Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are of interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. Umbilical cord blood (UCB) is reported to contain MSC, and therefore may be a useful source of these cells for clinical applications. METHODS: We evaluated protocols for isolating MSC from UCB and characterized the surface phenotype, differentiation potential and immunoregulatory properties of the cells obtained. RESULTS: Ten of 25 UCB units processed yielded MSC-like colonies, with depletion of lineage+ cells providing a higher efficiency. Only two of the cultures could be expanded satisfactorily; the remainder failed to proliferate. One culture generated transformed lines that were grossly aneuploid, had up-regulated TERT transcripts and had lost CD90 expression and the capacity to differentiate. The two propagated UCB-MSC lines were similar to those from bone marrow but were not identical to each other, with differences seen in expression of surface markers and cytoskeletal proteins. Both underwent osteogenesis, but at different rates and to different degrees, while neither generated adipocytes. When added as a third party to a mixed lymphocyte culture, both suppressed proliferation. DISCUSSION: MSC-like cells can be isolated from UCB, but at low efficiencies, and they exhibit a variety of morphologies, growth rates and differentiation potentials and can transform in culture.

Distribution of beta-amyloid precursor and B-cell lymphoma protooncogene proteins in the rat retina after optic nerve transection or vascular lesion.

The expression of beta-amyloid precursor protein (APP) and B-cell lymphoma protooncogene protein (Bcl-2) in retinal cells in the rat was studied using immunocytochemistry at different times after intraorbital optic nerve transection or vascular lesion. Three hours to one month after transection of the optic nerve, a significant increase in APP and Bcl-2 immunostaining was observed in retinal Müller glia but not in retinal neurons. In contrast, injury to blood vessels that supply the eye without cutting the optic nerve resulted in a complete loss of APP and Bcl-2 immunostaining in Müller cells and an increase in immunoreactivity in distinct populations of retinal neurons. The overall pattern of APP immunostaining in Müller cells and neurons was essentially the same as that of Bcl-2 under identical experimental conditions. These results suggest that the expression of APP and Bcl-2 in retinal cells is dependent on the nature and severity of injury, and that rapid and common mechanisms are involved in regulating the expression of these molecules.

Factors that affect the expression of beta-amyloid precursor protein immunoreactivity in the rat retina.

Expression of beta-amyloid precursor protein immunoreactivity in the rat retina was studied after intravitreal injection of substances known to influence neural function in different ways. The substances were the excitatory amino acid glutamate, the inflammatory agent lipopolysaccharide, the depolarizing agent potassium chloride, and the potassium channel blocker barium chloride. In comparison with controls, more beta-amyloid precursor protein immunoreactivity was observed in the radial process of Müller glial cells 24 hours after injection of glutamate or lipopolysaccharide. In contrast, administration of barium chloride greatly reduced immunostaining in Müller cells. Further, an increase in immunostaining was observed in the inner and outer plexiform layers in retinas treated with any of the 3 chemicals, and in blood vessels after injection of glutamate and lipopolysaccharide. These observations suggest that multiple but specific signaling pathways are involved in regulating expression of beta-amyloid precursor protein in distinct cell types and regions in the retina.

The origin and terminal arbors of individual uncrossed retinogeniculate fibers in rabbits.

The distribution and morphology of individual uncrossed retinogeniculate fibers in both normal and monocularly enucleated adult Dutch-belted rabbits were studied using horseradish peroxidase and wheat germ agglutinin conjugated to horseradish peroxidase as neuronal markers. The results showed that the uncrossed retinogeniculate fibers were distributed almost entirely in the ipsilateral segment of the dorsal nucleus of the lateral geniculate body, and the extent of terminal distribution of the fibers observed in rabbits with one eye enucleated during the young adult stage was essentially the same as that in the normal rabbit. Most of the uncrossed retinogeniculate fibers appeared to arise as collateral branches of optic tract fibers which were apparently destined for the pretectum or the superior colliculus. The uncrossed retinogeniculate fibers labeled by the wheat germ agglutinin conjugated to horseradish peroxidase passed through the dorsal nucleus of the lateral geniculate body without any branching until they reached the ipsilateral segment. There they could be divided into several morphological types although the possibility that they may represent different classes of a continuum cannot be ruled out.

