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Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Figure10

Excess stroma and cancer-associated fibroblasts (CAF) enhance cancer progression and facilitate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer, and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type gastric cancer stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5, and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic gastric cancer xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti–PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti–PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.Significance:Stromal targeting with PDGFRα/β dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer.See related commentary by Tauriello, p. 655

Supplementary Information

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogenesis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

In order to support bone marrow regeneration after myeloablation, hematopoietic stem cells (HSCs) actively divide to provide both stem and progenitor cells. However, the mechanisms regulating HSC function and cell fate choice during hematopoietic recovery remain unclear. We herein provide novel insights into HSC regulation during regeneration by focusing on mitochondrial metabolism and ATP citrate lyase (ACLY). After 5‐fluorouracil‐induced myeloablation, HSCs highly expressing endothelial protein C receptor (EPCR(high)) were enriched within the stem cell fraction at the expense of more proliferative EPCR(Low) HSCs. These EPCR(High) HSCs were initially more primitive than EPCR(Low) HSCs and enabled stem cell expansion by enhancing histone acetylation, due to increased activity of ACLY in the early phase of hematopoietic regeneration. In the late phase of recovery, HSCs enhanced differentiation potential by increasing the accessibility of cis‐regulatory elements in progenitor cell‐related genes, such as CD48. In conditions of reduced mitochondrial metabolism and ACLY activity, these HSCs maintained stem cell phenotypes, while ACLY‐dependent histone acetylation promoted differentiation into CD48(+) progenitor cells. Collectively, these results indicate that the dynamic control of ACLY‐dependent metabolism and epigenetic alterations is essential for HSC regulation during hematopoietic regeneration.

Hematopoietic stem cells (HSCs) and HSC-independent progenitors are generated from hemogenic endothelium. Yokomizo et al. show that Hlf expression distinguishes nascent HSCs from HSC-independent progenitors. HSC specification, regulated by the Evi-1/Hlf axis, is activated only within Hlf+ nascent hematopoietic clusters., Before the emergence of hematopoietic stem cells (HSCs), lineage-restricted progenitors, such as erythro-myeloid progenitors (EMPs), are detected in the embryo or in pluripotent stem cell cultures in vitro. Although both HSCs and EMPs are derived from hemogenic endothelium, it remains unclear how and when these two developmental programs are segregated during ontogeny. Here, we show that hepatic leukemia factor (Hlf) expression specifically marks a developmental continuum between HSC precursors and HSCs. Using the Hlf-tdTomato reporter mouse, we found that Hlf is expressed in intra-aortic hematopoietic clusters and fetal liver HSCs. In contrast, EMPs and yolk sac hematopoietic clusters before embryonic day 9.5 do not express Hlf. HSC specification, regulated by the Evi-1/Hlf axis, is activated only within Hlf+ nascent hematopoietic clusters. These results strongly suggest that HSCs and EMPs are generated from distinct cohorts of hemogenic endothelium. Selective induction of the Hlf+ lineage pathway may lead to the in vitro generation of HSCs from pluripotent stem cells.

Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a novel G0 marker (G0M), we narrowed down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis revealed NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by IRAK1/4 inhibitors together with imatinib eradicated mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuated PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eradicated CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors could eradicate CML LSCs through inhibiting NF-ΚB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.

Most of the hematopoietic stem cells (HSCs) within the bone marrow (BM) show quiescent state with a low mitochondrial membrane potential (ΔΨm). In contrast, upon stress hematopoiesis, HSCs actively start to divide. However, the underlying mechanism for the initiation of HSC division still remains unclear. To elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria in HSCs after BM suppression induced by 5-fluoruracil (5-FU). We found that HSCs initiate cell division after exhibiting enhanced ΔΨm as a result of increased intracellular Ca2+ level. Although further activation of Ca2+–mitochondria pathway led to loss of HSCs after cell division, the appropriate suppression of intracellular Ca2+ level by exogenous adenosine or Nifedipine, a Ca2+ channel blocker, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance. Collectively, our results indicate that the Ca2+–mitochondria pathway induces HSC division critically to determine HSC cell fate.

