Anna Ferri

Hippocampal development and neural stem cell maintenance require Sox2 -dependent regulation of Shh

Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.

Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner

The role of transcription factors in regulating the development of midbrain dopaminergic (mDA) neurons is intensively studied owing to the involvement of these neurons in diverse neurological disorders. Here we demonstrate novel roles for the forkhead/winged helix transcription factors Foxa1 and Foxa2 in the specification and differentiation of mDA neurons by analysing the phenotype of Foxa1 and Foxa2 single- and double-mutant mouse embryos. During specification, Foxa1 and Foxa2 regulate the extent of neurogenesis in mDA progenitors by positively regulating Ngn2 (Neurog2) expression. Subsequently, Foxa1 and Foxa2 regulate the expression of Nurr1 (Nr4a2) and engrailed 1 in immature neurons and the expression of aromatic l-amino acid decarboxylase and tyrosine hydroxylase in mature neurons during early and late differentiation of midbrain dopaminergic neurons. Interestingly, genetic evidence indicates that these functions require different gene dosages of Foxa1 and Foxa2. Altogether, our results demonstrate that Foxa1 and Foxa2 regulate multiple phases of midbrain dopaminergic neuron development in a dosage-dependent manner.

Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons

Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2+ ventricular zone progenitors into Nurr1+ postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.

Transcriptional repression coordinates the temporal switch from motor to serotonergic neurogenesis

In many regions of the developing CNS, distinct cell types are born at different times. The means by which discrete and stereotyped temporal switches in cellular identities are acquired remains poorly understood. To address this, we have examined how visceral motor neurons (VMNs) and serotonergic neurons, two neuronal subtypes, are sequentially generated from a common progenitor pool in the vertebrate hindbrain. We found that the forkhead transcription factor Foxa2, acting in progenitors, is essential for the transition from VMN to serotonergic neurogenesis. Loss-of-function and gain-of-function experiments indicated that Foxa2 activates the switch through a temporal cross-repressive interaction with paired-like homeobox 2b (Phox2b), the VMN progenitor determinant. This mechanism bears a marked resemblance to the cross-repression between neighboring domains of transcription factors that establish discrete progenitor identities along the spatial axes. Moreover, the subsequent differentiation of central serotonergic neurons required both the suppression of VMN neurogenesis and the induction of downstream intrinsic determinants of serotonergic identity by Foxa2.

Sox2 is required for embryonic development of the ventral telencephalon through the activation of the ventral determinants Nkx2.1 and Shh.

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE-derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction.

Rai is a new regulator of neural progenitor migration and glioblastoma invasion.

The invasive nature of glioblastoma (GBM) is one important reason for treatment failure. GBM stem/progenitor cells retain the migratory ability of normal neural stem/progenitor cells and infiltrate the brain parenchyma. Here, we identify Rai (ShcC/N‐Shc), a member of the family of Shc‐like adaptor proteins, as a new regulator of migration of normal and cancer stem/progenitor cells. Rai is expressed in neurogenic areas of the brain and its knockdown impairs progenitor migration to the olfactory bulb. Its expression is retained in GBM stem/progenitor cells where it exerts the same promigratory activity. Rai silencing in cancer stem/progenitor cells isolated from different patients causes significant decrease in cell migration and invasion, both in vitro and in vivo, providing survival benefit. Rai depletion is associated with alteration of multiple‐signaling pathways, yet it always leads to reduced expression of proinvasive genes. STEM CELLS 2012;30:817–832

Drug-releasing mesenchymal cells strongly suppress B16 lung metastasis in a syngeneic murine model

BackgroundMesenchymal stromal cells (MSCs) are considered an important therapeutic tool in cancer therapy. They possess intrinsic therapeutic potential and can also be in vitro manipulated and engineered to produce therapeutic molecules that can be delivered to the site of diseases, through their capacity to home pathological tissues. We have recently demonstrated that MSCs, upon in vitro priming with anti-cancer drug, become drug-releasing mesenchymal cells (Dr-MCs) able to strongly inhibit cancer cells growth.MethodsMurine mesenchymal stromal cells were loaded with Paclitaxel (Dr-MCsPTX) according to a standardized procedure and their ability to inhibit the growth of a murine B16 melanoma was verified by in vitro assays. The anti-metastatic activity of Dr-MCsPTX was then studied in mice injected i.v. with B16 melanoma cells that produced lung metastatic nodules. Lung nodules were counted under a dissecting stereomicroscope and metastasis investigated by histological analysis.ResultsWe found that three i.v. injections of Dr-MCsPTX on day 5, 10 and 15 after tumor injection almost completely abolished B16 lung metastasis. Dr-MCsPTX arrested into lung by interacting with endothelium and migrate toward cancer nodule through a complex mechanism involving primarily mouse lung stromal cells (mL-StCs) and SDF-1/CXCR4/CXCR7 axis.ConclusionsOur results show for the first time that Dr-MCsPTX are very effective to inhibit lung metastasis formation. Actually, a cure for lung metastasis in humans is mostly unlikely and we do not know whether a therapy combining engineered MSCs and Dr-MCs may work synergistically. However, we think that our approach using Dr-MCs loaded with PTX may represent a new valid and additive therapeutic tool to fight lung metastases and, perhaps, primary lung cancers in human.

