Nuclear factor I-C regulates TGF-{beta}-dependent hair follicle cycling.

Skin appendages such as teeth and hair share several common signaling pathways. The nuclear factor I C (NFI-C) transcription factor has been implicated in tooth development, but a potential role in hair growth had not been assessed. In this study we found that NFI-C regulates the onset of the hair growth cycle. NFI-C(-/-) mice were delayed in the transition from the telogen to anagen phase of the hair follicle cycle after either experimental depilation or spontaneous hair loss. Lack of NFI-C resulted in delayed induction of the sonic hedgehog, Wnt5a, and Lef1 gene expression, which are key regulators of the hair follicle growth initiation. NFI-C(-/-) mice also showed elevated levels of transforming growth factor β1 (TGF-β1), an inhibitor of keratinocyte proliferation, and of the cell cycle inhibitor p21 at telogen. Reduced expression of Ki67, a marker of cell proliferation, was noted at the onset of anagen, indicating impaired activation of the hair progenitor cells. These findings implicate NFI-C in the repression of TGF-β1 signaling during telogen stage, resulting in the delay of progenitor cell proliferation and hair follicle regeneration in NFI-C-deficient mice. Taken together with prior observations, these findings also designate NFI-C as a regulator of adult progenitor cell proliferation and of postnatal tissue growth or regeneration.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature.

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease.

Role of the microenvironment on epithelial stem cell fate

Stratified epithelia of mammals contain adult stem/progenitor cells that are instrumental for renewal, regeneration and repair. We have recently demonstrated, using clonal and functional analysis, that all stratified epithelia contain clonogenic stem cells that can respond to skin morphogenetic signals, while cells obtained from simple or pseudo-stratified epithelia cannot. A genome-wide expression analysis favors multilineage priming rather than reprogramming. Collectively, these observations are reminiscent of epithelial metaplasia, a phenomenon in which a cell adopts the phenotype of another epithelial cell, often in response to repeated environmental stress, e.g. smoking, alcohol and micro-traumatisms. Furthermore, they support the notion that metaplasia results from the expression of an unseen potency, revealed by an environmental deficiency. The thymus supposedly contains only progenitor epithelial cells but no stem cells. We have demonstrated that the thymus also contains a small population of clonogenic cells that can function as bona fide multipotent hair follicle stem cells in response to an inductive skin microenvironment and a genome-wide expression analysis indicates that it correlates with robust changes in the expression of genes important for thymus identity. Hence, multilineage priming or reprogramming can account for the fate change of epithelial stem/progenitor cells in response to a varying microenvironment.

High-dose sequential chemotherapy (ICE) under circulating progenitor cells (CPC) protection in patients with small cell lung carcinoma (SCLC). Preliminary report on a multicentric study

Starting in February 1994, 20 patients (pt) with a median age of 50 years(range 41-63) from 7 European centers have been included. Completedata were obtained in 16 patients so far. CPC were mobilized with chemo(Epirubicine 75 mg/m2 /d, 01 + 02) followed by G-CSF 5 p.gfkg/d for14 days. HD chemo consisted in 3 sequential courses of ICE regimen(UOs. 10 g/m2 , Carbo. 1200 mg/m2 and Etop. 1200 mg/m2 ) underCPC protection and G-CSF 5 p.g/kg/d. Out of the 16 pt, 12 completedfull program (3 cycles). One pt died of septic shock before receivingany ICE course. One pt died during the first ICE of renal insufficiency.Two pt had only 2 courses because of toxicity. Among the 16 pt, responserate (RR) was: 7 CR, 6 PR, 1 PO; 3 pt are not evaluable dueto early withdrawal (overall RR: 13/16 = 81 %). Thirty-nine cycles ofHD chemo were given with a median hematological recovery of 9 days(range 7-12) until neutro. counts> 1.0 x 109 /1 and 9 days (range 717)until thrombo. > 20 x 109 /1. No cumulative, hematological toxicitywas seen. Accrual of patients is still ongoing and updated results will bepresented.

