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Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. Vldlr high HSCs show higher erythroid potential, which is enhanced after acute anemia induction. Vldlr high HSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions., (© 2024. The Author(s).)

Erythroid terminal differentiation and maturation depend on an enormous energy supply. During periods of fasting, ketone bodies from the liver are transported into circulation and utilized as crucial fuel for peripheral tissues. However, the effects of fasting or ketogenesis on erythroid behavior remain unknown. Here, we generated a mouse model with insufficient ketogenesis by conditionally knocking out the gene encoding the hepatocyte-specific ketogenic enzyme hydroxymethylglutary-CoA synthase 2 (Hmgcs2 KO). Intriguingly, erythroid maturation was enhanced with boosted fatty acid synthesis in the bone marrow of a hepatic Hmgcs2 KO mouse under fasting conditions, suggesting that systemic ketogenesis has a profound effect on erythropoiesis. Moreover, we observed significantly activated fatty acid synthesis and mevalonate pathways along with reduced histone acetylation in immature erythrocytes under a less systemic ketogenesis condition. Our findings revealed a new insight into erythroid differentiation, in which metabolic homeostasis and histone acetylation mediated by ketone bodies are essential factors in adaptation toward nutrient deprivation and stressed erythropoiesis., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2023. Published by Elsevier Inc.)

Hematopoietic stem cells (HSCs) enable hematopoietic stem cell transplantation (HCT) through their ability to replenish the entire blood system. Proliferation of HSCs is linked to decreased reconstitution potential, and a precise regulation of actively dividing HSCs is thus essential to ensure long-term functionality. This regulation becomes important in the transplantation setting where HSCs undergo proliferation followed by a gradual transition to quiescence and homeostasis. Although mouse HSCs have been well studied under homeostatic conditions, the mechanisms regulating HSC activation under stress remain unclear. Here, we analyzed the different phases of regeneration after transplantation. We isolated bone marrow from mice at 8 time points after transplantation and examined the reconstitution dynamics and transcriptional profiles of stem and progenitor populations. We found that regenerating HSCs initially produced rapidly expanding progenitors and displayed distinct changes in fatty acid metabolism and glycolysis. Moreover, we observed molecular changes in cell cycle, MYC and mTOR signaling in both HSCs, and progenitor subsets. We used a decay rate model to fit the temporal transcription profiles of regenerating HSCs and identified genes with progressively decreased or increased expression after transplantation. These genes overlapped to a large extent with published gene sets associated with key aspects of HSC function, demonstrating the potential of this data set as a resource for identification of novel HSC regulators. Taken together, our study provides a detailed functional and molecular characterization of HSCs at different phases of regeneration and identifies a gene set associated with the transition from proliferation to quiescence., Competing Interests: Conflict of Interest Disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2023 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Decreased antigen presentation contributes to the ability of cancer cells to evade the immune system. We used the minimal gene regulatory network of type 1 conventional dendritic cells (cDC1) to reprogram cancer cells into professional antigen-presenting cells (tumor-APCs). Enforced expression of the transcription factors PU.1, IRF8, and BATF3 (PIB) was sufficient to induce the cDC1 phenotype in 36 cell lines derived from human and mouse hematological and solid tumors. Within 9 days of reprogramming, tumor-APCs acquired transcriptional and epigenetic programs associated with cDC1 cells. Reprogramming restored the expression of antigen presentation complexes and costimulatory molecules on the surfaces of tumor cells, allowing the presentation of endogenous tumor antigens on MHC-I and facilitating targeted killing by CD8 + T cells. Functionally, tumor-APCs engulfed and processed proteins and dead cells, secreted inflammatory cytokines, and cross-presented antigens to naïve CD8 + T cells. Human primary tumor cells could also be reprogrammed to increase their capability to present antigen and to activate patient-specific tumor-infiltrating lymphocytes. In addition to acquiring improved antigen presentation, tumor-APCs had impaired tumorigenicity in vitro and in vivo. Injection of in vitro generated melanoma-derived tumor-APCs into subcutaneous melanoma tumors delayed tumor growth and increased survival in mice. Antitumor immunity elicited by tumor-APCs was synergistic with immune checkpoint inhibitors. Our approach serves as a platform for the development of immunotherapies that endow cancer cells with the capability to process and present endogenous tumor antigens.

