Fms-like tyrosine kinase 3 is a regulator of the cardiac side population in mice.

Fms-like tyrosine kinase 3 (Flt3) is a regulator of hematopoietic progenitor cells and a target of tyrosine kinase inhibitors. Flt3-targeting tyrosine kinase inhibitors can have cardiovascular side effects. Flt3 and its ligand (Flt3L) are expressed in the heart, but little is known about their physiological functions. Here, we show that cardiac side population progenitor cells (SP-CPCs) from mice produce and are responsive to Flt3L. Compared with wild-type, flt3L-/- mice have less SP-CPCs with less contribution of CD45-CD34+ cells and lower expression of genes related to epithelial-to-mesenchymal transition, cardiovascular development and stem cell differentiation. Upon culturing, flt3L-/- SP-CPCs show increased proliferation and less vasculogenic commitment, whereas Akt phosphorylation is lower. Notably, proliferation and differentiation can be partially restored towards wild-type levels in the presence of alternative receptor tyrosine kinase-activating growth factors signaling through Akt. The lower vasculogenic potential of flt3L-/- SP-CPCs reflects in decreased microvascularisation and lower systolic function of flt3L-/- hearts. Thus, Flt3 regulates phenotype and function of murine SP-CPCs and contributes to cellular and molecular properties that are relevant for their cardiovasculogenic potential.

Adult stem cells in endometrial regeneration: Molecular insights and clinical applications.

Endometrial damage is an important cause of female reproductive problems, manifested as menstrual abnormalities, infertility, recurrent pregnancy loss, and other complications. These conditions are collectively termed "Asherman syndrome" (AS) and are typically associated with recurrent induced pregnancy terminations, repeated diagnostic curettage and intrauterine infections. Cancer treatment also has unexpected detrimental side effects on endometrial function in survivors independently of ovarian effects. Endometrial stem cells act in the regeneration of the endometrium and in repair through direct differentiation or paracrine effects. Nonendometrial adult stem cells, such as bone marrow-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells, with autologous and allogenic applications, can also repair injured endometrial tissue in animal models of AS and in human studies. However, there remains a lack of research on the repair of the damaged endometrium after the reversal of tumors, especially endometrial cancers. Here, we review the biological mechanisms of endometrial regeneration, and research progress and challenges for adult stem cell therapy for damaged endometrium, and discuss the potential applications of their use for endometrial repair after cancer remission, especially in endometrial cancers. Successful application of such cells will improve reproductive parameters in patients with AS or cancer. Significance: The endometrium is the fertile ground for embryos, but damage to the endometrium will greatly impair female fertility. Adult stem cells combined with tissue engineering scaffold materials or not have made great progress in repairing the injured endometrium due to benign lesions. However, due to the lack of research on the repair of the damaged endometrium caused by malignant tumors or tumor therapies, the safety and effectiveness of such stem cell-based therapies need to be further explored. This review focuses on the molecular insights and clinical application potential of adult stem cells in endometrial regeneration and discusses the possible challenges or difficulties that need to be overcome in stem cell-based therapies for tumor survivors. The development of adult stem cell-related new programs will help repair damaged endometrium safely and effectively and meet fertility needs in tumor survivors.

4,4'­Bond secalonic acid D targets SP cells and inhibits metastasis in hepatocellular carcinoma.

The existence of cancer stem cells (CSCs) is considered to be the main reason for chemoresistance, metastasis and the ultimate failure of treatment in hepatocellular carcinoma (HCC). However, there are a few chemical agents that may inhibit CSCs. The present study identified that 4,4'­bond secalonic acid D (4,4'­SAD), a compound isolated from the marine­derived fungus Penicillium oxalicum, inhibited the growth of side population (SP) cells isolated from human liver cancer cell lines PLC/PRF/5 and HuH­7 by attenuating the expression of ATP­binding cassette superfamily G member 2. Furthermore, the results of wound healing, Transwell, western blotting and reverse transcription­quantitative PCR assays demonstrated that 4,4'­SAD suppressed the invasion and migration of SP cells by downregulating matrix metallopeptidase 9 (MMP­9) and upregulating the antagonist tissue inhibitor of metalloproteinases 1 in vitro. Moreover, in vivo study results found that 4,4'­SAD had anti­lung metastasis efficacy via the decrease of MMP­9 expression in the H22 HCC model of Kunming mice. Therefore, the present study identified the potential of 4,4'­SAD as a promising candidate for the treatment of advanced liver cancer.

