Automated algorithm development to assess survival of human neurons using longitudinal single-cell tracking: Application to synucleinopathy.

The development of phenotypic assays with appropriate analyses is an important step in the drug discovery process. Assays using induced pluripotent stem cell (iPSC)-derived human neurons are emerging as powerful tools for drug discovery in neurological disease. We have previously shown that longitudinal single cell tracking enabled the quantification of survival and death of neurons after overexpression of α-synuclein with a familial Parkinson's disease mutation (A53T). The reliance of this method on manual counting, however, rendered the process labor intensive, time consuming and error prone. To overcome these hurdles, we have developed automated detection algorithms for neurons using the BioStation CT live imaging system and CL-Quant software. In the current study, we use these algorithms to successfully measure the risk of neuronal death caused by overexpression of α-synuclein (A53T) with similar accuracy and improved consistency as compared to manual counting. This novel method also provides additional key readouts of neuronal fitness including total neurite length and the number of neurite nodes projecting from the cell body. Finally, the algorithm reveals the neuroprotective effects of brain-derived neurotrophic factor (BDNF) treatment in neurons overexpressing α-synuclein (A53T). These data show that an automated algorithm improves the consistency and considerably shortens the analysis time of assessing neuronal health, making this method advantageous for small molecule screening for inhibitors of synucleinopathy and other neurodegenerative diseases.

A morphology-based assay platform for neuroepithelial-like cells differentiated from human pluripotent stem cells.

Cell morphology is recognized as an important hallmark of neural cells. During the differentiation of human pluripotent stem cells (hPSCs) into neural cells, cell morphology changes dynamically. Therefore, characterization of the morphology of cells during this period is important to improve our understanding of the differentiation and development of neural cells. General methods for the directed induction of hPSCs include the steps of multi-cellular aggregation or high-density cell culture, particularly at the early phase of neural differentiation, and therefore, the morphology of each differentiating cell is difficult to recognize. Here, we have developed a new method for the directed differentiation of neuroepithelial-like cells (NELCs) from hPSCs at a low cell density in an adherent monolayer culture, as well as an image-processing algorithm to evaluate the cell morphology of differentiating NELCs, in order to follow cell morphology during the differentiation of hPSCs into NELCs. Using these methods, the morphological transition of differentiating cells was observed in real time using phase contrast imaging and then quantified. Because cell morphology is also considered an inherent biological marker of neural cells cultured in vitro, this method is potentially useful to study the mechanisms underlying neural cell differentiation.

Fibroblast-led cancer cell invasion is activated by epithelial-mesenchymal transition through platelet-derived growth factor BB secretion of lung adenocarcinoma.

Cancer-associated fibroblast (CAF)-dependent local invasion is the process by which cancer cells invade the extracellular matrix using tracks that have been physically remodeled by CAFs. In the present study, we investigated the process by which the epithelial-mesenchymal transition (EMT) of cancer cells affect CAF-dependent local invasion. Using an in vitro collagen invasion assay, we showed cancer cells undergoing EMT to promote the matrix-remodeling ability of CAFs and thereby enhance CAF-dependent local cancer cell invasion. Platelet-derived growth factor (PDGF)-BB secretion was significantly elevated in cancer cells undergoing EMT, and this induced an increase in the invasion ability of both CAFs and cancer cells. Conversely, knockdown of PDGF-B expression in cancer cells undergoing EMT, or treatment with a PDGF-receptor inhibitor, decreased the invasion ability of both CAFs and cancer cells. By analyzing the gene expression profiles of 442 patients with lung adenocarcinomas, we established that high expression of PDGF-B and presentation of mesenchymal-like tumors were significantly associated with a high rate of disease recurrence and poor patient prognosis. Thus, cancer cells undergoing EMT may accelerate their own ability to invade local tissues via PDGF-BB secretion to promote CAF matrix remodeling. Therefore, targeting PDGF signaling between cancer cells undergoing EMT and CAFs is a promising therapeutic target to inhibit cancer progression and improve patient prognosis.

