Stem cells and their niche: a matter of fate

Embryonic stem cells provide an in vitro model for developmental biologists to study cell fate decisions during ontogenesis, while somatic stem cells allow physiologists to understand tissue homeostasis in the adult. The behavior of stem cells is dependent on an intimate relationship with a supportive niche. This brief review highlights some of the most important recent trends in stem cell biology, focusing in particular on the supportive microenvironments for both embryonic and adult stem cells. Known intrinsic and extrinsic molecular players from the best-characterized stem cell types are summarized, illuminating a number of shared environmental cues among tissues originating from all three embryonic germ layers. Received 6 October 2005; received after revision 27 December 2005; accepted 17 January 2006     

Extensive characterization of sphere models established from colorectal cancer cell lines

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.     

无血清分离人结直肠癌细胞的体外培养形态及标志物表达特征

目的探讨人结直肠肿瘤干细胞在体外分化过程中细胞形态和干细胞相关标志物CD133的表达变化,为进一步研究结直肠肿瘤干细胞分化走向提供实验依据。方法取来源于人结直肠癌的细胞系HCT116,无血清培养分离出CD133+细胞,加血清诱导分化,相差显微镜下观察其形态变化;在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞,利用流式细胞仪检测干细胞标志物CD133的表达量,采用激光共聚焦检测CD133表面标记分子的表达。结果 1)细胞形态:无血清培养分离的CD133+细胞,在生长过程中聚集成规则的细胞球,血清诱导后即贴壁生长,贴壁形态与同来源细胞形态一致,且再次无血清悬浮培养后聚集成球稳定生长。2)标志物变化:结直肠肿瘤干细胞未分化时CD133表面标记分子高表达,流式细胞仪检测未分化细胞CD133第7天表达率为(20.4±0.52)%,第14天表达率为(78.5±2.80)%,分化后表达率为(0.50±0.17)%。结论细胞形态和标志物表达改变均表明高表达CD133+的HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞,CD133+细胞经血清诱导后表达下调而使细胞失去干细胞特性。     

Single cell biology beyond the era of antibodies: relevance,challenges, and promises in biomedical research

Research of the past two decades has proved the relevance of single cell biology in basic research and translational medicine. Successful detection and isolation of specific subsets is the key to understand their functional heterogeneity. Antibodies are conventionally used for this purpose, but their relevance in certain contexts is limited. In this review, we discuss some of these contexts, posing bottle neck for different fields of biology including biomedical research. With the advancement of chemistry, several methods have been introduced to overcome these problems. Even though microfluidics and microraft array are newer techniques exploited for single cell biology, fluorescence-activated cell sorting (FACS) remains the gold standard technique for isolation of cells for many biomedical applications, like stem cell therapy. Here, we present a comprehensive and comparative account of some of the probes that are useful in FACS. Further, we illustrate how these techniques could be applied in biomedical research. It is postulated that intracellular molecular markers like nucleostemin (GNL3), alkaline phosphatase (ALPL) and HIRA can be used for improving the outcome of cardiac as well as bone regeneration. Another field that could utilize intracellular markers is diagnostics, and we propose the use of specific peptide nucleic acid probes (PNPs) against certain miRNAs for cancer surgical margin prediction. The newer techniques for single cell biology, based on intracellular molecules, will immensely enhance the repertoire of possible markers for the isolation of cell types useful in biomedical research.     

Molecular and Cellular Basis of Regeneration and Tissue Repair

The ability to produce differentiated cell types at will offers one approach to cell therapy and therefore the treatment and cure of degenerative diseases such as diabetes and liver failure. Until recently it was thought that differentiated cells could only be produced from embryonic or adult stem cells. However, we now know that this is not the case, and there is a growing body of evidence to show that one differentiated cell type can convert into a completely different phenotype (transdifferentiation). Understanding the cellular and molecular basis of transdifferentiation will allow us to reprogram cells for transplantation. This approach will complement the use of embryonic and adult stem cells in the treatment of degenerative disorders. In this review, we will focus on some well-documented examples of transdifferentiation.     

