Neurogenic differentiation of human adipose-derived stem cells: Relevance of different signaling molecules,transcription factors,and key marker genes

Since numerous diseases affect the central nervous system and it has limited self-repair capability, a great interest in using stem cells as an alternative cell source is generated. Previous reports have shown the differentiation of adipose-derived stem cells in neuron-like cells and it has also been proved that the expression pattern of patterning, proneural, and neural factors, such as Pax6, Mash1, Ngn2, NeuroD1, Tbr2 and Tbr1, regulates and defines adult neurogenesis. Regarding this, we hypothesize that a functional parallelism between adult neurogenesis and neuronal differentiation of human adipose-derived stem cells exists. In this study we differentiate human adipose-derived stem cells into neuron-like cells and analyze the expression pattern of different patterning, proneural, neural and neurotransmitter genes, before and after neuronal differentiation. The neuron-like cells expressed neuronal markers, patterning and proneural factors characteristics of intermediate stages of neuronal differentiation. Thus we demonstrated that it is possible to differentiate adipose-derived stem cells in vitro into immature neuron-like cells and that this process is regulated in a similar way to adult neurogenesis. This may contribute to elucidate molecular mechanisms involved in neuronal differentiation of adult human non-neural cells, in aid of the development of potential therapeutic tools for diseases of the nervous system.     

Expression of developmental genes during early embryogenesis of<Emphasis Type="Italic"> Hydra</Emphasis>

Hydra is a classical model to study key features of embryogenesis such as axial patterning and stem cell differentiation. In contrast to other organisms where these mechanisms are active only during embryonic development, in Hydra they can be studied in adults. The underlying assumption is that the machinery governing adult patterning mimics regulatory mechanisms which are also active during early embryogenesis. Whether, however, Hydra embryogenesis is governed by the same mechanisms which are controlling adult patterning, remains to be shown. In this paper, in precisely staged Hydra embryos, we examined the expression pattern of 15 regulatory genes shown previously to play a role in adult patterning and cell differentiation. RT-PCR revealed that most of the genes examined were expressed in rather late embryonic stages. In situ hybridization, nuclear run-on experiments, and staining of nucleolar organizer region-associated proteins indicated that genes expressed in early embryos are transcribed in the engulfed "nurse cells" (endocytes). This is the first direct evidence that endocytes in Hydra not only provide nutrients to the developing oocyte but also produce maternal factors critical for embryogenesis. Our findings are an initial step towards understanding the molecular machinery controlling embryogenesis of a key group of basal metazoans and raise the possibility that in Hydra there are differences in the mechanisms controlling embryogenesis and adult patterning.Edited by D. Tautz     

Neuroblast formation and patterning during early brain development in Drosophila

The Drosophila embryo provides a useful model system to study the mechanisms that lead to pattern and cell diversity in the central nervous system (CNS). The Drosophila CNS, which encompasses the brain and the ventral nerve cord, develops from a bilaterally symmetrical neuroectoderm, which gives rise to neural stem cells, called neuroblasts. The structure of the embryonic ventral nerve cord is relatively simple, consisting of a sequence of repeated segmental units (neuromeres), and the mechanisms controlling the formation and specification of the neuroblasts that form these neuromeres are quite well understood. Owing to the much higher complexity and hidden segmental organization of the brain, our understanding of its development is still rudimentary. Recent investigations on the expression and function of proneural genes, segmentation genes, dorsoventral-patterning genes and a number of other genes have provided new insight into the principles of neuroblast formation and patterning during embryonic development of the fly brain. Comparisons with the same processes in the trunk help us to understand what makes the brain different from the ventral nerve cord. Several parallels in early brain patterning between the fly and the vertebrate systems have become evident.     