Mouse hematopoietic stem cells (HSCs) have been extensively defined both molecularly and functionally at steady state, while regenerative stress induces immunophenotypical changes that limit high purity isolation and analysis. It is therefore important to identify markers that specifically label activated HSCs to gain further knowledge about their molecular and functional properties. Here, we assessed the expression of macrophage-1 antigen (MAC-1) on HSCs during regeneration following transplantation and observed a transient increase in MAC-1 expression during the early reconstitution phase. Serial transplantation experiments demonstrated that reconstitution potential was highly enriched in the MAC-1 + portion of the HSC pool. Moreover, in contrast to previous reports, we found that MAC-1 expression inversely correlates with cell cycling, and global transcriptome analysis showed that regenerating MAC-1 + HSCs share molecular features with stem cells with low mitotic history. Taken together, our results suggest that MAC-1 expression marks predominantly quiescent and functionally superior HSCs during early regeneration., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intercellular mitochondria transfer is a biological phenomenon implicated in diverse biological processes. However, the physiological role of this phenomenon remains understudied between erythroblasts and their erythroblastic island (EBI) macrophage niche. To gain further insights into the mitochondria transfer functions, we infused EBI macrophages in vivo into mice subjected to different modes of anemic stresses. Interestingly, we observed the occurrence of mitochondria transfer events from the infused EBI macrophages to early stages of erythroblasts coupled with enhanced erythroid recovery. Single-cell RNA-sequencing analysis on erythroblasts receiving exogenous mitochondria revealed a subset of highly proliferative and metabolically active erythroid populations marked by high expression of CD47. Furthermore, CD47 or Sirpα blockade leads to a decline in both the occurrence of mitochondria transfer events and their mediated erythroid recovery. Hence, these data indicate a significant role of mitochondria transfer in the enhancement of erythroid recovery from stress through the alteration of the bioenergetic profiles via CD47-Sirpα interaction in the early stages of erythroblasts., (© 2022 Yang et al.)

Enucleation is a crucial event during the erythropoiesis, implicating drastic morphologic and transcriptomic/proteomic changes. While many genes deletion lead to failed or impaired enucleation have been identified, directly triggering the erythroid maturation, particularly enucleation, is still challenging. Inducing enucleation at the desired timing is necessary to develop efficient methods to generate mature, fully functional red blood cells in vitro for future transfusion therapies. However, there are considerable differences between primary erythroid cells and cultured cell sources, particularly pluripotent stem cell-derived erythroid cells and immortalized erythroid cell lines. For instance, the difference in the proliferative status between those cell types could be a critical factor, as cell cycle exit is closely connected to the terminal maturation of primary. In this review, we will discuss previous findings on the enucleation machinery and current challengings to trigger the enucleation of infinite erythroid cell sources., (© 2022. Japanese Society of Hematology.)

Hematopoietic stem cells (HSC) give rise to all types of blood lineages, including red blood cells (RBC). Hematopoietic stem/progenitor cells (HSPC) are known to be functionally diverse in terms of their self-renewal potential and lineage output. Consequently, investigation of molecular heterogeneity in the differentiation potential of HSPC is vital to identify novel regulators that affect generation of specific cell types, especially RBC. Here, we compared the erythroid potential of CD34 + hematopoietic stem and progenitor cells from 50 different umbilical cord blood (UCB) donors and discovered that those donors gave rise to diverse frequencies of Glycophorin-A + erythroid cells after in vitro differentiation, despite having similar frequencies of phenotypic HSC initially. RNA sequencing revealed that genes involved in G protein-coupled receptor (GPCR) signaling were significantly up-regulated in the high-erythroid output donors. When we chemically modified two main signaling elements in this pathway, adenylyl cyclase (AC) and phosphodiesterase (PDE), we observed that inhibition of PDE led to 10 times higher yield of Glycophorin-A + cells than activation of AC. Our findings suggest that GPCR signaling, and particularly the cAMP-related pathway, contributes to the diversity of erythroid potential among UCB donors., (© 2022. Japanese Society of Hematology.)