Roles of innervation in developing and regenerating orofacial tissues

The head is innervated by 12 cranial nerves (I–XII) that regulate its sensory and motor functions. Cranial nerves are composed of sensory, motor, or mixed neuronal populations. Sensory neurons perceive generally somatic sensations such as pressure, pain, and temperature. These neurons are also involved in smell, vision, taste, and hearing. Motor neurons ensure the motility of all muscles and glands. Innervation plays an essential role in the development of the various orofacial structures during embryogenesis. Hypoplastic cranial nerves often lead to abnormal development of their target organs and tissues. For example, Möbius syndrome is a congenital disease characterized by defective innervation (i.e., abducens (VI) and facial (VII) nerves), deafness, tooth anomalies, and cleft palate. Hence, it is obvious that the peripheral nervous system is needed for both development and function of orofacial structures. Nerves have a limited capacity to regenerate. However, neural stem cells, which could be used as sources for neural tissue maintenance and repair, have been found in adult neuronal tissues. Similarly, various adult stem cell populations have been isolated from almost all organs of the human body. Stem cells are tightly regulated by their microenvironment, the stem cell niche. Deregulation of adult stem cell behavior results in the development of pathologies such as tumor formation or early tissue senescence. It is thus essential to understand the factors that regulate the functions and maintenance of stem cells. Yet, the potential importance of innervation in the regulation of stem cells and/or their niches in most organs and tissues is largely unexplored. This review focuses on the potential role of innervation in the development and homeostasis of orofacial structures and discusses its possible association with stem cell populations during tissue repair.     

DNA repair mechanisms in embryonic stem cells

Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.     

Studies on the cytochemical localization of adenylate-cyclase activity in Dugesia lugubris s.I.

The localization of adenylate-cyclase activity in Dugesia lugubris s.1. has been investigated cytochemically using 5'-adenylyl-imidodiphosphate as substrate. The enzyme was localized in mucous gland cells, in rhabdite cells, in intercellular spaces and also in nerve endings of this planarian. The presence of adenylate-cyclase on the membrane suggests that it might mediate different stimulus-secretion coupling by increasing cyclic AMP synthesis in specialized areas of the planarian.     

Breast cancer stem cell: the roles and therapeutic implications

Breast cancers have been increasingly recognized as malignancies displaying frequent inter- and intra-tumor heterogeneity. This heterogeneity is represented by diverse subtypes and complexity within tumors, and impinges on response to therapy, metastasis, and prognosis. Cancer stem cells (CSCs), a subpopulation of cancer cells endowed with self-renewal and differentiation capacity, have been suggested to contribute to tumor heterogeneity. The CSC concept posits a hierarchical organization of tumors, at the apex of which are stem cells that drive tumor initiation, progression, and recurrence. In breast cancer, CSCs have been proposed to contribute to malignant progression, suggesting that targeting breast cancer stem cells (BCSCs) may improve treatment efficacy. Currently, several markers have been reported to identify BCSCs. However, there is objective variability with respect to the frequency and phenotype of BCSCs among different breast cancer cell lines and patients, and the regulatory mechanisms of BCSCs remain unclear. In this review, we summarize current literature about the diversity of BCSC markers, the roles of BCSCs in tumor development, and the regulatory mechanisms of BCSCs. We also highlight the most recent advances in BCSC targeting therapies and the challenges in translating the knowledge into clinical practice.     

Multidrug-resistant cancer cells and cancer stem cells hijack cellular systems to circumvent systemic therapies,can natural products reverse this?

Chemotherapy is one of the most effective and broadly used approaches for cancer management and many modern regimes can eliminate the bulk of the cancer cells. However, recurrence and metastasis still remain a major obstacle leading to the failure of systemic cancer treatments. Therefore, to improve the long-term eradication of cancer, the cellular and molecular pathways that provide targets which play crucial roles in drug resistance should be identified and characterised. Multidrug resistance (MDR) and the existence of tumor-initiating cells, also referred to as cancer stem cells (CSCs), are two major contributors to the failure of chemotherapy. MDR describes cancer cells that become resistant to structurally and functionally unrelated anti-cancer agents. CSCs are a small population of cells within cancer cells with the capacity of self-renewal, tumor metastasis, and cell differentiation. CSCs are also believed to be associated with chemoresistance. Thus, MDR and CSCs are the greatest challenges for cancer chemotherapy. A significant effort has been made to identify agents that specifically target MDR cells and CSCs. Consequently, some agents derived from nature have been developed with a view that they may overcome MDR and/or target CSCs. In this review, natural products-targeting MDR cancer cells and CSCs are summarized and clustered by their targets in different signaling pathways.     

