Generation of male germ cells from induced pluripotent stem cells （iPS cells）： an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem （iPS） cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitrodifferentiation and in vivotransplantation. Embryoid bodies （EBs） were obtained from iPS cells using leukaemia inhibitor factor （LIF）-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in StraSand Vasa mRNA in the EBs derived from iPS cells, iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells （SSCs）, as evidenced by their expression of VASA, as well as CDH1 and GFRal, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Derivation of male germ cells from induced pluripotent stem （iPS） cells： a novel and crucial source for generating male gametes

One of the most significant findings in recent stem cell research is the establishment of the induced pluripotent stem （iPS） cells, because they could have critical implications in both regenerative and repro- ductive medicine. Male gametes play a crucial role in transmitting genetic information to subsequent generations, and notably there are more and more patients with azoospermia, due to genetic and environmental factors. Recent advancements on generation of male gametes from human iPS cells would bring great promise to produce patient own male gametes for treating male infertility and provide an excellent platform for unveiling molecular mechanisms of male germ cell development.

膝骨关节炎软骨基质刚度对线粒体形态异质性的影响

目的研究膝骨关节炎(osteoarthritis,OA)胫骨平台不同区域基质刚度的差异,及其对软骨和线粒体形态的影响。方法获取OA胫骨平台软骨标本用于纳米压痕测试、透射电镜拍摄以及组织学分析。利用纳米压痕检测OA胫骨平台不同区域软骨基质刚度,通过透射电镜观察不同区域软骨线粒体形态,定量分析线粒体平面面积、形状、嵴体积密度的变化。通过组织学染色观察OA胫骨平台不同区域软骨损伤情况。结果OA胫骨平台软骨损伤具有区域异质性,内侧胫骨平台软骨和线粒体损伤更重,同时基质刚度更高。OA评分与基质刚度呈正相关。OA评分与线粒体形态之间也有显著的相关性:OA评分越高,线粒体平面面积越大、越圆以及嵴体积密度越低。结论胫骨平台不同区域的差异揭示了软骨基质刚度、OA评分和线粒体形态学参数之间的相关性,软骨基质刚度增加可能是造成软骨细胞线粒体损伤的主要原因,进而加重了OA进展。

Vitamin E in the management of pancreatic cancer: A scoping review

Pancreatic cancer is the leading cause of cancer mortality worldwide.Research investigating effective management strategies for pancreatic cancer is ongoing.Vitamin E,consisting of both tocopherol and tocotrienol,has demonstrated debatable effects on pancreatic cancer cells.Therefore,this scoping review aims to summarize the effects of vitamin E on pancreatic cancer.In October 2022,a literature search was conducted using PubMed and Scopus since their inception.Original studies on the effects of vitamin E on pancreatic cancer,including cell cultures,animal models and human clinical trials,were considered for this review.The literature search found 75 articles on this topic,but only 24 articles met the inclusion criteria.The available evidence showed that vitamin E modulated proliferation,cell death,angiogenesis,metastasis and inflammation in pancreatic cancer cells.However,the safety and bioavailability concerns remain to be answered with more extensive preclinical and clinical studies.More in-depth analysis is necessary to investigate further the role of vitamin E in the management of pancreatic cancers.