Protein production is tightly regulated in cells because the accumulation of un-/misfolded proteins triggers cellular responses, particularly in the endoplasmic reticulum (ER). Recently, several studies have reported the implications of unfolded protein response (UPR) and ER stress in hematopoiesis, particularly in hematopoietic stem cells (HSCs). The majority of HSCs are maintained in a dormant state under physiological conditions in the adult body, and their protein synthesis rate is also maintained at a low level. Once HSC proliferation is activated, the protein synthesis rate is elevated, and therefore, newly synthesized peptides have to be efficiently folded to prevent the induction of UPR. Importantly, UPR can expand the ER capacity that enables increased protein production and eliminates cells accumulating abnormal proteins; thus, blocking the UPR signal could rather be hazardous for the cells. Thus, understanding how protein quality control is properly controlled and developing methods to manipulate the regulatory mechanisms are imperative to maximize the potential role of HSC.

Ex vivo manufactured red blood cells (RBC) generated from immortalized erythroid cell lines which can continuously grow are expected to become a significant alternative in future transfusion therapies. The ectopic expression of human papilloma virus (HPV) E6/E7 gene has successfully been employed to establish these cell lines. To induce differentiation and maturation of the immortalized cell lines, terminating the HPV-E6/E7 expression through a gene induction system has been believed to be essential. Here, we report that erythroid cell lines established from human bone marrow using simple expression of HPV-E6/E7 are capable of normal erythroid differentiation, without turning gene expression off. Through simply changing cell culture conditions, a newly established cell line, Erythroid Line from Lund University (ELLU), is able to differentiate toward mature cells, including enucleated reticulocytes. ELLU is heterogeneous and, unexpectedly, clones expressing adult hemoglobin rapidly differentiate and produce fragile cells. Upon differentiation, other ELLU clones shift from fetal to adult hemoglobin expression, giving rise to more mature cells. Our findings propose that it is not necessary to employ gene induction systems to establish immortalized erythroid cell lines sustaining differentiation potential and describe novel cellular characteristics for desired functionally competent clones., (© 2021. The Author(s) under exclusive licence to Japan Human Cell Society.)

Isolation of long-term hematopoietic stem cell (HSC) is possible by utilizing flow cytometry with multiple cell surface markers. However, those cell surface phenotypes do not represent functional HSCs after in vitro culture. Here we show that cultured HSCs express mast cell-related genes including Cd244 . After in vitro culture, phenotypic HSCs were divided into CD244 - and CD244 + subpopulations, and only CD244 - cells that have low mast cell gene expression and maintain HSC-related genes sustain reconstitution potential. The result was same when HSCs were cultured in an efficient expansion medium containing polyvinyl alcohol. Chemically induced endoplasmic reticulum (ER) stress signal increased the CD244 + subpopulation, whereas ER stress suppression using a molecular chaperone, TUDCA, decreased CD244 + population, which was correlated to improved reconstitution output. These data suggest CD244 is a potent marker to exclude non-functional HSCs after in vitro culture thereby useful to elucidate mechanism of functional decline of HSCs during ex vivo treatment., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Immortalized erythroid cell lines are expected to be a promising source of ex vivo manufactured red blood cells (RBCs), however the induction of enucleation in these cell lines is inefficient at present. We utilized an imaging-based high-throughput system to identify chemical compounds that trigger enucleation of human erythroid cell lines. Among >3,300 compounds, we identified multiple histone deacetylase inhibitors (HDACi) inducing enucleated cells from the cell line, although an increase in membrane fragility of enucleated cells was observed. Gene expression profiling revealed that HDACi treatment increased the expression of cytoskeletal genes, while an erythroid-specific cell membrane protein, SPTA1, was significantly down-regulated. Restoration of SPTA1 expression using CRISPR-activation partially rescued the fragility of cells and thereby improved the enucleation efficiency. Our observations provide a potential solution for the generation of mature cells from erythroid cell lines, contributing to the future realization of the use of immortalized cell lines for transfusion therapies.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs in vivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chemotherapeutic agents can reduce bone marrow (BM) activity, causing myelosuppression, a common life-threatening complication of cancer treatment. It is challenging to predict the patients in whom prolonged myelosuppression will occur, resulting in a delay or discontinuation of the treatment protocol. An early indicator of recovery from myelosuppression would thus be highly beneficial in clinical settings. In this study, bile acids (BAs) were highly increased in the systemic circulation as a natural response during recovery from myelosuppression, supporting regeneration of BM cells. BA levels in the blood of pediatric cancer patients and mice treated with chemotherapeutic agents were increased, in synchrony with early proliferation of BM cells and recovery from myelosuppression. In a mouse model of altered BA composition, Cyp8b1 knockout mice, a subset of mice recovered poorly after chemotherapy. The poor recovery correlated with low levels and changes in composition of BAs in the liver and systemic circulation. Conversely, BA supplementation in chemotherapy-treated wild-type mice resulted in significantly improved recovery. The results suggest that part of the mechanism by which BAs support recovery is the suppression of endoplasmic reticulum stress pathways in expanding and recovering hematopoietic cells. The findings propose a novel role of BAs as early markers of recovery and active components of the recovery process after chemotherapy., (© 2020 by The American Society of Hematology.)

