Role of the Ubi-p63E gene in the germline stem cell niche of the Drosophila testis / 医学研究生学报

Objective Whether the Ubi-p63E gene regulates spermatogenesis and tumorigenesis remains unclear. This study aimed to explore the regulatory effect of Ubi-p63E on germline stem cells (GSC) in the GSC niche of the Drosophila testis. Methods We used the UAS-Gal4 system for knockdown of the Ubi-p63E gene in the specific GSCs of the Drosophila testis and divided the male flies for this experiment into three groups: control (wild-type W1118 flies), nos>Ubi-p63E RNAi (knockdown of the Ubi-p63E gene in the early germ cells), and tj>Ubi-p63E RNAi (knockdown of the Ubi-p63E gene in the cystoblasts). We determined the fertility rate of the flies by fertility tests and examined the effect of Ubi-p63E on the Drosophila testis in the GSC niche by immunofluorescence staining. Results Fertility tests manifested a significantly lower rate of fertility in the nos>Ubi-p63E RNAi and tj>Ubi-p63E RNAi groups than in the control (0.00% and 4.12% vs 97.26%, P < 0.01). Morphologically abnormal testes were observed in the nos>Ubi-p63E RNAi and tj>Ubi-p63E RNAi groups, only 22.77% and 18.86% as long as the testes of the control flies. Immunofluorescence staining revealed no morphologically normal testes in the tj>Ubi-p63E RNAi group, but quite a few masses of abnormal cells, and mostly Vasa-positive cells. Conclusion The Ubi-p63E gene affects the self-renewal ability of GSCs in the GSC niche of the Drosophila testis as well as the differentiation of GSCs via cystoblasts, and consequently leads to the formation of germ cell tumors.

Regulatory factors and modulation mechanisms of functions of ovarian germline stem cell niche / 上海交通大学学报（医学版）

With increasing age in women, the ovarian function declines, which leads to decreased follicle generation, declined female fertility and age-related diseases ultimately. Female germline stem cells are epithelial cells existing on the ovarian surface, which can divide into new stem cells symmetrically and differentiate into germ cells and granulosa cells asymmetrically. The discovery of female germline stem cells brings much hope for the post-natal renewal of oocytes and solving female infertility problems. Ovarian germline stem cell niche in which female germline stem cells live is the surrounding microenvironment which plays an essential role in maintaining the function of female germline stem cells. Many factors including nutrition supply, protein, cytokines and signaling pathways can control the biological characters of female germline stem cells, and also influence their proliferation and differentiation. This paper reviewed the knowledge about the influencing factors and regulatory mechanisms of the function of ovarian germline stem cell niche.

Regulatory factors and modulation mechanisms of functions of ovarian germline stem cell niche / 上海交通大学学报（医学版）

With increasing age in women, the ovarian function declines, which leads to decreased follicle generation, declined female fertility and age-related diseases ultimately. Female germline stem cells are epithelial cells existing on the ovarian surface, which can divide into new stem cells symmetrically and differentiate into germ cells and granulosa cells asymmetrically. The discovery of female germline stem cells brings much hope for the post-natal renewal of oocytes and solving female infertility problems. Ovarian germline stem cell niche in which female germline stem cells live is the surrounding microenvironment which plays an essential role in maintaining the function of female germline stem cells. Many factors including nutrition supply, protein, cytokines and signaling pathways can control the biological characters of female germline stem cells, and also influence their proliferation and differentiation. This paper reviewed the knowledge about the influencing factors and regulatory mechanisms of the function of ovarian germline stem cell niche.

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny / Uma visão supramolecular sobre a regulação microambiental de células tronco epiteliais limbais e a diferenciação de sua progênie

ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation) can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase) nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

RESUMO Muitas abordagens têm sido utilizadas para ampliar entendimentos sobre a regulação microambiental das células tronco epiteliais limbais. Neste contexto, pesquisadores têm exaustivamente investigado a participação de fatores de crescimento, fatores de sobrevida, citocinas, enzimas e moléculas permeáveis secretadas pelas células limbais. Entretanto, evidências recentes sugerem que o destino (ie. autorrenovação ou recrutamento para a via de diferenciação) das células tronco também sofre influência de estímulos biofísicos ou mecânicos relacionados à organização supramolecular e à natureza liquido-cristalina (mesofases) da matriz extracelular estromal. Esses estímulos podem ser percebidos e traduzidos pelas células tronco em sinais bioquímicos que geram respostas funcionais, através de um processo designado de mecanotransdução. Objetiva-se, com a presente revisão, oferecer ao leitor perspectivas supramoleculares sobre a regulação microambiental das células tronco epiteliais limbais e a diferenciação de sua progênie.