Osteoclast differentiation is a dynamic differentiation process, which is accompanied by dramatic changes in metabolic status as well as in gene expression. Recent findings have revealed an essential connection between metabolic reprogramming and dynamic gene expression changes during osteoclast differentiation. However, the upstream regulatory mechanisms that drive these metabolic changes in osteoclastogenesis remain to be elucidated. Here, we demonstrate that induced deletion of a tumor suppressor gene, Folliculin (Flcn), in mouse osteoclast precursors causes severe osteoporosis in 3 weeks through excess osteoclastogenesis. Flcn-deficient osteoclast precursors reveal cell autonomous accelerated osteoclastogenesis with increased sensitivity to receptor activator of NF-κB ligand (RANKL). We demonstrate that Flcn regulates oxidative phosphorylation and purine metabolism through suppression of nuclear localization of the transcription factor Tfe3, thereby inhibiting expression of its target gene Pgc1. Metabolome studies revealed that Flcn-deficient osteoclast precursors exhibit significant augmentation of oxidative phosphorylation and nucleotide production, resulting in an enhanced purinergic signaling loop that is composed of controlled ATP release and autocrine/paracrine purinergic receptor stimulation. Inhibition of this purinergic signaling loop efficiently blocks accelerated osteoclastogenesis in Flcn-deficient osteoclast precursors. Here, we demonstrate an essential and novel role of the Flcn-Tfe3-Pgc1 axis in osteoclastogenesis through the metabolic reprogramming of oxidative phosphorylation and purine metabolism. © 2018 The Authors Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

Recently, we reported that bacterial incorporation induces cellular transdifferentiation of human fibroblasts. However, the bacterium-intrinsic cellular- transdifferentiation factor remained unknown. Here, we found that cellular transdifferentiation is caused by ribosomes. Ribosomes, isolated from both prokaryotic and eukaryotic cells, induce the formation of embryoid body-like cell clusters. Numerous ribosomes are incorporated into both the cytoplasm and nucleus through trypsin-activated endocytosis, which leads to cell-cluster formation. Although ribosome-induced cell clusters (RICs) express several stemness markers and differentiate into derivatives of all three germ layers in heterogeneous cell populations, RICs fail to proliferate, alter the methylation states of pluripotent genes, or contribute to teratoma or chimera formation. However, RICs express markers of epithelial–mesenchymal transition without altering the cell cycle, despite their proliferation obstruction. These findings demonstrate that incorporation of ribosomes into host cells induces cell transdifferentiation and alters cellular plasticity.

