Harnessing stem cells for health needs in India

While industrialized countries' stem cell research will be transferable to the developing world, research conducted by developing countries offers the potential to target innovation to local context, make treatments more affordable, and aid in economic development. India demonstrates that stem cell research and development (R&D) is not confined to industrialized countries and has begun to harness stem cells to address its own health needs.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Cell-interactive 3D-scaffold; advances and applications

Culturing cells ex vivo that differentiate and maintain in vivo characteristics holds great promise not only for the pragmatic revelations of cell function but also in tissue engineering and regenerative medicine. Lack of de-novo extra-cellular matrix (ECM) milieu, which plays a crucial role in generating physical and chemical signals besides providing structural support is attributed to be the major hurdle in normal cell growth in vitro. Hence, to comprehend the outcome of cell biology research in clinical context, it is important that the cell culture based models should incorporate both the three dimensional (3D) organization and multi cellular complexity of an organ while allowing experimental interventions in a desirable manner. This calls for the development of ECM-mimicking 3D scaffold, which can be integrated with relevant ECM cues to offer cell interactive versatility for different medical and non-medical applications. Present review discusses the status of ECM mimicking for 3D cell culture and its diverse implications.     

Quantitation of 3D ureteric branching morphogenesis in cultured embryonic mouse kidney

The growth and branching of the epithelial ureteric tree is critical for development of the permanent kidney (metanephros). Current methods of analysis of ureteric branching are mostly qualitative. We have developed a method for measuring the length of individual branches, and thereby the total length of the ureteric tree in 3 dimensions (3D). The method involves confocal microscopy of whole-mount immunostained metanephroi and computer-based image segmentation, skeletonisation and measurement. The algorithm performs semi-automatic segmentation of a set of confocal images and skeletonisation of the resulting binary object. Length measurements and number of branch points are automatically obtained. The final representation can be reconstructed providing a fully rotating 3D perspective of the skeletonised tree. After 36 h culture of E12 mouse metanephroi, the total length of the ureteric tree was 6103 +/- 291 microm (mean +/- SD), a four-fold increase compared with metanephroi cultured for just 6 h (1522 +/- 149 microm). Ureteric duct length increased at a rate of 153 microm/h over the first 30 h period and was maximal between 18 and 24 h at 325 microm/h. The distribution of branch lengths at the six time points studied was similar, suggesting tight control of ureteric lengthening and branching. This method will be of use in analysing ureteric growth in kidneys cultured in the presence of specific molecules suspected of regulating ureteric growth. The method can also be used to analyse in vivo kidneys and to quantify branching morphogenesis in other developing organs.     

In Vivo Wall Shear Measurements within the Developing Zebrafish Heart

Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.     

Role of melatonin in photoperiodic time measurement in the migratory redheaded bunting (Emberiza bruniceps) and the nonmigratory Indian weaver bird (Ploceus philippinus)

In the present study, we asked the question whether physiological responses to day length of migratory redheaded bunting (Emberiza bruniceps) and nonmigratory Indian weaver bird (Ploceus philippinus) are mediated by the daily rhythm of melatonin. Melatonin was given either by injection at certain times of the day or as an implant. In series I experiments on the redheaded bunting, melatonin was administered by subcutaneous injections daily at zeitgeber time (ZT) 4 (morning) or ZT10 (evening) and by silastic capsules in photosensitive unstimulated buntings that were held in natural day lengths (NDL) at 27 degrees N beginning from mid February, and in artificial day lengths (ADL, 12L:12D and 14L:10D). Melatonin did not affect the photoperiod-induced cycles of gain and loss in body mass and testicular growth-involution, but there was an effect on temporal phasing of the growth-involution cycle of testes in some groups. For example, the rate of testicular growth and development was faster in birds that received melatonin injection at ZT4 in NDL, and was slower in birds that carried melatonin implants both in NDL and ADL. In series II experiments on Indian weaver birds, melatonin was given in silastic capsules in the first week of September when they still had large gonads. Birds were exposed for 12 weeks to short day length (8L:16D; group 1), to long day length (eight weeks of 16L:8D and four weeks of 18L:6D; group 2), or to both short and long day lengths (four weeks each of 8L:16D, 16L:8D, and 18L:6D; groups 3 and 4). Whereas groups 1 to 3 carried melatonin or empty implant from the beginning, group 4 received one after four weeks. All birds underwent testicular regression during the first four weeks irrespective of the photoperiod they were exposed to or the implant they carried in, and there was a slight re-initiation of testis growth in some birds during the next eight weeks of long day lengths. However, with the exception of group 2, there was no difference in mean testis volume during the period of experiment between the melatonin- and empty-implant birds. The data on androgen-dependent beak color also supported the observations on testes. Together, these results do not support the idea that the daily rhythm of melatonin is involved in the photoperiodic time measurement in birds. However, there may still be a role of melatonin in temporal phasing of the annual reproductive cycle in birds.     

