Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids.

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13-CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

Identification of a gene set that maintains tumorigenicity of the hepatocellular carcinoma cell line Li-7.

The identification and development of therapeutic targets in cancer stem cells that lead to tumor development, recurrence, metastasis, and drug resistance is an important goal in cancer research. The hepatocellular carcinoma cell line Li-7 contains functionally different types of cells. Cells with tumor-forming activity are enriched in cancer stem cell-like CD13+CD166- cells and this cell population gradually decreases during culture in conventional culture medium (RPMI1640 containing 10% fetal bovine serum). When Li-7 cells are cultured in mTeSR1, a medium developed for human pluripotent stem cells, CD13+CD166- cells, and their tumorigenicity is maintained. Here, we sought to identify the mechanisms of tumorigenicity in this sub-population. We compared gene expression profiles of CD13+CD166- cells with other cell sub-populations and identified nine overexpressed genes (ENPP2, SCGN, FGFR4, MCOLN3, KCNJ16, SMIM22, SMIM24, SERPINH1, and TMPRSS2) in CD13+CD166- cells. After transfer from mTeSR1 to RPMI1640 containing 10% fetal bovine serum, the expression of these nine genes decreased in Li-7 cells and they lost tumorigenicity. In contrast, when these genes of Li-7 cells were forcibly expressed in cultures using RPMI1640 containing 10% fetal bovine serum, Li-7 cells maintained tumorigenicity. A metabolome analysis using capillary electrophoresis-mass spectrometry showed that two metabolic pathways, "Alanine, aspartate and glutamate metabolism" and "Arginine biosynthesis" were activated in cancer stem-cell-like cells. Our analyses here showed potential therapeutic target genes and metabolites for treatment of cancer stem cells in hepatocellular carcinoma.

Chemically defined cytokine-free expansion of human haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases1,2. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation3. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo4. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.

Human induced pluripotent stem cell formation and morphology prediction during reprogramming with time-lapse bright-field microscopy images using deep learning methods.

BACKGROUND AND OBJECTIVE: Human induced pluripotent stem cells (hiPSCs) represent an ideal source for patient specific cell-based regenerative medicine; however, efficiency of hiPSC formation from reprogramming cells is low. We use several deep-learning results from time-lapse brightfield microscopy images during culture, to early detect the cells potentially reprogramming into hiPSCs and predict the colony morphology of these cells for improving efficiency of culturing a new hiPSC line. METHODS: Sets of time-lapse bright-field images are taken to track reprogramming process of CD34+ cells biologically identified as just beginning reprogramming. Prior the experiment, 9 classes of templates with distinct cell features clipped from microscopy images at various reprogramming stages are used to train a CNN model. The CNN is then used to classify a microscopy image as probability images of these classes. Probability images of some class are used to train a densely connected convolutional network for extracting regions of this class on a microscopy image. A U-net is trained to segment cells on the time-lapse images in early reprogramming stage during culture. The segmented cells are classified by the extracted regions to count various types of cells appearing in the early reprogramming stage for predicting the identified cells potentially forming hiPSCs. The probability images of hiPSC classes are also used to train a spatiotemporal RNN for predicting the future hiPSC colony morphology of the potential cells. RESULTS: Experimental results show the prediction (before 7 days after of beginning of the reprogramming) achieved 0.8 accuracy, and 66% of the identified cells under different culture conditions, predicted as forming, finally formed hiPSCs. The predicted hiPSC images and extracted colonies on the images show the prediction for future 1.5 days achieved high accuracy of hiPSC colony areas and image similarity. CONCLUSIONS: Our study proposes a method using several deep learning models to efficiently select the reprogramming cells possibly forming hiPSCs and to predict the shapes of growing hiPSC colonies. The proposed method is expected to improve the efficiency when establishing a new hiPSC line culture.

Prediction of Human Induced Pluripotent Stem Cell Formation Based on Deep Learning Analyses Using Time-lapse Brightfield Microscopy Images.