Harnessing epithelial stem cell potency

Increase in potency of adult stem/progenitor cells holds great expectations for regenerative medicine; reprogramming is achieved by manipulating the genome or indirectly by manipulating the microenvironment. However, the genetic approach, which can result in lineage conversion up to ground pluripotent embryonic state, will certainly face strict regulatory constraints and consequently translation to the clinic may be difficult. Manipulating stem cell fate without altering the genome of adult stem cells is a promising alternative. My laboratory has demonstrated that non hairy squamous epithelia e.g. the cornea, the oral cavity, the oesophagus, the vagina, contain clonogenic stem cells that can respond to skin morphogenetic signals and form epidermis, cycling hair follicles and sebaceous glands. This capacity is maintained in serial transplantation, crosses primary germ line boundaries and is intrinsic to the stem cells, as cells which have never been exposed to cell culture behave in a similar fashion. Even more surprising, the thymus contains a population of clonogenic epithelial cells of endodermal origin that maintain a thymic identity in culture and have the capacity to incorporate into a thymic network, but can acquire the functionality of bona fide multipotent stem cells of the skin when exposed to proper developmental signals. Thymic epithelial cells exposed to a skin microenvironment exhibit a down-regulation or silencing of transcription factors important for thymic function. Hence, it is possible to reveal unsuspected potency and even to robustly reprogram stem cells by solely manipulating the microenvironment.

Stem cell transplantation in brain tumors: a new field for molecular imaging?

Neural stem cells have been proposed as a new and promising treatment modality in various pathologies of the central nervous system, including malignant brain tumors. However, the underlying mechanism by which neural stem cells target tumor areas remains elusive. Monitoring of these cells is currently done by use of various modes of molecular imaging, such as optical imaging, magnetic resonance imaging and positron emission tomography, which is a novel technology for visualizing metabolism and signal transduction to gene expression. In this new context, the microenvironment of (malignant) brain tumors and the blood-brain barrier gains increased interest. The authors of this review give a unique overview of the current molecular-imaging techniques used in different therapeutic experimental brain tumor models in relation to neural stem cells. Such methods for molecular imaging of gene-engineered neural stem/progenitor cells are currently used to trace the location and temporal level of expression of therapeutic and endogenous genes in malignant brain tumors, closing the gap between in vitro and in vivo integrative biology of disease in neural stem cell transplantation.

Roles of aldehyde dehydrogenase (ALDH) isoforms and retinoic acids in the growth and differentiation potential of human adult cardiac progenitor cells

Aim: We have studied human adult cardiac progenitor cells (CPCs) based on high aldehyde dehydrogenase activity (ALDH-hi), a property shared by many stem cells across tissues and organs. However, the role of ALDH in stem cell function is poorly known. In humans, there are 19 ALDH isoforms with different biological activities. The isoforms responsible for the ALDH-hi phenotype of stem cells are not well known but they may include ALDH1A1 and ALDH1A3 isoforms, which function in all-trans retinoic acid (RA) cell signaling. ALDH activity has been shown to regulate hematopoietic stem cell function via RA. We aimed to analyze ALDH isoform expression and the role of RA in human CPC function. Methods: Human adult CPCs were isolated from atrial appendage samples from patients who underwent heart surgery for coronary artery or valve disease. Atrial samples were either cultured as primary explants or enzymatically digested and sorted for ALDH activity by FACS. ALDH isoforms were determined by qRT-PCR. Cells were cultured in the presence or absence of the specific ALDH inhibitor DEAB, with or without RA. Induction of cardiac-specific genes in cells cultured in differentiation medium was measured by qRT-PCR. Results: While ALDH-hi CPCs grew in culture and could be expanded, ALDH-low cells grew poorly. CPC isolated as primary explant outgrowths expressed high levels of ALDH1A3 but not of other isoforms. CPCs isolated from cardiospheres expressed relatively high levels of all the 11 isoforms tested. In contrast, expanded CPCs and cardiosphere-derived cells expressed low levels of all ALDH isoforms. DEAB inhibited CPC growth in a dose-dependent manner, whereas RA rescued CPC growth in the presence of DEAB. In differentiation medium, ALDH-hi CPCs expressed approximately 300-fold higher levels of cardiac troponin T compared with their ALDH-low counterparts. Conclusions: High ALDH activity identifies human adult cardiac cells with high growth and cardiomyogenic potential. ALDH1A3 and, possibly, ALDH1A1 isoforms account for high ALDH activity and RA-mediated regulation of CPC growth.