Visualization of morphological categories of colonies for monitoring of effect on induced pluripotent stem cell culture status.

From the recent advances, there are growing expectations toward the mass production of induced pluripotent stem cells (iPSCs) for varieties of applications. For such type of industrial cell manufacturing, the technology which can stabilize the production efficiency is strongly required. Since the present iPSC culture is covered by delicate manual operations, there are still quality differences in produced cells from same culture protocols. To monitor the culture process of iPSCs with the quantified data to evaluate the culture status, we here introduce image-based visualization method of morphological diversity of iPSC colonies. We have set three types of experiments to evaluate the influential factors in iPSC culture technique that may disturb the undifferentiation status of iPSC colonies: (Exp. 1) technical differences in passage skills, (Exp. 2) technical differences in feeder cell preparation, and (Exp. 3) technical differences in maintenance skills (medium exchange frequency with the combination of manual removal of morphologically irregular colonies). By measuring the all existing colonies from real-time microscopic images, the heterogenous change of colony morphologies in the culture vessel was visualized. By such visualization with morphologically categorized Manhattan chart, the difference between technical skills could be compared for evaluating appropriate cell processing.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control.

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of 'hPSC colony morphology', permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

Cancer cell invasion driven by extracellular matrix remodeling is dependent on the properties of cancer-associated fibroblasts.

PURPOSE: As one form of tumor invasion, cancer cells can invade the extracellular matrix (ECM) through tracks that have been physically remodeled by cancer-associated fibroblasts (CAFs). However, CAFs are a heterogeneous population with diverse matrix-remodeling capacities. The purpose of this study was to investigate how CAFs with various matrix-remodeling capacities influence cancer cell invasion. METHODS: We established single-cell-derived clones from three primary cultures of CAFs from lung adenocarcinoma patients (Case 1, 5 clones; Case 2, 5 clones; and Case 3, 7 clones). Using a co-culture model, we evaluated the correlations between the number of invaded cancer cells and the remodeling areas generated by CAF clones in each case. RESULTS: When A549 lung adenocarcinoma cells and CAF clones were co-cultured, both the numbers of invaded cancer cells and the remodeling areas generated by the CAF clones varied greatly. The number of invaded cancer cells was moderately and strongly correlated with the remodeling areas generated by each CAF clone originating from Cases 1 and 2 (R(2) value = 0.53 and 0.68, respectively), suggesting that the remodeling areas in the ECM may determine the number of invaded cancer cells. In contrast, the number of invaded cancer cells was not correlated with the remodeling areas generated by CAF clones originating from Case 3, suggesting that factors other than the remodeling areas might determine the number of invading cancer cells. CONCLUSIONS: These findings showed two types of fibroblast-dependent cancer cell invasion that are dependent on and independent of the remodeling areas generated by CAFs.

Development of a Monitoring Method for Nonlabeled Human Pluripotent Stem Cell Growth by Time-Lapse Image Analysis.

UNLABELLED: : Cell growth is an important criterion for determining healthy cell conditions. When somatic cells or cancer cells are dissociated into single cells for passaging, the cell numbers can be counted at each passage, providing information on cell growth as an indicator of the health conditions of these cells. In the case of human pluripotent stem cells (hPSCs), because the cells are usually dissociated into cell clumps of ∼50-100 cells for passaging, cell counting is time-consuming. In the present study, using a time-lapse imaging system, we developed a method to determine the growth of hPSCs from nonlabeled live cell phase-contrast images without damaging these cells. Next, the hPSC colony areas and number of nuclei were determined and used to derive equations to calculate the cell number in hPSC colonies, which were assessed on time-lapse images acquired using a culture observation system. The relationships between the colony areas and nuclei numbers were linear, although the equation coefficients were dependent on the cell line used, colony size, colony morphology, and culture conditions. When the culture conditions became improper, the change in cell growth conditions could be detected by analysis of the phase-contrast images. This method provided real-time information on colony growth and cell growth rates without using treatments that can damage cells and could be useful for basic research on hPSCs and cell processing for hPSC-based therapy. SIGNIFICANCE: This is the first study to use a noninvasive method using images to systemically determine the growth of human pluripotent stem cells (hPSCs) without damaging or wasting cells. This method would be useful for quality control during cell culture of clinical hPSCs.

