Imaging hematopoietic precursor division in real time

SUMMARYStem cells are thought to balance self-renewal and differentiation through asymmetric and symmetric divisions, but whether such divisions occur during hematopoietic development remains unknown. Using a Notch reporter mouse, in which GFP acts as a sensor for differentiation, we image hematopoietic precursors and show that they undergo both symmetric and asymmetric divisions. In addition we show that the balance between these divisions is not hardwired but responsive to extrinsic and intrinsic cues. Precursors in a prodifferentiation environment preferentially divide asymmetrically, whereas those in a prorenewal environment primarily divide symmetrically. Oncoproteins can also influence division pattern: although BCR-ABL predominantly alters the rate of division and death, NUP98-HOXA9 promotes symmetric division, suggesting that distinct oncogenes subvert different aspects of cellular function. These studies establish a system for tracking division of hematopoietic precursors and show that the balance of symmetric and asymmetric division can be influenced by the microenvironment and subverted by oncogenes.     

Asymmetric Distribution of UCH‐L1 in Spermatogonia Is Associated With Maintenance and Differentiation of Spermatogonial Stem Cells

Asymmetric division of germline stem cells in vertebrates was proposed a century ago; however, direct evidence for asymmetric division of mammalian spermatogonial stem cells (SSCs) has been scarce. Here, we report that ubiquitin carboxy‐terminal hydrolase 1 (UCH‐L1) is expressed in type A (A_s, A_pr, and A_al) spermatogonia located at the basement membrane (BM) of seminiferous tubules at high and low levels, but not in differentiated germ cells distant from the BM. Asymmetric segregation of UCH‐L1 was associated with self‐renewal versus differentiation divisions of SSCs as defined by co‐localization of UCH‐L1^high and PLZF, a known determinant of undifferentiated SSCs, versus co‐localization of UCH‐L1^low/? with proteins expressed during SSC differentiation (DAZL, DDX4, c‐KIT). In vitro, gonocytes/spermatogonia frequently underwent asymmetric divisions characterized by unequal segregation of UCH‐L1 and PLZF. Importantly, we could also demonstrate asymmetric segregation of UCH‐L1 and PLZF in situ in seminiferous tubules. Expression level of UCH‐L1 in the immature testis where spermatogenesis was not complete was not affected by the location of germ cells relative to the BM, whereas UCH‐L1‐positive spermatogonia were exclusively located at the BM in the adult testis. Asymmetric division of SSCs appeared to be affected by interaction with supporting somatic cells and extracelluar matrix. These findings for the first time provide direct evidence for existence of asymmetric division during SSCs self‐renewal and differentiation in mammalian spermatogenesis. J. Cell. Physiol. 220: 460–468, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular mechanisms of asymmetric divisions in mammary stem cells

Stem cells have the remarkable ability to undergo proliferative symmetric divisions and self‐renewing asymmetric divisions. Balancing of the two modes of division sustains tissue morphogenesis and homeostasis. Asymmetric divisions of Drosophila neuroblasts (NBs) and sensory organ precursor (SOP) cells served as prototypes to learn what we consider now principles of asymmetric mitoses. They also provide initial evidence supporting the notion that aberrant symmetric divisions of stem cells could correlate with malignancy. However, transferring the molecular knowledge of circuits underlying asymmetry from flies to mammals has proven more challenging than expected. Several experimental approaches have been used to define asymmetry in mammalian systems, based on daughter cell fate, unequal partitioning of determinants and niche contacts, or proliferative potential. In this review, we aim to provide a critical evaluation of the assays used to establish the stem cell mode of division, with a particular focus on the mammary gland system. In this context, we will discuss the genetic alterations that impinge on the modality of stem cell division and their role in breast cancer development.     

Letter from the Guest Editor

Understanding the mechanisms of stem cell proliferation, self-renewal and differentiation is fundamental for stem cell biology. Stem cells proliferate by either symmetric division or asymmetric division. Through asymmetric division, stem cells self-renew and differentiate to mature cells. Stem cells could also divide symmetrically to give rise to differentiated cells. Besides intrinsic cues, proliferation and self-renewal of most stem cell types also rely on extrinsic signals from niche or surrounding cells. Failure in any of these factors may result in disturbed stem cell proliferation, self-renewal or differentiation and/or generate cancer stem cells that drive cancer development.     