Neuroreplacement therapy and stem cell biology under disease conditions

Recent advances in stem cell technology are expanding our ability to replace a variety of cells throughout the body. In the past, neurological diseases caused by the degeneration of neuronal cells were considered incurable because of a long-held 'truism'; neurons do not regenerate during adulthood. However, this statement has been challenged, and we have now found much evidence that the brain is indeed capable of regenerating neurons after maturing. Based on this new concept, researchers have shown neural differentiation of stem cells and recovery of function following transplantation of these cells into the brain. These results may promise a bright future for clinical applications of stem cell strategies in neurological diseases; however, we must consider the pathophysiological environments of individual diseases that may affect stem cell biology. Before we begin to develop clinical applications, we must consider environmental factors that have not been discussed in the current preclinical studies. Here, we study cases of Alzheimer's disease and schizophrenia and discuss the effects of environmental factors under disease conditions.Received 15 January 2003; received after revision 7 April 2003; accepted 8 April 2003     

东亚三角涡虫肌球蛋白轻链（MLC）融合蛋白原核表达载体的构建及其在大肠杆菌中的表达

构建了涡虫肌球蛋白轻链融合蛋白原核表达载体,并进行表达,根据涡虫基因文库中肌球蛋白轻链（Mlc）基因完整的ORF序列设计合成特异性51物,通过PCR扩增涡虫Mlc基因,并插入到融合蛋白原核表达载体PET-28a中,转化宿主菌B121（DE3）细胞．0．4mMol／L的IPTG诱导表达MLc蛋白．重组质粒测序和酶切结果显示Mlc基因已正确插入PET-28a中,重组蛋白经SDS—PAGE在18．2KD处有一条明显的蛋白表达条带。western blot检测得到同样大小的条带。结果表明,涡虫His-MLC融合蛋白已成功表达。     

Role of p75 neurotrophin receptor in stem cell biology: more than just a marker

p75^NTR, the common receptor for both neurotrophins and proneurotrophins, has been widely studied because of its role in many tissues, including the nervous system. More recently, a close relationship between p75^NTR expression and pluripotency has been described. p75^NTR was shown to be expressed in various types of stem cells and has been used to prospectively isolate stem cells with different degrees of potency. Here, we give an overview of the current knowledge on p75^NTR in stem cells, ranging from embryonic to adult stem cells, and cancer stem cells. In an attempt to address its potential role in the control of stem cell biology, the molecular mechanisms underlying p75^NTR signaling in different models are also highlighted. p75^NTR-mediated functions include survival, apoptosis, migration, and differentiation, and depend on cell type, (pro)neurotrophin binding, interacting transmembrane co-receptors expression, intracellular adaptor molecule availability, and post-translational modifications, such as regulated proteolytic processing. It is therefore conceivable that p75^NTR can modulate cell-fate decisions through its highly ramified signaling pathways. Thus, elucidating the potential implications of p75^NTR activity as well as the underlying molecular mechanisms of p75^NTR will shed new light on the biology of both normal and cancer stem cells.     

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM⁺/CD44⁺ population accounts for 4.5% of tumor cells. EpCAM⁺/CD44⁺ gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM^?/CD44^?, EpCAM⁺/CD44^? and EpCAM^?/CD44⁺ cells failed to do so. Xenografts of EpCAM⁺/CD44⁺ gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM⁺/CD44⁺, but not EpCAM^?/CD44^?, EpCAM⁺/CD44^? or EpCAM^?/CD44⁺ cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM⁺/CD44⁺ cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM⁺/CD44⁺ subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.