In comparison to other human cancer types, malignant melanoma exhibits the greatest amount of heterogeneity. After DNA-based detection of the BRAF V600E mutation in melanoma patients, targeted inhibitor treatment is the current recommendation. This approach, however, does not take the abundance of the therapeutic target, i.e., the B-raf V600E protein, into consideration. As shown by immunohistochemistry, the protein expression profiles of metastatic melanomas clearly reveal the existence of inter- and intra-tumor variability. Nevertheless, the technique is only semi-quantitative. To quantitate the mutant protein there is a fundamental need for more precise techniques that are aimed at defining the currently non-existent link between the levels of the target protein and subsequent drug efficacy. Using cutting-edge mass spectrometry combined with DNA and mRNA sequencing, the mutated B-raf protein within metastatic tumors was quantitated for the first time. B-raf V600E protein analysis revealed a subjacent layer of heterogeneity for mutation-positive metastatic melanomas. These were characterized into two distinct groups with different tumor morphologies, protein profiles and patient clinical outcomes. This study provides evidence that a higher level of expression in the mutated protein is associated with a more aggressive tumor progression. Our study design, comprised of surgical isolation of tumors, histopathological characterization, tissue biobanking, and protein analysis, may enable the eventual delineation of patient responders/non-responders and subsequent therapy for malignant melanoma.

The distinct lineage potential is a key feature of hematopoietic stem cell (HSC) heterogeneity, but a subset of HSCs specialized for a single lymphoid compartment has not been identified. Here we report that HSCs expressing junctional adhesion molecule 2 (Jam2) at a higher level (Jam2 high HSCs) have a greater T cell reconstitution capacity. Jam2 high HSCs are metabolically dormant but preferentially differentiate toward lymphocytes, especially T cell lineages. Jam2 high HSCs uniquely express T cell-related genes, and the interaction with Jam1 facilitates the Notch/Delta signaling pathway. Frequency of Jam2 high HSCs changes upon T cell depletion in vivo, potentially suggesting that Jam2 expression may reflect scarcity of T cells and requirement of T cell replenishment. Our findings highlight Jam2 as a potential marker for a subfraction of HSCs with an extensive lymphopoietic capacity, mainly in T lymphopoiesis., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

Hematopoietic stem cells (HSCs) play a central role in hematopoietic regeneration, which has been demonstrated by thorough studies. In contrast, the cell cycle status and metabolic condition of HSCs define these cells as dormant. Recent studies have also revealed that protein metabolism is quite unique, as dormant HSCs have a lower protein synthesis rate and folding capacity. Under proliferative conditions, upon hematopoietic stress, HSCs need to deal with higher requirements of protein production to achieve fast and effective blood replenishment. In such cases, increased protein synthesis could exceed the capacity of precise protein quality control, leading to the accumulation of unfolded and misfolded proteins. In turn, this triggers endoplasmic reticulum (ER) stress as a part of the unfolded protein response (UPR). Since ER stress is a multi-layered, bidirectional cellular response that contains both positive (survival) and negative (death) reactions, proper management of UPR and ER stress signals is crucial for HSCs and also for maintaining the healthy hematopoietic system. In this review, we introduce the latest findings in this emerging field within hematopoiesis and HSC regulation.