Novel testis germ cell-specific transcript of the CREB gene contains an alternatively spliced exon with multiple in-frame stop codons.

The gene encoding the cAMP-responsive transcription factor CREB consists of multiple small exons some of which undergo alternative RNA splicing. We describe the finding of a novel transcript of the CREB gene expressed at high levels in the germ cells of the rat testis. The transcript contains an alternatively spliced exon inserted within the sequence encoding the transcriptional transactivation domain of CREB and this exon contains multiple in-frame stop codons. Furthermore, the exon is conserved in both rat and human genes (75% nucleotide identity). Although the function(s) of this RNA or the truncated CREB protein predicted to result from the translation of this unusual transcript is unknown, the high level of expression in the testicular germ cells and remarkable conservation of sequences in rat and human suggests that it may have a unique biological function in these cells.

Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279.

The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.

Development of Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells.

Ewing's sarcoma is a member of Ewing's family tumors (EFTs) and the second most common solid bone and soft tissue malignancy of children and young adults. It is associated in 85% of cases with the t(11;22)(q24:q12) chromosomal translocation that generates fusion of the 5' segment of the EWS gene with the 3' segment of the ETS family gene FLI-1. The EWS-FLI-1 fusion protein behaves as an aberrant transcriptional activator and is believed to contribute to EFT development. However, EWS-FLI-1 induces growth arrest and apoptosis in normal fibroblasts, and primary cells that are permissive for its putative oncogenic properties have not been discovered, hampering basic understanding of EFT biology. Here, we show that EWS-FLI-1 alone can transform primary bone marrow-derived mesenchymal progenitor cells and generate tumors that display hallmarks of Ewing's sarcoma, including a small round cell phenotype, expression of EFT-associated markers, insulin like growth factor-I dependence, and induction or repression of numerous EWS-FLI-1 target genes. These observations provide the first identification of candidate primary cells from which EFTs originate and suggest that EWS-FLI-1 expression may constitute the initiating event in EFT pathogenesis.

Adult human adipose tissue contains several types of multipotent cells.

Multipotent mesenchymal stromal cells (MSCs) are a type of adult stem cells that can be easily isolated from various tissues and expanded in vitro. Many reports on their pluripotency and possible clinical applications have raised hopes and interest in MSCs. In an attempt to unify the terminology and the criteria to label a cell as MSC, in 2006 the International Society for Cellular Therapy (ISCT) proposed a standard set of rules to define the identity of these cells. However, MSCs are still extracted from different tissues, by diverse isolation protocols, are cultured and expanded in different media and conditions. All these variables may have profound effects on the selection of cell types and the composition of heterogeneous subpopulations, on the selective expansion of specific cell populations with totally different potentials and ergo, on the long-term fate of the cells upon in vitro culture. Therefore, specific molecular and cellular markers that identify MSCs subsets as well as standardization of expansion protocols for these cells are urgently needed. Here, we briefly discuss new useful markers and recent data supporting the rapidly emerging concept that many different types of progenitor cells are found in close association with blood vessels. This knowledge may promote the necessary technical improvements required to reduce variability and promote higher efficacy and safety when isolating and expanding these cells for therapeutic use. In the light of the discussed data, particularly the identification of new markers, and advances in the understanding of fundamental MSC biology, we also suggest a revision of the 2006 ISCT criteria.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.