Tcf7l2 is required for left-right asymmetric differentiation of habenular neurons.

BACKGROUND: Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown. RESULTS: In a forward genetic screen for mutations that result in loss of habenular asymmetry, we identified two mutant alleles of tcf7l2, a gene that encodes a transcriptional regulator of Wnt signaling. In tcf7l2 mutants, most neurons on both sides differentiate with dHbl identity. Consequently, the habenulae develop symmetrically, with both sides adopting a pronounced leftward character. Tcf7l2 acts cell automously in nascent equipotential neurons, and on the right side, it promotes dHbm and suppresses dHbl differentiation. On the left, the parapineal prevents this Tcf7l2-dependent process, thereby promoting dHbl differentiation. CONCLUSIONS: Tcf7l2 is essential for lateralized fate selection by habenular neurons that can differentiate along two alternative pathways, thereby leading to major neural circuit asymmetries.

Cultivation in human serum reduces adipose tissue-derived mesenchymal stromal cell adhesion to laminin and endothelium and reduces capillary entrapment.

The increasing use of mesenchymal stromal cells (MSC) in clinical cellular therapy requires a safe and controlled production process compliant with Good Manufacturing Practice guidelines. Pooled blood group AB human serum (HS) has been used to replace fetal bovine serum (FBS), critically rated by the regulatory agencies, since it can support the expansion of adipose tissue-derived mesenchymal stromal cells (ASC). However, it remains unknown whether the choice of serum affects application-relevant characteristics of ASC. A microarray-based screen has revealed differentially expressed adhesion and extracellular matrix-associated molecules in HS- and FBS-ASC. Since cell therapy relies on the cells' efficacy to home and engraft, HS- and FBS-ASC were compared by analyzing adhesion, migration, and transmigration as well as short-term homing in vivo. HS-cultivated ASC demonstrated a higher adhesion to plastic, but reduced adhesion to extracellular matrix molecules, that is, laminin, and to endothelial cells both under static and flow conditions. Migration and transmigration assays confirmed the attraction of ASC by the tumor conditioned medium irrespective of the supplement. Coinjecting differently labeled HS- and FBS-ASC into nonobese diabetic, severe combined immunodeficiency mice revealed reduced numbers of HS-ASC in lungs and liver. This has been interpreted as reduced capillary entrapment. Our data indicate that varying the serum supplement may alter application-relevant characteristics of ASC, such as adhesion, as well as lung entrapment after infusion. Appropriate injury models and further molecular analyses are required to provide mechanistic insight into the differential effects of HS versus FBS on ASC cultures.

A comparative analysis of the adipogenic potential in human mesenchymal stromal cells from cord blood and other sources.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) from umbilical cord blood (CB) attract attention by significantly impaired or absent adipogenic differentiation compared with MSCs derived from bone marrow (BM) and adipose tissue (AT). The diverging adipogenic propensity between the developmentally younger CB-MSCs and MSCs of the adult AT and BM resembles the age-dependent process in the BM, where adipose tissue increases with advancing age, accompanied by loss of bone stability. Thus, MSCs appeal as an attractive model to study the adipogenic process with respect to tissue sources and developmental ages. METHODS: We followed the expression of main adipogenic transcription factors, genes and protein markers in CB-, BM- and AT-MSCs under adipogenic induction, after silencing of preadipocyte factor 1 (Pref-1, PREF1) and after incubation with CB-plasma supplemented adipogenic media. RESULTS: An inverse relation in the expression of adipogenesis-associated markers and PREF1 in CB-MSCs suggested an inhibitory role of Pref-1 toward adipogenesis. However, Pref-1 protein was rarely detected in CB-MSCs, and siRNA silencing of Pref-1 failed to induce adipogenic differentiation in CB-MSCs. Thus, the impaired adipogenic differentiation of CB-MSCs in vitro was unrelated to endogenous Pref-1 protein expression. Nevertheless CB-plasma containing Pref-1 protein revealed an anti-adipogenic effect on AT-MSCs. CONCLUSIONS: Because Pref-1 is vastly abundant in CB-plasma and confers anti-adipogenic properties, Pref-1 in association with the ontogenic milieu probably induces long-lasting unresponsiveness toward adipogenic stimuli in CB-MSCs.