The transcription factor hepatic leukemia factor (HLF) is strongly expressed in hematopoietic stem cells (HSCs) and is thought to influence both HSC self-renewal and leukemogenesis. However, the physiological role of HLF in hematopoiesis and HSC function is unclear. Here, we report that mice lacking Hlf are viable with essentially normal hematopoietic parameters, including an intact HSC pool during steady-state hematopoiesis. In contrast, when challenged through transplantation, Hlf-deficient HSCs showed an impaired ability to reconstitute hematopoiesis and became gradually exhausted upon serial transplantation. Transcriptional profiling of Hlf-deficient HSCs revealed changes associated with enhanced cellular activation, and cell-cycle analysis demonstrated a significant reduction of quiescent HSCs. Accordingly, toxic insults targeting dividing cells completely eradicated the HSC pool in Hlf-deficient mice. In summary, our findings point to HLF as a critical regulator of HSC quiescence and as an essential factor for maintaining the HSC pool during regeneration., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pigment epithelium derived factor (PEDF), a ubiquitously expressed 50 kDa secreted glycoprotein, was recently discovered to regulate self-renewal of neural stem cells and have a supportive effect on human embryonic stem cell growth. Here, we analyzed expression of PEDF in the murine hematopoietic stem cell (HSC) compartments and found that PEDF is highly expressed in primary long-term HSCs. Therefore, we characterized the hematopoietic system in a knockout mouse model for PEDF and using this model we surprisingly found that PEDF is dispensable for HSC regulation. PEDF knockout mice exhibit normal hematopoiesis in steady state conditions and the absence of PEDF lead to normal regeneration capacity in a serial competitive transplantation setting. Additionally, PEDF-deficient cells exhibit unaltered lineage distribution upon serial transplantations. When human cord blood stem and progenitor cells were cultured in media supplemented with recombinant PEDF they did not show changes in growth potential. Taken together, we report that PEDF is not a critical regulatory factor for HSC function during regeneration in vivo or growth of human stem/progenitor cells in vitro.

Bmi1 was originally identified as a gene that contributes to the development of mouse lymphoma by inhibiting MYC-induced apoptosis through repression of Ink4a and Arf. It codes for the Polycomb group protein BMI-1 and acts primarily as a transcriptional repressor via chromatin modifications. Although it binds to a large number of genomic regions, the direct BMI-1 target genes described so far do not explain the full spectrum of BMI-1-mediated effects. Here we identify the putative tumor suppressor gene EphA7 as a novel direct BMI-1 target in neural cells and lymphocytes. EphA7 silencing has been reported in several different human tumor types including lymphomas, and our data suggest BMI1 overexpression as a novel mechanism leading to EphA7 inactivation via H3K27 trimethylation and DNA methylation., Competing Interests: The authors declare no conflicts of interest.

Adult hematopoietic stem cells (HSCs) reside in bone marrow and are maintained in a dormant state within a special microenvironment, their so-called "niche". Detaching from the niche induces cell cycle progression, resulting in a reduction of the reconstitution capacity of HSCs. In contrast, fetal liver HSCs actively divide without losing their stem cell potentials. Thus, it has been unclear what types of cellular responses and metabolic changes occur in growing HSCs. We previously discovered that HSCs express relatively low levels of endoplasmic reticulum (ER) chaperone proteins governing protein folding, making HSCs vulnerable to an elevation of stress signals caused by accumulation of un-/misfolded proteins (ER stress) upon in vitro culture. Interestingly, fetal liver HSCs do not show ER stress elevation despite unchanged levels of chaperone proteins. Our latest studies utilizing multiple mouse models revealed that in the fetal liver bile acids as chemical chaperones play a key role supporting the protein folding which results in the suppression of ER stress induction. These findings highlight the importance of ER stress regulations in hematopoiesis.