Correlation between the visual callosal connections and the retinotopic organization in striate-peristriate border region in the hamster: an anatomical and physiological study.

The correlation between the retinotopic organization of receptive fields in the striate-peristriate border region and the distribution pattern of the visual callosal projections was investigated in hamsters with corpus callosum transected 2 days before recording. The results showed that in all the animals studied, the V1/V2 border defined by reversal of receptive fields at the vertical meridian was located in the dense central portion of the visual callosal projection which terminated in cortical regions bordering areas 17 and 18a. These results indicate that there is a close relationship between the striate-peristriate border determined by anatomical and physiological methods. In addition, these data strengthen the suggestion that the pattern of visual callosal projections is a useful and reliable reference system for delineating boundaries of different visual areas in the golden hamster.

Cell-specific expression of beta-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes.

The abnormal accumulation of beta-amyloid (A beta) in senile plaques appears to be a central pathological process in Alzheimer's disease. A beta is formed by proteolysis of beta-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A beta region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was approximately 90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A beta is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.

Alteration of Bcl-2 expression in the nigrostriatal system after kainate injection with or without melatonin co-treatment.

In order to understand further the role of the anti-apoptotic Bcl-2 proto-oncogene protein in excitotoxin-induced brain injury and possible interaction between Bcl-2 and the antioxidant melatonin, the expression of Bcl-2 in various brain parts was studied after intrastriatal injection of kainate (KA, 2.5 nmol) with or without co-treatment of melatonin (10 mg/kg, intraperitoneally (i.p.)). Three days after unilateral injection of KA to the striatum in the rat, a dramatic direct cytotoxic effect was observed, as indicated an expression of Bcl-2 immunoreactivity in TUNEL- and OX-42-positive cells in the KA-injected striatum and traumatized cortical region. A less severe detrimental effect was also observed in the ipsilateral substantia nigra and peritraumatic cortex, as reflected by an upregulation of Bcl-2-immunostained neurons. Surprisingly, a reduction in Bcl-2-immunoreactive neurons that was accompanied by a less severe loss of tyrosine hydroxylase-immunoreactive neurons in the nigrostriatal pathway was observed after co-treatment with melatonin. Western blot analysis confirmed that Bcl-2 expression is elevated in striatum and cortex on the lesioned side, and that its expression was attenuated substantially after systemic administration of melatonin. The results showing an upregulation of Bcl-2 in nigral neurons and reactive microglia after KA lesion are consistent with the view that Bcl-2 is protective in function in the central nervous system.

Expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in rat retinas after intravitreal injection of aminoadipic acid.

In order to investigate the role of glia in relation to factors that affect the expression of beta-amyloid precursor protein (betaAPP) and B cell lymphoma oncogene protein (Bcl-2) in the central nervous tissue, the patterns of expression of betaAPP and Bcl-2 in developing and mature rat retinas were studied immunocytochemically after intravitreal injection of alpha-aminoadipic acid (alpha-AAA), a glutamate analogue and gliotoxin that is known to cause injury of retinal Müller glial cells. In normal developing retinas, betaAPP and Bcl-2 were expressed primarily but transiently in a small number of neurons in the ganglion cell layer during the first postnatal week. Immunoreactivity of betaAPP and Bcl-2 appeared in the endfeet and proximal part of the radial processes of Müller glial cells from the second postnatal week onwards. In rats that received intravitreal injection of alpha-AAA at birth, there was a loss of immunoreactivity to vimentin, and a delayed expressed on betaAPP or Bcl-2 in Muller glial cells until 3-5 weeks post-injection. Immunoreactive neurons were also observed in the inner retina especially in the ganglion cell layer from 5 to 35 days after injection. A significant reduction in numerical density of cells with large somata in the ganglion cell layer was observed in the neonatally injected retinas at P56, which was accompanied by an increased immunostaining in radial processes of Müller glial cells. In contrast, no detectable changes in the expression of betaAPP and Bcl-2 were observed in retina that received alpha-AAA as adults. These results indicate that the gliotoxin alpha-AAA has long lasting effects on the expression of betaAPP and Bcl-2 in Müller glial cells as well as neurons in the developing but not mature retinas. The loss of vimentin and delayed expression of betaAPP and Bcl-2 in developing Müller glial cells suggests that the metabolic integrity of Müller cells was temporarily compromised, which may have adverse effects on developing neurons that are vulnerable or dependent on trophic support from the Müller glial cells.

