In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that ste...In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.展开更多
In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few region...In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .展开更多
The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells （NSCs）, are important for learning and memory as well as m...The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells （NSCs）, are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associatedcognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has garnered significant interest because targeting it could be a novel potential therapeutic strategy for these disorders. However, if we are to use nenrogenesis to halt or reverse hippocampal-related pathology, we need to understand better the core molecular machinery that governs NSC and their progeny. In this review, we summarize a wide variety of mouse models used in adult neurogenesis field, present their advantages and disadvantages based on specificity and efficiency of labeling of different cell types, and review their contribution to our understanding of the biology and the heterogeneity of different cell types found in adult neurogenic niches.展开更多
Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, r...Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.展开更多
During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the prolif...During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the proliferation of cardiomyocytes, while the postnatal atrium is primarily shaped by the increase in the volume of cardiomyocytes. Cell proliferation and differentiation of atrial development is the basis for its functions such as “blood reservoir” and “supplementary pump”. Deep understanding the cellular mechanism of atrial development is imperative to explore the causes of common congenital arrhythmia heart diseases such as atrial fibrillation. We used genetically engineered mouse reproduction knowledge, lineage tracing method based on CreloxP system, molecular biology and immunofluorescence technology to track the cardiomyocyte lineage of Nppa-GFP mouse line with stereo fluorescence microscope and ultra-high-speed confocal microscope. Besides the atrium of Nppa-CreER;Rosa26 tdTomato mouse was examined during embryonic (E10.5 - E18.5) and postnatal (P0, P3, P5, P7, P14, P28, P8w) stage. Immunofluorescence results revealed that Nppa-positive cells labeled TNNI3-positive cardiomyocytes and protruded into the atrial cavity at the beginning of E11.5 - E12.0 and during subsequent development to form Nppa-positive myocardial trabeculae. Thick comb-shaped myocardium was observed after birth, and we suspect that this was particularly important for the normal contractile activity and pumping function of the atrium. Additionally, non-single origin of Nppa-positive trabecular myocardiocytes were revealed through Tamoxifen-induced lineage tracing experiment. Our findings reveal proliferation dynamics and non-comprehensive fate decisions of cardiomyocytes that produce the distinct architecture of the atrium chamber.展开更多
MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cr...MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes--green GFP and red tdTomato -- and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.展开更多
CRISPR/Cas系统是细菌和古生菌的自适应免疫系统,其对基因组高效精准的编辑,极大地推动了发育生物学、表观遗传学、药物开发、疾病治疗等多个学科和研究领域的发展.CRISPR/Cas9系统诱导基因组DNA产生双链断裂,以非同源末端连接(non-homo...CRISPR/Cas系统是细菌和古生菌的自适应免疫系统,其对基因组高效精准的编辑,极大地推动了发育生物学、表观遗传学、药物开发、疾病治疗等多个学科和研究领域的发展.CRISPR/Cas9系统诱导基因组DNA产生双链断裂,以非同源末端连接(non-homologous end joining,NHEJ)的方式进行修复,因此会在剪切位置随机地引入短的插入和删除(insertions and deletions,indels).这些引入的indels作为区分细胞的标签,被称为条形码.细胞条形码技术已经被用于谱系追踪、基因组功能筛选等.而测序技术的飞速发展和成本的大幅度降低以及单细胞转录组测序技术的出现,可以在时间和空间层面同时对数百万个单细胞进行谱系追踪,记录细胞活动.本综述讨论了CRISPR/Cas系统的工作原理、细胞条形码技术和单细胞测序技术(scRNA-seq),以及两者结合产生的单细胞谱系追踪技术.展开更多
Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vas...Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.展开更多
There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a consider...There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear.展开更多
Regeneration of many cell types found in adult organs relies upon the presence of relatively small pools of undifferentiated stem cells. Initial studies that attempted to isolate stem cells and propagate them in vitro...Regeneration of many cell types found in adult organs relies upon the presence of relatively small pools of undifferentiated stem cells. Initial studies that attempted to isolate stem cells and propagate them in vitro have been complemented by analysis of stem cells in their endogenous tissues where they are subject to a variety of regulatory cues. This has been facilitated by the advent of new methods for lineage tracing and genetic manipulation of stem cells and their associated niche cells. The picture that is emerging is that different stem cell populations utilize diverse processes to ensure maintenance of the stem cell pool accompanied by production of cells committed to regenerate differentiated cells.展开更多
基金Supported by Grants from the Wellcome Trust,No.088876/Z/09/Zthe UK Biotechnology and Biological Sciences Research Council,No.BB/J015172/1 and No.BB/J015237/1
