Adult Mammalian Tissues Research Articles

Skeletal myogenic progenitors in the endothelium of lung and yolk sac

We previously showed that clonable skeletal myogenic cells can be derived from the embryonic aorta but become very rare in the more mature and structured fetal aorta. The aim of this study was to investigate whether, during fetal and postnatal development, these myogenic progenitors progressively disappear or may rather associate with the microvascular district, being thus distributed to virtually all tissues. To test this hypothesis, we used F1 embryos (or mice) from a transgenic line expressing a striated muscle-specific reporter gene (LacZ) crossed with a transgenic line expressing a different endothelial-specific reporter genes (GFP). Endothelial cells were isolated from yolk sac (at E11) and lung (at E11, E17, P1, P10, and P60), two organs embryologically unrelated to paraxial mesoderm, rich in vessels, and devoid of skeletal muscle. Endothelial cells, purified by magnetic bead selection (CD31/PECAM-1 +) or cell sorting (Tie2-GFP +) were then challenged for their skeletal myogenic potential in vitro and in vivo. The results demonstrated that both yolk sac and lung contain progenitor cells, which express endothelial markers and are endowed with a skeletal myogenic potential that they reveal when in the presence of differentiating myoblasts, in vitro, and regenerating muscle, in vivo. The number (or potency to generate skeletal muscle) of these vessels associated cells decreases rapidly with age and is very low in mature animals, possibly correlating with reduced regenerative capacity of adult mammalian tissues.

Kir channels in the CNS: emerging new roles and implications for neurological diseases.

Inwardly rectifying potassium (Kir) channels have long been regarded as transmembrane proteins that regulate the membrane potential of neurons and that are responsible for [K(+)] siphoning in glial cells. The subunit diversity within the Kir channel family is growing rapidly and this is reflected in the multitude of roles that Kir channels play in the central nervous system (CNS). Kir channels are known to control cell differentiation, modify CNS hormone secretion, modulate neurotransmitter release in the nigrostriatal system, may act as hypoxia-sensors and regulate cerebral artery dilatation. The increasing availability of genetic mouse models that express inactive Kir channel subunits has opened new insights into their role in developing and adult mammalian tissues and during the course of CNS disorders. New aspects with respect to the role of Kir channels during CNS cell differentiation and neurogenesis are also emerging. Dysfunction of Kir channels in animal models can lead to severe phenotypes ranging from early postnatal death to an increased susceptibility to develop epileptic seizures. In this review, we summarize the in vivo data that demonstrate the role of Kir channels in regulating morphogenetic events, such as the proliferation, differentiation and survival of neurons and glial cells. We describe the way in which the gating of Kir channel subunits plays an important role in polygenic CNS diseases, such as white matter disease, epilepsy and Parkinson's disease.

Effect of hyaluronan on calcification-nodule formation from human periodontal ligament cell culture.

The periodontal ligament (PDL) has been found to house progenitor elements which may give rise to a new periodontium when properly triggered. Complex molecular mechanisms are involved in guiding such cells towards their final regenerative task. Hyaluronan (HA), an extracellular matrix molecule abundantly present during early tissue development, is usually found at high concentrations in all adult mammalian PDL tissue. In this study, PDL cells were cultured on HA coated experimental plastic Petri's dishes (E.D.) and mineralized-nodules formation, at the microscopical level, was evaluated in comparison to HA-free control dishes (C.D.) after 21 days. Little colony formation occurred in presence of HA, whereas CD showed normal cell confluence with mineralization of nodules even at 10 days. However, when harvesting was performed from both dishes after only 12 hours, before cell aggregation occurred, cell confluence and nodule formation could be seen with no apparent difference between test and control. This study shows that, with respect to PDL cells, hyaluronan seems to inhibit cell adhesion, although with no interference with cell aggregates calcifying potentials. This latter finding may: a) provide further insight in analyzing the properties of extracellular matrix substrates in the PDL environment, b)guide research to possible explanations on how the PDL maintains its characteristics of uncalcified tissue and c) lead to theorize regarding utilization in tissue regeneration. (Journal of Applied Biomaterial & Biomechanics 2003; 1: 84-90).

Somatic stem cells and the kinetics of mutagenesis and carcinogenesis.

