment of BKV and JCV-associated diseases. We have successfully used this optimized protocol to generate BKV-specific T cells for treating two patients who were unresponsive to standard clinical management. Patient 1 who had hemorrhagic cystitis was injected with 4 doses of autologous BKV-specific T cells and Patient 2 who had BK virus associated nephropathy was administered with 10 doses of HLA-matched third party BKV-specific T cells. Both patients completely cleared BKV in both urine and serum with no further reactivation. Conclusion Development of a novel HLA-defined peptide platform for BKV and JCV provides an unique opportunity to offer rapid autologous or “off-the-shelf” allogenic T cell therapy. More importantly, preliminary clinical studies indicates that the adoptive T cell therapy manufactured using this pepmix offers safe and effective clinical responses in untreatable disease settings.

Generation of iPS clones using automated live-imaging, picking and in-process quality controls

Background & Aim Induced pluripotent stem (iPS) cells have great potential as tools for regenerative medicine, disease modeling and drug screening applications. However, current manufacturing remain highly dependent on manual tasks and subjective human decisions about which colonies to choose and progress in large-scale cell expansion process. The goal of this work is to develop automated workflow options using the Cell X™ robotic platform for: 1) Cell Source Selection based on quantitative reproducible quality attributes, 2) Precision Automation of iPS clone “picking”, “weeding”, passage and expansion, and 3) quantitative documentation of cell and clone attributes over time to define critical process parameters and critical quality attributes that can predict future performance. Methods, Results & Conclusion The Cell X™ robotic platform was used to develop and characterize imaging and analysis tools relevant to iPS populations using the DF6-9-9T.B hiPSC cell line, reprogramming of human skin fibroblasts. The Cell X platform (Fig. 1) is a multifaceted robotic device that enables: automated large field of view (LFOV) imaging using phase contrast or fluorescence; serial imaging (videomicroscopy); quantitative LFOV image analysis (Colonyze™); media changes; cell and colony “picking” (isolation and transplantation of desired cells), “weeding” (removal of undesired cells); or “biopsy (sampling of defined cells for analysis). Feasibility was demonstrated in the capture LFOV images over the reprogramming phase to track quantitative metrics of evolution and morphology of individual iPS clones (e.g. size, shape, area, perimeter roughness, texture, growth rate) (Fig. 2 & 3). Automated image processing tools were defined to segment regions of undifferentiated iPS cells from regions of differentiating and potentially differentiating cells, which could be used to guide “picking”, “weeding” or “biopsy” interventions. Picking operations were effective, and transplanted cells continued to proliferate effectively with preservation of iPS morphology. The integrated Cell X™ platform enables both fully automated iPS clone “picking” and expansion in a hands free, reproducible, and environmentally controlled setting. This system represent a potential valuable tool to automate and scale iPS fabrication with reduced expense and variation, and with rigorous documentation. Cell X™ methods can be refined for lab-specific application, core facility, and integrated in fully automated industrial GMP manufacturing facility.