Abcg2-expressing side population cells contribute to cardiomyocyte renewal through fusion.

The adult mammalian heart has a limited regenerative capacity. Therefore, identification of endogenous cells and mechanisms that contribute to cardiac regeneration is essential for the development of targeted therapies. The side population (SP) phenotype has been used to enrich for stem cells throughout the body; however, SP cells isolated from the heart have been studied exclusively in cell culture or after transplantation, limiting our understanding of their function in vivo. We generated a new Abcg2-driven lineage-tracing mouse model with efficient labeling of SP cells. Labeled SP cells give rise to terminally differentiated cells in bone marrow and intestines. In the heart, labeled SP cells give rise to lineage-traced cardiomyocytes under homeostatic conditions with an increase in this contribution following cardiac injury. Instead of differentiating into cardiomyocytes like proposed cardiac progenitor cells, cardiac SP cells fuse with preexisting cardiomyocytes to stimulate cardiomyocyte cell cycle reentry. Our study is the first to show that fusion between cardiomyocytes and non-cardiomyocytes, identified by the SP phenotype, contribute to endogenous cardiac regeneration by triggering cardiomyocyte cell cycle reentry in the adult mammalian heart.

Melanocyte Chitosan/Gelatin Composite Fabrication with Human Outer Root Sheath-Derived Cells to Produce Pigment.

The hair follicle serves as a melanocyte reservoir for both hair and skin pigmentation. Melanocyte stem cells (MelSCs) and melanocyte progenitors reside in the bulge/sub-bulge region of the lower permanent portion of the hair follicle and play a vital role for repigmentation in vitiligo. It would be beneficial to isolate MelSCs in order to further study their function in pigmentary disorders; however, due to the lack of specific molecular surface markers, this has not yet been successfully accomplished in human hair follicles (HuHF). One potential method for MelSCs isolation is the "side population" technique, which is frequently used to isolate hematopoietic and tumor stem cells. In the present study, we decided to isolate HuHF MelSCs using "side population" to investigate their melanotic function. By analyzing mRNA expression of TYR, SOX10, and MITF, melanosome structure, and immunofluorescence with melanocyte-specific markers, we revealed that the SP-fraction contained MelSCs with an admixture of differentiated melanocytes. Furthermore, our in vivo studies indicated that differentiated SP-fraction cells, when fabricated into a cell-chitosan/gelatin composite, could transiently repopulate immunologically compromised mice skin to regain pigmentation. In summary, the SP technique is capable of isolating HuHF MelSCs that can potentially be used to repopulate skin for pigmentation.

Differentiation of CD45­/CD31+ lung side population cells into endothelial and smooth muscle cells in vitro.

Side population (SP) cells are a small subpopulation of cells found in many mammalian tissues and organs, identified by their capacity to efflux Hoechst 33342 dye. They are enriched for stem/progenitor cell activity. SP cells isolated from the adult mouse lung can be separated into a CD45+ subset (bone marrow­derived) and a CD45­ subset that can be subdivided into CD31­ and CD31+ subpopulations. CD45­/CD31­ lung SP (LSP) cells are known to be mesenchymal stem cells. However, CD45­/CD31+ LSP cells are not fully characterized. In the present study, it was found that CD45­/CD31+ LSP cells were able to form colonies. Based on the expression of vascular endothelial growth factor receptor 2 (VEGFR2), these cells were separated into VEGFR2­ and VEGFR2+ cells. The CD45­/CD31+/VEGFR2­ LSP cells expressed genes characteristic of smooth muscle and endothelial progenitors, and were able to differentiate into smooth muscle and endothelial cells in vitro. The CD45­/CD31+/VEGFR2+ LSP cells expressed genes characteristic of endothelial progenitors and gave rise to endothelial cells, although not smooth muscle, in vitro. The data demonstrate that CD45­/CD31+/VEGFR2­ LSP cells differentiated into CD45­/CD31+/VEGFR2+ LSP cells and then endothelial cells, indicating that CD45­/CD31+/VEGFR2+ LSP cells are likely to be derived from CD45­/CD31+/VEGFR2­ LSP cells. Taken together, the results suggest that CD45­/CD31+ LSP cells can be separated into CD45­/CD31+/VEGFR2­ LSP cells, which may be progenitors of endothelial and smooth muscle, whereas CD45­/CD31+/VEGFR2+ LSP cells may serve as late commitment endothelial progenitors in the adult mouse lung.