During development, hematopoietic stem cells (HSCs) undergo a rapid expansion in the fetal liver (FL) before settling in the adult bone marrow. We recently reported that proliferating adult HSCs are vulnerable to ER stress caused by accumulation of mis-folded proteins. Here, we find that FL-HSCs, despite an increased protein synthesis rate and a requirement for protein folding, do not upregulate ER chaperones. Instead, bile acids (BAs), secreted from maternal and fetal liver, coordinate to serve as chemical chaperones. Taurocholic acid, the major BA in FL, supports growth of HSCs in vitro by inhibiting protein aggregation. In vivo, reducing BA levels leads to ER stress elevation and accumulation of aggregated proteins and significantly decreases the number of FL-HSCs. Taken together, these findings reveal that BA alleviation of ER stress is a mechanism required for HSC expansion during fetal hematopoiesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pericytes are located on the abluminal side of endothelial cells lining the microvasculature in all organs. They have been identified as multipotent progenitor cells in several tissues of the body including the human brain. New evidence suggests that pericytes contribute to tissue repair, but their role in the injured brain is largely unknown. Here, we investigate the role of pericytes in ischemic stroke. Using a pericyte-reporter mouse model, we provide unique evidence that regulator of G-protein signaling 5 expressing cells are activated pericytes that leave the blood vessel wall, proliferate and give rise to microglial cells after ischemic brain injury. Consistently, we show that activated pericytes express microglial markers in human stroke brain tissue. We demonstrate that human brain-derived pericytes adopt a microglial phenotype and upregulate mRNA specific for activated microglial cells under hypoxic conditions in vitro. Our study indicates that the vasculature is a novel source of inflammatory cells with a microglial phenotype in brain ischemia and hence identifies pericytes as an important new target for the development of future stroke therapies.

Developmental pluripotency-associated 5 (Dppa5) is an RNA binding protein highly expressed in undifferentiated pluripotent stem cells. Here, we demonstrate that Dppa5 is a regulator of hematopoietic stem cells (HSCs) that critically governs reconstitution capacity after bone marrow transplantation. Ectopic expression of Dppa5 followed by in vitro culture robustly increased HSC reconstitution levels through suppression of endoplasmic reticulum (ER) stress and apoptosis. Remarkably, a chemical chaperone that decreases ER stress in HSCs also increases HSC engraftment. Conversely, knockdown of Dppa5 impaired the long-term reconstitution ability of HSCs due to elevated ER stress levels, suggesting that ER stress regulation is physiologically important for proper HSC function in vivo. Thus, Dppa5 represents a pivotal connection between ER stress regulation and stem cell properties in HSCs. The findings also demonstrate that protein quality control is critical for the maintenance, survival, and function of HSCs in vivo and ex vivo., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Prospective isolation is critical for understanding the cellular and molecular aspects of stem cell heterogeneity. Here, we identify the cell surface antigen CD9 as a positive marker that provides a simple alternative for hematopoietic stem cell isolation at high purity. Crucially, CD9 affords the capture of all hematopoietic stem cells in murine bone marrow in the absence of contaminating populations that lack authentic stem cell function. Using CD9 as a tool to subdivide hematopoietic stem-cell-containing populations, we provide evidence for heterogeneity at the cellular, functional, and molecular levels., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Cripto has been known as an embryonic stem (ES)- or tumor-related soluble/cell membrane protein. In this study, we demonstrated that Cripto has a role as an important regulatory factor for hematopoietic stem cells (HSCs). Recombinant Cripto sustained the reconstitution ability of HSCs in vitro. Flow cytometry analysis uncovered that GRP78, one of the candidate receptors for Cripto, was expressed on a subset of HSCs and could distinguish dormant/myeloid-biased HSCs and active/lymphoid-biased HSCs. Cripto is expressed in hypoxic endosteal niche cells where GRP78(+) HSCs mainly reside. Proteomics analysis revealed that Cripto-GRP78 binding stimulates glycolytic metabolism-related proteins and results in lower mitochondrial potential in HSCs. Furthermore, conditional knockout mice for HIF-1α, a master regulator of hypoxic responses, showed reduced Cripto expression and decreased GRP78(+) HSCs in the endosteal niche area. Thus, Cripto-GRP78 is a novel HSC regulatory signal mainly working in the hypoxic niche., (© 2012 New York Academy of Sciences.)