There is now strong experimental evidence that epithelial stem cells arrange their sister chromatids at mitosis such that the same template DNA strands stay together through successive divisions; DNA labeled with tritiated thymidine in infancy is still present in the stem cells of adult mice even though these cells are incorporating (and later losing) bromodeoxyuridine [Potten, C. S., Owen, G., Booth, D. & Booth, C. (2002) J. Cell Sci.115, 2381-2388]. But a cell that preserves "immortal strands" will avoid the accumulation of replication errors only if it inhibits those pathways for DNA repair that involve potentially error-prone resynthesis of damaged strands, and this appears to be a property of intestinal stem cells because they are extremely sensitive to the lethal effects of agents that damage DNA. It seems that the combination, in the stem cell, of immortal strands and the choice of death rather than error-prone repair makes epithelial stem cell systems resistant to short exposures to DNA-damaging agents, because the stem cell accumulates few if any errors, and any errors made by the daughters are destined to be discarded. This paper discusses these issues and shows that they lead to a model that explains the strange kinetics of mutagenesis and carcinogenesis in adult mammalian tissues. Coincidentally, the model also can explain why cancers arise even though the spontaneous mutation rate of differentiated mammalian cells is not high enough to generate the multiple mutations needed to form a cancer and why loss of nucleotide-excision repair does not significantly increase the frequency of the common internal cancers.

Non-neural adult stem cells: tools for brain repair?

Stem cells isolated from adult mammalian tissues may provide new approaches for the autologous treatment of disease and tissue repair. Although the potential of adult stem cells has received much attention, it has also recently been brought into question. This article reviews the recent work describing the ability of non-hematopoietic stem cells derived from adult bone marrow to form neural derivatives and their potential for brain repair. Earlier transplantation experiments imply that grafted adult stem cells can differentiate into neural derivatives. Recent reports suggest, however, that such findings may be misleading and grafted cells acquiring different identities may merely be explained by their fusion with host cells and not the result of radical changes to their program of cellular differentiation. Nonetheless, in vitro studies have shown that neural development by bone-marrow-derived stem cells also appears possible. Understanding the molecular mechanisms that specify the neural lineage will lead to the development of tools for the targeted production of neural cell types in vitro that may ultimately provide a source of material to treat specific neurological deficits.

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1.

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of DeltaFosB. In hippocampus of rat brain, expression of DeltaFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue.

We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

Support of axonal regrowth by endogenous mechanisms following spinal cord injury in adult rats.

Because of its non-invasive nature and ease of regulation, a closely monitored cryogenic method of tissue injury was used to create a degree of spinal cord injury within which there would be an extended regrowth of axons. The parameters of cooling used in the present study resulted in an injury length of 1 cm through which 3 mm of measured axonal regrowth and 8 mm of observed regrowth occurred over a 56-day period in the ascending fibers of the dorsal column of the mature rat. This was associated with the development of a cellular matrix consisting of macrophages, macroglia and Schwann cells which gradually expands within the injured area initially dominated by macrophages. It is the authors' impression that the presence of a substantial microglial component within the macrophage population may be a significant factor in the success of the axonal regrowth. Under this influence and that of the invading axons, the astrocyte, which provides the immediate cell support to the growing axon, can be maintained in a functional state that is supportive and not obstructive to the axon, presumably through the recruitment of astrocyte precursors from an indigenous stem cell population. These tissue changes indicate that adult mammalian spinal cord tissue does have the capacity to develop on its own a matrix capable of supporting the regrowth of axons.

Mammary Fetal Gland: Identification of New Oncofetal Antigens by Monoclonal Antibodies B72.3, MM1.80 and 4.36

The B72.3, MM1.80 and 4.63 monoclonal antibodies directed against tumor-associated antigens react in human breast also with metaplastic, preneoplastic and metastatic cells, whereas they do not react with normal adult mammary tissue. The aim of our study was to point out the expression of these antigens during mammary gland development, since tumor-associated antigens are known to represent antigens of differentiation. Fifty female fetal breasts between 20 and 40 weeks of gestational age were studied. Mammary tissue was identified only in 15 cases. B72.3, MM1.80 and 4.36 monoclonal antibodies reacted with epithelial antigens and maintained the same location and intensity in the various gestational ages. Immunoreactivity was weak, cytoplasmic and widespread for the 4.36 monoclonal antibody, intense and cytoplasmatic in a large number of cells for the B72.3 monoclonal antibody, and intense and luminal for the MM1.80 monoclonal antibody. Such data further support the hypothesis that the normal process of development and differentiation can occur during tumor progression processes. Identification of these new oncofetal markers could offer a new perspective able to recognize the different phases of neoplastic progression and could be useful for prevention.

Mammalian Prohibitin Proteins Respond to Mitochondrial Stress and Decrease during Cellular Senescence

The two prohibitin proteins, Phb1p and Phb2pBAP37, have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.

Multilineage differentiation from human embryonic stem cell lines.

