Pub Date : 2024-04-08DOI: 10.1007/s00441-024-03890-x
Wataru Kudo, Retsu Mitsui, Hikaru Hashitani
The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl− channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential −60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl− concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.
{"title":"Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis","authors":"Wataru Kudo, Retsu Mitsui, Hikaru Hashitani","doi":"10.1007/s00441-024-03890-x","DOIUrl":"https://doi.org/10.1007/s00441-024-03890-x","url":null,"abstract":"<p>The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca<sup>2+</sup>-activated Cl<sup>−</sup> channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential −60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl<sup>−</sup> concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca<sup>2+</sup>-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα<sup>+</sup>, ANO1<sup>+</sup> specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca<sup>2+</sup> release from intracellular Ca<sup>2+</sup> store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca<sup>2+</sup> channels are activated to develop SPCs.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140568159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The endoplasmic reticulum (ER) extends throughout a cell and plays a critical role in maintaining cellular homeostasis. Changes in ER shape could provide a clue to explore the mechanisms that underlie the fate determination of neurons after axon injury because the ER drastically changes its morphology under neuronal stress to maintain cellular homeostasis and recover from damage. Because of their tiny structures and richness in the soma, the detailed morphology of the ER and its dynamics have not been well analysed. In this study, the focused ion beam/scanning electron microscopy (FIB/SEM) analysis was performed to explore the ultra-structures of the ER in the somata of motor neuron with axon regenerative injury models. In normal motor neurons, ER in the somata is abundantly localised near the perinucleus and represents lamella-like structures. After injury, analysis of the ER volume and ER branching points indicated a collapse of the normal distribution and a transformation from lamella-like structures to mesh-like structures. Furthermore, accompanied by ER accumulation near the plasma membrane (PM), the contact between the ER and PM (ER-PM contacts) significantly increased after injury. The accumulation of extended-synaptotagmin 1 (E-Syt1), a tethering protein of the ER and PM that regulates Ca2+-dependent lipid transfer, was also identified by immunohistochemistry and quantitative Real-time PCR after injury. These morphological alterations of ER and the increase in ER-PM contacts may be crucial events that occur in motor neurons as a resilient response for the survival after axonal injury.
{"title":"Dynamic changes in endoplasmic reticulum morphology and its contact with the plasma membrane in motor neurons in response to nerve injury.","authors":"Mahmoud Elgendy, Hiromi Tamada, Takaya Taira, Yuma Iio, Akinobu Kawamura, Ayusa Kunogi, Yuka Mizutani, Hiroshi Kiyama","doi":"10.1007/s00441-024-03858-x","DOIUrl":"10.1007/s00441-024-03858-x","url":null,"abstract":"<p><p>The endoplasmic reticulum (ER) extends throughout a cell and plays a critical role in maintaining cellular homeostasis. Changes in ER shape could provide a clue to explore the mechanisms that underlie the fate determination of neurons after axon injury because the ER drastically changes its morphology under neuronal stress to maintain cellular homeostasis and recover from damage. Because of their tiny structures and richness in the soma, the detailed morphology of the ER and its dynamics have not been well analysed. In this study, the focused ion beam/scanning electron microscopy (FIB/SEM) analysis was performed to explore the ultra-structures of the ER in the somata of motor neuron with axon regenerative injury models. In normal motor neurons, ER in the somata is abundantly localised near the perinucleus and represents lamella-like structures. After injury, analysis of the ER volume and ER branching points indicated a collapse of the normal distribution and a transformation from lamella-like structures to mesh-like structures. Furthermore, accompanied by ER accumulation near the plasma membrane (PM), the contact between the ER and PM (ER-PM contacts) significantly increased after injury. The accumulation of extended-synaptotagmin 1 (E-Syt1), a tethering protein of the ER and PM that regulates Ca<sup>2+</sup>-dependent lipid transfer, was also identified by immunohistochemistry and quantitative Real-time PCR after injury. These morphological alterations of ER and the increase in ER-PM contacts may be crucial events that occur in motor neurons as a resilient response for the survival after axonal injury.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139680723","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-02-13DOI: 10.1007/s00441-024-03859-w
M Dieris, D Kowatschew, T Hassenklöver, I Manzini, S I Korsching
The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish.