Simultaneous immunohistochemical detection of IUdR and BrdU infused intravenously to cancer patients

Cell cycle kinetics of solid tumors in the past have been restricted to an in vitro labeling index (LI) measurement. Two thymidine analogues, bromodeoxyuridine (BrdU) and iododeoxyuridine (IUdR), can be used to label S-phase cells in vivo because they can be detected in situ by use of monoclonal antibodies (MAb) against BrdU (Br-3) or IUdR (3D9). Patients with a variety of solid tumors (lymphoma, brain, colon cancers) received sequential intravenous IUdR and BrdU. Tumor tissue removed at the end of infusion was embedded in plastic and treated with MAb Br-3 and 3D9 sequentially, using a modification of a previously described method. Clearly single and double labeled cells were visible, which enabled us to determine the duration of S-phase (Ts) and the total cell cycle time (Tc), in addition to the LI in these tumors. Detailed control experiments using tissue culture cell lines as well as bone marrow cells from leukemic patients are described, including the comparison of this double label technique with our previously described BrdU-tritiated thymidine technique. We conclude that the two methods are comparable and that the IUdR/BrdU method permits rapid and reliable cell cycle measurements in solid tumors.     

Light-sheet microscopy-based 3D single-cell tracking reveals a correlation between cell cycle and the start of endoderm cell internalization in early zebrafish development

Controlling the initiation of cell migration plays a fundamental role in shaping the tissue during embryonic development. During gastrulation in zebrafish, some mesendoderm cells migrate inward to form the endoderm as the innermost germ layer along the yolk syncytial layer. However, how the initiation of inward migration is regulated is poorly understood. In this study, we performed light-sheet microscopy-based 3D single-cell tracking consisting of (a) whole-embryo time-lapse imaging with light-sheet microscopy and (b) three-dimensional single cell tracking in the zebrafish gastrula in which cells are marked with histone H2A-mCherry (nuclei) and the sox17:EGFP transgene (expressed in endoderm cells). We analyzed the correlation between the timing of cell internalization and cell division. Most cells that differentiated into endoderm cells began to internalize during the first half of the cell cycle, where the length of a cell cycle was defined by the period between two successive cell divisions. By contrast, the timing of other internalized cells was not correlated with a certain phase of the cell cycle. These results suggest the possibility that cell differentiation is associated with the relationship between cell cycle progression and the start of internalization. Moreover, the 3D single-cell tracking approach is useful for further investigating how cell migration is integrated with cell proliferation to shape tissues in zebrafish embryos.     

PAR proteins direct asymmetry of the cell cycle regulators Polo-like kinase and Cdc25

Cell cycle lengths vary widely among different cells within an animal, yet mechanisms of cell cycle length regulation are poorly understood. In the Caenorhabditis elegans embryo, the first cell division produces two cells with different cell cycle lengths, which are dependent on the conserved partitioning-defective (PAR) polarity proteins. We show that two key cell cycle regulators, the Polo-like kinase PLK-1 and the cyclin-dependent kinase phosphatase CDC-25.1, are asymmetrically distributed in early embryos. PLK-1 shows anterior cytoplasmic enrichment and CDC-25.1 shows PLK-1-dependent enrichment in the anterior nucleus. Both proteins are required for normal mitotic progression. Furthermore, these asymmetries are controlled by PAR proteins and the muscle excess (MEX) proteins MEX-5/MEX-6, and the latter is linked to protein degradation. Our results support a model whereby the PAR and MEX-5/MEX-6 proteins asymmetrically control PLK-1 levels, which asymmetrically regulates CDC-25.1 to promote differences in cell cycle lengths. We suggest that control of Plk1 and Cdc25 may be relevant to regulation of cell cycle length in other developmental contexts.     

Signalling molecules,growth regulators and cell cycle control in Drosophila

During the development of a given organ or tissue within a multicellular organism, growth and patterning are controlled in a coordinated manner by the activity of a discrete number of signalling molecules and their corresponding pathways to give rise to a well formed structure with a particular size, shape and pattern. Understanding how cells of different tissues or organs translate in a context dependent manner the activity of these pathways into an activation or repression of the cell cycle machinery is one of the most intriguing questions in developmental and cancer biology nowadays. Here we revise the different roles of the signalling molecules Notch and Wingless in the regulation of cell cycle progression in the developing eye and wing imaginal discs of Drosophila and propose that depending on how growth regulators are regulated in a context dependent manner by the activity of these pathways, signalling molecules might have tumour suppressor or oncogene activity.     

PTP-Pez: A novel regulator of TGFβ signaling

The TGF-betas are a family of pleiotropic cytokines that mediate diverse effects including the regulation of cell cycle progression, apoptosis, tissue remodelling and epithelial-mesenchymal transition (EMT). These diverse effects allow the TGF-betas to play multiple and even opposing roles in different contexts during embryonal development, tissue homeostasis and cancer progression. We recently reported that the protein tyrosine phosphatase Pez is a novel inducer of TGF-beta signaling, regulating EMT and organogenesis in developing zebrafish embryos, and leading to TGF-beta mediated EMT when over-expressed in vitro in epithelial MDCK cells. A number of mutations in Pez have been shown to be associated with breast and colorectal cancers, although the effect of these mutations on Pez function and their contribution to cancer progression remains unclear. Our finding that Pez regulates TGF-beta signaling is therefore of interest not only in the context of identifying a novel upstream regulator of TGF-beta signaling, but also in implicating the dysregulation of TGF-beta signaling as a possible link between Pez mutation and cancer progression. Here we discuss the implications of our research, in the context of dysregulation of TGF-beta signaling in cancer and other human pathologies.     