We use deep learning methods to predict human induced pluripotent stem cell (hiPSC) formation using time-lapse brightfield microscopy images taken from a cell identified as the beginning of entered into the reprogramming process. A U-net is used to segment cells and a CNN is used to classify the segmented cells into eight types of cells during the reprogramming and hiPSC formation based on cellular morphology on the microscopy images. The numbers of respective types of cells in cell clusters before the hiPSC formation stage are used to predict if hiPSC regions can be well formed lately. Experimental results show good prediction by the criteria using the numbers of different cells in the clusters. Time-series images with respective types of classified cells can be used to visualize and quantitatively analyze the growth and transition among dispersed cells not in cell clusters, various types of cells in the clusters before the hiPSC formation stage and hiPSC cells.

Polyvinyl alcohol hydrolysis rate and molecular weight influence human and murine HSC activity ex vivo.

Ex vivo expansion of hematopoietic stem cells (HSCs) is one of the most promising strategies to increase the availability of transplantable HSCs and improve bone marrow transplantation outcomes. We recently demonstrated that mouse HSCs could be efficiently expanded in polyvinyl alcohol (PVA)-containing culture medium using only recombinant stem cell factor and thrombopoietin cytokines. However, the behavior of human HSCs in these simple PVA-based media was not fully elucidated. In this study, we analyzed the compatibility of PVA of different hydrolysis rates (HR) and molecular weights (MW) to support functional human and mouse HSCs ex vivo. Human and mouse HSCs proliferated more frequently in media containing PVA with lower HR than with higher HR, but both PVA types supported HSC multilineage reconstitution potential. Importantly, human HSCs cultured in PVA-containing media engrafted not only in irradiated recipients but also in non-irradiated recipients. Our results demonstrate that human HSCs can be maintained ex vivo using PVA-based culture systems and suggest approaches for future optimization of human HSC expansion.

Prediction for Morphology and States of Stem Cell Colonies using a LSTM Network with Progressive Training Microscopy Images.

We present a new LSTM (P-LSTM: Progressive LSTM) network, aiming to predict morphology and states of cell colonies from time-lapse microscopy images. Apparent short-term changes occur in some types of time-lapse cell images. Therefore, long-term-memory dependent LSTM networks may not predict accurately. The P-LSTM network incorporates the images newly generated from cell imaging progressively into LSTM training to emphasize the LSTM short-term memory and thus improve the prediction accuracy. The new images are input into a buffer to be selected for batch training. For real-time processing, parallel computation is introduced to implement concurrent training and prediction on partitioned images.Two types of stem cell images were used to show effectiveness of the P-LSTM network. One is for tracking of ES cell colonies. The actual and predicted ES cell images possess similar colony areas and the same transitions of colony states (moving, merging or morphology changing), although the predicted colony mergers may delay in several time-steps. The other is for prediction of iPS cell reprogramming from the CD34+ human cord blood cells. The actual and predicted iPS cell images possess high similarity evaluated by the PSNR and SSIM similarity evaluation metrics, indicating the reprogramming iPS cell colony features and morphology can be accurately predicted.

Author Correction: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

A robust culture method for maintaining tumorigenic cancer stem cells in the hepatocellular carcinoma cell line Li-7.

Cancer tissues contain small populations of highly tumorigenic cells termed cancer stem cells (CSCs). Immortalized cell lines containing CSCs are valuable and powerful experimental tools for research into the characteristics of these stem cells. We previously reported that the hepatocellular carcinoma cell line Li-7 includes abundant CD13+ CD166- CSCs; however, the number of these cells decreases after long-term culture as a result of differentiation to non-CSC populations. To ensure consistent and reproducible results in experiments using Li-7 cells, it is important that the CSC population is maintained stably regardless of culture duration and passage. In the present study, we found that a commercially available culture medium for maintenance of embryonic stem cells and induced pluripotent stem cells, mTeSR1, effectively prevented spontaneous differentiation by CD13+ CD166- cells to CD13- CD166+ cells and therefore maintained the CSC population in Li-7 cell cultures. CD13+ CD166- CSCs maintained using this culture medium retained high tumorigenicity after transplantation into mice; they also showed the ability to differentiate in vitro into non-CSC populations in RPMI-1640 with 10% FBS medium. We analyzed gene expression profiles of CSC and non-CSC populations in Li-7 cultures using an RNA sequencing method. Genes such as FGFR, NOTCH1, and JAG1, that are associated with tumorigenicity and stemness, were upregulated in the CSC population. Our results suggest that CSCs can be maintained in immortalized cancer cell lines cultured over an extended period using a medium developed for culture of embryonic/induced pluripotent stem cells.