PKCδ and ε regulate the morphological integrity of the ER-Golgi intermediate compartment (ERGIC) but not the anterograde and retrograde transports via the Golgi apparatus.

The ER-Golgi intermediate compartment (ERGIC) is an organelle through which cargo proteins pass and are being transferred by either anterograde or retrograde transport between the endoplasmic reticulum (ER) and the Golgi apparatus. We examined the effect of 80 different kinase inhibitors on ERGIC morphology and found that rottlerin, a PKCδ inhibitor, induced the dispersion of the perinuclear ERGIC into punctate structures. Rottlerin also delayed anterograde transport of vesicular stomatitis virus G protein (VSVG) from the ER to the Golgi and retrograde transport of cholera toxin from cell surface to the ER via the Golgi. RNA interference revealed that knockdown of PKCδ or ε resulted in the dispersion of the ERGIC, but unexpectedly did not inhibit VSVG and cholera toxin transport. We also found that rottlerin depolarized the mitochondrial membrane potential, as does carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), an uncoupler, and demonstrated that a decrease in the intracellular adenosine triphosphate (ATP) levels by rottlerin might underlie the block in transports. These results suggest that PKCδ and ε specifically regulate the morphology of the ERGIC and that the maintenance of ERGIC structure is not necessarily required for anterograde and retrograde transports.

DNA microstructure based on self-assembly of 4-sticky-end Holliday junctions in aqueous solution.

Liverwort-like DNA microscale structures consist of 4-sticky-end Holiday junctions as DNA bricks that can be used in nanotechnology and nanobiotechnology to direct the self-assembly of nanomachines as well as DNA assembly. Previously it has not been possible to obtain such DNA microscale structural forms, but herein we report construction of a mesh-like material made up of 4 strands of 40-base DNA. Advanced bioimaging techniques such as fluorescence correlation spectroscopy (FCS), laser scanning microscopy (LSM), and atomic force microscopy (AFM) help us as ultrasensitive detection tools for examing structures in solutions. Combinations of these techniques allow us to survey various chemical conditions of materials and solutions.

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy.

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.

Quantification of size distribution of restriction fragments in mitochondrial genome using fluorescence correlation spectroscopy.

A crucial investigation is to quantify restriction fragment length polymorphisms without gel electrophoresis, as the distribution of fragment size is mainly evaluated on the gel, which cannot be easily quantified. We developed a method to determine the fragmentation of the mitochondrial genome caused by restriction enzymes using fluorescence correlation spectroscopy (FCS). Distribution of fragment size was evaluated by the decrease in amplitude of the fluorescence correlation function while the mitochondrial genome PCR product was digested with Hga I or Hae III. Using a multicomponent model, which was considered as a fragment length-weighted correlation function, we calculated the correlation amplitude theoretically expected and compared it to that measured by FCS. These amplitudes for Hga I were coincident, whereas the measured amplitude for Hae III was more than the theoretical one. Because of tetra-nucleotide recognition by Hae III, there were many more fragments than with Hga I. Therefore, the amplitude measured by FCS would be a very useful index for primary screening for alterations in the entire mitochondrial genome with restriction enzymes that have several polymorphic restriction sites in the genome.

Specifically associated PCR products probed by coincident detection of two-color cross-correlated fluorescence intensities in human gene polymorphisms of methylene tetrahydrofolate reductase at site C677T: a novel measurement approach without follow-up mathematical analysis.