Expression of C19MC miRNAs in HCC associates with stem-cell features and the cancer-testis genes signature.

BACKGROUND: Intratumor heterogeneity of hepatocellular carcinoma (HCC) and, among HCC cell subsets, the cancer stem cell population (hCSC), is responsible for therapeutic resistance and disease relapse. AIMS: To characterize hCSC-enriched HCCs at the molecular level. METHODS: Side population (SP) was used to identify the hCSCs in multiple tumor sampling from different patients and primary HCCs cultures. FACS was used to immunoprofile cultures. miRNAs were profiled in samples and correlated to SP. The Cancer Genome Atlas (TCGA) HCC dataset was analyzed to search for signatures associated with C19MC miRNAs expression. Results were confirmed by immunohistochemistry. RESULTS: The miRNA cluster on chromosome 19 (C19MC) was enriched in SP and in HCCs with a high SP fraction. At the molecular level, an elevated C19MC was correlated with expression of precursor transcripts. In TCGA-HCC series, high C19MC expression identified a subset of patients with poorer prognosis, advanced disease and overexpression of the cancer-testis (CT) antigens. These data were confirmed in an independent cohort of HCCs and at the protein level. CONCLUSION: C19MC miRNAs and CT antigens overexpression represents a novel oncogenic pathway in a subset of hCSC-enriched HCCs with dismal prognosis. CT antigens are promising immunotherapy targets. Therefore, these molecular signatures could identify HCCs who could benefit from immunotherapy.

Cellular stress induces cancer stem-like cells through expression of DNAJB8 by activation of heat shock factor 1.

In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

Characterization of the Human Pancreas Side Population as a Potential Reservoir of Adult Stem Cells.

OBJECTIVES: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells. METHODS: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture. RESULTS: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity. CONCLUSIONS: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.

[Differentiation of Cultivated CD45-/CD31+ Mouse Lung Side Population Cells into Vascular Smooth Muscle Cells in vitro].

OBJECTIVES: To investigate the characteristics of differentiation of lung side population cells (LSP cells)in vitro. METHODS: CD45-/CD31+ LSP cells sorted by flow cytometry were taken from mouse lung tissues and cultured for 14 d. The cultured LSP cells were observed with colony formation assay and flow cytometryin vitro. The mRNA expressions of ATP-binding cassette transporter G2 (ABCG2), smooth muscle actin (SMA) and α-smooth muscle tropomyosin (α-SMT) in both freshly isolated LSP cells and cultured LSP cells were examined. The expressions of ABCG2 and stem cell antigen 1 (Sca1) in LSP cells were detected using immunofluorescence. RT-PCR tests were performed to detect the expressions of ABCG2, SMA and α-SMT in LSP cells. RESULTS: The isolated CD45-/CD31+ lung side population cells expressed ABCG2, SMA and Sca1, but not α-SMT. A large number of LSP in aggregated state were observed after 14 d of culture. Before induction of differentiation, the CD45-/CD31+ LSP cells expressed ABCG2 and SMA, but not α-SMT. After induction of differentiation, the CD45-/CD31+ lung side population cells expressed α-SMT and SMA, but not ABCG2. CONCLUSIONS: CD45-/CD31+ LSP cells might be progenitor cells of vascular smooth muscle cells, possessing the characteristics of stem cell differentiations.

Targeting the aryl hydrocarbon receptor nuclear translocator complex with DMOG and Stemregenin 1 improves primitive hematopoietic stem cell expansion.