Stem cells are unique cell populations with the ability to undergo both self-renewal and differentiation. A wide variety of adult mammalian tissues harbors stem cells, yet "adult" stem cells may be capable of developing into only a limited number of cell types. In contrast, embryonic stem (ES) cells, derived from blastocyst-stage early mammalian embryos, have the ability to form any fully differentiated cell of the body. Human ES cells have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential, providing the possibility for unlimited expansion in culture. Furthermore, they can differentiate into derivatives of all three embryonic germ layers when transferred to an in vivo environment. Data are now emerging that demonstrate human ES cells can initiate lineage-specific differentiation programs of many tissue and cell types in vitro. Based on this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms underlying many facets of human development. Because they have the dual ability to proliferate indefinitely and differentiate into multiple tissue types, human ES cells could potentially provide an unlimited supply of tissue for human transplantation. Though human ES cell-based transplantation therapy holds great promise to successfully treat a variety of diseases (e.g., Parkinson's disease, diabetes, and heart failure) many barriers remain in the way of successful clinical trials.

Young neurons from medial ganglionic eminence disperse in adult and embryonic brain.

In this study, we identified neuronal precursors that can disperse through adult mammalian brain tissue. Transplanted neuronal precursors from embryonic medial ganglionic eminence (MGE), but not from lateral ganglionic eminence (LGE) or neocortex, dispersed and differentiated into neurons in multiple adult brain regions. In contrast, only LGE cells were able to migrate efficiently from the adult subventricular zone to the olfactory bulb. In embryonic brain slices, MGE cells migrated extensively toward cortex. Our results demonstrate that cells in different germinal regions have unique migratory potentials, and that adult mammalian brain can support widespread dispersion of specific populations of neuronal precursors. These findings could be useful in repair of diffuse brain damage.

Identification of megalin as the sole rat kidney sialoglycoprotein containing poly alpha2,8 deaminoneuraminic acid.

Recently, poly alpha2,8 deaminoneuraminic acid (poly alpha2,8 KDN) was demonstrated in various embryonic and adult mammalian tissues. This study reports the purification and characterization of the single poly alpha2,8 KDN-bearing glycoprotein from rat kidney. Amino acid sequences of proteolytic fragments shared homology with megalin, a member of the LDL receptor family. Immunochemical analysis supported this finding, since immunoprecipitated poly alpha2,8 KDN-bearing glycoprotein was immunoreactive with anti-megalin antibodies in Western blotting and conversely immunoprecipitated megalin was immunoreactive with the monoclonal anti-poly alpha2,8 KDN antibody. Furthermore, receptor-associated protein affinity-purified megalin reacted with the anti-poly alpha2,8 KDN antibody. By immunoelectron microscopy, labeling for both poly alpha2,8 KDN and megalin coincided in the brush border, endocytic invaginations and vesicles, and apical dense tubules of proximal convoluted tubules. Immunoreactivity for poly alpha2,8 KDN on purified megalin was abolished by beta-elimination reaction but not by N-glycosidase F treatment. These data identified megalin as the sole glycoprotein of rat kidney, which contains poly alpha2,8 KDN present on O-glycosidically linked oligosaccharides. Furthermore, this study shows that megalin carries N-glycosidically linked hybrid and complex-type oligosaccharides terminating with sialic acid. Both poly alpha2,8 KDN and sialic acids on megalin may contribute to the binding of Ca2+ and cationic ligands.

Regulated expression of p14 (cofactor A) during spermatogenesis.

The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.

Mom, dad, clone: implications for reproductive privacy.

On 5 July 1996 a sheep named Dolly was born in Scotland, the result of the transfer of the nucleus of an adult mammary tissue cell to the enucleated egg cell of an unrelated sheep, and gestation in a third, surrogate mother sheep. Although for the past ten years scientists have routinely cloned sheep and cows from embryo cells, this was the first cloning experiment that apparently succeeded using the nucleus of an adult cell.

Isolation of Two Novel WNT Genes, WNT14 and WNT15, One of Which (WNT15) Is Closely Linked to WNT3 on Human Chromosome 17q21

TheWntgene family consists of at least 15 structurally related genes that encode secreted extracellular signaling factors. Wnt proteins function in a range of critical developmental processes in both vertebrates and invertebrates and are implicated in regulation of cell growth and differentiation in certain adult mammalian tissues, including the mammary gland. We have isolated a number of WNT sequences from human genomic DNA, two of which, designated WNT14 and WNT15, represent novel members of theWntgene family. We also isolated WNT sequences from human mammary cDNA and present evidence that WNT13 is expressed in human breast tissue, in addition to those previously described. WNT14 and WNT15 appear to have originated from an ancestral branch of theWntgene family that also includes theWnt9sequences found in jawless and cartilaginous fishes. AWnt14cDNA was also isolated from chicken and a partialWnt15sequence from mouse. We show that human WNT14 maps to chromosome 1 and that WNT15 maps distal to BRCA1 on chromosome 17q21, where it lies within 125 kb of another WNT family member, WNT3.