Hypoxia Enhances Cell Properties of Human Mesenchymal Stem Cells

Atmospheric (in vitro) oxygen pressure is around 150 mm Hg (20% O₂), whereas physiologic (in vivo) oxygen pressure ranges between 5 and 50 mm Hg (0.7–7% O₂). The normoxic environment in cell culture does not refer to a physiological stem cell niche. The aim of this study is to investigate the effect of oxygen concentration on cell properties of human mesenchymal stem cells (MSCs). We analyzed cell proliferation rate, senescence, immunophenotype, stemness gene expression and differentiation potency with human urine stem cells (USCs), dental pulp stem cells (DPSCs), amniotic fluid stem cells (AFSCs), and bone marrow stromal cells (BMSCs). USCs, DPSCs, AFSCs and BMSCs were cultured under either 5% O₂ hypoxic or 20% O₂ normoxic conditions for 5 days. MSCs cultured under hypoxia showed significantly increased proliferation rate and high percentage of S-phase cells, compared to normoxic condition. In real-time PCR assay, the cells cultured under hypoxia expressed higher level of Oct4, C-Myc, Nanog, Nestin and HIF-1α. In immunophenotype analysis, MSCs cultured under hypoxia maintained higher level of the MSC surface markers, and lower hematopoietic markers. Senescence was inhibited under hypoxia. Hypoxia enhances osteogenic differentiation efficiency compared to normoxia. Hypoxia showed enhanced cell proliferation rate, retention of stem cell properties, inhibition of senescence, and increased differentiation ability compared to normoxia.

Microambiente medular en la leucemia mieloide crónica: su relación con la enfermedad y la respuesta al tratamiento / Bone marrow microenvironment in chronic myeloid leukemia: Implications for disease physiopathology and response to treatment

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm related to the presence of the BCR-ABL1 fusion gene, linked to t (9;22) (q34;q11). It is originated from an abnormal hematopoietic stem cell, which is characterized as its normal counterparts by long-term self-renewal and multi-lineage differentiation. Both leukemic and quiescent normal hematopoietic stem cells preferentially reside in the osteoblastic niche. Mesenchymal stromal cells (MSC) are located near them, playing a critical role in their regulation. Currently, with tyrosine kinase inhibitor (TKI) therapy, long term clinical responses are achieved in most CML cases. However, late treatment failures may be observed related to the persistence of leukemic stem cells. The interactions between the leukemic stem cell and the microenvironment may be responsible in part for these events. We review the interactions between the leukemic stem cell and BM stroma and its potential clinical and therapeutic implications.

Mapeamento de potencial nicho neurogênico no lobo temporal humano / Mapping of potential neurogenic niche in the human temporal lobe