ASGR1在肝细胞癌中的意义及机制研究

目的·探究去唾液酸糖蛋白受体1(asialoglycoprotein receptor 1,ASGR1)在肝细胞癌(hepatocellular carcinoma,HCC)中的意义及潜在机制。方法·通过R语言分析癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中ASGR1在肝癌患者中的表达情况并绘制相关生存曲线。利用人类蛋白质图谱(The Human Protein Atlas,HPA)数据库获得人体正常肝组织和肝癌组织的免疫组织化学(immunohistochemistry,IHC)数据来分析ASGR1的蛋白表达情况。利用流体动力学尾静脉注射(hydrodynamic tail vein injection,HTVI)递送方法,在免疫完全的小鼠肝脏中敲除Asgr1探究其在体内的致瘤功能,并通过蛋白免疫印迹法(Western blotting,WB)验证基因敲除效率。利用R语言进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析及相关性分析,利用基因探针富集(Gene Set Enrichment Analysis,GSEA)软件进行GSEA hallmark相关通路分析,利用实时荧光定量PCR(quantitative real-time PCR,qPCR)在小鼠肝癌组织中验证糖酵解关键基因表达水平。结果·ASGR1在肝癌组织中显著低表达,在肝癌患者中ASGR1的低表达与患者较差的总体生存期(overall survival,OS)、无疾病间隔(disease free interval,DFI)、无进展间隔期(progression free interval,PFI)和疾病特异性生存期(disease specific survival,DSS)相关;肿瘤分级程度越高的肝癌患者ASGR1基因表达水平越低。人体正常肝组织ASGR1蛋白的表达显著高于肝癌组织。在免疫完全的肝细胞癌小鼠模型中,小鼠内源性Asgr1敲除可增加肝组织中肿瘤结节的大小和数量。TCGA数据库中ASGR1低表达组肝癌患者富集到多条癌症及代谢相关通路,ASGR1表达与部分糖酵解关键基因表达呈负相关,Asgr1敲除组的小鼠肝癌组织中糖酵解水平高于对照组,提示ASGR1低表达很可能促进肝癌的生长发展,加强代谢重编程促进肿瘤的合成代谢发展。结论·ASGR1在肝癌患者中表达显著降低,与患者的预后呈正相关;小鼠体内敲除Asgr1可促进肝细胞癌的发生发展;ASGR1可以作为肝癌预后不良的潜在生物标志物和潜在治疗新靶点。

EDA基因新剪切突变导致X连锁少汗性外胚层发育不良

目的·检测一X连锁少汗性外胚层发育不良家系EDA基因突变位点。方法·提取先证者及其家系共13位成员外周血基因组DNA,PCR扩增EDA基因编码区的8个外显子及其2端侧翼序列并测序,明确突变位点。结果·先证者及其患病哥哥EDA基因6号内含子剪切供体发生T>A突变,而家系无其他外胚层发育不良临床表现成员,均无该位点突变。结论·该家系中IVS 6+2T>A(g.69250372,Xq22.3)突变为剪切致病突变,属国内外首报,是X连锁少汗性外胚层发育不良的新致病突变。该突变可用于遗传咨询和产前诊断,减少出生缺陷。

组蛋白变体macroH2A1调控肿瘤增殖的研究进展

组蛋白变体不仅为染色质提供独特的功能,而且通过特殊的沉积和清除机制参与基因表达调控。macroH2A1是主要的组蛋白变体之一,早期发现其与女性X染色体失活和转录抑制有关,但目前研究发现其在特定的条件下可通过调控细胞衰老和代谢抑制肿瘤增殖。该文基于目前研究进展阐述macroH2A1调控基因表达的分子机制及其在肿瘤进展中的作用。

Targeting ferroptosis suppresses osteocyte glucolipotoxicity and alleviates diabetic osteoporosis

Diabetic osteoporosis(DOP) is the leading complication continuously threatening the bone health of patients with diabetes. A key pathogenic factor in DOP is loss of osteocyte viability. However, the mechanism of osteocyte death remains unclear. Here, we identified ferroptosis, which is iron-dependent programmed cell death, as a critical mechanism of osteocyte death in murine models of DOP. The diabetic microenvironment significantly enhanced osteocyte ferroptosis in vitro, as shown by the substantial lipid peroxidation, iron overload, and aberrant activation of the ferroptosis pathway. RNA sequencing showed that heme oxygenase-1(HO-1) expression was notably upregulated in ferroptotic osteocytes. Further findings revealed that HO-1 was essential for osteocyte ferroptosis in DOP and that its promoter activity was controlled by the interaction between the upstream NRF2 and c-JUN transcription factors. Targeting ferroptosis or HO-1 efficiently rescued osteocyte death in DOP by disrupting the vicious cycle between lipid peroxidation and HO-1 activation, eventually ameliorating trabecular deterioration. Our study provides insight into DOP pathogenesis, and our results provide a mechanism-based strategy for clinical DOP treatment.

Plasticity of male germline stem cells and their applications in reproductive and regenerative medicine

Spermatogonial stem cells （SSCs）, also known as male germline stem cells, are a small subpopulation of type A spermatogonia with the potential of self-renewal to maintain stem cell pool and differentiation into spermatids in mammalian testis. SSCs are previously regarded as the unipotent stem cells since they can only give rise to sperm within the seminiferous tubules. However, this concept has recently been challenged because numerous studies have demonstrated that SSCs cultured with growth factors can acquire pluripotency to become embryonic stem-like cells. The in vivo and in vitro studies from peers and us have clearly revealed that SSCs can directly transdifferentiate into morphologic, phenotypic, and functional cells of other lineages. Direct conversion to the cells of other tissues has important significance for regenerative medicine. SSCs from azoospermia patients could be induced to differentiate into spermatids with fertilization and developmental potentials. As such, SSCs could have significant applications in both reproductive and regenerative medicine due to their unique and great potentials. In this review, we address the important plasticity of SSCs, with focuses on their self-renewal, differentiation, dedifferentiation, transdifferentiation, and translational medicine studies.