Cell lineage and cell fate specification in the embryonic CNS of Drosophila

The Drosophila CNS derives from a population of neural stem cells, called neuroblasts (NBs), which delaminate individually from the neurogenic region of the ectoderm. In the embryonic ventral nerve cord each NB can be uniquely identified and gives rise to a specific lineage consisting of neurons and/or glial cells. This 'NB identity' is dependent on the position of the progenitor cells in the neuroectoderm before delamination. The positional information is provided by the products of segment polarity and dorsoventral (D/V) patterning genes. Subsequently, 'cell fate genes' like huckebein (hkb) and eagle (eg) contribute to the generation of specific NB lineages. These genes act downstream of segment polarity and D/V patterning genes and regulate different processes such as the generation of glial cells and the determination of serotonergic neurons.     

Epithelial stem cells

New molecular markers for epidermal stem cells have enabled their isolation both in vitro and from the epidermis lying between hair follicles. Micro-dissection experiments have localised a second population of stem cells within hair follicles. Epidermal stem cells have a patterned distribution in vivo. The patterning can be reconstituted in vitro, showing that it is generated by interactions between keratinocytes and that the differentiation of epidermal stem cells is regulated by signals from other keratinocytes. Recent evidence from transgenic mice suggests that stem cell behaviour in the gut may be regulated by similar cell-cell interactions in vivo. Candidate genes for mediating these interactions are the homologues of Drosophila cell fate patterning genes such as Notch and Wingless and the Cadherin family of cell-cell adhesion molecules. The roles of stem cells and of mutations of the Patched gene in epithelial carcinogenesis are discussed.     

Mouse chimeras as a system to investigate development,cell and tissue function,disease mechanisms and organ regeneration

Chimeras are organisms composed of at least two genetically distinct cell lineages originating from different zygotes. In the laboratory, mouse chimeras can be produced experimentally; various techniques allow combining different early stage mouse embryos with each other or with pluripotent stem cells. Identification of the progeny of the different lineages in chimeras permits to follow cell fate and function, enabling correlation of genotype with phenotype. Mouse chimeras have become a tool to investigate critical developmental processes, including cell specification, differentiation, patterning and the function of specific genes. In addition, chimeras can also be generated to address biological processes in the adult, including mechanisms underlying diseases or tissue repair and regeneration. This review summarizes the different types of chimeras and how they have been generated and provides examples of how mouse chimeras offer a unique and powerful system to investigate questions pertaining to cell and tissue function in the developing and adult organism.Key words: chimera, developmental chimera, aggregation, blastocyst injection, embryonic stem cells, induced pluripotent stem cells, complementation, regeneration     

Evolutionary conservation of mechanisms for neural regionalization, proliferation and interconnection in brain development

Comparative studies of brain development in vertebrate and invertebrate model systems demonstrate remarkable similarities in expression and action of developmental control genes during embryonic patterning, neural proliferation and circuit formation in the brain. Thus, comparable sets of developmental control genes are involved in specifying the early brain primordium as well as in regionalized patterning along its anteroposterior and dorsoventral axes. Furthermore, similar cellular and molecular mechanisms underlie the formation and proliferation of neural stem cell-like progenitors that generate the neurons in the central nervous systems. Finally, neural identity and some complex circuit interconnections in specific brain domains appear to be comparable in vertebrates and invertebrates and may depend on similar developmental control genes.     

Hydra, a niche for cell and developmental plasticity

The silencing of genes whose expression is restricted to specific cell types and/or specific regeneration stages opens avenues to decipher the molecular control of the cellular plasticity underlying head regeneration in hydra. In this review, we highlight recent studies that identified genes involved in the immediate cytoprotective function played by gland cells after amputation; the early dedifferentiation of digestive cells into blastema-like cells during head regeneration, and the early late proliferation of neuronal progenitors required for head patterning. Hence, developmental plasticity in hydra relies on spatially restricted and timely orchestrated cellular modifications, where the functions played by stem cells remain to be characterized.     