No final do século 19, o neurônio foi descrito como a unidade funcional básica do sistema nervoso e sua formação era considerada inexistente na fase adulta, explicando a ausência de recuperação significativa em doenças neurológicas. Evidências de geração de neurônios em mamíferos adultos surgiram na década de 1960 e foram confirmadas três décadas depois. Atualmente, predomina a visão de que mamíferos adultos possuem dois nichos neurogênicos independentes: a zona subventricular (ZSV) e a zona subgranular (ZSG) do giro denteado. No entanto, a existência de nichos neurogênicos em humanos adultos é controversa. Nossa hipótese foi de que o mapeamento de nichos neurogênicos no lobo temporal humano poderia esclarecer aspectos sobre a neurogênese adulta. A detecção destes nichos foi buscada em 28 lobos temporais através de imuno-histoquímica para nestina, o marcador mais comum de células-tronco neurais, que são aquelas capazes de se autorrenovar e de gerar novas células neurais. A neurogênese foi pesquisada no hipocampo pelo uso de DCX (do inglês "doublecortin"), o principal marcador de neuroblastos e neurônios imaturos. Nestina foi observada em uma camada contínua formada pela ZSV, zona subpial do lobo temporal medial e ZSG, terminando no subículo. A partir do subículo, uma intensa expressão de DCX ocorreu através da principal via eferente do hipocampo até a fímbria. A visão panorâmica das marcações por nestina e DCX mostrava em conjunto uma linha que circundava as estruturas límbicas do lobo temporal. Por isto, foi denominada linha externa de células do sistema límbico (LECEL). Uma possível explicação para os resultados é que a LECEL seja um nicho neurogênico no qual a ZSV, a zona subpial do lobo temporal medial e a ZSG formam uma unidade contendo células-tronco neurais que se diferenciam em neurônios no subículo. Curiosamente, a área identificada previamente como sendo a corrente migratória rostral humana (formada por células neurais imaturas migrando a partir da...

At the end of the 19th century, the neuron was described as the basic functional unit of the nervous system. The formation of neurons was thought to be absent in adulthood, thus explaining the lack of significant recovery from neurological diseases. Evidence for the generation of neurons in adult mammals was reported in the 1960s and confirmed three decades later. Currently, the prevailing view is that adult mammals harbour two neurogenic niches: the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ). Nonetheless, the existence of these niches in adult humans is controversial. We hypothesised that mapping neurogenic niches in the human temporal lobe could clarify this issue. The presence of neurogenic niches was examined in 28 temporal lobes via immunostaining for nestin, the most common marker for neural stem cells, which are cells with the capacities of self-renewal and the generation of neural cells. The presence of neurogenesis was examined in the hippocampus with doublecortin (DCX), a prominent marker for neuroblasts and immature neurons. Nestin was observed in a continuous layer that was formed by the SVZ, the subpial zone of the medial temporal lobe and the SGZ, terminating in the subiculum. In the subiculum, remarkable DCX expression was observed through the principal efferent pathway of the hippocampus to the fimbria. A panoramic view of nestin and DCX staining collectively displayed a line that surrounded the limbic structures of the temporal lobe. Hence, we termed it the external line of cells of the limbic system (EXCEL). A possible explanation for the results is that the EXCEL is a neurogenic niche, in which the SVZ, the subpial zone of the medial temporal lobe and the SGZ form a unit containing neural stem cells that differentiate into neurons in the subiculum. Curiously, the area previously identified as the human rostral migratory stream (formed by immature neural cells that migrate from the SVZ of the frontal horn)...

Tracking the Fate of Muscle-derived Stem Cells: an Insight into the Distribution and Mode of Action

PURPOSE: To examine the fate of muscle-derived stem cells (MDSC) after injection into different host conditions and provide an insight for their mechanism of action. MATERIALS AND METHODS: MDSCs differentiated in vitro towards the endothelial lineage and transfected with lentivirus tagged with green fluorescent protein (GFP) were injected into two animal models mimicking vascular diseases: hindlimb ischemia and carotid injury models. Injected cells were tracked at the site of injection and in remote organs by harvesting the respective tissues at different time intervals and performing immunofluorescent histological analyses. Stem cell survival was quantified at the site of injection for up to 4 weeks. RESULTS: MDSCs were successfully tagged with fluorescent material GFP and showed successful implantation into the respective injection sites. These cells showed a higher affinity to implant in blood vessel walls as shown by double fluorescent co-stain with CD31. Quantification of stem cell survival showed a time-dependent decrease from day 3 to 4 weeks (survival rate normalized against day 3 was 72.0% at 1 week, 26.8% at 2 weeks and 2.4% at 4 weeks). Stem cells were also fo und in distant organs, especially the kidneys and liver, which survived up to 4 weeks. CONCLUSION: MDSCs were successfully tracked in different vascular disease models, and their fate was assessed in terms of cell survival and distribution. Better understanding of the donor cell properties, including their interaction with the host conditions and their mechanism of action, are needed to enhance cell survival and achieve improved outcomes.