Human Induced Pluripotent Stem Cell Reprogramming Prediction in Microscopy Images using LSTM based RNN.

We present a LSTM (Long Short-Term Memory) based RNN (recurrent neural network) method for predicting human induced Pluripotent Stem (hiPS) cells in the reprogramming process. The method uses a trained LSTM network by time-lapse microscopy images to predict growth and transition of reprogramming processes of CD34+ human cord blood cells into hiPS cells. The prediction can be visualized by output time-series probability images. The growth and transition are thus analyzed quantitatively by region areas of distinct cells emerged during the iPS formation processes. The experimental results show that our LSTM network is a potentially powerful tool to predict the cells at the distinct phases of the reprogramming to hiPS cells. This method should be extremely useful not only for basic biology of iPS cells but also detection of the reprogramming cells that will become genuine hiPS cells even at early stages of hiPS formation. Such predictive power should greatly reduce cost, labor and time required for establishment of the genuine hiPS cells, thereby accelerating the practical use of hiPS cells in regenerative medicine.

Human induced pluripotent stem cell region recognition in microscopy images using Convolutional Neural Networks.

We present a deep learning architecture Convolutional Neural Networks (CNNs) for automatic classification and recognition of reprogramming and reprogrammed human Induced Pluripotent Stem (iPS) cell regions in microscopy images. The differentiated cells that possibly undergo reprogramming to iPS cells can be detected by this method for screening reagents or culture conditions in iPS induction. The learning results demonstrate that our CNNs can achieve the Top-1 and Top-2 error rates of 9.2% and 0.84%, respectively, to produce probability maps for the automatic analysis. The implementation results show that this automatic method can successfully detect and localize the human iPS cell formation, thereby yield a potential tool for helping iPS cell culture.

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

Establishment of immortalized human erythroid progenitor cell lines able to produce enucleated red blood cells.

Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs.

Gene expression profiles of cryopreserved CD34(+) human umbilical cord blood cells are related to their bone marrow reconstitution abilities in mouse xenografts.

Human umbilical cord blood (UCB) cells are an alternative source of hematopoietic stem cells for treatment of leukemia and other diseases. It is very difficult to assess the quality of UCB cells in the clinical situation. Here, we sought to assess the quality of UCB cells by transplantation to immunodeficient mice. Cryopreserved CD34(+) UCB cells from twelve different human donors were transplanted into sublethally irradiated NOD/shi-scid Jic mice. In parallel, the gene expression profiles of the UCB cells were determined from oligonucleotide microarrays. UCB cells from three donors failed to establish an engraftment in the host mice, while the other nine succeeded to various extents. Gene expression profiling indicated that 71 genes, including HOXB4, C/EBP-beta, and ETS2, were specifically overexpressed and 23 genes were suppressed more than 2-fold in the successful UCB cells compared to those that failed. Functional annotation revealed that cell growth and cell cycle regulators were more abundant in the successful UCB cells. Our results suggest that hematopoietic ability may vary among cryopreserved UCB cells and that this ability can be distinguished by profiling expression of certain sets of genes.

Human hematopoietic stem cells can survive in vitro for several months.

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells.

BACKGROUND: The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. METHODOLOGY/PRINCIPAL FINDINGS: We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells.

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.

Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells.

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.

Mesenchymal progenitors able to differentiate into osteogenic, chondrogenic, and/or adipogenic cells in vitro are present in most primary fibroblast-like cell populations.

MSCs and mesenchymal progenitor cells (MPCs) are studied for their potential in regenerative medicine. MSCs in particular have great potential, because various reports have shown that they can differentiate into many different cell types. However, the difference between mesenchymal stem/progenitor cells and so-called fibroblasts is unclear. In this study, we found that most of the distinct populations of primary fibroblast-like cells derived from various human tissues, including lung, skin, umbilical cord, and amniotic membrane, contained cells that were able to differentiate into at least one mesenchymal lineage, including osteoblasts, chondrocytes, and adipocytes. We therefore propose that primary fibroblast-like cell populations obtained from various human tissues do not comprise solely fibroblasts, but rather that they also include at least MPCs and possibly MSCs, to some extent. Disclosure of potential conflicts of interest is found at the end of this article.