水生生物的气味空间必然与呼吸空气的动物有很大不同。远洋鱼类公认的气味类别包括氨基酸、胆汁酸、生殖激素、核苷酸和数量有限的多胺。相反,鱼类嗅觉受体的很大一部分是由痕量胺相关受体组成的,一般认为这些受体负责检测胺。斑马鱼拥有一百多种这样的受体,但嗅觉神经元对胺的反应至今仍不得而知。在此,我们采用钙成像技术,在细胞水平上研究了斑马鱼嗅上皮外植体的气味反应。我们发现胺能引起嗅觉神经元的强烈反应,其反应时间与 ATP 反应(基底)细胞的反应时间截然不同。层高分布的定量分析显示,胺反应细胞与嗅觉标志蛋白阳性的纤毛神经元没有区别。这种分布与瞬时受体电位通道 2 阳性的微绒毛神经元和增殖细胞核抗原阳性的基底细胞的分布明显不同。我们的研究结果表明,胺类是长尾鳍鱼类的一个重要气味类别。
{"title":"Calcium imaging of adult olfactory epithelium reveals amines as important odor class in fish.","authors":"M Dieris, D Kowatschew, T Hassenklöver, I Manzini, S I Korsching","doi":"10.1007/s00441-024-03859-w","DOIUrl":"10.1007/s00441-024-03859-w","url":null,"abstract":"<p><p>The odor space of aquatic organisms is by necessity quite different from that of air-breathing animals. The recognized odor classes in teleost fish include amino acids, bile acids, reproductive hormones, nucleotides, and a limited number of polyamines. Conversely, a significant portion of the fish olfactory receptor repertoire is composed of trace amine-associated receptors, generally assumed to be responsible for detecting amines. Zebrafish possess over one hundred of these receptors, but the responses of olfactory sensory neurons to amines have not been known so far. Here we examined odor responses of zebrafish olfactory epithelial explants at the cellular level, employing calcium imaging. We report that amines elicit strong responses in olfactory sensory neurons, with a time course characteristically different from that of ATP-responsive (basal) cells. A quantitative analysis of the laminar height distribution shows amine-responsive cells undistinguishable from ciliated neurons positive for olfactory marker protein. This distribution is significantly different from those measured for microvillous neurons positive for transient receptor potential channel 2 and basal cells positive for proliferating cell nuclear antigen. Our results suggest amines as an important odor class for teleost fish.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997700/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139721787","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-02-08DOI: 10.1007/s00441-024-03864-z
Christine T Nguyen, Carolina Chávez-Madero, Erik Jacques, Brennen Musgrave, Ting Yin, Kejzi Saraci, Penney M Gilbert, Bryan A Stewart
3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples, or from pluripotent stem cells, can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity, and the appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD-, and iPSC-derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.
三维生物工程骨骼肌大组织对细胞生物学研究和治疗药物开发越来越重要。从患者样本或多能干细胞中获得的永生化细胞组织可与运动神经元共同培养,从而在培养中创建人类神经肌肉接头模型。在本研究中,我们介绍了三维培养肌肉超微结构的基础工作,包括有运动神经元和无运动神经元的培养,这得益于新型共培养平台的开发。我们的研究结果表明,与健康供体的组织相比,杜氏肌营养不良症患者的组织结构较差,而运动神经元的存在则无一例外地改善了肌节的组织结构。电子显微照片显示,在存在运动神经元的情况下,健康、DMD 和 iPSC 衍生肌肉组织中的肌丝方向性、带状模式、z 盘连续性以及推测 SSR 和 T 管轮廓的出现均有所改善。进一步确定 DMD 组织紊乱的潜在缺陷以及运动神经元支持肌肉的机制,很可能会产生治疗杜兴氏肌营养不良症患者的潜在新疗法。
{"title":"Electron microscopic analysis of the influence of iPSC-derived motor neurons on bioengineered human skeletal muscle tissues.","authors":"Christine T Nguyen, Carolina Chávez-Madero, Erik Jacques, Brennen Musgrave, Ting Yin, Kejzi Saraci, Penney M Gilbert, Bryan A Stewart","doi":"10.1007/s00441-024-03864-z","DOIUrl":"10.1007/s00441-024-03864-z","url":null,"abstract":"<p><p>3D bioengineered skeletal muscle macrotissues are increasingly important for studies of cell biology and development of therapeutics. Tissues derived from immortalized cells obtained from patient samples, or from pluripotent stem cells, can be co-cultured with motor-neurons to create models of human neuromuscular junctions in culture. In this study, we present foundational work on 3D cultured muscle ultrastructure, with and without motor neurons, which is enabled by the development of a new co-culture platform. Our results show that tissues from Duchenne muscular dystrophy patients are poorly organized compared to tissues grown from healthy donor and that the presence of motor neurons invariably improves sarcomere organization. Electron micrographs show that in the presence of motor neurons, filament directionality, banding patterns, z-disc continuity, and the appearance of presumptive SSR and T-tubule profiles all improve in healthy, DMD-, and iPSC-derived muscle tissue. Further work to identify the underlying defects of DMD tissue disorganization and the mechanisms by which motor neurons support muscle are likely to yield potential new therapeutic approaches for treating patients suffering from Duchenne muscular dystrophy.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139701972","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-02-28DOI: 10.1007/s00441-024-03878-7
Jingjing Zhao, Xiaoping Yu, Xuping Shentu, Danting Li
Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.