MP29-17 A DECELLULARIZED SKELETAL MUSCLE-DERIVED ECM FOR IN SITU MUSCLE REGENERATION

INTRODUCTION AND OBJECTIVE: Cell-based tissue engineering strategies have gained attention in restoring normal tissue function for reconstructive procedures; however, these approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation. In order to avoid this limitation, we developed a cell-free muscle-specific scaffolding system that consists of a skeletal muscle-derived decellularized extracellular matrix (dECM) and a myogenic factor, insulin growth factor-1 (IGF-1). We hypothesized that muscle-derived dECM biomaterials combined with IGF-1 could provide a muscle-specific microenvironment for in situ muscle tissue regeneration. We developed a novel dECM-based scaffolding system containing IGF-1 and characterized its rheological properties and in vitro biological properties for tissue reconstruction. METHODS: We developed a novel dECM-based scaffolding system containing IGF-1 and characterized its rheological properties and in vitro biological properties. In addition, we investigated the feasibility of using this dECM-based scaffolding system for in situ muscle tissue regeneration in a rabbit TA muscle defect model. RESULTS: The cell viability in all scaffolds had over 90% at 1, 3, and 7 days in culture. The cell proliferation in the IGF-1/dECM was significantly increased when compared with other groups. More importantly, the IGF-1/dECM strongly supported the myogenic differentiation in the scaffold as confirmed by MHC immunofluorescence. We also investigated the feasibility in a rabbit tibialis anterior (TA) muscle defect model. The IGF-1/dECM had a significantly greater number of myofibers when compared to both collagen and dECM groups at 1 and 2 months after implantation. CONCLUSIONS: We developed the muscle-specific dECM-based scaffolding system and investigated the synergistic effect of the dECM with IGF-1 for in situ muscle regeneration. The dECM was obtained by the decellularization of skeletal muscle tissue, followed by the solubilization. Combining with IGF-1, the dECM showed high in vitro cellular activities, including cell viability, proliferation, and differentiation. Moreover, in vivo implantation of IGF-1/dECM scaffold in the rabbit TA muscle defect model showed an acceleration in the new muscle formation. We demonstrated that this novel muscle-specific scaffolding system could enhance the body’s regenerative capability for the in situ muscle tissue regeneration, which may have potential utility in reconstruction. Source of Funding: This study was supported by the Musculoskeletal Transplant Foundation (MTF).

Comparative Proteomics of Root Apex and Root Elongation Zones Provides Insights into Molecular Mechanisms for Drought Stress and Recovery Adjustment in Switchgrass.

Switchgrass plants were grown in a Sandwich tube system to induce gradual drought stress by withholding watering. After 29 days, the leaf photosynthetic rate decreased significantly, compared to the control plants which were watered regularly. The drought-treated plants recovered to the same leaf water content after three days of re-watering. The root tip (1cm basal fragment, designated as RT1 hereafter) and the elongation/maturation zone (the next upper 1 cm tissue, designated as RT2 hereafter) tissues were collected at the 29th day of drought stress treatment, (named SDT for severe drought treated), after one (D1W) and three days (D3W) of re-watering. The tandem mass tags mass spectrometry-based quantitative proteomics analysis was performed to identify the proteomes, and drought-induced differentially accumulated proteins (DAPs). From RT1 tissues, 6156, 7687, and 7699 proteins were quantified, and 296, 535, and 384 DAPs were identified in the SDT, D1W, and D3W samples, respectively. From RT2 tissues, 7382, 7255, and 6883 proteins were quantified, and 393, 587, and 321 proteins DAPs were identified in the SDT, D1W, and D3W samples. Between RT1 and RT2 tissues, very few DAPs overlapped at SDT, but the number of such proteins increased during the recovery phase. A large number of hydrophilic proteins and stress-responsive proteins were induced during SDT and remained at a higher level during the recovery stages. A large number of DAPs in RT1 tissues maintained the same expression pattern throughout drought treatment and the recovery phases. The DAPs in RT1 tissues were classified in cell proliferation, mitotic cell division, and chromatin modification, and those in RT2 were placed in cell wall remodeling and cell expansion processes. This study provided information pertaining to root zone-specific proteome changes during drought and recover phases, which will allow us to select proteins (genes) as better defined targets for developing drought tolerant plants. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD017441.

Organ-wide and ploidy-dependent regulation both contribute to cell-size determination: evidence from a computational model of tomato fruit.

The development of a new organ is the result of coordinated events of cell division and expansion, in strong interaction with each other. This study presents a dynamic model of tomato fruit development that includes cell division, endoreduplication, and expansion processes. The model is used to investigate the potential interactions among these developmental processes within the context of the neo-cellular theory. In particular, different control schemes (either cell-autonomous or organ-controlled) are tested and compared to experimental data from two contrasting genotypes. The model shows that a pure cell-autonomous control fails to reproduce the observed cell-size distribution, and that an organ-wide control is required in order to get realistic cell-size variations. The model also supports the role of endoreduplication as an important determinant of the final cell size and suggests that a direct effect of endoreduplication on cell expansion is needed in order to obtain a significant correlation between size and ploidy, as observed in real data.