Stem cell niche: Dynamic neighbor of stem cells

Stem cell niche is a specialized and dynamic microenvironment around the stem cells which plays a critical role in maintaining the stemness properties of stem cells. Over the years, advancement in the research activity has revealed the various important aspects of stem cell niche including cell-cell interaction, cell-extracellular matrix interaction, a large number of soluble signaling factors and various biochemical and biophysical cues (such as oxygen tension, flow, and shear and pore size). Stem cells have the potential to be a powerful tool in regenerative medicine due to their self-renewal property and immense differentiation potential. Recent progresses in in vitro culture conditions of embryonic stem cells, adult stem cells and induced pluripotent stem cells have enabled the researchers to investigate and understand the role of the microenvironment in stem cell properties. The engineered artificial stem cell niche has led to a better execution of stem cells in regenerative medicine. Here we elucidate the key components of stem cell niche and their role in niche engineering and stem cell therapeutics.     

Stomaching Notch

The self‐renewal and differentiation of tissue stem cells must be tightly controlled. Unrestrained self‐renewal leads to over‐proliferation of stem cells, which may cause tumor formation, while uncontrolled differentiation leads to depletion of the stem cell pool. In this issue of The EMBO Journal, Demitrack et al (2015) show that the Notch pathway is a key regulator of Lgr5 antral stem cell self‐renewal and differentiation. Notch signaling controls the proliferation and differentiation of stem cells as well as gastric tissue growth, while uncontrolled Notch activity in stem cells leads to polyp formation.     

Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.     

Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.     

Cell shape and Wnt signaling redundantly control the division axis of C. elegans epithelial stem cells

Tissue-specific stem cells combine proliferative and asymmetric divisions to balance self-renewal with differentiation. Tight regulation of the orientation and plane of cell division is crucial in this process. Here, we study the reproducible pattern of anterior-posterior-oriented stem cell-like divisions in the Caenorhabditis elegans seam epithelium. In a genetic screen, we identified an alg-1 Argonaute mutant with additional and abnormally oriented seam cell divisions. ALG-1 is the main subunit of the microRNA-induced silencing complex (miRISC) and was previously shown to regulate the timing of postembryonic development. Time-lapse fluorescence microscopy of developing larvae revealed that reduced alg-1 function successively interferes with Wnt signaling, cell adhesion, cell shape and the orientation and timing of seam cell division. We found that Wnt inactivation, through mig-14 Wntless mutation, disrupts tissue polarity but not anterior-posterior division. However, combined Wnt inhibition and cell shape alteration resulted in disordered orientation of seam cell division, similar to the alg-1 mutant. Our findings reveal additional alg-1-regulated processes, uncover a previously unknown function of Wnt ligands in seam tissue polarity, and show that Wnt signaling and geometric cues redundantly control the seam cell division axis.     

Proteomic profiling of distinct clonal populations of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) have attracted immense research interest in the field of regenerative medicine due to their ability to be cultured for successive passages and multi‐lineage differentiation. The molecular mechanisms governing MSC self‐renewal and differentiation remain largely unknown. The development of sophisticated techniques, in particular clinical proteomics, has enabled researchers in various fields to identify and characterize cell specific biomarkers for therapeutic purposes. This study seeks to understand the cellular and sub‐cellular processes responsible for the existence of stem cell populations in bone marrow samples by revealing the whole cell proteome of the clonal cultures of bone marrow‐derived MSCs (BMSCs). Protein profiling of the MSC clonal populations was conducted by Two‐Dimensional Liquid Chromatography/Matrix‐Assisted Laser Desorption/Ionisation (MALDI) Mass Spectrometry (MS). A total of 83 proteins were identified with high confidence of which 11 showed differential expression between subpopulations, which included cytoskeletal and structural proteins, calcium binding proteins, cytokinetic proteins, and members of the intermediate filament family. This study generated a proteome reference map of BMSCs from the clonal populations, which will be valuable to better understand the underlying mechanism of BMSC self‐renewal and differentiation. J. Cell. Biochem. 106: 776–786, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of asymmetric cell division in the epidermis

