Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions.

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

MITOL deficiency triggers hematopoietic stem cell apoptosis via ER stress response.

Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.

Downregulation of 15-PGDH enhances MASH-HCC development via fatty acid-induced T-cell exhaustion.

Background & Aims: Hepatocellular carcinoma (HCC) mainly develops from chronic hepatitis. Metabolic dysfunction-associated steatohepatitis (MASH) has gradually become the main pathogenic factor for HCC given the rising incidence of obesity and metabolic diseases. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) degrades prostaglandin 2 (PGE2), which is known to exacerbate inflammatory responses. However, the role of PGE2 accumulation caused by 15-PGDH downregulation in the development of MASH-HCC has not been determined. Methods: We utilised the steric animal model to establish a MASH-HCC model using wild-type and 15-Pgdh+/- mice to assess the significance of PGE2 accumulation in the development of MASH-HCC. Additionally, we analysed clinical samples obtained from patients with MASH-HCC. Results: PGE2 accumulation in the tumour microenvironment induced the production of reactive oxygen species in macrophages and the expression of cell growth-related genes and antiapoptotic genes. Conversely, the downregulation of fatty acid metabolism in the background liver promoted lipid accumulation in the tumour microenvironment, causing a decrease in mitochondrial membrane potential and CD8+ T-cell exhaustion, which led to enhanced development of MASH-HCC. Conclusions: 15-PGDH downregulation inactivates immune surveillance by promoting the proliferation of exhausted effector T cells, which enhances hepatocyte survival and proliferation and leads to the development of MASH-HCC. Impact and implications: The suppression of PGE2-related inflammation and subsequent lipid accumulation leads to a reduction in the severity of MASH and inhibition of subsequent progression toward MASH-HCC.

Stromal Reprogramming through Dual PDGFRα/ß Blockade Boosts the Efficacy of Anti-PD-1 Immunotherapy in Fibrotic Tumors.

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 (PDGFRß) 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/ß blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/ß 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/ß dual blockade reverses the immunosuppressive microenvironment and enhances the efficacy of immune checkpoint inhibitors in fibrotic cancer. See related commentary by Tauriello, p. 655.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

ATP citrate lyase controls hematopoietic stem cell fate and supports bone marrow regeneration.

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 (EPCRhigh ) were enriched within the stem cell fraction at the expense of more proliferative EPCRLow HSCs. These EPCRHigh HSCs were initially more primitive than EPCRLow 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.

Intracellular MUC20 variant 2 maintains mitochondrial calcium homeostasis and enhances drug resistance in gastric cancer.

BACKGROUND: Signet ring cell carcinoma (SRCC) is a particular histologic variant of gastric cancer (GC). However, the critical factor related to the aggressive characteristics of SRCC has not been determined. METHODS: We collected surgically resected tissues from 360 GC patients in the Kumamoto University cohort and generated survival curves via the Kaplan-Meier method. In vitro, we identified the specific transcript variant of MUC20 in SRCC cells by direct sequencing and investigated the role of MUC20 in GC progression using GC cells with MUC20 silencing and forced expression. In vivo, we examined chemoresistance using MUC20 variant 2 (MUC20v2)-overexpressing non-SRCC cells to construct a xenograft mouse model. RESULTS: We analyzed a comprehensive GC cell line database to identify the specifically expressed genes in gastric SRCC. We focused on MUC20 and investigated its role in GC progression. Survival analysis revealed that GC patients with high MUC20 expression exhibited a poor prognosis and that MUC20 expression was significantly correlated with SRCC histological type. Moreover, we found that gastric SRCC cells specifically expressed MUC20v2, which was dominantly expressed in the cytoplasm. Silencing MUC20v2 caused cell death with characteristic morphological changes in gastric SRCC cells. To further determine the types of cell death, we examined apoptosis, pyroptosis and ferroptosis by detecting cleaved PARP, gasdermin E-N-terminal (GSDME-N), and lipid reactive oxygen species (ROS) levels, respectively. We found that apoptosis and pyroptosis occurred in MUC20-silenced gastric SRCC cells. In addition, MUC20v2-overexpressing GC cells exhibited chemoresistance to cisplatin (CDDP) and paclitaxel (PTX). RNA sequencing revealed that the pathways involved in intracellular calcium regulation were significantly upregulated in MUC20v2-overexpressing GC cells. Notably, forced expression of MUC20v2 in the cytoplasm of GC cells led to the maintenance of mitochondrial calcium homeostasis and mitochondrial membrane potential (MMP), which promoted cell survival and chemoresistance by suppressing apoptosis and pyroptosis. Finally, we investigated the significance of MUC20v2 in a xenograft model treated with CDDP and showed that MUC20v2 overexpression caused chemoresistance by inhibiting cell death. CONCLUSION: These findings highlight the novel functions of MUC20v2, which may confer cell survival and drug resistance in GC cells. SIGNIFICANCE: MUC20v2 protects GC cells from apoptosis and pyroptosis by maintaining mitochondrial calcium levels and mitochondrial membrane potential and subsequently induces drug resistance.