Isolation and Characterization of a Human Fetal Mesenchymal Stem Cell Population: Exploring the Potential for Cell Banking in Wound Healing Therapies

Various cell-based therapies are in development to address chronic and acute skin wound healing, for example for burns and trauma patients. An off-the-shelf source of allogeneic dermal cells could be beneficial for innovative therapies accelerating the healing in extensive wounds where the availability of a patient’s own cells is limited. Human fetal-derived dermal fibroblasts (hFDFs) show high in vitro division rates, exhibit low immunological rejection properties, and present scarless wound healing in the fetus, and previous studies on human fetal tissue-derived cell therapies have shown promising results on tissue repair. However, little is known about cell lineage stability and cell differentiation during the cell expansion process, required for any potential therapeutic use. We describe an isolation method, characterize a population, and investigate its potential for cell banking and thus suitability as a potential product for cell grafting therapies. Our results show hFDFs and a bone marrow-derived mesenchymal stem cell (BM-MSC) line shared identification markers and in vitro multilineage differentiation potential into osteogenic, chondrogenic, and adipogenic lineages. The hFDF population exhibited similar cell characteristics as BM-MSCs while producing lower pro-inflammatory cytokine IL-6 levels and higher levels of the wound healing factor hepatocyte growth factor. We demonstrate in vitro differentiation of hFDFs, which may be a problem in maintaining long-term lineage stability, potentially limiting their use for cell banking and therapy development.

A novel decellularized skeletal muscle-derived ECM scaffolding system for in situ muscle regeneration.

The cell-based tissue engineering strategies have gained attention in restoring normal tissue function after skeletal muscle injuries; however, these approaches require a donor tissue biopsy and extensive cell expansion process prior to implantation. In order to avoid this limitation, we developed a novel cell-free muscle-specific scaffolding system that consisted of a skeletal muscle-derived decellularized extracellular matrix (dECM) and a myogenic factor, insulin growth factor-1 (IGF-1). Rheological, morphological, and biological properties of this muscle-specific scaffold (IGF-1/dECM) as well as collagen and dECM scaffolds were examined. The cell viability in all scaffolds had over 90% at 1, 3, and 7 days in culture. The cell proliferation in the IGF-1/dECM was significantly increased when compared with other groups. More importantly, the IGF-1/dECM strongly supported the myogenic differentiation in the scaffold as confirmed by myosin heavy chain (MHC) immunofluorescence. We also investigated the feasibility in a rabbit tibialis anterior (TA) muscle defect model. The IGF-1/dECM had a significantly greater number of myofibers when compared to both collagen and dECM groups at 1 and 2 months after implantation. We demonstrated that this novel muscle-specific scaffolding system could effectively promote the muscle tissue regeneration in situ.

New concepts in regenerative medicine approaches to the treatment of female stress urinary incontinence.

Update on recent regenerative medicine approaches to the treatment of stress urinary incontinence (SUI) caused by intrinsic sphincter deficiency (ISD). In the treatment of female SUI/ISD, results using different types of cellular therapy have been disappointing, and new approaches are desirable. To advance our regenerative medicine approaches to SUI/ISD, it is critical to utilize animal models that best parallel the pathophysiology of this disease in women. Many current animal models mimic acute SUI/ISD. However, SUI/ISD in women is usually a chronic condition resulting from previous muscle and nerve sphincter damage during parturition or muscle loss during aging. Similar to women, a nonhuman primate (NHP) model of chronic SUI/ISD has demonstrated only modest response to cell therapy. However, treatment with stromal cell-derived factor 1 (SDF1), also known as C-X-C motif chemokine 12 (CXCL12) restored continence in this model. As a potential therapeutic approach, the use of a well characterized chemokine, such as CXCL12, may by-pass the lengthy and expensive process of cell isolation, expansion, and injection. Recent findings in this new NHP model of chronic SUI/ISD may open up the field for noncell-based treatments.

Kinetic modeling of human induced pluripotent stem cell expansion in suspension culture.