Injury-resistant retinal ganglion cells that are rich in cytochrome oxidase.

Previous studies have demonstrated that the vast majority of retinal ganglion cells (RGCs) die one month after optic nerve transection. However, a small percentage do not degenerate. The present study examined one aspect of the chemical nature of these surviving RGCs using cytochrome oxidase (CO) as a neuronal marker in whole-mounted retinae. In the normal retina, 4.3% of the total population of RGCs show high CO activity. One month after optic nerve transection, 37% of the CO-positive RGCs counted in the control retinae survive and, because they stain with CO, must be metabolically active. Previous studies have shown that only up to 10% of the total RGC population survive optic nerve transection. The implication of our results is that the CO-positive RGCs, as a subpopulation, are more resistant to injury than the general population of RGCs.

Parasagittal patches in the granular layer of the developing and adult rat cerebellum as demonstrated by NADPH-diaphorase histochemistry.

The development of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the rat cerebellum was studied histochemically. NADPH-d reactivity was observed in the internal granular layer and some Purkinje cells by the end of first postnatal week (PW1). By PW2, the staining was localized in the granular layer (GL) and the molecular layer (ML), but not in the Purkinje cell layer. The staining in GL and ML increased further, and parasagittally organized NADPH-d patches in GL were recognizable by PW3. These patches were more distinct and almost adult-like by PW4. These results provide the first evidence of a patchy organization of NADPH-d activity in GL and suggest that NADPH-d plays an important role in the maturation and function of the cerebellum.

Effects of bFGF and TGFbeta on the expression of amyloid precursor and B-cell lymphoma protooncogene proteins in the rat retina.

The pattern of immunoreactivity for amyloid precursor (APP) and B-cell lymphoma protooncogene (Bcl-2) proteins in the rat retina was studied after intravitreal injection of basic fibroblast growth factor (bFGF) or transforming growth factor-beta (TGFbeta). In normal control retinas, intense immunostaining of APP and Bcl-2 was observed primarily in the endfeet and proximal part of radial processes of Müller glial cells. A dose-dependent reduction in immunostaining of APP and Bcl-2 in Müller cells was observed after injection of bFGF and TGFbeta. These results provide the first evidence that APP and Bcl-2 can be down-regulated by cytokines in vivo.

Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion.

Immunoreactivity to beta-amyloid precursor protein (APP) was present in the inner plexiform, ganglion cell and optic fibre layers, as well as in blood vessels, at birth in normally developing rat retinas. In the inner plexiform layer immunoreactivity disappeared by postnatal day (P) 14. A small population of ganglion cells was immunoreactive at birth, but none were visible at P7. From P14 onwards, however, there was weak immunoreactivity in ganglion cells again, and strong staining in Müller glia. Retinas affected by neonatal optic tract lesions contained more immunoreactive ganglion cells at P4 than did controls, but by P14 there was a severe loss of ganglion cells. These observations are consistent with APP being involved in retinal differentiation, including maturation of glia and neurones, synaptogenesis and possibly neuronal survival.

Enlargement of uncrossed retinal projections in the albino rat: additive effects of neonatal eye removal and thalamectomy.

The present study in the albino rat investigates the effects of neonatal unilateral eye removal and/or thalamectomy upon the number of ipsilaterally projecting retinal ganglion cells (IPRGCs), using retrograde fluorescent labelling and electron microscopy. The results show that neonatal unilateral eye removal and thalamectomy result in a significant increase in the number of aberrant IPRGCs as compared to that observed in normal rats, and the effects of the two different neonatal lesions appear to be additive. These findings strongly suggest that there are at least two subpopulations of IPRGCs, which normally do not exist in mature albino rats, but which can be preserved into adulthood by neonatal enucleation or thalamectomy. The data also suggest that about 70% of the entire population of IPRGCs, most of which normally exist only transiently in neonatal retinas, can be retained in rats which receive both unilateral eye removal and thalamectomy at the neonatal stage.