An arterially-perfused trunk-hindquarters preparation of adult mouse in vitro

We describe a preparation of arterially-perfused spinal cord with attached hindquarters, taken from adult mouse. This is the first preparation of adult mammalian spinal cord tissue to have the advantages of an in vitro approach whilst retaining intact intraspinal circuitry, sensory inputs, and somatic and sympathetic segmental outputs. The functional integrity of the preparation has been demonstrated by the motor and sympathetic reflexes that can readily be evoked by peripheral noxious thermal, mechanical and electrical stimuli, and also by bladder distension. The mechanical stability of the preparation allows intracellular recordings to be made from spinal dorsal or ventral horn neurones. The intact connectivity permits synaptic responses to be evoked by stimulation of functionally-defined peripheral sensory receptors. The preparation is relatively quick to set up and remains viable for more than 6 h. This model offers the opportunity to perform complex electrophysiological and pharmacological studies on functionally characterised synaptic responses of mature spinal neurones. The choice of the mouse will furthermore permit studies to be performed on genetically mutant strains.

Fibroblast growth factor receptor (FGFR)-4, but not FGFR-3 is expressed in the pregnant ovary

The intraovarian mechanisms for follicle recruitment, growth, maturation, and ovulation are not well understood. The data suggest that fibroblast growth factor (FGF)-2 is expressed in granulosa and theca cells of growing and mature follicles and in luteal cells during pregnancy. Exogenous FGF-2 modulates steroidogenesis, stimulates tissue plasminogen activator (tPA), and induces germinal vesicle breakdown (GVBD) in cultured follicles. Previously, we have reported that another FGF ligand, FGF-4, is expressed in ovulated mouse oocytes. Two studies have examined the expression of receptors (FGFR) for FGF ligands in the ovary. These prior reports have been limited to FGFR-1, one of the four isoforms that are variably expressed in adult mammalian tissues. This study evaluates FGFR-4 and FGFR-3 mRNA expression in the ovary. Granulosa cells from several follicular stages express the receptor for FGFR-4 mRNA as assayed by in situ hybridization. FGFR-4 mRNA is not expressed in theca cells or the oocyte. FGFR-3 mRNA is not detected in the ovary by in situ hybridization. These results suggest that FGFR-4 may play a role in mediating the effects of FGF ligands in follicular development in the ovary.

Frzb-1 Is a Secreted Antagonist of Wnt Signaling Expressed in the Spemann Organizer

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10 −10 M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Mitochondrial creatine kinase isoform expression does not correlate with its mode of action.

In adult mammalian ventricular tissue, mitochondrial creatine kinase (mi-CK), which is bound to the outer surface of the mitochondrial inner membrane, is functionally coupled to oxidative phosphorylation. This is shown, in saponin-permeabilized rat ventricular fibres, by a shift in the apparent K(m) of mitochondrial respiration for ADP from 300 +/- 56 microM to 111 +/- 40 microM (P < 0.001) on the addition of 25 mM creatine, due to a local accumulation of ADP close to the ATP/ADP translocator. We have found that in atrial fibres, the apparent K(m) for ADP is high, but is not decreased by creatine, suggesting an absence of coupling in this tissue, as has previously been observed in smooth muscle. mi-CK is encoded by two different genes, which are expressed in a tissue-specific manner: the sarcomeric isoform is expressed in ventricular and skeletal muscles, while the ubiquitous isoform is expressed in smooth muscle, brain and other tissues. In order to determine whether a specific function can be attributed to the expression of a specific isoform, we investigated mi-CK mRNA expression by Northern blot analysis. Hybridization with synthetic oligonucleotides specific for each mi-CK isoform showed the expression of only the sarcomeric isoform in rat atria. This result was confirmed by PCR using primers specific for each isoform. In addition, electrophoretic analysis of CK isoforms showed no difference in the octamer/dimer ratio of mi-CK in the atria and ventricles. In atria, unlike the soleus or ventricles, the maximum potential rate of mitochondrial phosphocreatine synthesis was lower than the maximal rate of ATP production by the mitochondria. The total CK/adenylate kinase ratio was also lower in atria than in the other tissues, suggesting a greater contribution of adenylate kinase to adenine nucleotide compartmentation in this tissue. The functional differences between mi CK in the two cardiac tissues seem to imply a specific arrangement of the proteins in the intermembrane space rather than the expression of specific isoforms.