成像技术可实现生物结构和过程的可视化,在推动生物研究方面发挥了举足轻重的作用。传统的电子显微镜(EM)可生成二维图像,而现在的新兴技术可对原位标本进行高分辨率的三维(3D)表征,满足了分子和细胞生物学日益增长的需求。将透射电子显微镜(TEM)与连续切片相结合,开创了三维成像技术,吸引了生物学家探索细胞超微结构,推动了三维电磁重建技术的发展。三维透射电子显微镜(TEM)重建技术全面而精确地呈现了细胞内部结构和分布,为人们深入了解细胞和分子的超微结构提供了无与伦比的视角,在阐明结构-功能关系和广泛推动结构生物学发展方面具有巨大价值。在此,我们首先介绍用传统的 TEM 方法进行细胞和组织三维重建的原理,然后讨论利用 TEM 和基于扫描电子显微镜(SEM)的新技术以及冷冻电子显微镜(cryo-EM)技术的现代技术。我们研究了序列切片、电子断层扫描(ET)和最新的单粒子分析(SPA)的三维重建技术;讨论了用于大体积三维重建的聚焦离子束扫描电子显微镜(FIB-SEM)、序列块面扫描电子显微镜(SBF-SEM)和自动集带车床超微切片机(ATUM-SEM)。最后,我们回顾了这些技术在生命科学领域面临的挑战和发展前景。本书旨在为生物研究人员提供信息参考。
{"title":"The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.","authors":"Jingjing Zhao, Xiaoping Yu, Xuping Shentu, Danting Li","doi":"10.1007/s00441-024-03878-7","DOIUrl":"10.1007/s00441-024-03878-7","url":null,"abstract":"<p><p>Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139982488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-02-19DOI: 10.1007/s00441-024-03868-9
Neeti Gandhi, Lauren Wills, Kyle Akers, Yiqi Su, Parker Niccum, T M Murali, Padmavathy Rajagopalan
Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.
{"title":"Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes.","authors":"Neeti Gandhi, Lauren Wills, Kyle Akers, Yiqi Su, Parker Niccum, T M Murali, Padmavathy Rajagopalan","doi":"10.1007/s00441-024-03868-9","DOIUrl":"10.1007/s00441-024-03868-9","url":null,"abstract":"<p><p>Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139899404","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-01Epub Date: 2024-02-27DOI: 10.1007/s00441-024-03877-8
Maria Luigia Vommaro, Sandro Donato, Simone Caputo, Raffaele G Agostino, Aurora Montali, Gianluca Tettamanti, Anita Giglio
In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.