Intercollicular fibers terminating in the superficial layers of the superior colliculus: a WGA-HRP study in the rat.

The intercollicular pathway in the albino rat was studied using a lectin, wheat germ agglutinin, conjugated to horseradish peroxidase (WGA-HRP) as a pathway tracer and a modified HRP histochemistry method. The results showed that a small number of intercollicular fibers were observed in the rostral half of the superficial layers and the caudal half of the intermediate layers of the superior colliculus, while the majority of the intercollicular fibers were distributed in the intermediate and deep layers in the rostral half of the colliculus. The fibers which terminated in the superficial layers of the colliculus were studied in some detail and were subdivided into 4 different morphological types. These results suggest that the intercollicular projection is more broadly distributed in the superior colliculus than has been reported previously in most of the mammalian species studied, and the optic portion of the colliculus receives direct input from different morphological types of intercollicular neurons located on the opposite side of the midbrain.

Experimentally induced enlargement of the uncrossed retinotectal pathway in rats.

The present experiments investigate the effects of neonatal lesions upon projection patterns of the uncrossed retinotectal pathway in albino rats. The results indicate that enlargement of the terminal field from one eye can be induced either by a contralateral optic tract lesion or by removal of the opposite eye at birth. The extent of the enlargement is more prominent in the latter case. If an optic tract lesion is accompanied by eye enucleation on the side ipsilateral to the tract lesion, the uncrossed retinotectal projection from the remaining eye will undergo further enlargement. However, optic fiber counts show that such an enlargement of the terminal field is not due to a significant increase in the number of uncrossed optic axons which contribute to the enlarged projection, but rather to an increased terminal arbor of individual axons (as shown by results from fiber counts). While a severe ganglion cell loss was observed in the retina contralateral to a tract lesion, a substantial population of cells persists in the ganglion cell layer and the number of cells appear much higher than the number of uncrossed optic axons arising from the same eye. The implications of these findings are discussed in relation to results reported in previous studies.

Intertectal crossing of optic axons after tectal fusion in neonatal rats.

Surgically-induced fusion of the superior colliculi along the midline was performed in newborn rats to provide a bridge for optic axons from one eye to cross the tectal midline. The results indicate that although tectal fusion is a necessary condition of optic axons to cross from one side of the midbrain to the other, it is not sufficient by itself to induce crossing unless the optic input from the opposite side is also removed. This finding is discussed in relation to tectal lesion studies and is compared with our previous observations of the corticotectal pathway.

An autoradiographic study of the retinofugal projections in the shark, Hemiscyllium plagiosum.

The retinofugal projections in the shark, Hemiscyllium plagiosum were studied after unilateral eye injection of tritiated proline. The results showed that each eye in this animal projects not only to the contralateral side of the brain but also to several ipsilateral visual centers including the nucleus suprachiasmaticus, ventrolateral nucleus of the optic tract and the optic tectum. These results suggest that some of the visual centers in this animal receive direct retinal inputs from both eyes as is the case in many other animals and most notably in mammals.

An anterograde HRP study of the retinocollicular pathways in normal hamsters and hamsters with one eye enucleated at birth.

The present anterograde HRP study indicated that the uncrossed retinal projection to the superior colliculus in both normal hamsters and hamsters with one eye enucleated at birth was more extensive than previously reported. In addition, a small retinal projection to the contralateral inferior colliculus was also observed in all animals studied.

The normal and abnormal postnatal development of retinogeniculate projections in golden hamsters: an anterograde horseradish peroxidase tracing study.

