Christian Dani

Autocrine Fibroblast Growth Factor 2 Signaling Is Critical for Self‐Renewal of Human Multipotent Adipose‐Derived Stem Cells

Adipose tissue‐derived stem cells offer tremendous potential for regenerative medicine. However, characterization of their self‐renewal ability has not been performed yet, although it is a crucial feature for in vitro expansion of undifferentiated cells and in vivo maintenance of stem cell pools. We have undertaken the identification of molecular events that are involved in in vitro self‐renewal of human multipotent adipose‐derived stem (hMADS) cells from young donors, by assessing their proliferation rate, their ability to grow at the single‐cell level (clonogenicity), and their differentiation potential. As hMADS cells are propagated in culture, cell morphology changes dramatically, concomitantly to a progressive decrease in proliferation, clonogenicity, and differentiation potential. This decrease is associated with a decrease in fibroblast growth factor 2 (FGF2) expression and can be circumvented by chronic treatment with exogenous FGF2. Moreover, analysis of FGF2 secretion revealed that it is exported to hMADS cell surface without being released into the culture medium, suggesting a strictly autocrine loop. Indeed, treatment of FGF2‐expressing hMADS cells with PD173074, a specific FGF receptor inhibitor, decreases dramatically their clonogenicity and differentiation potential. Thus, hMADS cells express a functional autocrine FGF loop that allows maintenance of their self‐renewal ability in vitro. Finally, inhibition of mitogen‐activated protein kinase kinase 1 reduces the clonogenic potential of hMADS cells but does not affect their differentiation potential, indicating that the extracellular signal‐related kinases 1/2 signaling pathway is partly involved in FGF2‐mediated self‐renewal. Together, our data clearly identify the key function of FGF2 in the maintenance of self‐renewal of adipose tissue‐derived stem cells.

Reconstituted Skin from Murine Embryonic Stem Cells

Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.

Impaired ossification in mice lacking the transcription factor Sp3.

Sp3 is a ubiquitously expressed member of the Sp family of transcription factors. Recently, the mouse Sp3 gene has been disrupted by homologous recombination. Sp3 null mice die immediately after birth due to respiratory failure. In addition, these mice show a pronounced defect in late tooth formation. Here we show that Sp3 is also required for proper skeletal ossification. Both endochondral and intramembranous ossification are impaired in E18.5 Sp3-/- embryos. The delay in ossification is reflected by reduced expression of the osteoblast-specific marker gene osteocalcin. The transcription factor - core binding factor 1 (Cbfa1)--that is essential for bone formation, however, is expressed at normal levels. In vitro differentiation studies using Sp3-/- ES cells further support the conclusion that Sp3 is needed for correct bone formation. The capacity of Sp3-/- cells to undergo osteogenic differentiation in vitro is reduced and osteocalcin expression is significantly diminished. Our studies establish Sp3 as an essential transcription factor for late bone development.

Hedgehog Signaling Alters Adipocyte Maturation of Human Mesenchymal Stem Cells

Human stem cells are powerful tools by which to investigate molecular mechanisms of cell growth and differentiation under normal and pathological conditions. Hedgehog signaling, the dysregulation of which causes several pathologies, such as congenital defects and cancer, is involved in several cell differentiation processes and interferes with adipocyte differentiation of rodent cells. The present study was aimed at investigating the effect of Hedgehog pathway modulation on adipocyte phenotype using different sources of human mesenchymal cells, such as bone marrow stromal cells and human multipotent adipose‐derived stem cells. We bring evidence that Hedgehog signaling decreases during human adipocyte differentiation. Inhibition of this pathway is not sufficient to trigger adipogenesis, but activation of Hedgehog pathway alters adipocyte morphology as well as insulin sensitivity. Analysis of glycerol‐3‐phosphate dehydrogenase activity and expression of adipocyte marker genes indicate that activation of Hedgehog signaling by purmorphamine impairs adipogenesis. In sharp contrast to reports in rodent cells, the maturation process, but not the early steps of human mesenchymal stem cell differentiation, is affected by Hedgehog activation. Hedgehog interferes with adipocyte differentiation by targeting CCAAT enhancer‐binding protein α and peroxisome proliferator‐activated receptor (PPAR) γ2 expression, whereas PPARγ1 level remains unaffected. Although Hedgehog pathway stimulation does not modify the total number of adipocytes, adipogenesis appears dramatically impaired, with reduced lipid accumulation, a decrease in adipocyte‐specific markers, and acquisition of an insulin‐resistant phenotype. This study indicates that a decrease in Hedgehog signaling is necessary but not sufficient to trigger adipocyte differentiation and unveils a striking difference in the adipocyte differentiation process between rodent and human mesenchymal stem cells.