在全代谢昆虫中,蛹期会发生广泛的组织和细胞重组。传统上,许多昆虫物种都是通过组织学或超微结构学方法来研究变态过程中外部外骨骼和内部器官的重塑。本研究展示了同步辐射 X 射线相位对比显微计算机断层扫描技术作为一种强大的非破坏性工具,用于对两种天牛甲虫(即 Tribolium castaneum 和 Tenebrio molitor)蛹期的解剖结构进行原位形态学观察。我们对蛹早期、中期和晚期的雌雄甲虫进行了虚拟切片和三维重建。通过该数据集,我们观察到了黄粉虫和红粉甲虫在不同蛹龄时肠道和神经系统的重塑,以及雌性和雄性生殖系统的形成。此外,我们还观察到不同物种器官发育的时间和持续时间模式各不相同,这可能与物种特有的前形象阶段对环境条件的适应有关,而环境条件最终会影响它们的生命周期。这项研究提供了有关全代谢昆虫蛹期形态变化的新知识,并提供了一套有关甲虫变态的基础信息,可为今后的法医学、生理学和生态学研究以及教育用途的图像图集提供支持。
{"title":"Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.","authors":"Maria Luigia Vommaro, Sandro Donato, Simone Caputo, Raffaele G Agostino, Aurora Montali, Gianluca Tettamanti, Anita Giglio","doi":"10.1007/s00441-024-03877-8","DOIUrl":"10.1007/s00441-024-03877-8","url":null,"abstract":"<p><p>In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997553/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139971100","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5-/-) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.
{"title":"Nescient helix-loop-helix 1 (Nhlh1) is a novel activating transcription factor 5 (ATF5) target gene in olfactory and vomeronasal sensory neurons in mice.","authors":"Chiharu Ishii, Haruo Nakano, Riko Higashiseto, Yusaku Ooki, Mariko Umemura, Shigeru Takahashi, Yuji Takahashi","doi":"10.1007/s00441-024-03871-0","DOIUrl":"10.1007/s00441-024-03871-0","url":null,"abstract":"<p><p>Activating transcription factor 5 (ATF5) is a transcription factor that belongs to the cAMP-response element-binding protein/ATF family and is essential for the differentiation and survival of sensory neurons in mouse olfactory organs. However, transcriptional target genes for ATF5 have yet to be identified. In the present study, chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) experiments were performed to verify ATF5 target genes in the main olfactory epithelium and vomeronasal organ in the postnatal pups. ChIP-qPCR was conducted using hemagglutinin (HA)-tagged ATF5 knock-in olfactory organs. The results obtained demonstrated that ATF5-HA fusion proteins bound to the CCAAT/enhancer-binding protein-ATF response element (CARE) site in the enhancer region of nescient helix-loop-helix 1 (Nhlh1), a transcription factor expressed in differentiating olfactory and vomeronasal sensory neurons. Nhlh1 mRNA expression was downregulated in ATF5-deficient (ATF5<sup>-/-</sup>) olfactory organs. The LIM/homeobox protein transcription factor Lhx2 co-localized with ATF5 in the nuclei of olfactory and vomeronasal sensory neurons and bound to the homeodomain site proximal to the CARE site in the Nhlh1 gene. The CARE region of the Nhlh1 gene was enriched by the active enhancer marker, acetyl-histone H3 (Lys27). The present study identified Nhlh1 as a novel target gene for ATF5 in murine olfactory organs. ATF5 may upregulate Nhlh1 expression in concert with Lhx2, thereby promoting the differentiation of olfactory and vomeronasal sensory neurons.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139930231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-03-19DOI: 10.1007/s00441-024-03876-9
Roberta De Mori, Silvia Tardivo, Lidia Pollara, Silvia Clara Giliani, Eltahir Ali, Lucio Giordano, Vincenzo Leuzzi, Rita Fischetto, Blanca Gener, Santo Diprima, Marco J. Morelli, Maria Cristina Monti, Virginie Sottile, Enza Maria Valente
Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the “molar tooth sign.” Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.
{"title":"Joubert syndrome-derived induced pluripotent stem cells show altered neuronal differentiation in vitro","authors":"Roberta De Mori, Silvia Tardivo, Lidia Pollara, Silvia Clara Giliani, Eltahir Ali, Lucio Giordano, Vincenzo Leuzzi, Rita Fischetto, Blanca Gener, Santo Diprima, Marco J. Morelli, Maria Cristina Monti, Virginie Sottile, Enza Maria Valente","doi":"10.1007/s00441-024-03876-9","DOIUrl":"https://doi.org/10.1007/s00441-024-03876-9","url":null,"abstract":"<p>Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the “molar tooth sign.” Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an <i>in vitro</i> neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (<i>AHI1, CPLANE1</i>, <i>TMEM67</i>, and <i>CC2D2A</i>) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant <i>in vitro</i> model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.</p>","PeriodicalId":9712,"journal":{"name":"Cell and Tissue Research","volume":null,"pages":null},"PeriodicalIF":3.6,"publicationDate":"2024-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140169149","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}