To date, practical application of mathematical models for model-based design of stem cell expansion processes is limited. Nevertheless, the first attempts show vast potential of this approach for the improvement of expansion process performance. This article presents the developed dynamic kinetic model of the human induced pluripotent stem cell expansion process in suspension culture. The model predicts cell growth, consumption of glucose and production of lactic acid, as well as the average aggregate size. The latter process variable is of particular importance for achieving high cell density. By adding botulinum hemagglutinin, an E-cadherin inhibitor and subsequent aggregate break-up, one can significantly increase performance of cell expansion process. After defining the appropriate optimization criteria and additional modification of the model, the latter can be further applied for model-based optimization of the final cell concentration by calculating optimal aggregate break-up and glucose/glutamine feeding strategies.

Nectary size is a pollination syndrome trait in Penstemon.

Summary Evolution of complex phenotypes depends on the adaptive importance of individual traits, and the developmental changes required to modify traits. Floral syndromes are complex adaptations to pollinators that include color, nectar, and shape variation. Hummingbird‐adapted flowers have evolved a remarkable number of times from bee‐adapted ancestors in Penstemon, and previous work demonstrates that color over shape better distinguishes bee from hummingbird syndromes. Here, we examined the relative importance of nectar volume and nectary development in defining Penstemon pollination syndromes.We tested the evolutionary association of nectar volume and nectary area with pollination syndrome across 19 Penstemon species. In selected species, we assessed cellular‐level processes shaping nectary size. Within a segregating population from an intersyndrome cross, we assessed trait correlations between nectar volume, nectary area, and the size of stamens on which nectaries develop.Nectar volume and nectary area displayed an evolutionary association with pollination syndrome. These traits were correlated within a genetic cross, suggesting a mechanistic link. Nectary area evolution involves parallel processes of cell expansion and proliferation.Our results demonstrate that changes to nectary patterning are an important contributor to pollination syndrome diversity and provide further evidence that repeated origins of hummingbird adaptation involve parallel developmental processes in Penstemon.

Enhanced metabolic activities for ATP production and elevated metabolic flux via pentose phosphate pathway contribute for better CIK cells expansion.

ObjectiveEx vivo expansion is an effective way to produce cytokine‐induced killer (CIK) cells needed for clinical trials. Here, ex vivo expansion and metabolism characters of CIK cells in static and dynamic cultures and the relationship between cell expansion and metabolism were investigated.Materials and methodsOxygen transfer efficiency was assessed by computational fluid dynamics technique. Cell phenotype, apoptosis and of transporter expression were determined by flow cytometry and Western blotting. Metabolites and enzyme activities were assessed by biochemical methods.ResultsDynamic cultures favoured better CIK cell expansion without impairing their phenotype and cytotoxicity, enhanced oxygen transfer efficiency. The glucose metabolism flux of cells in dynamic cultures was enhanced by upregulating surface glucose transporter 1 expression and phosphofructokinase activity. Moreover, pentose phosphate pathway (PPP) metabolic flux was enhanced through upregulating glucose‐6‐phosphate dehydrogenase activity. Glutaminolysis was also accelerated via boosting glutamine transporters expression, glutaminase (GLS) and glutamate dehydrogenase activities. Together with higher oxygen consumption rate and extracellular acidification rate, it was suggested that cells in dynamic cultures were in a more vigorous metabolic state for ATP production.ConclusionDynamic cultures accelerated glucose and glutamine metabolic flux to promote ATP production, elevated glucose metabolic flux through PPP to promote biosynthesis for better cell expansion. These findings may provide the basis for ex vivo CIK cell expansion process optimization.

Divide, expand, differentiate - new insights on plant organ growth through cytokinin signaling.