Caudal genes in blood development and leukemia

Members of the caudal gene family (in mice and humans: Cdx1, Cdx2, and Cdx4) have been studied during early development as regulators of axial elongation and anteroposterior patterning. In the adult, Cdx1 and Cdx2, but not Cdx4, have been intensively explored for their function in intestinal tissue homeostasis and the pathogenesis of gastrointestinal cancers. Involvement in embryonic hematopoiesis was first demonstrated in zebrafish, where cdx genes render posterior lateral plate mesoderm competent to respond to genes specifying hematopoietic fate, and compound mutations in cdx genes thus result in a bloodless phenotype. Parallel studies performed in zebrafish embryos and murine embryonic stem cells (ESCs) delineate conserved pathways between fish and mammals, corroborating a BMP/Wnt-Cdx-Hox axis during blood development that can be employed to augment derivation of blood progenitors from pluripotent stem cells in vitro. The molecular regulation of Cdx genes appears complex, as more recent data suggest involvement of non-Hox-related mechanisms and the existence of auto- and cross-regulatory loops governed by morphogens. Here, we will review the role of Cdx genes during hematopoietic development by comparing effects in zebrafish and mice and discuss their participation in malignant blood diseases.     

Chimeric mice reveal clonal development of pancreatic acini, but not islets

Intestinal crypt stem cells establish clonal descendants. To determine whether the pancreas is patterned by a similar process, we used embryonic stem (ES) cell chimeric mice, in which male ES cells were injected into female blastocysts. Fluorescence in situ hybridization for the Y chromosome (Y-FISH) revealed clonal patterning of ES-derived cells in the adult mouse small intestine and pancreas. Intestinal crypts were entirely male or entirely female. Villi contained columns of male or female epithelial cells, consistent with upward migration of cells from the crypts which surround them. Within the exocrine pancreas, acini were entirely male or entirely female, consistent with patterning from a single stem/progenitor cell. Pancreatic islets contained a mixture of male and female cells, consistent with patterning from multiple progenitors. Male-female chimeric mice demonstrate that the adult mouse exocrine pancreatic acinus is patterned from a single stem/progenitor cell, while the endocrine pancreas arises from multiple progenitors.     

Stem-cell niches: nursery rhymes across kingdoms

Despite the large evolutionary distance between the plant and animal kingdoms, stem cells in both reside in specialized cellular contexts called stem-cell niches. Although stem-cell-specification factors have been recruited from plant-specific gene families, maintenance factors that repress stem-cell differentiation are conserved between plants and animals. Recent evidence indicates that stem cells in multicellular organisms can be specified by kingdom-specific patterning mechanisms that connect to a related core of epigenetic stem-cell factors.     

Mouse chimeras as a system to investigate development,cell and tissue function,disease mechanisms and organ regeneration

Chimeras are organisms composed of at least two genetically distinct cell lineages originating from different zygotes. In the laboratory, mouse chimeras can be produced experimentally; various techniques allow combining different early stage mouse embryos with each other or with pluripotent stem cells. Identification of the progeny of the different lineages in chimeras permits to follow cell fate and function, enabling correlation of genotype with phenotype. Mouse chimeras have become a tool to investigate critical developmental processes, including cell specification, differentiation, patterning, and the function of specific genes. In addition, chimeras can also be generated to address biological processes in the adult, including mechanisms underlying diseases or tissue repair and regeneration. This review summarizes the different types of chimeras and how they have been generated and provides examples of how mouse chimeras offer a unique and powerful system to investigate questions pertaining to cell and tissue function in the developing and adult organism.     

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.     

Opposing FGF and retinoid pathways control ventral neural pattern, neuronal differentiation, and segmentation during body axis extension

Vertebrate body axis extension involves progressive generation and subsequent differentiation of new cells derived from a caudal stem zone; however, molecular mechanisms that preserve caudal progenitors and coordinate differentiation are poorly understood. FGF maintains caudal progenitors and its attenuation is required for neuronal and mesodermal differentiation and to position segment boundaries. Furthermore, somitic mesoderm promotes neuronal differentiation in part by downregulating Fgf8. Here we identify retinoic acid (RA) as this somitic signal and show that retinoid and FGF pathways have opposing actions. FGF is a general repressor of differentiation, including ventral neural patterning, while RA attenuates Fgf8 in neuroepithelium and paraxial mesoderm, where it controls somite boundary position. RA is further required for neuronal differentiation and expression of key ventral neural patterning genes. Our data demonstrate that FGF and RA pathways are mutually inhibitory and suggest that their opposing actions provide a global mechanism that controls differentiation during axis extension.     

