Neural induction and neural stem cell development

Embryonic stem (ES) cells are a pluripotent and renewable cellular resource with tremendous potential for broad applications in regenerative medicine. Arguably the most important consideration for stem cell-based therapies is the ability to precisely direct the differentiation of stem cells along a preferred cellular lineage. During development, lineage commitment is a multistep process requiring the activation and repression of sets of genes at various stages, from an ES cell identity to a tissue-specific stem cell identity and beyond. Thus, the challenge is to ensure that the pattern of genomic regulation is recapitulated during the in vitro differentiation of ES cells into stem/progenitor cells of the appropriate tissue in a robust, predictable and stable manner. To address this issue, we must understand the ontogeny of tissue-specific stem cells during normal embryogenesis and compare the ontogeny of tissue-specific stem cells in ES cell models. Here, we discuss the issue of directed differentiation of pluripotent ES cells into neural stem cells, which is fundamentally linked to two early events in the development of the mammalian nervous system: the 'decision' of the ectoderm to acquire a neural identity (neural determination) and the origin of neural stem cells within this neural-committed population of cells. A clearer understanding of the molecular and cellular mechanisms that govern mammalian neural cell fate determination will lead to improved ES technology applications in neural regeneration.     

Molecular Pathways Governing Development of Vascular Endothelial Cells from ES/iPS Cells

Assembly of complex vascular networks occurs in numerous biological systems through morphogenetic processes such as vasculogenesis, angiogenesis and vascular remodeling. Pluripotent stem cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells can differentiate into any cell type, including endothelial cells (ECs), and have been extensively used as in vitro models to analyze molecular mechanisms underlying EC generation and differentiation. The emergence of these promising new approaches suggests that ECs could be used in clinical therapy. Much evidence suggests that ES/iPS cell differentiation into ECs in vitro mimics the in vivo vascular morphogenic process. Through sequential steps of maturation, ECs derived from ES/iPS cells can be further differentiated into arterial, venous, capillary and lymphatic ECs, as well as smooth muscle cells. Here, we review EC development from ES/iPS cells with special attention to molecular pathways functioning in EC specification.     

Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro.

The epidermis develops from a stem cell population in the surface ectoderm that feeds a single vertical terminal differentiation pathway. To date, however, the limited capacity for the isolation or purification of epidermal stem or precursor cells has hampered studies on early commitment and differentiation events. We have developed a two-step culture scheme in which pluripotent mouse embryonic stem (ES) cells are induced first to a surface ectoderm phenotype and then are positively selected for putative epidermal stem cells. We show that the earliest stages of epidermal development follow an ordered sequence that is similar to that observed in vivo (expression of keratin 8, keratin 19, keratin 17, and keratin 14), suggesting that ES cell-derived surface ectoderm-like cells can be induced to follow the epidermal developmental pathway. At a low frequency, keratin 14-positive early epidermal cells progressed to keratin 1-positive and terminally differentiated cells producing a cornified envelope. This culturing protocol provides an invaluable system in which to study both the mechanisms that direct stem cells along the epidermal pathway as well as those that influence their subsequent epidermal differentiation.     

The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.     

Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses

Efforts to study the development and function of the human cerebral cortex in health and disease have been limited by the availability of model systems. Extrapolating from our understanding of rodent cortical development, we have developed a robust, multistep process for human cortical development from pluripotent stem cells: directed differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to cortical stem and progenitor cells, followed by an extended period of cortical neurogenesis, neuronal terminal differentiation to acquire mature electrophysiological properties, and functional excitatory synaptic network formation. We found that induction of cortical neuroepithelial stem cells from human ES cells and human iPS cells was dependent on retinoid signaling. Furthermore, human ES cell and iPS cell differentiation to cerebral cortex recapitulated in vivo development to generate all classes of cortical projection neurons in a fixed temporal order. This system enables functional studies of human cerebral cortex development and the generation of individual-specific cortical networks ex vivo for disease modeling and therapeutic purposes.     