High-mobility group AT-hook 2 (Hmga2), an epigenetic modifier, opens the chromatin and modulate the transcription. Hmga2 is highly expressed in fetal and adult hematopoietic stem cells (HSCs). Hmga2 over-expression has been shown to promote self-renewal of HSC, however the molecular mechanism of how Hmga2 enhanced the self-renewal of HSC is still unclear. In this study, we assessed the function of Hmga2 in HSCs in steady and stress conditions by utilizing new Hmga2 conditional knock-in (KI) mouse and Hmga2 conditional knock-out mouse, which were crossed with either Cre-ERT2 mouse or Vav1-iCre mouse. Hmga2 KI mice showed a mild elevation in platelet counts, but did not develop malignancies in one year observation period. We performed a competitive transplantation assay by using purified HSCs, and found that wild-type HSCs diminished the repopulating capacity at the tertiary transplantation, Hmga2 KI HSCs maintained higher chimerism in myeloid cells and platelets in the PB and HSCs in the BM. We found that Hmga2 KO cells reduced the repopulating capacity, compared to wild-type cells. Thus, the expression of Hmga2 is critical for the self-renewal of HSC upon the transplantation. By performing RNA-sequencing of HSCs in homeostatic condition, we found that Hmga2 KI HSCs showed positive enrichments in cell cycle and proliferative signature, but maintained a stem cell signature, compared to wild-type HSCs. Since Hmga2 has been shown to globally open the chromatin in neural stem cells, we performed ATAC-sequence analysis in HSCs. Notably, Hmga2 KI HSCs showed 539 opened and 387 closed chromatin in H3K27ac-marked active regulatory regions, compared to wild-type HSCs. Among these opened genes by Hmga2, we generated a virus vector for fifteen genes, which were highly expressed in Hmga2 KI HSCs, and found that ectopic expression of Igf2bp2, an RNA binding protein, increased self-renewal capacity of HSC, but did not induce the enhanced production of myeloid cells and platelets that were observed in Hmga2 KI cells, in in vitro and in vivo settings. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was directly bound to a proximal region of the Igf2bp2 gene, and CRISPR/Cas9-mediated deletion of the Igf2bp2 gene canceled the enhanced self-renewal capacity of Hmga2 KI HSCs, indicating that the Hmga2-Igf2bp2 axis is critical for the self-renewal of HSC. We next assessed function of Hmga2 in stress hematopoiesis after in vivo treatment of 5-FU. Hmga2 KI mice showed faster recoveries of reduction of platelets in the PB and increased CD41+HSCs and megakaryocyte progenitors in the BM in twelve days, in which WT mice reduced numbers of those cells in this condition. RNA-sequencing revealed that Hmga2 KI HSCs maintained expression levels of genes in stem cell- and proliferation-signatures at 3 days and 6 days post 5-FU injection, compared to WT HSCs that reduced expression of stem cell genes but activated inflammatory response genes. In contrast, Hgma2 KO HSCs enhanced expression of inflammatory response genes post the 5-FU injection, indicating Hmga2 represses expression of inflammatory response genes in the stress condition. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was bound to larger numbers of genes involved in inflammatory responses in Kit+ cells post the 5-FU treatment from those in the control cells. Given increased expression of and a post-translational modification of Hmga2 protein in Kit+ cells post the 5-FU treatment, the remodeling of Hmga2 binding regions was appeared to depend on the modification of Hmga2 at downstream of the stress signal. Thus, Hmga2 directly activates Igf2bp2 to enhance the self-renew of HSC, but also represses the inflammatory response, leading to the enhanced megakaryopoiesis in the stress condition. Disclosures No relevant conflicts of interest to declare.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generated a G0 marker (G0M) mouse line that visualizes quiescent cells. G0M identifies a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-Seq analyses showed that the gene expression profiles of these populations were nearly identical, yet differed in their Cdk4/6 activity. Furthermore high-throughput small molecule screening revealed that high concentrations of cytoplasmic calcium ([Ca2+]c) were linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations., Wellcome Trust, Takeda Science Foundation, The Tokyo Biochemical Research Foundation

Key Points HSCs can be separated based on high or low mitochondrial mass. Higher mitochondrial mass is associated with quiescence and greater reconstitution capacity of HSCs.

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generated a G0 marker mouse line that visualizes quiescent cells. The G0 marker identifies a population of active HSCs (G0 markerlow) which are rare and distinct from dormant HSCs (G0 markerhigh) within the conventional quiescent HSC fraction. Single-cell RNA-sequencing analyses showed that the gene expression profiles of these populations were nearly identical, yet differed in their Cdk4/6 activity. Furthermore high-throughput small molecule screening revealed that high levels of basal cytoplasmic calcium (Ca2+) protected the dormancy of HSCs from cell cycle activation induced by calcium influx. These findings indicate that the G0 marker separates dormant and active adult HSCs, which are regulated by Cdk4/6 and Ca2+ levels. This G0 marker mouse line represents a useful new resource for investigating physiologically important stem cell subpopulations.