In adult hamsters, the dorsal lateral geniculate nucleus (LGd), which lacks a noticeable pattern of cellular lamination, receives fibers predominantly from the contralateral eye except for a medial segment which receives fibers from the ipsilateral eye. Using the method of anterograde transport of horseradish peroxidase (HRP), it is shown in this study that the contralateral and ipsilateral retinogeniculate fibers innervate the LGd by day 0 with the development of the contralateral fibers slightly ahead of the ipsilateral ones. The entire contralateral LGd is filled with retinal fibers by day 1. The ipsilateral LGd is almost completely covered with retinal fibers on day 2 but with the fiber density much higher in the dorsal half of the nucleus. Thus, fibers from both eyes overlap with each other completely beginning on day 2 in the LGd. The segregation of these fibers becomes obvious on day 6 as indicated by a decrease in the density of ipsilateral fibers in the ventral portion of the LGd while the ipsilateral projection continues to concentrate in the dorsal half of the nucleus. A low density area in the dorsomedial part of the contralateral LGd is observed on day 7. By day 8, the segregation of the contralateral and ipsilateral projections has achieved an adult-like pattern. Thus, there seems to be two phases for the normal development of the retinogeniculate fibers. In the first phase, axons from both eyes grow in and occupy the entire LGd. In the second phase, those axons occupying inappropriate areas of the LGd are eliminated to form the adult pattern. The effect of unilateral eye removal at birth on the development of the retinogeniculate projection from the remaining eye was also studied with the anterograde HRP method. The ipsilateral fibers in the experimental animals are distributed in the lateral portion of the nucleus in the first two postnatal days. The entire LGd is not filled with ipsilateral fibers until day 4. From day 6 onwards, the ipsilateral fibers are more extensive than those of the normal animals. In addition to a dense projection to the dorsal two-thirds of the LGd, moderate amount of ipsilateral axons can be detected in the remaining ventral portion of the nucleus in day 6 and older experimental animals. The development of the contralateral retinal fibers in the experimental animals is similar to that of the normal from day 1 to day 6, i.e. the entire LGd is densely filled with crossed optic axons.(ABSTRACT TRUNCATED AT 400 WORDS)

The postnatal development of retinocollicular projections in normal hamsters and in hamsters following neonatal monocular enucleation: a horseradish peroxidase tracing study.

The pattern of distribution of the retinal projections to the superior colliculus (SC) has been studied in developing normal hamsters and in hamsters following unilateral eye enucleation at birth, using the anterograde horseradish peroxidase (HRP) method. The results show that in normal hamsters the contralateral retinocollicular projection has already reached the caudal pole of the SC on the day of birth, and covered the entire SC by day 1. The ipsilateral retinocollicular projection is distributed only to the rostrolateral portion of the SC on day 0, but has covered the entire area of the SC on day 1. The innervation of the SC by the ipsilateral projection increases gradually until it reaches its maximum density on day 3 or day 4. Beginning on day 6, the density of the ipsilateral projection decreases markedly except in areas where there is a distinct clumping of retinal fibers. The normal adult pattern, which consists of dense clumps of ipsilateral retinal projections in the rostral half of the SC and a sparse ipsilateral retinal projection distributing in almost the entire extent of the SC, is established on day 10. In animals in which one eye was removed on the day of birth, the ipsilateral projection is observed in the rostral two-thirds of the SC on day 1, and innervates the entire extent of the colliculus on day 2. On day 3 or day 4, this projection is denser than that found in normal animals of the same age. The SC remains heavily innervated by ipsilateral fibers on and after day 6. The abnormal adult pattern, which consists of dense ipsilateral retinal projections in most parts of the SC, is observed on day 10. The anomalous ipsilateral retinocollicular projection which develops in eye-enucleated animals suggests that there is a competitive interaction between fibers from the two eyes during development which is critical in shaping the normal adult pattern of the ipsilateral retinocollicular projection. The early development of the contralateral projection is in advance of the ipsilateral projection, and removal of the contralateral fibers by eye enucleation at birth seems to result in a further delay in the development of the ipsilateral projection. This suggests that in the hamster the contralateral fibers may play a role in guiding the ipsilateral fibers to reach their target region.(ABSTRACT TRUNCATED AT 400 WORDS)