Crucial roles of mesodermal cell lineages in a murine embryonic stem cell-derived in vitro liver organogenesis system

Recent studies in the field of regenerative medicine have exploited the pluripotency of embryonic stem (ES) cells to generate a variety of cell lineages. However, the target has always been only a single lineage, which was isolated from other differentiated cell populations. In the present study, we selected sublines with a high capability for differentiation to contracting cardiomyocytes and also produced germ-line chimeric mice from a parent ES line. We also succeed in establishing embryoid bodies prepared from the ES cells that differentiated into not only hepatocytes but also at least two mesodermal lineages: cardiomyocytes that supported liver development and endothelial cells corresponding to sinusoids. This allowed the development of an in vitro system using murine ES cells that approximated the events of liver development in vivo. The expression of albumin was significantly higher in cardiomyocytes that had arisen in differentiated ES cells than in those that had not. Our in vitro system for liver organogenesis consists of a blood/sinusoid vascular-like network and hepatocyte layers and shows higher levels of hepatic function, such as albumin production and ammonia degradation, than hepatic cell lines and primary cultures of murine adult hepatocytes. This innovative system will lead to the development of second-generation regenerative medicine techniques using ES cells and is expected to be useful for the development of bioartificial liver systems and drug-metabolism assays.     

Embryonic stem cells

Stem cells are unique cell populations with the ability to choose between self-renewal and differentiation. Embryonic stem (ES) cells have the ability to form any fully differentiated cell of the body. To date, only three species of mammals have yielded long-term cultures of self-renewing ES cells- mice, monkeys, and humans. These cells have some special requirements to maintain their undifferentiated state in culture, e.g., presence of feeder cells, serum, or cytokines. Many scientific studies have tried to manipulate the growth and differentiation conditions with varied success. Studies of development in model systems, such as mice help our efforts to manipulate human stem cells in vitro. Data are now emerging that ES cells can be directed toward lineage-specific differentiation programs. On the basis of this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms of human development. Recent advances in culturing ES cells and success in exploiting their pluripotency brings great hope for using human ES cell-based reparative therapy in future.     

Cripto as a target for improving embryonic stem cell-based therapy in Parkinson's disease

Embryonic stem (ES) cells have been suggested as candidate therapeutic tools for cell replacement therapy in neurodegenerative disorders. However, limitations for the use of these cells lie in our restricted knowledge of the molecular mechanisms involved in their specialized differentiation and in the risk of tumor formation. Recent findings suggest that the EGF-CFC protein Cripto is a key player in the signaling pathways controlling neural induction in ES cells. Here we show that in vitro differentiation of Cripto(-/-) ES cells results in increased dopaminergic differentiation and that, upon transplantation into Parkinsonian rats, they result in behavioral and anatomical recovery with no tumor formation. The use of knockout ES cells that can generate dopamine cells while eliminating tumor risk holds enormous potential for cell replacement therapy in Parkinson's disease.     

Gene expression profiling of mouse embryonic stem cell progeny differentiated by Lumelsky's protocol

Successful conversion of embryonic stem (ES) cells into insulin-producing cells has been reported by Lumelsky et al. (Science 2001;292:1389-1394); however, it remains controversial. In this study, we investigated the properties of ES cell progeny-induced differentiation according to Lumelsky's protocol by immunocytochemistry, oligonucleotide microarray and real-time RT-PCR. Insulin-positive cells were observed at stages 3, 4 and 5. Microarray analysis demonstrated upregulation and appearance of some genes involved in pancreatic development but not beta-cell-specific functional genes in cells at stage 5. Similarly, real-time RT-PCR revealed that expression of beta-cell-specific functional genes such as islet amyloid polypeptide, insulin I and II was not increased in cells at stage 5. These results suggest that terminal differentiation of ES cell progeny toward functional pancreatic beta-cell is insufficient. This study also demonstrates the usefulness of multiple time-course expression profiles for validating differentiation fates of ES cell progeny.     

Double labeling of mRNA and protein markers in cultured embryoid bodies

Summary In vitro suspension cultures of embryonal carcinoma or embryonic stem cells (EC/ES) generate cell aggregates termed as embryoid bodies (EBs). EBs have been analyzed to study the mechanisms of cellular differentiation in vitro. The multipotency of EC/ES cells to differentiate into various cell types as well as the expression of many marker genes provides a valuable in vitro model system to study the mechanisms of cellular differentiation. Here we present a procedure for a mRNA detection of a specific gene using double labeling-mRNA probe and an antibody against cellular marker proteins. This double labeling analysis in combination with a culture of EBs provides a useful approach to analyze several mechanisms of cellular differentiation from multipotent EC/ES cells.     