Summary: The tumor suppressor folliculin (FLCN) suppresses nuclear translocation of TFE3, a master transcription factor for lysosomal biogenesis, via regulation of amino-acid-sensing Rag GTPases. However, the importance of this lysosomal regulation in mammalian physiology remains unclear. Following hematopoietic-lineage-specific Flcn deletion in mice, we found expansion of vacuolated phagocytes that accumulate glycogen in their cytoplasm, phenotypes reminiscent of lysosomal storage disorder (LSD). We report that TFE3 acts in a feedback loop to transcriptionally activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. Tfe3 deletion in Flcn knockout mice reduces the number of phagocytes and ameliorates LSD-like phenotypes. We further reveal that TFE3 stimulates glycogenesis by promoting the expression of glycogenesis genes, including Gys1 and Gyg, upon loss of Flcn. Taken together, we propose that the FLCN-TFE3 feedback loop acts as a rheostat to control lysosome activity and prevents excessive glycogenesis and LSD-like phagocyte activation. : Endoh et al. show that hematopoietic-lineage-specific FLCN deletion in mice induces expansion of phagocytes accumulating cytoplasmic glycogen. TFE3 acts in a feedback loop to activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. TFE3 deletion in FLCN knockout mice blocks aberrant glycogenesis and ameliorates the phenotypes. Keywords: Lysosome, Lysosomal storage disease, glycogen, glycogenesis, gluconeogenesis, Birt-Hogg-Dubé Syndrome, Folliculin, hemophagocytosis

Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between "dormant" and "active" HSCs remains unresolved. Here we generate a G

Real-time reverse transcription polymerase chain reaction (RT-PCR) is regarded as one of the most useful and powerful tools for characterizing hematopoietic stem cells (HSCs), because samples of extremely small cell numbers can be analyzed. The expression levels determined by RT-PCR are based on relative quantification; therefore, the selection of an appropriate reference gene with a relatively stable expression level under most conditions is crucial. Here, we determined that beta2-microglobulin (B2m) is an appropriate reference gene for analyzing mouse HSCs by a novel method using single-cell RT-PCR. Clonally sorted HSCs were subjected to RT reactions with exogenous RNA fragments and then to real-time PCR. Next, the relative gene expression levels of 4 well-known housekeeping genes were quantified in each single cell sample based on the threshold cycle of exogenous RNA. The analysis revealed that B2m expression was reproducibly detected in almost all HSCs and that B2m had the most stable expression level among the compared genes, even after the cells had been cultured under various conditions. Thus, our results indicate that B2m can reliably be used as a reference gene for the relative quantification of expression levels in HSCs across various conditions. Furthermore, our work proposes a novel method for the selection of appropriate reference genes.

Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41+ cells in the lineage Sca1+cKit+ (LSK) fraction (subCD41+LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41+LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3+CD41−LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41+LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41+LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41+CD42b+LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196–2207

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.

Thready stripe-patterned thermo-responsive surfaces were prepared and their surface properties were characterized. Prepared 3 μm wide stripe-patterned surfaces were evaluated by observing the adhesions and detachments of three types of cells: HeLa cells (HeLas), human umbilical vein endothelial cells (HUVECs), and NIH-3T3 cells (3T3s). Although cell adhesion and detachment in response to temperature were observed on all cells on a conventional thermo-responsive surface without patterns, the thermo-responsive surface with a 3 μm striped-pattern exhibited various cell adhesion properties. HeLas hardly adhered to the patterned surface even at 37 °C. On the other hand, although HUVECs adhered on the patterned surface at 12 h after incubation at 37 °C, the adhered HUVECs detached themselves after another 12 h incubation at 37 °C. 3T3s adhered to the patterned surface at 37 °C and detached themselves after reducing temperature to 20 °C. A mixture of HeLa, HUVEC and 3T3 was separated using their different specific cell-adhesion properties, and the composition of cells was analyzed by a flow-cytometry. As a result, the conventional thermo-responsive surface with a stripe-pattern was found to function as a cell-separating interface by using specific cell adhesion properties.

Rodent incisors grow throughout the animal's lives, and the tooth-forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre-ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61(-/-) ), also known as integrin β3, were significantly shorter than those of the wild-type mice at 8-week-old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre-ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2-week-old CD61(-/-) mice. The proliferation of pre-ameloblasts was reduced in CD61(-/-) ameloblasts. The siRNA-mediated suppression of CD61 (siCD61) reduced the proliferation of pre-ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre-ameloblastic cells through Lgr5.