For proper tissue morphogenesis, cell divisions and cell fate decisions must be tightly and coordinately regulated. One elegant way to accomplish this is to couple them with asymmetric cell divisions. Progenitor cells in the developing epidermis undergo both symmetric and asymmetric cell divisions to balance surface area growth with the generation of differentiated cell layers. Here we review the molecular machinery implicated in controlling asymmetric cell division. In addition, we discuss the ability of epidermal progenitors to choose between symmetric and asymmetric divisions and the key regulatory points that control this decision.     

microRNA在多能干细胞向心肌细胞分化中的作用

microRNAs(miRNAs)是长约22 nt的非编码RNAs,广泛参与细胞的增殖、分化、病变、修复和凋亡等多种生命活动．多能干细胞（pluripotent stem cells）是指体外具有自我更新和多向分化潜能的细胞,在一定条件下可被定向诱导分化为多种细胞类型．miRNAs在多能干细胞中表达丰富,并通过调控基因表达影响其自我更新及分化．由多能干细胞向心肌细胞分化的方法主要有3种,即拟胚体形成法、与内胚层细胞共培养法和特定诱导物添加法．虽然这3种方法均可成功诱导多能干细胞向心肌细胞分化,但重复率很低. 所以,人们把研究的视野逐渐转向miRNAs--这个广泛参与细胞生命活动的小分子物质．大量研究表明,在多能干细胞中,不同的miRNAs可通过打靶不同基因影响其向心肌细胞分化．在间充质干细胞中,miR-1、miR-133 和miR-499可分别打靶Hes-1、SRF和Pdcd4| 而在胚胎干细胞中,miR-1和miR-499分别打靶 Hand2和Pacs2促进其向心肌细胞分化．miRNAs在多能干细胞向心肌分化作用机制的研究必将促进再生医学在心脏疾病治疗上的应用．     

Stem cells' centrosomes: How can organelles identified 130 years ago contribute to the future of regenerative medicine?

At the core of regenerative medicine lies the expectation of repair or replacement of damaged tissues or whole organs. Donor scarcity and transplant rejection are major obstacles, and exactly the obstacles that stem cell based therapy promises to overcome. These therapies demand a comprehensive understanding of the asymmetric division of stem cells, i.e. their ability to produce cells with identical potency or differentiated cells. It is believed that with better understanding, researchers will be able to direct stem cell differentiation. Here, we describe extraordinary advances in manipulating stem cell fate that show that we need to focus on the centrosome and the centrosome-derived primary cilium. This belief comes from the fact that this organelle is the vehicle that coordinates the asymmetric division of stem cells. This is supported by studies that report the significant role of the centrosome/cilium in orchestrating signaling pathways that dictate stem cell fate. We anticipate that there is sufficient evidence to place this organelle at the center of efforts that will shape the future of regenerative medicine.     

多细胞生物干细胞不对称分裂的内在调节机制研究进展

多细胞生物的发育是从一个受精卵分化成多种类型细胞的过程。细胞多样性形成的基础是不等分裂,不等分裂是干细胞自我更新和自我维持的关键。干细胞不等分裂有细胞内和细胞外两种调节机制。果蝇神经干细胞增殖和分化、植物胚胎发育、表皮气孔形成及根内皮层的分化,是研究不等细胞分裂调节机制最多的发育背景。本综述介绍了果蝇神经干细胞和植物胚胎发育早期、表皮气孔发生及根皮层内皮层中细胞不等分裂内在调节机制的研究进展。     

Symmetric versus asymmetric cell division during neurogenesis in the developing vertebrate central nervous system

The type and number of cell divisions of neuronal progenitors determine the number of neurons generated during the development of the vertebrate central nervous system. Over the past several years, there has been substantial progress in characterizing the various kinds of neuronal progenitors and the types of symmetric and asymmetric divisions they undergo. The understanding of the cell-biological basis of symmetric versus asymmetric progenitor cell division has been consolidated, and the molecular machinery controlling these divisions is beginning to be unravelled. Other recent advances include comparative studies of brain development in rodents and primates, as well as the identification of gene mutations in humans that affect the balance between the various types of cell division of neuronal progenitors.     