Angiogenic and anti-inflammatory properties of micro-fragmented fat tissue and its derived mesenchymal stromal cells.

BackgroundAdipose-derived mesenchymal stromal cells (Ad-MSCs) are a promising tool for advanced cell-based therapies. They are routinely obtained enzymatically from fat lipoaspirate (LP) as SVF, and may undergo prolonged ex vivo expansion, with significant senescence and decline in multipotency. Besides, these techniques have complex regulatory issues, thus incurring in the compelling requirements of GMP guidelines. Hence, availability of a minimally manipulated, autologous adipose tissue would have remarkable biomedical and clinical relevance. For this reason, a new device, named Lipogems® (LG), has been developed. This ready-to-use adipose tissue cell derivate has been shown to have in vivo efficacy upon transplantation for ischemic and inflammatory diseases. To broaden our knowledge, we here investigated the angiogenic and anti-inflammatory properties of LG and its derived MSC (LG-MSCs) population.MethodsHuman LG samples and their LG-MSCs were analyzed by immunohistochemistry for pericyte, endothelial and mesenchymal stromal cell marker expression. Angiogenesis was investigated testing the conditioned media (CM) of LG (LG-CM) and LG-MSCs (LG-MSCs-CM) on cultured endothelial cells (HUVECs), evaluating proliferation, cord formation, and the expression of the adhesion molecules (AM) VCAM-1 and ICAM-1. The macrophage cell line U937 was used to evaluate the anti-inflammatory properties, such as migration, adhesion on HUVECs, and release of RANTES and MCP-1.ResultsOur results indicate that LG contained a very high number of mesenchymal cells expressing NG2 and CD146 (both pericyte markers) together with an abundant microvascular endothelial cell (mEC) population. Substantially, both LG-CM and LG-MSC-CM increased cord formation, inhibited endothelial ICAM-1 and VCAM-1 expression following TNFα stimulation, and slightly improved HUVEC proliferation. The addition of LG-CM and LG-MSC-CM strongly inhibited U937 migration upon stimulation with the chemokine MCP-1, reduced their adhesion on HUVECs and significantly suppressed the release of RANTES and MCP-1.ConclusionsOur data indicate that LG micro-fragmented adipose tissue retains either per se, or in its embedded MSCs content, the capacity to induce vascular stabilization and to inhibit several macrophage functions involved in inflammation.

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

Establishment, characterization and long-term culture of human endocrine pancreas-derived microvascular endothelial cells.

BACKGROUND In vitro primary cultures of microvascular endothelial cells from endocrine pancreas are difficult to obtain, but can be a very helpful tool for studies of islet biology, transplantation and regenerative medicine. METHODS We applied a protocol recently described for the isolation and culture of brain microvascular endothelial cells (EC) on human pancreatic islets. EC obtained were characterized in terms of morphological (light and transmission electron microscopy), phenotypical (by immunofluorescence and flow cytometry) and functional (cord formation assay and protein secretion by multiplex bead-based assay) characteristics. RESULTS EC were obtained from 25% of islet preparations processed. Two primary endothelial cell lines showed high proliferative potential and were deeply characterized: they presented endothelial cell morphology and expressed CD31, CD49a, CD49e, CD34, von Willebrand Factor (vWF), Vascular Endothelial CAdherin (VE-CAD), Tyrosine Kinase with Ig and EGF Homology Domains-2 (TIE2), Vascular Endothelial Growth Factor Receptor 1 (VEGFR1), Ulex lectin and the endothelium endocrine-specific marker nephrin. Besides, they were able to form cordons in vitro and secreted factors involved in the process of angiogenesis such as Vascular Endothelial Growth Factor (VEGF), Monocyte Chemotactic Protein 1 (MCP-1), interleukin (IL)-8 and Melanoma Growth Stimulatory Activity Alpha (GROα). These cell lines were termed Human Islet Microvascular Endothelial Cells (HIMEC). DISCUSSION This study establishes a simple and effective strategy for isolation and long-term culture of EC derived from human pancreatic islet. HIMEC in culture preserve phenotype and functional properties and are, therefore, a useful tool for future experiments of in vitro pancreas modelling, co-transplantation with pancreatic islets, re-vascularization of scaffold or matrix for regenerative medicine purposes.