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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 tumorforming 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 are 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.

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 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.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.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.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.

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.

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. An albumin-free culture system for the long-term ex vivo expansion of mouse haematopoietic stem cells produces 236- to 899-fold expansion, and generates cultures that robustly engraft in recipient mice without toxic pre-conditioning.

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.

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.

CNN classifiers using microscopy images to pick up induced pluripotent stem (iPS) cells from a lot of non-iPS cells ware proposed to improves efficiency of human iPS cell production. By selecting iPS-forming cells at early culture stage, it should be more promising to reduce a considerable amount of work. This paper proposes a CNN reprogramming stage classifier trained by respective types (grown into high-, medium, and low-growth status of iPS-like cells) of time-lapse images taken from the completed cell culturing process. These trained CNNs are used to classify a time-lapse image taken in cell culturing as one stage (a number of consecutive frames) of a respective set of trained time-lapse images. The differences between the actual stage with respective classified stages are than calculated to decide which stage of the completed cell this time-step image taken in cell culturing is near to, then to predict the iPS cell growth status that the culturing cells finally grow into. The method achieved the 85% accuracy to predict the growth status of culturing cells two days later, thus can be applicable for early selection suggestion of good iPS-forming cells.

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.

Summary 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., Graphical Abstract, Highlights • Different BSA lots alter how HSCs respond to cytokines • RSA can replace BSA to provide HSC maintenance culture with minimal variability • By comparing the protein profiles of “good” and “bad” BSAs, HPX was identified • HPX reduces HSC intracellular reactive ROS and is expressed by BM Schwann cells, In this article, Yamazaki, Nakauchi, and colleagues demonstrate that BSA batches have unique protein profiles and vary widely in their ability to maintain mouse HSCs ex vivo. By replacing BSA with recombinant serum albumin, they developed a standardized HSC culture platform that can be used to identify novel maintenance and expansion factors.

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.

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.

The self-renewal of multipotent hematopoietic stem cells (HSCs) is key for life-long maintenance of the blood system and the curative capacity of clinical HSC transplantation. However, while in vivo HSC self-renewal capacity has been well-described, existing culture conditions poorly support ex vivo HSC self-renewal and afford only a very limited window to study or modify HSCs in-a-dish. By taking a reductionist optimization approach, we have developed a simple culture platform that supports functional mouse HSCs ex vivo over 1-2 months. Importantly, we identified the synthetic polymer polyvinyl alcohol as a superior, inexpensive, and chemically-defined alternative to serum albumin supplements, which have long represented a major source of biological contaminants and batch-to-batch variability in HSC cultures. Limiting dilution transplantation analysis of day-28 HSC cultures estimated a ∼900-fold expansion of functional HSCs (based on >1% multilineage engraftment at 16-weeks post-transplantation) with secondary transplantation analysis estimating >200-fold expansion of serially-engraftable long-term HSCs. HSCs could also be expanded clonally using this system, demonstrating bona fide ex vivo HSC self-renewal. The large numbers of functional HSCs generated by this long-term ex vivo expansion protocol even enabled HSC transplantation in nonconditioned immunocompetent and immunodeficient recipients. Finally, this albumin-free and GMP-compatible culture system also supported human HSCs ex vivo, as determined by long-term engraftment in NSG mice. Thus, our novel HSC culture platform provides an important new tool to interrogate HSC self-renewal and lineage commitment, and also suggests a novel approach in clinical HSC transplantation.

We have developed an ultrabroadband multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopic system using a supercontinuum (SC) seeded by sub-100-ps (85 ps) laser pulses with a sub-MHz (0.82 MHz) repetition rate. Because of the high peak power and ultrabroadband spectral profile of the SC, we can efficiently generate multiplex CARS signals in the spectral range of 600–3600 cm−1, which covers the entire molecular fingerprint region, as well as the C—H and O—H stretching regions. Due to the high peak power of the new laser source, the exposure time (pixel dwell time) for CARS imaging of polymer beads was reduced to less than 1 ms (0.8 ms), which was limited by the readout time of a CCD camera. Owing to the improvement in CARS spectral quality, clear molecular fingerprinting was achieved for living HeLa cells at different phases in the cell cycle.

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.

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.

Blood transfusion is indispensable for many clinical applications. However, the supply of transfusable material is insufficient in many countries. Human cord blood contains many hematopoietic stem and progenitor cells, providing a promising resource for the production of transfusable material in vitro. In this study, we have refined a protocol to produce abundant red blood cells (RBC) from human cord blood in an in vitro culture system. We found that erythropoietin and interleukin-3 were most effective when they were added to the culture medium sequentially rather than simultaneously. Although insulin-like growth factor-I (IGF-1) has been reported to function as a positive regulator of RBC production in some in vitro culture systems, we found that IGF-1 had a negative effect upon RBC production. However, IGF-II appeared to function as a positive regulator of RBC production. Finally, stem cell factor functioned to both expand and accelerate the differentiation of immature erythroid cells.