Decreased p53 is associated with a decline in asymmetric stem cell self‐renewal in aged human epidermis

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self‐renewal in multiple murine tissues. The fate of SC self‐renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self‐renewal has been observed in inflammatory‐mediated hyperproliferation, while increased symmetric SC self‐renewal has been observed in cancers. We analyzed SC self‐renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age‐dependent decrease in epidermal basal layer divisions. The balance of SC self‐renewal shifted toward symmetric SC self‐renewal, with a decline in asymmetric SC self‐renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self‐renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin‐3 (a p53 activator) returned p53 activity as well as asymmetric SC self‐renewal divisions to adult levels. Nutlin‐3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin‐3 (p53)‐induced asymmetric SC self‐renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53‐induced asymmetric SC divisions.     

Universal rules for division plane selection in plants

Coordinated cell divisions and cell expansion are the key processes that command growth in all organisms. The orientation of cell divisions and the direction of cell expansion are critical for normal development. Symmetric divisions contribute to proliferation and growth, while asymmetric divisions initiate pattern formation and differentiation. In plants these processes are of particular importance since their cells are encased in cellulosic walls that determine their shape and lock their position within tissues and organs. Several recent studies have analyzed the relationship between cell shape and patterns of symmetric cell division in diverse organisms and employed biophysical and mathematical considerations to develop computer simulations that have allowed accurate prediction of cell division patterns. From these studies, a picture emerges that diverse biological systems follow simple universal rules of geometry to select their division planes and that the microtubule cytoskeleton takes a major part in sensing the geometric information and translates this information into a specific division outcome. In plant cells, the division plane is selected before mitosis, and spatial information of the division plane is preserved throughout division by the presence of reference molecules at a distinct region of the plasma membrane, the cortical division zone. The recruitment of these division zone markers occurs multiple times by several mechanisms, suggesting that the cortical division zone is a highly dynamic region.     

Increased SCF/c‐kit by hypoxia promotes autophagy of human placental chorionic plate‐derived mesenchymal stem cells via regulating the phosphorylation of mTOR

Hypoxia triggers physiological and pathological cellular processes, including proliferation, differentiation, and death, in several cell types. Mesenchymal stem cells (MSCs) derived from various tissues have self‐renewal activity and can differentiate towards multiple lineages. Recently, it has been reported that hypoxic conditions tip the balance between survival and death by hypoxia‐induced autophagy, although the underlying mechanism is not clear. The objectives of this study are to compare the effect of hypoxia on the self‐renewal of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and placental chorionic plate‐derived mesenchymal stem cells (CP‐MSCs) and to investigate the regulatory mechanisms of self‐renewal in each MSC type during hypoxia. The expression of self‐renewal markers (e.g., Oct4, Nanog, Sox2) was assessed in both cell lines. PI3K and stem cell factor (SCF) expression gradually increased in CP‐MSCs but were markedly downregulated in BM‐MSCs by hypoxia. The phosphorylation of ERK and mTOR was augmented by hypoxia in CP‐MSCs compared to control. Also, the expression of LC3 II, a component of the autophagosome and the hoof‐shaped autophagosome was detected more rapidly in CP‐MSCs than in BM‐MSCs under hypoxia. Hypoxia induced the expression of SCF in CP‐MSCs and increased SCF/c‐kit pathway promotes the self‐renewal activities of CP‐MSCs via an autocrine/paracrine mechanism that balances cell survival and cell death events by autophagy. These activities occur to a greater extent in CP‐MSCs than in BM‐MSCs through regulating the phosphorylation of mTOR. These findings will provide useful guidelines for better understanding the function of SCF/c‐kit in the self‐renewal and autophagy‐regulated mechanisms that promote of MSC survival. J. Cell. Biochem. 114: 79–88, 2012. © 2012 Wiley Periodicals, Inc.