文摘In this review we evaluate evidence for three different hypotheses that explain how the corneal epithelium is maintained. The limbal epithelial stem cell(LESC)hypothesis is most widely accepted. This proposes that stem cells in the basal layer of the limbal epithelium, at the periphery of the cornea, maintain themselves and also produce transient(or transit) amplifying cells(TACs). TACs then move centripetally to the centre of the cornea in the basal layer of the corneal epithelium and also replenish cells in the overlying suprabasal layers. The LESCs maintain the corneal epithelium during normal homeostasis and become more active to repair significant wounds. Second, the corneal epithelial stem cell(CESC) hypothesis postulates that, during normal homeostasis, stem cells distributed throughout the basal corneal epithelium, maintain the tissue. According to this hypothesis, LESCs are present in the limbus but are only active during wound healing. We also consider a third possibility, that the corneal epithelium is maintained during normal homeostasis by proliferation of basal corneal epithelial cells without any input from stem cells. After reviewing the published evidence, we conclude that the LESC and CESC hypotheses are consistent with more of the evidence than the third hypothesis, so we do not consider this further. The LESC and CESC hypotheses each have difficulty accounting for one main type of evidence so we evaluate the two key lines of evidence that discriminate between them. Finally, we discuss how lineage-tracing experiments have begun to resolve the debate in favour of the LESC hypothesis. Nevertheless, it also seems likely that some basal corneal epithelial cells can act as long-term progenitors if limbal stem cell function is compromised. Thus, this aspect of the CESC hypothesis may have a lasting impact on our understanding of corneal epithelial maintenance, even if it is eventually shown that stem cells are restricted to the limbus as proposed by the LESC hypothesis.
文摘In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .
文摘The mammalian hippocampus shows a remarkable capacity for continued neurogenesis throughout life. Newborn neurons, generated by the radial neural stem cells （NSCs）, are important for learning and memory as well as mood control. During aging, the number and responses of NSCs to neurogenic stimuli diminish, leading to decreased neurogenesis and age-associatedcognitive decline and psychiatric disorders. Thus, adult hippocampal neurogenesis has garnered significant interest because targeting it could be a novel potential therapeutic strategy for these disorders. However, if we are to use nenrogenesis to halt or reverse hippocampal-related pathology, we need to understand better the core molecular machinery that governs NSC and their progeny. In this review, we summarize a wide variety of mouse models used in adult neurogenesis field, present their advantages and disadvantages based on specificity and efficiency of labeling of different cell types, and review their contribution to our understanding of the biology and the heterogeneity of different cell types found in adult neurogenic niches.
文摘Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.
文摘During the development of mammalian heart, the left and right atria play an important role in cardiovascular circulation. The embryonic atrium is mainly formed by the differentiation of progenitor cells and the proliferation of cardiomyocytes, while the postnatal atrium is primarily shaped by the increase in the volume of cardiomyocytes. Cell proliferation and differentiation of atrial development is the basis for its functions such as “blood reservoir” and “supplementary pump”. Deep understanding the cellular mechanism of atrial development is imperative to explore the causes of common congenital arrhythmia heart diseases such as atrial fibrillation. We used genetically engineered mouse reproduction knowledge, lineage tracing method based on CreloxP system, molecular biology and immunofluorescence technology to track the cardiomyocyte lineage of Nppa-GFP mouse line with stereo fluorescence microscope and ultra-high-speed confocal microscope. Besides the atrium of Nppa-CreER;Rosa26 tdTomato mouse was examined during embryonic (E10.5 - E18.5) and postnatal (P0, P3, P5, P7, P14, P28, P8w) stage. Immunofluorescence results revealed that Nppa-positive cells labeled TNNI3-positive cardiomyocytes and protruded into the atrial cavity at the beginning of E11.5 - E12.0 and during subsequent development to form Nppa-positive myocardial trabeculae. Thick comb-shaped myocardium was observed after birth, and we suspect that this was particularly important for the normal contractile activity and pumping function of the atrium. Additionally, non-single origin of Nppa-positive trabecular myocardiocytes were revealed through Tamoxifen-induced lineage tracing experiment. Our findings reveal proliferation dynamics and non-comprehensive fate decisions of cardiomyocytes that produce the distinct architecture of the atrium chamber.
文摘MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes--green GFP and red tdTomato -- and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.