Little is known about the manner in which hematopoietic stem cells (HSCs) self-renew. To address this issue, we used a serum-free single-cell culture, followed by transplantation of cultured cells into lethally irradiated mice. CD34-negative or low, c-Kit-positive, Sca-1-positive, lineage marker-negative (CD34-KSL) cells are highly enriched for murine bone marrow HSCs. Successful long-term reconstitution with a single CD34-KSL cell enabled us to study in vitro self-renewal of HSC at clonal level. Using this clonal cell transplantation system, we examined the effect of various cytokines on CD34-KSL cells. Among the cytokines examined, stem cell factor (SCF) and thrombopoietin (TPO) were minimum cytokines to induce cell division of CD34-KSL cells most efficiently. Similarly, multilineage repopulating activity was detected in the cells derived from a significant portion of single cells after culture in the presence of TPO and SCF. However, SCF + IL-3, SCF + IL-6, or SCF + IL-11 + FL appeared to be less effective for self-renewal of HSCs. The activity of HSCs as indicated by repopulation unit (RU) remaining after culture with SCF and TPO was not so different from that of freshly isolated HSCs. However, there was a substantial loss of HSC number in these cultured cells. Taken together, this study provides definitive proof that one HSC can generate at least one HSC in vitro.

How hematopoietic stem cells (HSCs) commit to a particular lineage is unclear. A high degree of HSC purification enabled us to address this issue at the clonal level. Single-cell transplantation studies revealed that 40% of the CD34−/low, c-Kit+, Sca-1+, and lineage marker− (CD34−KSL) cells in adult mouse bone marrow were able, as individual cells, to reconstitute myeloid and B- and T-lymphoid lineages over the long-term. Single-cell culture showed that >40% of CD34−KSL cells could form neutrophil (n)/macrophage (m)/erythroblast (E)/megakaryocyte (M) (nmEM) colonies. Assuming that a substantial portion of long-term repopulating cells can be detected as nmEM cells within this population, we compared differentiation potentials between individual pairs of daughter and granddaughter cells derived in vitro from single nmEM cells. One of the two daughter or granddaughter cells remained an nmEM cell. The other showed a variety of combinations of differentiation potential. In particular, an nmEM cell directly gave rise, after one cell division, to progenitor cells committed to nm, EM, or M lineages. The probability of asymmetric division of nmEM cells depended on the cytokines used. These data strongly suggest that lineage commitment takes place asymmetrically at the level of HSCs under the influence of external factors.

Objective Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector–mediated gene transfer into murine CD34 −/low c-Kit + Sca-1 + Lin − (CD34 − KSL) cells that are highly enriched for HSCs. Materials and Methods FACS-sorted CD34 − KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1–based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP + KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS. Results GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26–58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP + cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP + KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB. Conclusions Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.

Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34−/low c-Kit+Sca-1+ lineage marker–negative (CD34−KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs.In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34−KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.

Objective To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. Materials and Methods Human bone fragments were implanted into non-obese diabetes / severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. Results After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. Conclusion Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.

Members of the lipocalin protein family are typically small, secreted proteins that possess a variety of functions. Although the physiological role of lipocalin 2 remains to be fully elucidated, a few pivotal functions have recently been reported, e.g., regulation of the apoptosis of leukocytes. Unexpectedly, lipocalin 2 is abundantly expressed in erythroid progenitor cells. An in vitro culture experiment demonstrated that lipocalin 2 induces apoptosis and inhibits differentiation of erythroid progenitor cells. During acute anemia the expression of lipocalin 2 was reduced in erythroid cells by a feedback system. Furthermore, injection of recombinant lipocalin 2 into mice suffering from acute anemia retarded the recovery of red blood cell (RBC) numbers, suggesting the importance of reduced expression of lipocalin 2 for the efficient recovery of RBC numbers. These results indicate that lipocalin 2 suppresses RBC production in an autocrine fashion. Hence, anemia arising from pathological conditions, such as chronic inflammation, might be partly due to increased levels of lipocalin 2 secreted from expanded leukocytes and/or macrophages. Also, anemia arising from malignancies might be partly due to the abundant secretion of lipocalin 2 from tumor cells. Thus, lipocalin 2 may represent an attractive therapeutic target for anemia under certain pathological conditions.

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.

Electron doping into Cd3TeO6 with a 1:1 ordered perovskite-type structure was carried out by two methods: the introduction of oxygen vacancies, and the atomic substitution of In for Cd. Both methods turned polycrystalline samples into metallic conductors while single crystals with metallic conductivity were obtained by the second method. The resistivity and thermoelectric power of the single-crystal indium-substituted sample were 0.6 mΩcm and -50 µVK-1, respectively, giving a power factor of 4×10-4 WK-2m-1, which is the highest among nontransition metal oxides.

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.

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.

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.

Objective Induction of hematopoietic cells from human embryonic stem (ES) cells has been reported recently. However, before cells derived from human ES cells can be used in the clinic, preclinical studies using these cells in experimental primates will be necessary. Therefore, we attempted to establish a method to induce hematopoietic cells robustly and abundantly from primate ES cells. Methods A primate ES cell line, CMK-6, derived from the cynomolgus monkey was used in this study. We adapted a method to induce hematopoiesis from CMK-6 cells on feeder cells, and tested the effectiveness of three kinds of feeder cell lines (OP9, C2C12, and C3H10T1/2). In addition, we tested the effect of vascular endothelial growth factor (VEGF) and insulin-like growth factor-II (IGF-II) on hematopoiesis induction from CMK-6 cells. Results VEGF and IGF-II showed an extremely strong synergistic effect to induce hematopoiesis from CMK-6 cells. C3H10T1/2 cells proved to be very useful for the induction of hematopoiesis from CMK-6 cells, and the production of blood cells on C3H10T1/2 cells has been maintained as long as 5 months. During this long period, ES cell derivatives continuously produced mature blood cells, including terminally differentiated cells. Conclusion We have developed an original method to produce enriched blood cells abundantly from primate ES cells for an extremely long period. This method may represent a good in vitro model for studying primate hematopoiesis and related diseases. Furthermore, our method may be useful for preclinical studies of transfusion therapy using blood cells derived from ES cells in experimental primate systems.