Effects of pulsed electromagnetic fields on homing genes of mouse osteoblasts / 国际生物医学工程杂志

Objective To investigate the effects of pulsed electromagnetic fields (PEMF) on homing and proliferation-related genes of mouse osteoblasts.Methods 9 week-old C57BL/6 mice were treated with PEMF (70 Hz,1 mT) for 4～5 weeks,while mice in control group didn't not receive PEMF.Bone marrow cells of femurs and tibias were flushed out,and the bones were minced and incubated at 37 ℃ with a type Ⅰ collagenase.Bone associated mononuclear cells (MNCs) were isolated via density centrifugation with Lymphoprep.Magnetic cell sorting was used before flow-cytometric sorting,and the ALCAM+Sca-1-cells were collected.The homing and proliferation-related genes expressed in ALCAM+Sca-1-cells were detected with high throughput microarray and RT-PCR.Results The expression of Jag1 and Ang-1 in mouse osteoblasts increased under the effects of PEMF.Conclusions PEMF may have regulation effects on HSC (hematopoietic stem cell) survival through modulating the homing and proliferationrelated genes in ALCAM+Sca-1-osteoblasts.

Stem cells and their niches: importance in tissue engineering applied to dentistry / Células-tronco e seus nichos: importância na engenharia de tecidos aplicada à odontologia

Niches are special microenvironments in tissue where stem cells are located. At these sites, which are a compound of stromal cells, extracellular matrix and soluble factors, complex molecular interactions that maintain the essential properties of stem cells occur, such as self-renewal and differentiation into multiple lineages, according to the organism?s needs. Some adult stem cell niches have already been described, but the majority of them remain unclear, including the dental pulp stem cell niches. Dental pulp stem cells have been isolated from deciduous and permanent teeth and have the potential to self-renew and differentiate. However, little is known about the exact anatomic location of these cells, and the relationship between stem cells and surrounding cells in dental pulp. Understanding how stem cells behave in the niche is extremely important in order to extract these cells from their natural habitat, expand them in vitro and transplant the stem cells back to the patient, to repair and/or regenerate tissues and organs, with no risks to the individual?s integrity. Likewise, the knowledge of stem cell biology is crucial to the development of stem cell therapies, based on tissue engineering applied to dentistry, seeking the regeneration of dental tissues damaged or lost by caries, trauma or genetic diseases.

Os nichos são microambientes especiais nos tecidos onde células-tronco de várias origens estão localizadas. Nestes sítios específicos, formados por vários tipos de células, matriz extracelular e fatores solúveis, complexas interações moleculares ocorrem para que a célulatroncomantenha sua capacidade de autorrenovação e permaneça no seu estado indiferenciado ou se especialize em determinada linhagemcelular, atendendo desta maneira as necessidades do organismo. Alguns nichos de células-tronco adultas já foram descritos, embora a maioriapermaneça desconhecida, como o das células-tronco pulpares. As células-tronco pulpares, já foram isoladas tanto de dentes decíduos comode permanentes e apresentam as características essenciais de uma célula-tronco, como capacidade de autorrenovação e multi-diferenciação.Apesar disso, pouco se sabe a respeito da localização anatômica destas células na polpa, assim como as possíveis interações funcionais entreas células-tronco pulpares e as células do estroma circundante. O entendimento de como as células-tronco interagem com o microambienteonde estão inseridas é essencial para que se possa extrair as mesmas do seu habitat natural, cultivá-las in vitro e aplicá-las em diferentessítios para que promovam o reparo e/ou regeneração de tecidos e órgãos, sem que isso represente um risco à integridade do organismo. Da mesma forma, o conhecimento de como estas células se comportam e respondem ao meio é fundamental para o desenvolvimento de terapias baseadas na utilização de células-tronco, que através da engenharia de tecidos aplicada à odontologia, visa à reestruturação de tecidos dentários danificados e/ou perdidos por cárie, trauma ou distúrbios genéticos.

