Lina Ning

A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm

Author(s): Peng, Hongying; Shi, Junchao; Zhang, Ying; Zhang, He; Liao, Shangying; Li, Wei; Lei, Li; Han, Chunsheng; Ning, Lina; Cao, Yujing; Zhou, Qi; Chen, Qi; Duan, Enkui

NASA-Approved Rotary Bioreactor Enhances Proliferation of Human Epidermal Stem Cells and Supports Formation of 3D Epidermis-Like Structure

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a “stimulatory” environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

The Cytokine Gene CXCL14 Restricts Human Trophoblast Cell Invasion by Suppressing Gelatinase Activity

Well-controlled trophoblast invasion into uterine decidua is a critical process for the normal development of placenta, which is tightly regulated by various factors produced within the trophoblast-endometrial microenvironment. CXCL14 is involved in tumor growth and metastasis, and its expression in placenta is temporally regulated during pregnancy. However, the role of CXCL14 in trophoblast function during human pregnancy is not clear. In this study, by using RT-PCR through human pregnancy, we found that CXCL14 was selectively expressed at early but not late pregnancy. Immunostaining revealed that CXCL14 proteins were strongly expressed in villous cytotrophoblasts and moderately in decidualized stromal cells but very weakly in syncytiotrophoblasts and extravillous trophoblasts. The effect of CXCL14 on trophoblast invasion were examined by using human villous explants cultured on Matrigel and further proved by invasion and migration assay of primary trophoblast cells and trophoblast cell line HTR-8/SVneo. Our data showed that CXCL14 significantly inhibited outgrowth of villous explant in vitro; this effect is due to suppression of trophoblast invasion and migration through regulating matrix metalloproteinases activities, whereas the trophoblast proliferation was not affected. Moreover, because a receptor for CXCL14 has not been identified, we performed further cell-specific CXCL14 binding activities with regard to different cell types within the maternal-fetal interface. Our data revealed that CXCL14 could specifically bind to trophoblast cells but not decidual cells from the maternal-fetal interface. These results suggest that CXCL14 plays an important role in regulating trophoblast invasion through an autocrine/paracrine manner during early pregnancy.

Transient β2-Adrenoceptor Activation Confers Pregnancy Loss by Disrupting Embryo Spacing at Implantation

Pregnancy loss is a serious social and medical issue, with one important cause associated with aberrant embryo implantation during early pregnancy. However, whether and how the process of embryo implantation is affected by environmental factors such as stress-induced sympathetic activation remained elusive. Here we report an unexpected, transient effect of β2-adrenoreceptor (β2-AR) activation (day 4 postcoitus) in disrupting embryo spacing at implantation, leading to substantially increased midterm pregnancy loss. The abnormal embryo spacing could be prevented by pretreatment of β2-AR antagonist or genetic ablation of β-AR. Similar β2-AR activation at day 5 postcoitus, when implantation sites have been established, did not affect embryo spacing or pregnancy outcome, indicating that the adverse effect of β2-AR activation is limited to the preimplantation period before embryo attachment. In vitro and in vivo studies demonstrated that the transient β2-AR activation abolished normal preimplantation uterine contractility without adversely affecting blastocyst quality. The contractility inhibition is mediated by activation of the cAMP-PKA pathway and accompanied by specific down-regulation of lpa3, a gene previously found to be critical for uterine contraction and embryo spacing. These results indicated that normal uterine contraction-mediated correct intrauterine embryo distribution is crucial for successful ongoing pregnancy. Abnormal β2-AR activation at early pregnancy provided a molecular clue in explaining how maternal stress at early stages could adversely affect the pregnancy outcome.

Identification and characterization of an ancient class of small RNAs enriched in serum associating with active infection

Author(s): Zhang, Yunfang; Zhang, Ying; Shi, Junchao; Zhang, He; Cao, Zhonghong; Gao, Xuan; Ren, Wanhua; Ning, Yunna; Ning, Lina; Cao, Yujing; Chen, Yongchang; Ji, Weizhi; Chen, Zi-Jiang; Chen, Qi; Duan, Enkui

mTOR signaling promotes stem cell activation via counterbalancing BMP-mediated suppression during hair regeneration

Hair follicles (HFs) undergo cycles of degeneration (catagen), rest (telogen), and regeneration (anagen) phases. Anagen begins when the hair follicle stem cells (HFSCs) obtain sufficient activation cues to overcome suppressive signals, mainly the BMP pathway, from their niche cells. Here, we unveil that mTOR complex 1 (mTORC1) signaling is activated in HFSCs, which coincides with the HFSC activation at the telogen-to-anagen transition. By using both an inducible conditional gene targeting strategy and a pharmacological inhibition method to ablate or inhibit mTOR signaling in adult skin epithelium before anagen initiation, we demonstrate that HFs that cannot respond to mTOR signaling display significantly delayed HFSC activation and extended telogen. Unexpectedly, BMP signaling activity is dramatically prolonged in mTOR signaling-deficient HFs. Through both gain- and loss-of-function studies in vitro, we show that mTORC1 signaling negatively affects BMP signaling, which serves as a main mechanism whereby mTORC1 signaling facilitates HFSC activation. Indeed, in vivo suppression of BMP by its antagonist Noggin rescues the HFSC activation defect in mTORC1-null skin. Our findings reveal a critical role for mTOR signaling in regulating stem cell activation through counterbalancing BMP-mediated repression during hair regeneration.