Characterization, isolation, and culture of spermatogonial stem cells in Macaca fascicularis

Spermatogonial stem cells(SSCs)have great applications in both reproductive and regenerative medicine.Primates including monkeys are very similar to humans with regard to physiology and pathology.Nevertheless,little is known about the isolation,the characteristics,and the culture of primate SSCs.This study was designed to identify,isolate,and culture monkey SSCs.Immunocytochemistry was used to identify markers for monkey SSCs.Glial cell line-derived neurotrophic factor family receptor alpha-1(GFRAl)-enriched spermatogonia were isolated from monkeys,namely Macaca fascicularis(M.fascicularis),by two-step enzymatic digestion and magnetic-activated cell sorting,and they were cultured on precoated plates in the conditioned medium.Reverse transcription-polymerase chain reaction(RT-PCR),immunocytochemistry,and RNA sequencing were used to compare phenotype and transcriptomes in GFRAl-enriched spermatogonia between 0 day and 14 days of culture,and xenotransplantation was performed to evaluate the function of GFRAl-enriched spermatogonia.SSCs shared some phenotypes with rodent and human SSCs.GFRAl-enriched spermatogonia with high purity and viability were isolated from M.fascicularis testes.The freshly isolated cells expressed numerous markers for rodent SSCs,and they were cultured for 14 days.The expression of numerous SSC markers was maintained during the cultivation of GFRAl-enriched spermatogonia.RNA sequencing reflected a 97.3%similarity in global gene profiles between 0 day and 14 days of culture.The xenotransplantation assay indicated that the GFRAl-enriched spermatogonia formed colonies and proliferated in vivo in the recipient c-Kitw/w(W)mutant mice.Collectively,GFRAl-enriched spermatogonia are monkey SSCs phenotypically both in vitro and in vivo.This study suggests that monkey might provide an alternative to human SSCs for basic research and application in human diseases.

Targeting pancreatic cancer immune evasion by inhibiting histone deacetylases

The immune system plays a vital role in maintaining the delicate balance between immune recognition and tumor development.Regardless,it is not uncommon that cancerous cells can intelligently acquire abilities to bypass the antitumor immune responses,thus allowing continuous tumor growth and development.Immune evasion has emerged as a significant factor contributing to the progression and immune resistance of pancreatic cancer.Compared with other cancers,pancreatic cancer has a tumor microenvironment that can resist most treatment modalities,including emerging immunotherapy.Sadly,the use of immunotherapy has yet to bring significant clinical breakthrough among pancreatic cancer patients,suggesting that pancreatic cancer has successfully evaded immunomodulation.In this review,we summarize the impact of genetic alteration and epigenetic modification(especially histone deacetylases,HDAC)on immune evasion in pancreatic cancer.HDAC overexpression significantly suppresses tumor suppressor genes,contributing to tumor growth and progression.We review the evidence on HDAC inhibitors in tumor eradication,improving T cells activation,restoring tumor immunogenicity,and modulating programmed death 1 interaction.We provide our perspective in targeting HDAC as a strategy to reverse immune evasion in pancreatic cancer.

PUMA facilitates EMI 1-promoted cytoplasmic Rad51 ubiquitination and inhibits DNA repair in stem and progenitor cells

Maintenance of genetic stability via proper DNA repair in stem and progenitor cells is essential for the tissue repair and regeneration,while preventing cell transformation after damage.Loss of PUMA dramatically increases the survival of mice after exposure to a lethal dose of ionizing radiation(IR),while without promoting tumorigenesis in the long-term survivors.This finding suggests that PUMA(p53 upregulated modulator of apoptosis)may have a function other than regulates apoptosis.Here,we identify a novel role of PUMA in regulation of DNA repair in embryonic or induced pluripotent stem cells(PSCs)and immortalized hematopoietic progenitor cells(HPCs)after IR.We found that PUMA-deficient PSCs and HPCs exhibited a significant higher doublestrand break(DSB)DNA repair activity via Rad51-mediated homologous recombination(HR).This is because PUMA can be associated with early mitotic Inhibitor 1(EMI1)and Rad51 in the cytoplasm to facilitate EMI1-mediated cytoplasmic Rad51 ubiquitination and degradation,thereby inhibiting Rad51 nuclear translocation and HR DNA repair.Our data demonstrate that PUMA acts as a repressor for DSB DNA repair and thus offers a new rationale for therapeutic targeting of PUMA in regenerative cells in the context of DNA damage.