Molecular Culprits Generating Brain Tumor Stem Cells

Despite current advances in multimodality therapies, such as surgery, radiotherapy, and chemotherapy, the outcome for patients with high-grade glioma remains fatal. Understanding how glioma cells resist various therapies may provide opportunities for developing new therapies. Accumulating evidence suggests that the main obstacle for successfully treating high-grade glioma is the existence of brain tumor stem cells (BTSCs), which share a number of cellular properties with adult stem cells, such as self-renewal and multipotent differentiation capabilities. Owing to their resistance to standard therapy coupled with their infiltrative nature, BTSCs are a primary cause of tumor recurrence post-therapy. Therefore, BTSCs are thought to be the main glioma cells representing a novel therapeutic target and should be eliminated to obtain successful treatment outcomes.

Study on cardiac stem cell niche and its biological characteristics in vitro / 中华胸心血管外科杂志

Objective Endogenous cadiac stem cells become the famous star in the cardiac regeneration field in recent years.But the biological behavior of cardiac stem cells has not been fully explained clearly.This experiment intends to research the cardiac stem cell niche and its characteristics,as well as the succinct method for cardiac stem cells (Cardiospheres derived cells) culture in vitro.Methods Hearts from four 8-week-old SD rats were cut into pieces less than I mm3.Then,the small tissues were digested by trypsogen and collagenase in turn.And the undigested myocardium was collected and cultured in media until the de novo cells bespread the pertri-dish bottom.The 3rd generation of cardiac stem cells were harvested and prepared for immunofluorescence to detect the expression of related molecular markers,so as to identify the differentiation of these stem cells.Then,the cultural tissues were collected and embedded in paraffin,and the H-E staining,Masson staining,immunoflnorescence and TUNEL apoptosis assay were carried out.In addition,EdU was added into the medium and cuhured for 72 hours before the cultural tissues were harvested so as to label the proliferative cells.Results After 7 ～ 10 days in culture,some small,round and phase-bright cells aresed from the adherent plated tissue.These cells had the spontaneous differentiation potency in vitro culture.The immunofluorescence shows mitotic phase cells were c-kit positive,and most of passage cells were α-sarcomeric actin 、cardiac troponin T、flk-1 and vemintin positive while CD90 negative,but few of them were α-smooth muscle actin 、conexin-43 、CD31 and CD45 positive.There were lots of newborn cells grow exuberantly while accompanied apoptosis of myocardial cells in the paraffin section.The myocardial collagen remodels in the cultural myocardial tissues at the same time.The proliferative cells in the cultural tissues were EdU positive.Throe myocardium within the cultural tissues were MMP-2、MMP-9 and TGF-β1 positive,On the contrary,the proliferative cells were almost negative when stained with those antibodies.Conclusion Conclusions It's a convenient way to harvest cardiac stem cells by the use of myocarlial? tissues cultured in vitro.Those stem cells grow and proliferate in the cultural myocardial tissues accompanied with the degradation of extracellular matrix.Cardiospheres derived stem cells have the spontaneous differentiation potency when cultured in vito,and should be a promising? seed cells for stem cell therapy.

Intramarrow injection of beta-catenin-activated, but not naive mesenchymal stromal cells stimulates self-renewal of hematopoietic stem cells in bone marrow

Bone marrow mesenchymal stromal cells (MSCs) have been implicated in the microenvironmental support of hematopoietic stem cells (HSCs) and often co-transplanted with HSCs to facilitate recovery of ablated bone marrows. However, the precise effect of transplanted MSCs on HSC regeneration remains unclear because the kinetics of HSC self-renewal in vivo after co-transplantation has not been monitored. In this study, we examined the effects of intrafemoral injection of MSCs on HSC self-renewal in rigorous competitive repopulating unit (CRU) assays using congenic transplantation models in which stromal progenitors (CFU-F) were ablated by irradiation. Interestingly, naive MSCs injected into femur contributed to the reconstitution of a stromal niche in the ablated bone marrows, but did not exert a stimulatory effect on the in-vivo self-renewal of co-transplanted HSCs regardless of the transplantation methods. In contrast, HSC self-renewal was four-fold higher in bone marrows intrafemorally injected with beta-catenin-activated MSCs. These results reveal that naive MSCs lack a stimulatory effect on HSC self-renewal in-vivo and that stroma must be activated during recoveries of bone marrows. Stromal targeting of wnt/beta-catenin signals may be a strategy to activate such a stem cell niche for efficient regeneration of bone marrow HSCs.