Cell-cell interaction modulates neuroectodermal specification of embryonic stem cells

The controlled differentiation of embryonic stem (ES) cells is of utmost interest to their clinical, biotechnological, and basic science use. Many investigators have combinatorially assessed the role of specific soluble factors and extracellular matrices in guiding ES cell fate, yet the interaction between neighboring cells in these heterogeneous cultures has been poorly defined due to a lack of conventional tools to specifically uncouple these variables. Herein, we explored the role of cell-cell interactions during neuroectodermal specification of ES cells using a microfabricated cell pair array. We tracked differentiation events in situ, using an ES cell line expressing green fluorescent protein (GFP) under the regulation of the Sox1 gene promoter, an early marker of neuroectodermal germ cell commitment in the adult forebrain. We observed that a previously specified Sox1-GFP+ cell could induce the specification of an undifferentiated ES cell. This induction was modulated by the two cells being in contact and was dependent on the age of previously specified cell prior to coculture. A screen of candidate cell adhesion molecules revealed that the expression of connexin (Cx)-43 correlated with the age-dependent effect of cell contact in cell pair experiments. ES cells deficient in Cx-43 showed aberrant neuroectodermal specification and lineage commitment, highlighting the importance of gap junctional signaling in the development of this germ layer. Moreover, this study demonstrates the integration of microscale culture techniques to explore the biology of ES cells and gain insight into relevant developmental processes otherwise undefined due to bulk culture methods.     

Hepatic differentiation of mouse embryonic stem cells and induced pluripotent stem cells during organoid formation in hollow fibers

We have focused on pluripotent stem cells as a potential source of a hybrid-type artificial liver (HAL) and tried to develop a method for differentiating the pluripotent stem cells into cells of a hepatic lineage. In this study, we investigated the hepatic differentiation of mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells by applying hollow fiber (HF)/organoid culture method, in which cultured cells form a cellular aggregate called an "organoid" in the lumen of the HF. ES and iPS cells were injected into HFs to induce organoid formation, and cells were cultured. To induce hepatic differentiation, we added differentiation-promoting agents to the culture medium. The expression levels of differentiation-related genes were up-regulated, with cell proliferation and organoid formation inside HFs. Since we were able to achieve a high cell density in culture, the maximum levels of liver-specific functions per unit volume in the differentiating ES and iPS cells reached a level comparable to or better than that of primary mouse hepatocytes. In conclusion, ES and iPS cells have the potential to be a cell source for a HAL, and the HF/organoid culture method, therefore, has promise as a basic technology for the development of a HAL.     

Recent advances in the derivation of germ cells from the embryonic stem cells

In recent years, considerable progress has been made in the establishment and differentiation of human embryonic stem (ES) cell lines. The primordial germ cells (PGCs) and embryonic germ (EG) cells derived from them share many of their properties with ES cells. ES cell lines have now been derived from different stages of germ cell development and they have differentiated into gametes and shown embryonic development in mice, including the production of live pups. Conversely, germ cells can also be derived from ES cells. It has been demonstrated that murine (m) ES cells can differentiate into PGCs and subsequently into early gametes (oocytes and sperms) and blastocysts. Recently, immature sperm cells derived from mES cells in culture have produced live offspring. Preliminary research has indicated that human (h) ES cells probably have the potential to differentiate into germ cells. Adult stem cells have been reported to differentiate into mature germ cells in vitro. Therefore, stem cells may offer a valuable in vitro model for the investigation of germ cell development and the early stages of human gametogenesis, including epigenetic modifications of the germ line. This review discusses recent developments in the derivation and specification of mammalian germ cells from ES cells and describes some of the mechanisms of germ cell development.     

Lentiviral rescue of vascular endothelial growth factor receptor-2 expression in flk1-/- embryonic stem cells shows early priming of endothelial precursors

The vascular endothelial growth factor (VEGF) family and its receptors are important for vascular development and maintenance of blood vessels, as well as for angiogenesis, the formation of new vessels. Loss of VEGF receptor-2 (VEGFR-2; designated Flk-1 in mouse) results in arrest of vascular and hematopoietic development in vivo. We used lentiviral transduction to reconstitute VEGFR-2 expression in flk1-/- embryonic stem (ES) cells. VEGF-induced vasculogenesis and sprouting angiogenesis were rescued in transduced ES cultures differentiating in vitro as EBs. Although the transgene was expressed in the pluripotent stem cells and lacked linage restriction during differentiation, the extent of endothelial recruitment was similar to that in wild-type EBs. Reconstitution of VEGFR-2 in flk1-/- ES cells allowed only precommitted precursors to differentiate into functional endothelial cells able to organize into vascular structures. Chimeric EB cultures composed of wild-type ES cells mixed with flk1-/- ES cells or reconstituted VEGFR-2-expressing ES cells were created. In the chimeric cultures, flk1-/- endothelial precursors were excluded from wild-type vessel structures, whereas reconstituted VEGFR-2-expressing precursors became integrated together with wild-type endothelial cells to form chimeric vessels. We conclude that maturation of endothelial precursors, as well as organization into vascular structures, requires expression of VEGFR-2. Disclosure of potential conflicts of interest is found at the end of this article.     