Identification and characterization of leukemia-initiating cells (LICs) is important to understand leukemogenesis and develop novel therapies for leukemia. In this issue of Cell Stem Cell, Gibbs et al. (2012) demonstrate that common active signaling pathways in LICs may be targeted to treat acute myeloid leukemia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The hematopoietic stem cells that are present in bone marrow and umbilical cord blood are promising materials for in vitro production of red blood cells (RBCs). In particular, umbilical cord blood cells are likely to be readily available since they are generally discarded after parturition. Provided the mother of the neonate consents to the use of the umbilical cord blood, this material can provide a useful resource without any further complicating critical or ethical concerns. Here, we describe a method that does not require feeder cells but provides an efficient approach to the production of enucleated RBCs from the hematopoietic stem and progenitor cells in umbilical cord blood.

Hematopoietic stem cells (HSCs) are maintained in hypoxic niches in endosteal regions of bones. Here we demonstrate that Cripto and its receptor GRP78 are important regulators of HSCs in the niche. Flow cytometry analyses revealed two distinct subpopulations of CD34(-)KSL cells based on the expression of GRP78, and these populations showed different reconstitution potential in transplantation assays. GRP78(+)HSCs mainly reside in the endosteal area, are more hypoxic, and exhibit a lower mitochondrial potential, and their HSC capacity was maintained in vitro by Cripto through induction of higher glycolytic activity. Additionally, HIF-1α KO mice have decreased numbers of GRP78(+)HSCs and reduced expression of Cripto in the endosteal niche. Furthermore, blocking GRP78 induced a movement of HSCs from the endosteal to the central marrow area. These data suggest that Cripto/GRP78 signaling is an important pathway that regulates HSC quiescence and maintains HSCs in hypoxia as an intermediary of HIF-1α., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If immortalized erythroid progenitor cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. We have developed a robust method to establish immortalized erythroid progenitor cell lines following the induction of hematopoietic differentiation of mouse embryonic stem (ES) cells and have established many immortalized erythroid progenitor cell lines so far. Although their precise characteristics varied among cell lines, 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. Considering the number of human ES cell lines that have been established so far and the number of induced pluripotent stem cell lines that will be established in future, the intensive testing of a number of these lines for establishing immortalized erythroid progenitor cell lines may allow the establishment of such cell lines similar to the mouse erythroid progenitor cell lines.

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If transfusable RBCs could be produced abundantly from certain resources, it would be very useful. Our group has developed a method to produce enucleated RBCs efficiently from hematopoietic stem/progenitor cells present in umbilical cord blood. More recently, it was reported that enucleated RBCs could be abundantly produced from human embryonic stem (ES) cells. The common obstacle for application of these methods is that they require very high cost to produce sufficient number of RBCs that are applicable in the clinic. If erythroid cell lines (immortalized cell lines) able to produce transfusable RBCs ex vivo were established, they would be valuable resources. Our group developed a robust method to obtain immortalized erythroid cell lines able to produce mature RBCs. To the best of our knowledge, this was the first paper to show the feasibility of establishing immortalized erythroid progenitor cell lines able to produce enucleated RBCs ex vivo. This result strongly suggests that immortalized human erythroid progenitor cell lines able to produce mature RBCs ex vivo can also be established.

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5, we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly, concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However, Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover, although BMP receptor expression is increased in fetal liver, fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion, canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis, despite its crucial role in the initial patterning of hematopoiesis in early embryonic development.

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.

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., ((c) 2007 Wiley-Liss, Inc.)

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.

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.

Erythroblast enucleation is thought to be largely dependent on signals mediated by other cells, such as macrophages. In an attempt to improve the in vitro production of red blood cells (RBCs) from immature hematopoietic progenitor cells, we have developed a method to produce enucleated RBCs efficiently in the absence of feeder cells. Our method may represent an efficient way to produce transfusable RBCs on a large scale from hematopoietic progenitors.

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.