Eliminating chronic myeloid leukemia stem cells by IRAK1/4 inhibitors.

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 G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate 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.

Autophagy is dispensable for the maintenance of hematopoietic stem cells in neonates.

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.

Immuno-Modulation of Hematopoietic Stem and Progenitor Cells in Inflammation.

Lifelong blood production is maintained by bone marrow (BM)-residing hematopoietic stem cells (HSCs) that are defined by two special properties: multipotency and self-renewal. Since dysregulation of either may lead to a differentiation block or extensive proliferation causing dysplasia or neoplasia, the genomic integrity and cellular function of HSCs must be tightly controlled and preserved by cell-intrinsic programs and cell-extrinsic environmental factors of the BM. The BM had been long regarded an immune-privileged organ shielded from immune insults and inflammation, and was thereby assumed to provide HSCs and immune cells with a protective environment to ensure blood and immune homeostasis. Recently, accumulating evidence suggests that hemato-immune challenges such as autoimmunity, inflammation or infection elicit a broad spectrum of immunological reactions in the BM, and in turn, influence the function of HSCs and BM environmental cells. Moreover, in analogy with the emerging concept of "trained immunity", certain infection-associated stimuli are able to train HSCs and progenitors to produce mature immune cells with enhanced responsiveness to subsequent challenges, and in some cases, form an inflammatory or infectious memory in HSCs themselves. In this review, we will introduce recent findings on HSC and hematopoietic regulation upon exposure to various hemato-immune stimuli and discuss how these challenges can elicit either beneficial or detrimental outcomes on HSCs and the hemato-immune system, as well as their relevance to aging and hematologic malignancies.

A FLCN-TFE3 Feedback Loop Prevents Excessive Glycogenesis and Phagocyte Activation by Regulating Lysosome Activity.

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.

Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium.

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 G0 marker (G0M) mouse line that visualizes quiescent cells and identify 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 show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are 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.

Hlf marks the developmental pathway for hematopoietic stem cells but not for erythro-myeloid progenitors.

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.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

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.

Ca2+-mitochondria axis drives cell division in hematopoietic stem cells.

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.

Folliculin Regulates Osteoclastogenesis Through Metabolic Regulation.

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).

Ribosome Incorporation into Somatic Cells Promotes Lineage Transdifferentiation towards Multipotency.

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.

Integrin αvß3 enhances the suppressive effect of interferon-γ on hematopoietic stem cells.

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 αvß3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin ß3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro-inflammatory cytokine interferon-γ (IFNγ) and ß3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvß3 suppressed HSC function in the presence of IFNγ and impaired integrin ß3 signaling mitigated IFNγ-dependent negative action on HSCs. During IFNγ stimulation, integrin ß3 signaling enhanced STAT1-mediated gene expression via serine phosphorylation. These findings show that integrin ß3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvß3 within the BM niche acts as a context-dependent signal modulator to regulate the HSC function under both steady-state and inflammatory conditions.

Characterization of rabbit limbal epithelial side population cells using RNA sequencing and single-cell qRT-PCR.

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.