Noggin and chordin have distinct activities in promoting lineage commitment of mouse embryonic stem (ES) cells

To examine the role of secreted signaling molecules and neurogenic genes in early development, we have developed a culture system for the controlled differentiation of mouse embryonic stem (ES) cells. In the current investigation, two of the earliest identified BMP antagonists/neural-inducing factors, noggin and chordin, were expressed in pluripotent mouse ES cells. Neurons were present as early as 24 h following transfection of ES cells with a pCS2/noggin expression plasmid, with differentiation peaking at 72 h. With neuronal differentiation, stem cell marker genes were down-regulated and neural determination genes expressed. Coculture experiments and exposure to noggin-conditioned medium produced similar neuronal differentiation of control ES cells, while addition of BMP-4 to noggin expressants strikingly inhibited neuronal differentiation. Transfection of ES cells with a pCS2/chordin expression vector or exposure to chordin-conditioned medium produced a more complex pattern of differentiation; ES cells formed neurons, mesenchymal cells as well as N-CAM-positive, nestin-positive neuroepithelial progenitors. These data suggest that, consistent with their different expression fields, noggin and chordin may play distinct roles in patterning the early mouse embryo.     

Multiple roles of Activin/Nodal,bone morphogenetic protein,fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification.     

Spots and stripes: Pleomorphic patterning of stem cells via p-ERK-dependent cell chemotaxis shown by feather morphogenesis and mathematical simulation

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction-diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large.     

Geminin Coordinates Cell Cycle and Developmental Control

Growth and differentiation are two major themes in embryonic development. Numerous cell divisions have to be regulated on the path from a unicellular embryo, the zygote, to the multicellular structures of a mature being. Numerous functions, specializations and cellular identities have to be generated, in order to form a complex and mature animal. Numerous mechanisms have to control the correct assignment and acquisition of cellular fates, as well as the right timing and allocation of cells. Therefore, a strict coordination has to occur between embryonic patterning and the cell cycle. From this point of view, dual roles or mutual interactions of typical proliferation and developmental control genes are likely. Recently, new light was shed on these issues by identifying the nuclear protein Geminin as a molecular coordinator between the cell cycle and axial patterning. We summarize the role of Geminin in cell cycle, in the embryonic patterning controlled by Hox genes, providing insights into cell cycle regulators in embryonic development, and, conversely, typical developmental control genes in cell cycle regulation.     

Homology,limbs, and genitalia

SUMMARY Similarities in genetic control between the main body axis and its appendages have been generally explained in terms of genetic co-option. In particular, arthropod and vertebrate appendages have been explained to invoke a common ancestor already provided with patterned body outgrowths or independent recruitment in limb patterning of genes or genetic cassettes originally used for purposes other than axis patterning. An alternative explanation is that body appendages, including genitalia, are evolutionarily divergent duplicates (paramorphs) of the main body axis. However, are all metazoan limbs and genitalia homologous? The concept of body appendages as paramorphs of the main body axis eliminates the requirement for the last common ancestor of limb-bearing animals to have been provided with limbs. Moreover, the possibility for an animal to express complex organs ectopically demonstrates that positional and special homology may be ontogenetically and evolutionarily uncoupled. To assess the homology of animal genitalia, we need to take into account three different sets of mechanisms, all contributing to their positional and/or special homology and respectively involved (1) in the patterning of the main body axis, (2) in axis duplication, followed by limb patterning mechanisms diverging away from those still patterning the main body axis (axis paramorphism), and (3) in controlling the specification of sexual/genital features, which often, but not necessarily, come into play by modifying already developed and patterned body appendages. This analysis demonstrates that a combinatorial approach to homology helps disentangling phylogenetic and ontogenetic layers of homology.