Culture conditions used for the expansion of hematopoietic stem and progenitor cells (HSCs and HPCs, collectively HSPCs) should ideally favor the self renewal of long-term HSCs. At 20% O2, the synthesis of HIF-1α is balanced by its hydroxylation and proteasomal degradation. This favors HSPC differentiation, but can be prevented by culturing CD34+ cord blood cells in the presence of dimethyloxaloylglycine (DMOG). This differentiation may also be reduced by culturing the cells in the presence of Stemregenin 1, an antagonist of the aryl hydrocarbon receptor (AhR). The objective of this study was to investigate how hypoxia, DMOG and Stemregenin 1 might affect the expansion of HSPCs with the aim of identifying optimal conditions for expansion in culture. It was found that DMOG decreased proliferation but was effective in preserving the number of cells in the primitive hematopoietic sub-populations in vitro. The effect of DMOG was similar to hypoxia, although differences were observed with regard to the side population and CD34+ sub-populations. Stemregenin 1 on the other hand increased the size of the primitive as well as the other HSC sub-populations. The use of Stemregenin 1 with DMOG increased the proportion of primitive HSCs to 3.54% compared to 2.61% for Stemregenin 1 alone. In vivo engraftment studies confirmed these findings and showed that fewer cells (3710) are required for long-term engraftment when HSCs are grown in Stemregenin 1 together with hypoxia than in Stemregenin 1 under conditions of normoxia (13430).

A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Stem Cells Translational Medicine 2017;6:761-766.

An Adult Mouse Thyroid Side Population Cell Line that Exhibits Enriched Epithelial-Mesenchymal Transition.

BACKGROUND: Studies of thyroid stem/progenitor cells have been hampered due to the small organ size and lack of tissue, which limits the yield of these cells. A continuous source that allows the study and characterization of thyroid stem/progenitor cells is desired to push the field forward. METHOD: A cell line was established from Hoechst-resistant side population cells derived from mouse thyroid that were previously shown to contain stem/progenitor-like cells. Characterization of these cells were carried out by using in vitro two- and three-dimensional cultures and in vivo reconstitution of mice after orthotopic or intravenous injection, in conjunction with quantitative reverse transcription polymerase chain reaction, Western blotting, immunohisto(cyto)chemistry/immunofluorescence, and RNA seq analysis. RESULTS: These cells were named SPTL (side population cell-derived thyroid cell line). Under low serum culturing conditions, SPTL cells expressed the thyroid differentiation marker NKX2-1, a transcription factor critical for thyroid differentiation and function, while no expression of other thyroid differentiation marker genes were observed. SPTL cells formed follicle-like structures in Matrigel® cultures, which did not express thyroid differentiation marker genes. In mouse models of orthotopic and intravenous injection, the latter following partial thyroidectomy, a few SPTL cells were found in part of the follicles, most of which expressed NKX2-1. SPTL cells highly express genes involved in epithelial-mesenchymal transition, as demonstrated by RNA seq analysis, and exhibit a gene-expression pattern similar to anaplastic thyroid carcinoma. CONCLUSION: These results demonstrate that SPTL cells have the capacity to differentiate into thyroid to a limited degree. SPTL cells may provide an excellent tool to study stem cells, including cancer stem cells of the thyroid.

Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay.

The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFß treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.

LncRNA ANRIL is up-regulated in nasopharyngeal carcinoma and promotes the cancer progression via increasing proliferation, reprograming cell glucose metabolism and inducing side-population stem-like cancer cells.

Long noncoding RNAs play a vital role in diverse biological processes such as embryonic development, cell growth, and tumorigenesis. In this study, we report that LncRNA ANRIL, which encodes a 3834-nt RNA that contains 19 exons at the antisense orientation of the INK4B-ARF-INK4A gene cluster, generally up-regulated in nasopharyngeal carcinoma [1]. In a cohort of 88 NPC patients, ANRIL was highly expressed in advanced-stage cancer. Multivariate analyses revealed that ANRIL expression could serve as an independent predictor of overall survival (P = 0.027) and disease-free survival (P = 0.033). Further investigation showed that knockdown of ANRIL significantly repressed NPC cell proliferation and transformation. We also found that ANRIL could induce the percentage of side population cells (SP cells) in NPC. To meet the urgent needs of energy provision, ANRIL can also reprogram glucose metabolism via increasing glucose uptake for glycolysis, which was regulated by the mTOR signal pathway to affect the expression of essential genes in glycolysis. We concluded that ANRIL could promote NPC progression via increasing cell proliferation, reprograming cell glucose metabolism and inducing side-population stem-like cancer cells. Our results also suggested that ANRIL may serve as a novel diagnostic or prognostic biomarker and a candidate target for new therapies in NPC.