Recently, regenerative medicine has been focused on as next-generation definitive therapies for several diseases or injuries for which there are currently no effective treatments. These therapies have been rapidly developed through the effective fusion between different fields such as stem cell biology and biomaterials. So far, we have proposed “cell sheet engineering” through our core technology that simply applies alterations of the temperature which allows regulation of the attachment or detachment of living cells on the culture surfaces grafted with the temperature-responsive polymer “poly(N-isoproplyacrylamide)”. This technology enables us to construct bioengineered sheet-like tissues, termed “cell sheets”, without the need of using biodegradable scaffolds and protease treatments that are traditionally used. Therefore, our carrier-free cell sheets not only are independent of the issues observed in conventional methods, but also showed further advantages in the reconstruction of the corneal epithelium or endothelium (e.g. improvement of optical transparency and cell-cell interactions to host stroma in reconstructed tissues). Moreover, our approach does not have issues such as immune rejection or limited number of donors, due to the use of cell sheets derived from autologous limbal (in patients with unilateral disease) or oral mucosal epithelial cells (in patients with bilateral disorders). Indeed, we have successfully performed the clinical application for the reconstruction of ocular surfaces through the transplantation of our carrier-free corneal epithelial cell sheets, as evidenced by improved visual acuity as well as long-term maintenance of postoperative health conditions on ocular surfaces in all patients. We have also proposed a novel approach for the reconstruction of the corneal endothelium using corneal endothelial cell sheets by demonstrating its successful application. Thus, our cell sheet engineering technique provides a breakthrough in the field of regenerative medicine applied to the cornea.

Summary: During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage. : Nakamura-Ishizu et al. show that Thpo rapidly upregulates HSC mitochondrial activity in HSCs, an activity accompanied by preferential differentiation to an Mk lineage. Thpo-induced mitochondrial activity correlates with the tetraspanin CD9 expression pattern on HSCs. The findings suggest that Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage. Keywords: hematopoietic stem cell, thrombopoietin, mitochondria, megakaryocyte, lineage differentiation

Corneal epithelial stem cells reside in the limbus, a transitional zone between the cornea and conjunctiva, and are essential for maintaining homeostasis in the corneal epithelium. Although our previous studies demonstrated that rabbit limbal epithelial side population (SP) cells exhibit stem cell-like phenotypes with Hoechst 33342 staining, the different characteristics and/or populations of these cells remain unclear. Therefore, in this study, we determined the gene expression profiles of limbal epithelial SP cells by RNA sequencing using not only present public databases but also contigs that were created by de novo transcriptome assembly as references for mapping. Our transcriptome data indicated that limbal epithelial SP cells exhibited a stem cell-like phenotype compared with non-SP cells. Importantly, gene ontology analysis following RNA sequencing demonstrated that limbal epithelial SP cells exhibited significantly enhanced expression of mesenchymal/endothelial cell markers rather than epithelial cell markers. Furthermore, single-cell quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that the limbal epithelial SP population consisted of at least two immature cell populations with endothelial- or mesenchymal-like phenotypes. Therefore, our present results may propose the presence of a novel population of corneal epithelial stem cells distinct from conventional epithelial stem cells.

Corneal epithelial (CE) stem cells are believed to reside in the basal layer of the limbal epithelium but remain poorly understood due to the lack of an accepted in vivo reconstitution assay as well as definitive markers for epithelial stem cells. It has been reported that side-population (SP) cells with the ability to efflux the DNA-binding dye Hoechst 33342 have stem cell–like properties and that the SP phenotype accurately represents a quiescent and immature stem cell population in the adult bone marrow. In the present study, we investigated whether SP cells isolated from the limbal epithelium have stem cell–like properties. SP cells, separated by fluorescence-activated cell sorting, comprise approximately 0.4% of all limbal epithelial cells and have markedly higher expression of the stem cell markers ABCG2, Bmi-1, and nestin but no expression of markers for differentiated CE cells compared with non-SP cells. Cell-cycle and telomerase activity analyses revealed that SP cells are growth arrested and reside in the quiescent state. Moreover, limbal epithelial SP cells did not demonstrate proliferative capabilities under typical in vitro epithelial cell culture conditions using 3T3 feeder layers. These findings present the possibility that quiescent limbal epithelial SP cells may represent an extremely immature stem cell population compared with currently defined epithelial stem or progenitor cells.