脑胶质瘤与铁死亡研究进展

基于脑胶质瘤发病过程中的信号通路寻找治疗靶点成为探索脑胶质瘤新型治疗方式的重点领域。铁死亡是一种由脂质过氧化驱动的铁依赖性的细胞死亡方式,已被证实其参与多种肿瘤(包括脑胶质瘤)的发生及进展进程。全文回顾铁死亡的经典代谢途径,总结已有研究中常用的铁死亡诱导剂、抑制剂以及脂质过氧化的评估方法,梳理铁死亡与脑胶质瘤相关研究中的最新发现,包括铁死亡关键调节因子在脑胶质瘤中的表达情况、脑胶质瘤生理环境对铁死亡的影响及脑胶质瘤治疗药物与铁死亡的关系等,为脑胶质瘤的靶向治疗药物研发提供参考。

Aging hallmarks of the primate ovary revealed by spatiotemporal transcriptomics

The ovary is indispensable for female reproduction,and its age-dependent functional decline is the primary cause of infertility.However,the molecular basis of ovarian aging in higher vertebrates remains poorly understood.Herein,we apply spatiotemporal transcriptomics to benchmark architecture organization as well as cellular and molecular determinants in young primate ovaries and compare these to aged primate ovaries.From a global view,somatic cells within the non-follicle region undergo more pronounced transcriptional fluctuation relative to those in the follicle region,likely constituting a hostile microenvironment that facilitates ovarian aging.Further,we uncovered that inflammation,the senescent-associated secretory phenotype,senescence,and fibrosis are the likely primary contributors to ovarian aging(PCOA).Of note,we identified spatial co-localization between a PCOA-featured spot and an unappreciated MT2(Metallothionein 2)highly expressing spot(MT2^(high))characterized by high levels of inflammation,potentially serving as an aging hotspot in the primate ovary.Moreover,with advanced age,a subpopulation of MT2^(high)accumulates,likely disseminating and amplifying the senescent signal outward.Our study establishes the first primate spatiotemporal transcriptomic atlas,advancing our understanding of mechanistic determinants underpinning primate ovarian aging and unraveling potential biomarkers and therapeutic targets for aging and age-associated human ovarian disorders.

NODAL secreted by male germ cells regulates the proliferation and function of human Sertoli cells from obstructive azoospermia and nonobstructive azoospermia patients

This study was designed to explore the regulatory effects of male germ cell secreting factor NODAL on Sertoli cell fate decisions from obstructive azoospermia （OA） and nonobstructive azoospermia （NOA） patients. Human Sertoli cells and male germ cells were isolated using two-step enzymatic digestion and SATPUT from testes of azoospermia patients. Expression of NODAL and its multiple receptors in human Sertoli cells and male germ cells were characterized by reverse transcription-polymerase chain reaction （RT-PCRI and immunochemistry. Human recombinant NODAL and its receptor inhibitor SB431542 were employed to probe their effect on the proliferation of Sertoli cells using the CCK-8 assay. Quantitative PCR and Western blots were utilized to assess the expression of Sertoli cell functional genes and proteins. NODAL was found to be expressed in male germ cells but not in Sertoli cells, whereas its receptors ALK4, ALK7, and ACTR-IIB were detected in Sertoli cells and germ cells, suggesting that NODAL plays a regulatory role in Sertoli cells and germ cells via a paracrine and autocrine pathway, respectively. Human recombinant NODAL could promote the proliferation of human Sertoli cells. The expression of cell cycle regulators, including CYCLIN A, CYCLIN D1 and CYCLIN E, was not remarkably affected by NODAL signaling. NODAL enhanced the expression of essential growth factors, including GDNF, SCF, and BMP4, whereas SB431542 decreased their levels. There was not homogeneity of genes changes by NODAL treatment in Sertoli cells from OA and Sertoli cell-only syndrome （SCO） patients. Collectively, this study demonstrates that NODAL produced by human male germ cells regulates proliferation and numerous gene expression of Sertoli cells.