For centuries, the mechanisms underlying plant growth and morphogenesis have fascinated eminent thinkers who generated intriguing and still timely theories. It suffices to recall the theory on the directionality of cell division within a complex network of cells proposed by Hofmeister over 150 years ago and the theory on the trees’ organ shape proposed by Leonardo DaVinci over 500 years ago. Irrespective of the micro- or macro-dimension level of study, plant growth is ultimately the result of combined phenomena that include cell division, expansion, and differentiation within the constraints of genetic and physical interactions occurring in a growing network of cells. Though the molecular factors responsible for these basic cellular processes have surfaced and are becoming increasingly well characterized, the mechanisms and their precise spatial and temporal control are yet largely unknown. It is also questionable whether all the factors controlling plant growth have been identified. In their work, Skalák et al., 2019 addressed these questions in a study focused on understanding how cell division, expansion, and differentiation processes are coordinated through the action of cytokinines (CK) (Figure 1). It is well established that CKs are plant hormones controlling a plethora of developmental processes, including the determination of the final size and function of plant organs, such as leaves. Although zeatin, the first natural CK was isolated from unripe maize kernels by Letham in 1964, CKs signaling in the course of the transition from cell proliferation to cell expansion in early developing leaves or the differentiation of fully expanded leaf has been enigmatic. Recognizing the complexity of the plants inherent to the broad diversity of tissues, this research led by a collaboration of the Inzé and Brzobohatý groups focused on early leaf development to follow the transition from cell proliferation to cell expansion and differentiation. In their fascinating work, the authors queried the transcriptome and proteome of the leaf 3, being an established developmental model for morphogenesis analyses. Utilizing a suite of biomarkers for CK and inducible genetic tools to modulate CK abundance in situ, the authors were able to follow CK pool and signaling dynamics during the transition from cell proliferation to cell expansion and differentiation in leaf 3, and test their impact in the control of leaf development. The research led to a detailed time-course characterization of cell responses in relation to the CK pool during organ morphogenesis, an unanticipated relationship between CK pool and chloroplast biogenesis during the cell proliferation stage, and the findings supporting that a decrease of CK content at the end of the cell proliferation phase is necessary for the transition to cell expansion stimulation. In particular, a mere decrease in CK content triggered experimentally at the cell proliferation phase was sufficient to down- and up-regulate distinct sets of genes specific for the cell proliferation and cell expansion phase, respectively. Control by CK is novel for most of the genes found regulated during the transition from cell proliferation to cell expansion and differentiation. Intriguingly, genes involved in critical processes, such as cell wall biogenesis, very-long-chain fatty acid production and primary metabolism, were also identified, making several of the findings a springboard for testing the role of novel factors in controlling plant cell expansion. The work could have broader implications, as Brzobohatý states: “Plant biotechnologists might find our data useful when designing strategies to modify leaf size and structure in their efforts to generate novel plant cultivars for needs of sustainable agriculture”. The development of this work required tools and expertise developed in multiple labs. “Dirk's group has made a major contribution towards our understanding of molecular mechanisms governing leaf development. A direct motivation to initiate our collaboration came from re-examination of RNA-seq data sets obtained by transcriptome profiling of leaf 3 transition from cell proliferation to cell expansion phase (Andriankaja et al., 2012) which revealed down-regulation of a cytokinin activating gene (LOG3) during this process. Our transgenic plants enabling us to generate cytokinin excess and deficiency in specific developmental windows and our long-term interest in cytokinin biology together with Ondřej's expertise in hormonomics made it feasible to analyze cytokinin roles in leaf development”, Brzobohatý says. The experience and the tools developed to study leaf 3 development in relation to CK will allow the researchers to move forward to new horizons: “Having determined the multifaceted activity of CK in leaf development under standard conditions, we are now interested in CK roles in leaf development under various suboptimal growth conditions. We believe that by focusing our work on a particular leaf at various developmental windows we will be able to uncover molecular mechanisms, which might remain hidden when a whole rosette is analyzed as has been often the case”. With no doubts, many in the scientific community will be looking forward to the new research developments!

A Microrobotic System for Simultaneous Measurement of Turgor Pressure and Cell-Wall Elasticity of Individual Growing Plant Cells

Plant growth and morphogenesis is directed by cell division and the expansion of individual cells. How the tightly controlled process of cell expansion is regulated is poorly understood. We introduce a microrobotic platform able to separately measure the turgor pressure and cell wall elasticity of individual growing, turgid cells by combining microindentation with cell compression experiments. The system independently controls two indenters with geometries at different scales. Indentation measurements are performed automatically by deforming the cells with indenters with a spatial resolution in the nanometer range while recording force and displacement. The dual-indentation technique offers a noninvasive, high-throughput method to characterize the cytomechanics of single turgid cells by separately measuring elasticity and turgor pressure. In this way, the expansion regulation of growing cells can be investigated, as demonstrated here using Lilium longiflorum pollen tubes as an example.