Many types of organ-specific stem cells have been recently shown to exhibit a side population (SP) phenotype based on their ability to efflux Hoechst 33342 dye. Because stem cells from corneal epithelium reside in the basal layer of the limbal epithelium, the purpose of this study was to examine whether the limbal epithelium contains SP cells. The ATP-binding cassette transporter Bcrp1/ABCG2 is reported to contribute to the SP phenotype in cells from several diverse sources. Here we show data from fluorescence-activated cell sorting and real-time quantitative RT-PCR analysis showing that harvested limbal epithelial cells contain SP cells expressing ABCG2. Immunofluorescence revealed that a portion of limbal epithelial basal cells expressed ABCG2. Data indicate that ABCG2 positive limbal epithelial cells are putative corneal epithelial stem cells.

A stripe-patterned thermo-responsive surface is prepared to enable cell separation without labeling. The thermo-responsive surface containing a 3 μm striped pattern exhibits various cell adhesion and detachment properties. A mixture of three cell types is separated on the patterned surface based on their distinct cell-adhesion properties, and the composition of the cells is analyzed by flow cytometry.

Throughout life, hematopoietic stem cells (HSCs) sustain the blood cell supply through their capacities for self-renewal and multilineage differentiation. These processes are regulated within a specialized microenvironment termed the ‘niche’. Here, we show a novel mechanism for regulating HSC function that is mediated by nephroblastoma overexpressed (Nov/CCN3), a matricellular protein member of the CCN family. We found that Nov contributes to the maintenance of long-term repopulating (LTR) activity through association with integrin αvβ3 on HSCs. The resultant β3 integrin outside-in signaling is dependent on thrombopoietin (TPO), a crucial cytokine involved in HSC maintenance. TPO was required for Nov binding to integrin αvβ3, and stimulated Nov expression in HSCs. However, in the presence of IFNγ, a cytokine known to impair HSC function, not only was TPO-induced expression of Nov suppressed, but the LTR activity was conversely impaired by TPO-mediated ligation of integrin αvβ3 with exogenous ligands, including Nov, as well. Thus, Nov/integrin αvβ3-mediated maintenance of HSCs appears to be modulated by simultaneous stimulation by other cytokines. Our finding suggests that this system contributes to the regulation of HSCs within the bone marrow niche.

Rodent incisors grow throughout the animal's lives, and the tooth-forming cells are provided from proximal ends of the incisors where the tooth epithelium forms a stem cell niche called cervical loop. The committing cells in a cervical loop actively begin to proliferate (pre-ameloblasts), and differentiating into ameloblasts. This study showed that the lower incisors of mice null for CD61 (CD61(-/-) ), also known as integrin β3, were significantly shorter than those of the wild-type mice at 8-week-old. The protein and mRNA expressions levels of Fgfr2, Lgr5, and Notch1, which are known to be involved in pre-ameloblastic cell proliferation and stem cell maintenance, were reduced in the cervical loop of 2-week-old CD61(-/-) mice. The proliferation of pre-ameloblasts was reduced in CD61(-/-) ameloblasts. The siRNA-mediated suppression of CD61 (siCD61) reduced the proliferation of pre-ameloblastic cell line ALC, and the expression levels of Lgr5 and Notch1 were reduced by the transfection with siCD61. The suppression of Lgr5 by transfection with siLgr5 suppressed the proliferation of the ALC cells. These results suggested that CD61 signaling is required for the proper growth of the cervical loop and for the promotion of the proliferation of pre-ameloblastic cells through Lgr5.

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin β3, null mice ( CD61-/-) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61-/- mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61-/- mice.

Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.