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.     

Diversity of cell lengths in terminal portions of roots: implications to cell proliferation

Terminal meristems are responsible for all primary growth of roots. It has been asserted that all cells of root meristems are actively dividing (no cells cycle slowly or arrest in the cycle) and stem cell populations expand exponentially. Because cells do not slide relative to each other in roots, relative cell lengths may be used to determine relative cell cycle durations and/or proportions of cells actively dividing in root tissues. If all cells are cycling, no interphase cells should be longer than critical length (length of longest mitotic cell in the meristem) and cells should exhibit an exponential cell-age distribution. Lengths of all cells were obtained radially across entire median longitudinal root sections at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mm from the founder cell/root cap boundary for five plant species to estimate percentages of cells longer than critical length. For example, up to 15 and 90% of all interphase cells were longer than critical length in 0.5 and 2.0 mm tissues, respectively, indicating that slow-cycling and/or non-proliferative cells are present in such tissues. In order to determine if the distribution of cell lengths in 0.5 mm segments approximated an exponential cell-age distribution, lengths of interphase cells less than critical length were determined. Such interphase cells were placed into ten groups according to cell length and percentages of cells in each group were compared with percentages of cells in groups calculated from an exponential cell-age distribution. Percentages of cells were significantly different from predicted percentages of between 6 and 9 out of ten groups - cell lengths were not distributed exponentially. Because there are significant numbers of interphase cells longer than critical length and since lengths of interphase cells shorter than critical length do not resemble an exponential cell-age distribution, it must be concluded that not all cells in root segments from 0.5 to 3.0 mm root segments are actively dividing. Heretofore, no databases of cell lengths have been used to test these assertions.     

Multicellular spatial model of RNA virus replication and interferon responses reveals factors controlling plaque growth dynamics

Respiratory viruses present major public health challenges, as evidenced by the 1918 Spanish Flu, the 1957 H2N2, 1968 H3N2, and 2009 H1N1 influenza pandemics, and the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Severe RNA virus respiratory infections often correlate with high viral load and excessive inflammation. Understanding the dynamics of the innate immune response and its manifestations at the cell and tissue levels is vital to understanding the mechanisms of immunopathology and to developing strain-independent treatments. Here, we present a novel spatialized multicellular computational model of RNA virus infection and the type-I interferon-mediated antiviral response that it induces within lung epithelial cells. The model is built using the CompuCell3D multicellular simulation environment and is parameterized using data from influenza virus-infected cell cultures. Consistent with experimental observations, it exhibits either linear radial growth of viral plaques or arrested plaque growth depending on the local concentration of type I interferons. The model suggests that modifying the activity of signaling molecules in the JAK/STAT pathway or altering the ratio of the diffusion lengths of interferon and virus in the cell culture could lead to plaque growth arrest. The dependence of plaque growth arrest on diffusion lengths highlights the importance of developing validated spatial models of cytokine signaling and the need for in vitro measurement of these diffusion coefficients. Sensitivity analyses under conditions leading to continuous or arrested plaque growth found that plaque growth is more sensitive to variations of most parameters and more likely to have identifiable model parameters when conditions lead to plaque arrest. This result suggests that cytokine assay measurements may be most informative under conditions leading to arrested plaque growth. The model is easy to extend to include SARS-CoV-2-specific mechanisms or to use as a component in models linking epithelial cell signaling to systemic immune models.     

Sarcomere length changes in a 3D mathematical model of the pig ventricles

Measurements of the geometry and fibrous-sheet structure of the left and right ventricles of the pig heart are fitted with a finite element model. Mechanical changes during the heart cycle are computed by solving the equations of motion under specified ventricular boundary conditions and using experimentally defined constitutive laws for the active and passive material properties of myocardial tissue. The resulting patterns of deformation, such as axial torsion and changes in wall thickness and base-apex length, are consistent with experimental observations. The model can therefore be used to predict sarcomere length changes and other strain patterns throughout the myocardium and throughout the cardiac cycle. Here we present sarcomere length changes at a limited number of material points within the wall. Sarcomere length typically varies by 10% above and below the unloaded length; although under the boundary conditions imposed in the current model the midwall circumferentially oriented sarcomere lengths increased by up to 20% at end diastole. We provide web-access details for a downloadable software program designed to provide more extensive information on mechanical deformation, such as the principal strains and muscle fibre cross-sectional area changes during the cardiac cycle.     

肝癌组织中Cyclin D1及其相关基因研究的进展

细胞周期调控异常会导致肿瘤的发生和发展,Cyclin D1是细胞周期的重要调控元件之一,与肝癌发生、进展及预后关系密切。本文就Cyclin D1的结构、功能、作用机制、肝癌组织中Cyclin D1及其相关基因研究的进展情况予以综述。