Hair Follicle Stem Cells Derived from Single Rat Vibrissa via Organ Culture Reconstitute Hair Follicles in Vivo

Hair follicle stem cells (HFSCs) are potentially useful for the treatment of skin injuries and diseases. To achieve clinical application, a prerequisite must be accomplished: harvesting enough HFSCs from limited skin biopsy. The commonly used sorting approach for isolating HFSCs, however, suffers from its intrinsic disadvantages, such as requirement of large-scale skin biopsy. Here, we report an efficient organ culture method to isolate and expand rat HFSCs from limited skin biopsy and these HFSCs could reconstitute the epidermis and the hair follicles (HFs). Seventy-three percent of cultured HFs formed hair follicle stem cell colonies from the bulge, and a single hair follicle provided all the HFSCs used in this research, demonstrating the high efficiency of this method. Quantitative RT-PCR and immunofluorescent staining results revealed that these stem cells obtained from the bulge highly expressed basal layer markers K14 and alpha-6 integrin, epithelial stem cell marker P63, and bulge stem cell marker K15. After long-term culture in vitro, GFP-labeled hair follicle stem cells formed new hair follicles, epidermis, and sebaceous glands following xenotransplantation into the back of nude mice. This study indicated that multipotent hair follicle stem cells could be efficiently harvested through organ culture from limited skin material—even a single hair follicle—and reconstitute hair follicles in vivo after long-term expansion culture, providing the basis for future clinical applications.

GPR39 marks specific cells within the sebaceous gland and contributes to skin wound healing

G protein-coupled receptors (GPCRs) mediate multiple key biological processes in the body. The orphan receptor GPR39 has been reported to be involved in various pathophysiological events. However, the function of GPR39 in skin biology remains unknown. Using a genetically engineered mouse strain in which lacZ expression faithfully replaced endogenous Gpr39 expression, we discovered a unique expression pattern of Gpr39 in the sebaceous gland (SG). Using various methods, we confirmed that GPR39 marked a specific cell population at the opening of the SG and colocalised with the SG stem cell marker Blimp1. Further investigations showed that GPR39 was spatiotemporally expressed during skin wound repair. Although it was dispensable for skin development and homeostasis, GPR39 contributed positively to skin wound healing: its loss led to a delay in wound healing during the intermediate stage. The present study reveals a novel role of GPR39 in both dermatology and stem cell biology that has not been previously recognised.

Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles

R-spondin proteins are novel Wnt/β-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/β-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/β-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes (Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/β-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/β-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.

Senescence of human skin-derived precursors regulated by Akt-FOXO3-p27KIP1/p15INK4b signaling

Multipotent skin-derived precursors (SKPs) are dermal stem cells with the capacity to reconstitute the dermis and other tissues, such as muscles and the nervous system. Thus, the easily available human SKPs (hSKPs) hold great promises in regenerative medicine. However, long-term expansion is difficult for hSKPs in vitro. We previously demonstrated that hSKPs senesced quickly under routine culture conditions. To identify the underlying mechanisms so as to find an effective way to expand hSKPs, time-dependent microarray analysis of gene expression in hSKPs during in vitro culture was performed. We found that the senescence of hSKPs had a unique gene expression pattern that differs from reported typical senescence. Subsequent investigation ruled out the role of DNA damage and classical p53 and p16INK4a signaling in hSKP senescence. Examination of cyclin-dependent kinase inhibitors revealed the involvement of p15INK4b and p27KIP1. Further exploration about upstream signals indicated the contribution of Akt hypo-activity and FOXO3 to hSKP senescence. Forced activation of Akt and knockdown of FOXO3, p15INK4b and p27KIP1 effectively inhibited hSKP senescence and promoted hSKP proliferation. The unique senescent phenotype of human dermal stem cells and the role of Akt-FOXO3-p27KIP1/p15INK4b signaling in regulating hSKP senescence provide novel insights into the senescence and self-renewal regulation of adult stem cells. The present study also points out a way to propagate hSKPs in vitro so as to fulfill their promises in regenerative medicine.