Toxicity of Hoechst 33342: implication in side population analysis.

In the current study, we discovered that Hoechst 33342 (H342) could produce significant toxicity to the human neuroblastoma SK-N-SH cells at concentrations typically used to identify side population (SP) cells. At 3.75 and 5 µg/ml, H342 induced cell death and apoptosis. Flow cytometry analysis revealed cell cycle arrest at S-phase. Our findings caution against using H342 for SP analysis without considering differential susceptibility of different cells to H342. Specifically, concentration of H342 needs to be adjusted carefully to minimize toxicity for each cell line.

Constitutive expression of Wnt/ß­catenin target genes promotes proliferation and invasion of liver cancer stem cells.

Wnt/ß­catenin is an important signaling pathways involved in the tumorgenesis, progression and maintenance of cancer stem cells (CSCs). In the present study, the role of Wnt/ß­catenin signaling in CSC­mediated tumorigenesis and invasion in liver CSCs was investigated. A small population of cancer stem­like side population (SP) cells (3.6%) from liver cancer samples were identified. The cells were highly resistant to drug treatment due to the enhanced expression of drug efflux pumps, such as ABC subfamily G member 2, multidrug resistance protein 1 and ATP­binding cassette subfamily B member 5. Furthermore, using TOPflash and reverse transcription­quantitative polymerase chain reaction analysis, Wnt/ß­catenin signaling and the transcriptional regulation of Wnt/ß­catenin target genes including dickkopf Wnt signaling pathway inhibitor 1, axis inhibition protein 2 and cyclin D1 were observed to be markedly upregulated in liver cancer SP cells. As a consequence, SP cells possessed infinite cell proliferation potential and the ability to generating tumor spheres. In addition, upon reducing Wnt/ß­catenin signaling, the rates of proliferation, tumor sphere formation and tumor invasion of SP cells were markedly reduced. Therefore, these data suggest that Wnt/ß­catenin signaling is a potential therapeutic target to reduce CSC­mediated tumorigenicity and invasion in liver cancer.

A Synthetic Polymer Scaffold Reveals the Self-Maintenance Strategies of Rat Glioma Stem Cells by Organization of the Advantageous Niche.

Cancer stem cells (CSCs) are believed to be maintained within a microenvironmental niche. Here we used polymer microarrays for the rapid and efficient identification of glioma CSC (GSC) niche mimicries and identified a urethane-based synthetic polymer, upon which two groups of niche components, namely extracellular matrices (ECMs) and iron are revealed. In cultures, side population (SP) cells, defined as GSCs in the rat C6 glioma cell line, are more efficiently sustained in the presence of their differentiated progenies expressing higher levels of ECMs and transferrin, while in xenografts, ECMs are supplied by the vascular endothelial cells (VECs), including SP cell-derived ones with distinctively greater ability to retain xenobiotics than host VECs. Iron is stored in tumor infiltrating host macrophages (Mφs), whose protumoral activity is potently enhanced by SP cell-secreted soluble factor(s). Finally, coexpression of ECM-, iron-, and Mφ-related genes is found to be predictive of glioma patients' outcome. Our polymer-based approach reveals the intrinsic capacities of GSCs, to adapt the environment to organize a self-advantageous microenvironment niche, for their maintenance and expansion, which redefines the current concept of anti-CSC niche therapy and has the potential to accelerate cancer therapy development. Stem Cells 2016;34:1151-1162.

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.

Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells.

Previously, we demonstrated that hypoxia (1% O2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α), proliferating cell nuclear antigen (PCNA), cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.