Developmental origin of adipocytes: new insights into a pending question

The current epidemic of obesity has caused a surge of interest in the study of adipose tissue formation. Much progress has been made in defining the transcriptional networks controlling the terminal differentiation of preadipocytes into mature adipocytes. However, the mechanisms that direct MSCs (mesenchymal stem cells) down the adipocyte lineage remain largely unknown. Similarly, although adipocytes are generally described to derive from mesoderm, the study of the developmental origin of MSCs and adipose tissues has been largely disregarded until now. Functional variations do exist between different adipose tissues, which suggest possible differences in their developmental origin and might explain why some depots are more associated than other to metabolic disorders. This review summarizes the surprising findings that have recently emerged from both embryonic stem cells and lineage‐tracing studies in vivo, unravelling an unsuspected developmental origin for MSCs and adipocytes in the neural crest.

Embryonic Stem Cell-Derived Adipogenesis

Key events leading to terminal differentiation of preadipocytes into adipocytes have been characterized in the recent years. However, master genes that commit progression from multipotent mesenchymal stem cell to the adipoblast stage of development have not yet been identified. The use of embryonic stem (ES) cells as a route to study early events in adipogenesis and to characterize factors involved in the decision of stem cells to follow the adipogenic pathway is described in this paper. The capacity of lif–/– and lifr–/– ES cells to undergo adipocyte differentiation is reported as an application of mutant ES cells to study gene function during the development of adipose cells.

Coupling of growth arrest and expression of early markers during adipose conversion of preadipocyte cell lines

After growth arrest at the entry of the S phase of the cell cycle, Ob1771 and 3T3-F442A cells, but not 3T3-C2 cells, accumulate lipoprotein lipase and pOb24 mRNA that are early markers of adipose conversion. Removal of the single- or double-thymidine block when cultured cells are present at low density leads first to DNA synthesis and growth resumption, then to a continuous proliferation and a rapid disappearance of these markers. By contrast, growth-arrested Ob1771 cells reinoculated at high density undergo a single round of cell division, maintain high levels of early marker(s) and acquire with time both glycerol-3-phosphate dehydrogenase and lipids. Thus, depending upon the conditions in culture, growth-arrested cells can undergo either a dedifferentiation leading to a loss of early markers or a terminal differentiation leading to the acquisition of late markers.

Differentiation of embryonic stem cells for pharmacological studies on adipose cells

The ongoing global explosion in the incidence of obesity has focused attention on the development of adipose cells. Severe obesity is the result of an increase in fat cell size in combination with increased fat cell number. New fat cells arise from a pre-existing pool of adipose stem cells that are present irrespective of age. The development of established preadipocyte cell lines has facilitated the study of different steps leading to terminal differentiation. However, these systems are limited for studying early events of differentiation as they represent cells which are already determined for the adipogenic lineage. In vitro differentiation of mouse embryonic stem (ES) cells towards the adipogenic lineage provides an alternative source of adipocytes for study in tissue culture and offers the possibility to investigate regulation of the first steps of adipose cell development. In this review, we describe the sequential requirement of retinoic acid and PPARgamma during adipogenesis in ES cells. Stimulation of ES cells with synthetic retinoids which are selective ligands of the retinoic acid receptor isotypes allowed the investigation of the contribution of the different retinoic receptors on the RA-dependent differentiation. The effects of thiazolidinediones, a new class of pharmacological agents used for the treatment of type 2 diabetes, and of statins, drugs used in therapy for lowering cholesterol, on the differentiation of ES cells into adipocytes or osteoblasts are described. Finally, we propose a model in which PPARgamma plays a key role in the decision of stem cells to undergo differentiation into adipocytes or osteoblasts, two closely related lineages.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

Prostacyclin IP receptor up-regulates the early expression of C/EBPβ and C/EBPδ in preadipose cells

Prostacyclin (PGI(2)) and its stable analogue carbacyclin (cPGI(2)) are known to trigger the protein kinase A pathway after binding to the cell surface IP receptor and to promote or enhance terminal differentiation of adipose precursor cells to adipose cells. The early expression of C/EBPbeta and C/EBPdelta is known to be critical for adipocyte differentiation in vitro as well as in vivo. We report herein that in Ob1771 and 3T3-F442A preadipose cells, activation of the IP receptor by specific agonists (PGI(2), cPGI(2) and BMY 45778) is sufficient to up-regulate rapidly the expression of C/EBPbeta and C/EBPdelta. Cyclic AMP-elevating agents are able to substitute for IP receptor agonists, in agreement with the coupling of IP receptor to adenylate cyclase. Consistent with the fact that PGI(2) is released from preadipose cells and behaves as a paracrine/autocrine effector of adipose cell differentiation, the present results favor a key role of prostacyclin by means of the IP receptor and its intracellular signaling pathway in eliciting the critical early expression of both transcription factors.