Monitoring human mesenchymal stromal cell differentiation by electrochemical impedance sensing.

BACKGROUND AIMS: For their wide mesodermal differentiation potential, mesenchymal stromal/stem cells (MSC) are attractive candidates for tissue engineering. However, standardized quality control assays monitoring differentiation that are non-invasive and continuous over time are lacking. METHODS: We employed a non-invasive assay, using two different systems, to discriminate osteogenic and adipogenic differentiation of MSC by monitoring impedance. Fibroblasts and keratinocytes served as non-specific controls. Impedance profiles were recorded comparing MSC from bone marrow and adipose tissue, either non-induced or induced for osteogenesis or adipogenesis, for 5-14 days, and correlated with differentiation markers assessed by reverse transcription-quantitative polymerase chain reaction and Western blot. Additionally, differentiation modulating effects of extracellular matrix components were analyzed. RESULTS: Adhesion and growth-related impedance profiles of non-induced MSC roughly resembled those of fibroblasts, whereas keratinocytes differed significantly. Distinct from that, osteogenic induction of MSC revealed initially rapid and continuously rising impedance, corresponding to mineralized calcium matrix formation. Conversely, adipogenic induction caused shallower initial slopes and eventually declining profiles, corresponding to more compact, adipocyte-like cells with numerous lipid vacuoles. Pre-coating with either collagen type I or IV apparently favored osteogenesis and fibronectin adipogenesis. Impedance recordings correlated well with the extent of differentiation evaluated by histochemical staining and protein and gene expression. CONCLUSIONS: Overall, our data demonstrate that impedance profiling offers a basis for standardized real-time, non-invasive high-throughput screening of MSC properties. It enables further testing of the influence of diffusible factors or extracellular matrix composites on MSC differentiation or maintenance of stemness, thus substantiating therapeutic application.

All four zebrafish Wnt7 genes are expressed during early brain development.

Wnt-signalling is involved in a number of biological processes in the course of embryonic development, cell fate determination, proliferation, stem cell maintenance and oncogenesis. Wnt ligands are secreted glycoproteins and the number of Wnt isoforms varies between five in nematodes and 27 in fish. The highly conserved group of Wnt7 genes has been found to signal via at least three Wnt-signalling pathways dependent on the developmental context. These ligands have been identified as important regulators in a number of processes ranging from formation of bones, lungs, kidneys, reproductive organs and placenta to vasculogenesis and synaptogenesis in the brain. The importance of Wnt7 function is underscored by their implication in disease syndromes in man. Unlike the single Wnt7a and Wnt7b mammalian genes we find that the zebrafish genome contains two paralogues genes for each Wnt7 ligand. Here, we compare these four Wnt7 genes evolutionarily and analyse their expression during the first two days of embryonic development. We find Wnt7 genes mainly expressed in a number of CNS structures at developmental stages at which patterning and neural specification takes place. The timely and spatially overlapping as well as complementary gene expression suggests diverse as well as redundant involvements during brain development.

Mesenchymal stromal cells from human perinatal tissues: From biology to cell therapy.

Cell-based regenerative medicine is of growing interest in biomedical research. The role of stem cells in this context is under intense scrutiny and may help to define principles of organ regeneration and develop innovative therapeutics for organ failure. Utilizing stem and progenitor cells for organ replacement has been conducted for many years when performing hematopoietic stem cell transplantation. Since the first successful transplantation of umbilical cord blood to treat hematological malignancies, non-hematopoietic stem and progenitor cell populations have recently been identified within umbilical cord blood and other perinatal and fetal tissues. A cell population entitled mesenchymal stromal cells (MSCs) emerged as one of the most intensely studied as it subsumes a variety of capacities: MSCs can differentiate into various subtypes of the mesodermal lineage, they secrete a large array of trophic factors suitable of recruiting endogenous repair processes and they are immunomodulatory.Focusing on perinatal tissues to isolate MSCs, we will discuss some of the challenges associated with these cell types concentrating on concepts of isolation and expansion, the comparison with cells derived from other tissue sources, regarding phenotype and differentiation capacity and finally their therapeutic potential.