基金动物重要基因克隆及功能验证(2016ZX08009-003-006)The 111 Project(Project Grant No.B12008)
文摘CRISPR/Cas系统是细菌和古生菌的自适应免疫系统,其对基因组高效精准的编辑,极大地推动了发育生物学、表观遗传学、药物开发、疾病治疗等多个学科和研究领域的发展.CRISPR/Cas9系统诱导基因组DNA产生双链断裂,以非同源末端连接(non-homologous end joining,NHEJ)的方式进行修复,因此会在剪切位置随机地引入短的插入和删除(insertions and deletions,indels).这些引入的indels作为区分细胞的标签,被称为条形码.细胞条形码技术已经被用于谱系追踪、基因组功能筛选等.而测序技术的飞速发展和成本的大幅度降低以及单细胞转录组测序技术的出现,可以在时间和空间层面同时对数百万个单细胞进行谱系追踪,记录细胞活动.本综述讨论了CRISPR/Cas系统的工作原理、细胞条形码技术和单细胞测序技术(scRNA-seq),以及两者结合产生的单细胞谱系追踪技术.
基金supported by the National Institutes Health(grant Nos.5P20GM109095 and P20GM103408)Boise State University(to BEM)
文摘Understanding the contribution of endothelial cells to the progenitor pools of adult tissues has the potential to inform therapies for human disease.To address whether endothelial cells transdifferentiate into non-vascular cell types,we performed cell lineage tracing analysis using transgenic mice engineered to express a fluorescent marker following activation by tamoxifen in vascular endothelial cadherin promoter-expressing cells(VEcad-CreERT2;B6 Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze).Activation of target-cell labeling following 1.5 months of ad libitum feeding with tamoxifen-laden chow in 4–5 month-old mice resulted in the tracing of central nervous system and peripheral cells that include:cerebellar granule neurons,ependymal cells,skeletal myocytes,pancreatic beta cells,pancreatic acinar cells,tubular cells in the renal cortex,duodenal crypt cells,ileal crypt cells,and hair follicle stem cells.As Nestin expression has been reported in a subset of endothelial cells,Nes-CreERT2 mice were also utilized in these conditions.The tracing of cells in adult Nes-CreERT2 mice revealed the labeling of canonical progeny cell types such as hippocampal and olfactory granule neurons as well as ependymal cells.Interestingly,Nestin tracing also labeled skeletal myocytes,ileal crypt cells,and sparsely marked cerebellar granule neurons.Our findings provide support for endothelial cells as active contributors to adult tissue progenitor pools.This information could be of particular significance for the intravenous delivery of therapeutics to downstream endothelial-derived cellular targets.The animal experiments were approved by the Boise State University Institute Animal Care and Use Committee(approval No.006-AC15-018)on October 31,2018.
基金Supported by Grant AMANDA "Alterazioni metaboliche,stress cellulari e processi neurodegenerativi" from Regione Lombardia/CNR Project to P.V.Castelli A is a recipient of a fellowship from Fondazione Nicola del Roscio
文摘There is wide agreement that cell fusion is a physiological process in cells in mammalian bone, muscle and placenta. In other organs, such as the cerebellum, cell fusion is controversial. The liver contains a considerable number of polyploid cells: They are commonly believed to originate by genome endoreplication, although the contribution of cell fusion to polyploidization has not been excluded. Here, we address the topic of cell fusion in the liver from a historical point of view. We discuss experimental evidence clearly supporting the hypothesis that cell fusion occurs in the liver, specifically when bone marrow cells were injected into mice and shown to rescue genetic hepatic degenerative defects. Those experiments-carried out in the latter half of the last century-were initially interpreted to show "transdifferentiation", but are now believed to demonstrate fusion between donor macrophages and host hepatocytes, raising the possibility that physiologically polyploid cells, such as hepatocytes, could originate, at least partially, through homotypic cell fusion. In support of the homotypic cell fusion hypothesis, we present new data generated using a chimera-based model, a much simpler model than those previously used. Cell fusion as a road to polyploidization in the liver has not been extensively investigated, and its contribution to a variety of conditions, such as viral infections, carcinogenesis and aging, remains unclear.
文摘Regeneration of many cell types found in adult organs relies upon the presence of relatively small pools of undifferentiated stem cells. Initial studies that attempted to isolate stem cells and propagate them in vitro have been complemented by analysis of stem cells in their endogenous tissues where they are subject to a variety of regulatory cues. This has been facilitated by the advent of new methods for lineage tracing and genetic manipulation of stem cells and their associated niche cells. The picture that is emerging is that different stem cell populations utilize diverse processes to ensure maintenance of the stem cell pool accompanied by production of cells committed to regenerate differentiated cells.