Whole blood samples of known methylene tetrahydrofolate reductase (MTHFR) genotypes from 24 individuals were examined at site C677T. Their amplified DNA products were assessed by two-color fluorescence cross-correlation measurements and agarose gel electrophoresis/capillary gel electrophoresis. DNA subpopulations were identified which were not associated with the proper genotype by primer combinations and cycling conditions called multiplexes. We confirmed that DNA analysis by two-color fluorescence cross-correlation measurements allowed the detection of fluorescence signals specifically associated with the proper genotypes in a mixture of amplified nontarget DNA molecules without DNA sizing. The measurement approach does not require complex, follow-up mathematical analysis and is applicable to any single nucleotide polymorphisms. The simple immunogenetic model showed how the approach works to reveal specific DNA target by preventing detection of nontarget DNA. Under those experimental conditions, a new ultrasensitive, and specific method for clinical immunologists is born.

DNA measurements by using fluorescence correlation spectroscopy and two-color fluorescence cross correlation spectroscopy.

FCS and FCCS measurements provide two important analytical parameters, the average number of molecules in the detection area and the translational diffusion constant of the molecules at the single molecule level. Considering these properties, FCS and FCCS have been applied to analysis of the cellular environment and dynamic processes of molecules in the living cell. More recently, a systematic approach for the analysis of macromolecule complex formation has focused on the new field of single molecule detection in the post-genome era. In this work, we tested the sensitivities of FCS and FCCS based on the distance between fluorophores in DNA as a model macromolecule complex. The results show that FCCS is not limited by the size of the macromolecular complex even in a very small detection area.

C677T single nucleotide polymorphisms of the human methylene tetrahydrofolate reductase and specific identification : a novel strategy using two-color cross-correlation fluorescence spectroscopy.

BACKGROUND: A methylene tetrahydrofolate reductase (MTHFR) deficiency at site C677T renders the enzyme thermolabile and consequently represents a risk factor for vascular disease, neural tube defects, preeclampsia, and thrombosis. Highly specific identification techniques for genotyping are mandatory to give guidance for the diagnosis and monitoring of this deficiency. METHODS: A new approach for performing genotyping has been introduced with the identification of single nucleotide polymorphisms of the human MTHFR. It is based on PCR followed by two-color cross-correlation fluorescence spectroscopy (FCS). Experiments were carried out with green- and red-tagged allele-specific primers, which were fully compatible with the two-color fluorescence cross-correlation setup at 488 nm and 633 nm excitation wavelengths. RESULTS: The measured data of the amplification mixes (tubes) were normalized as the maximum correlation amplitude of each tube. Correlated and uncorrelated data were optically separated in the amplification mixes by their characteristic correlation times, which significantly differed from each other. The correlated data were generated in the presence of the proper mutated genotype template, whereas uncorrelated data were due to the absence of the proper genotype template. Furthermore, the specific association of the two-color fluorescence correlated signals with the target DNA was experimentally proven. Using this novel two-color cross-correlation approach, the MTHFR genotypes, which were determined in 21 clinical samples, showed concordance with methods involving a PCR-based assay with hexachloro-6-carboxy-fluorescein (HEX)- and 6-carboxy-fluorescein (FAM)-tagged allele-specific primers and a subsequent separation step with capillary electrophoresis, yet are simpler to perform. There was no evidence of a central trend of false-positive or false-negative results. We demonstrated how the novel, ultrasensitive typing system could be applied to studies where researchers are trying to perfect their assays and are often working with the unknown, or application to problematic assays in a clinical environment for those involved in molecular diagnosis. CONCLUSIONS: We present an alternative method to those commonly used in genotyping. Two-color cross-correlation FCS allows the detection of the fluorescence signals specifically associated with the heterozygous mutated, the homozygous mutated, and normal individuals, as exemplified in this study. The presence of nonspecific amplification products, which interfere with subsequent DNA analysis, could therefore highlight the need for two-color cross-correlation FCS as a means of discriminating between specific association of the fluorescence signals with the target DNA and DNA not related to the target.