Role of Gap Junctions in Embryonic and Somatic Stem Cells

Stem cells provide an invaluable tool to develop cell replacement therapies for a range of serious disorders caused by cell damage or degeneration. Much research in the field is focused on the identification of signals that either maintain stem cell pluripotency or direct their differentiation. Understanding how stem cells communicate within their microenvironment is essential to achieve their therapeutic potentials. Gap junctional intercellular communication (GJIC) has been described in embryonic stem cells (ES cells) and various somatic stem cells. GJIC has been implicated in regulating different biological events in many stem cells, including cell proliferation, differentiation and apoptosis. This review summarizes the current understanding of gap junctions in both embryonic and somatic stem cells, as well as their potential role in growth control and cellular differentiation.     

Developmental platelet endothelial cell adhesion molecule expression suggests multiple roles for a vascular adhesion molecule

Platelet endothelial cell adhesion molecule (PECAM) is used extensively as a murine vascular marker. PECAM interactions have been implicated in both vasculogenesis and angiogenesis. To better understand the role of PECAM in mammalian development, PECAM expression was investigated during differentiation of murine embryonic stem (ES) cells and in early mouse embryos. Undifferentiated ES cells express PECAM, and as in vitro differentiation proceeds previously unidentified PECAM-positive cells that are distinct from vascular endothelial cells appear. PECAM expression is gradually restricted to endothelial cells and some hematopoietic cells of differentiated blood islands. In embryos, the preimplantation blastocyst contains PECAM-positive cells. PECAM expression is next documented in the postimplantation embryonic yolk sac, where clumps of mesodermal cells express PECAM before the development of mature blood islands. The patterns of PECAM expression suggest that undifferentiated cells, a prevascular cell type, and vascular endothelial cells express this marker during murine development. PECAM expression in blastocysts and by ES cells suggests that PECAM may function outside the vascular/hematopoietic lineage.     

Induction of cellular differentiation by retinoic acid in vitro

Cellular differentiation by the vitamin A derivative retinoic acid (RA) has been studied with undifferentiated pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells in vitro. Both cellular systems are suitable to study differentiation of various cell types, because they recapitulate early stages of mouse embryogenesis. In vivo, RA was identified as a morphogenic and teratogenic compound and furthermore as a signalling molecule influencing gene expression in a complex manner via a family of RA receptors. Here, we summarize in vitro studies with ES and EC cells in comparison to in vivo studies that have contributed to our understanding how RA influences differentiation and regulates gene expression. We demonstrate that modulation of ES cell differentiation in vitro by RA depends on the concentration and developmental stage of application which is comparable to its stage-dependent influence on embryonic development in vivo.     

昆明小鼠胚胎干细胞向心肌细胞的分化

目的饲养层制作及复苏培养昆明小鼠胚胎干细胞,探索体外诱导胚胎干细胞向心肌细胞分化。方法饲养层制作,复苏及体外培养胚胎干细胞,采用一步法消化贴壁胚胎干细胞,悬浮培养5d,形成拟胚体(EB),待EB贴壁后,加入淫羊藿苷(Icraiin,ICA)诱导液对其诱导并每天观察,免疫荧光检测心肌细胞特异性肌钙蛋白T(cTnT)和心室肌球蛋白轻链(MLC-2v)的表达。结果胚胎干细胞悬浮聚集5d,形成类似球状的拟胚体,将拟胚体贴壁诱导8d,发现拟胚体中出现跳动,诱导后10d拟胚体跳动率达65%,显著高于空白对照组和阴性对照组,一个分化拟胚体中一般出现1-3个跳动点,节律约为50-80times/min,在诱导10d时跳动的合胞体,免疫荧光表明cTnT和MLC-2v表达为阳性。结论胚胎干细胞经悬浮聚集培养5d后经10-7mol/L淫羊藿苷诱导,得到了可以跳动的心肌细胞团。