Ginsenoside Rb1 inhibits oxidative stress-induced ovarian granulosa cell injury through Akt-FoxO1 interaction

Ginsenoside Rb1 shows a strong antioxidant effect and has potential activation effects on Akt.The aim of the present study was to investigate the protective effect of Rb1 on age-related ovarian granulosa cell injury.Ovarian granulosa cells(GCs)were obtained from 50 young women(≤30 years)and 50 aged women(≥38 years)at an IVF center.Young and aged ICR mice were administered with or without Rb1(10 mg kg^(-1),i.p.)for 2 weeks.The protective effects of Rb1 were investigated and the role of Rb1 on the modulation of Akt-FoxO1 interaction was determined with immunofluorescence,Western blotting,immunoprecipitation,si RNA silencing and pharmacological inhibitor.Rb1 effectively decreased LDH and MDA,and reversed the apoptotic-related protein levels in h GL cells from old patients.Similar results were found in mice.In addition,the mitochondrial membrane potential was restored and the overaccumulation of ROS was reversed by Rb1.Rb1 preserved peroxide-impaired Akt activation,to some extent,by increasing phosphorylation at Ser473.Rb1 also facilitated p-Akt binding to FoxO1 and promoted the phosphorylation of FoxO1.Si RNA silencing of Akt,Akt inhibitor LY294002,and FoxO1 inhibitor AS1842856 attenuated the effects of Rb1.Ginsenoside Rb1 inhibits age-related GCs oxidative damage by activating Akt phosphorylation at Ser473 and by further interaction with FoxO1.

Non-catalytic roles for TET1 protein negatively regulating neuronal differentiation through srGAP3 in neuroblastoma cells

The methylcytosine dioxygenases TET proteins （TET1, TET2, and TET3） play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA （valproic acid）-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neu- ronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demon- strated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.

脂肪间充质干细胞治疗糖尿病及其慢性并发症的研究进展

糖尿病是常见的慢性病之一,持续的高血糖状态可以引起大血管系统和微血管系统的多种慢性并发症,是造成糖尿病患者死亡的主要原因。脂肪间充质干细胞(ADSCs)具有取材方便、分离简单和易于培养等特点,在治疗糖尿病及其慢性并发症方面表现出广阔应用潜力,现将近年来ADSCs在糖尿病及其慢性并发症治疗中取得的研究进展进行综述。

源于干细胞的囊胚样结构:研究现状与展望

由于样本和伦理的限制,人类早期胚胎发育中仍有许多未完全阐明的问题.而类囊胚的出现使得构建体外模拟胚胎发育,尤其是植入前、植入和早期植入后过程的类器官系统成为可能.但初始方案的类囊胚构建效率较低,且对受精囊胚的模拟还存在较大差异,因此研究人员探索利用不同类型细胞以及不同诱导体系,提高类囊胚的构建效率,并获得了在形态学和转录组水平上更类似于受精囊胚的模型.除了转录组分析,蛋白组和翻译后修饰的组学也被用于探索类囊胚形成的分子机制和与受精囊胚的相似性.同时,类囊胚已经被作为研究模型探索不同基因组特征对胚胎发育的影响以及潜在作用机制.因此,我们总结了近期小鼠和人类的类囊胚模型的研究进展及新型全能样细胞参与构建类囊胚的未来前景.

Single-nucleus profiling unveils a geroprotective role of the Foxo3 in primate skeletal muscle aging

Age-dependent loss of skeletal muscle mass and function is a feature of sarcopenia,and increases the risk of many aging-related metabolic diseases.Here,we report phenotypic and single-nucleus transcriptomic analyses of non-human primate skeletal muscle aging.A higher transcriptional fluctuation was observed in myonuclei relative to other interstitial cell types,indicating a higher susceptibility of skeletal muscle fiber to aging.We found a downregulation of Foxo3 in aged primate skeletal muscle,and identi-fied FOxo3 as a hub transcription factor maintaining skeletal muscle homeostasis.Through the establishment of a complementary experimental pipeline based on a human pluripotent stem cell-derived myotube model,we revealed that silence of Foxo3 accelerates human myotube senescence,whereas genetic activation of endogenous FOxO3 alleviates human myotube aging.Altogether,based on a combination of monkey skeletal muscle and human myotube aging research models,we unraveled the pivotal role of the FOxO3 in safeguarding primate skeletal muscle from aging,providing a comprehensive resource for the development of clinical diagnosis and targeted therapeutic interventions against human skeletal muscle aging and the onset of sarcopenia along with aging-relateddisorders.