Divergent projection of individual corticospinal axons to motoneurons of multiple muscles in the monkey

Intracellular staining with horseradish peroxidase (HRP) of physiologically identified corticospinal (CS) axons originating from the monkey motor cortex revealed the intraspinal morphology of their branching patterns. CS collaterals spread in a delta-like fashion in the intermediate zone and lamina IX. Virtually all CS axons examined terminated in lamina IX, and it was shown by labeling motoneurons with retrograde transport of HRP that individual CS axons made direct contacts with dendrites of motoneurons of different muscle species.     

Glutamine synthetase is essential in early mouse embryogenesis.

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.     

胰岛素、可地松和已烯雌酚处理妊娠早期小鼠对其胚胎发育的影响

本文用不同剂量的胰岛素、可地松、已烯雌酚,作用于妊娠开始至80小时的昆明小鼠,然后观察胚胎早期(植入前)和胚胎晚期(妊娠18天)的胚胎数量、发育时期及其与黄体数相比成活率的变化。实验表明,用较大剂量激素处理后,早期胚胎的上述各项指标,均表现出十分明显的抑制效果。当剂量逐渐降低后,仍表现出不同程度的抑制作用,只是逐渐趋于正常。所用各种激素对晚期胚胎的影响,表现在除部分死亡外,存活者的生长和发育一直落后。说明妊娠早期小鼠体内某些激素的水平,不仅直接影响早期,而且也影响晚期胚胎的发育。     

bFGF对胚胎神经干细胞分化为神经元及神经胶质细胞的影响

本研究观察了碱性成纤维生长因子对胚胎神经干细胞生长和分化的影响。从孕 12 d大鼠胚胎神经管分离神经干细胞 ,进行体外培养 ,分为碱性成纤维生长因子组及对照组。培养过程中观察神经干细胞的生长 ,于培养第 3、5、10 d用免疫组化方法检测培养细胞神经元特异烯醇化酶和胶质纤维酸性蛋白的表达 ,以观察神经干细胞分化为神经元及神经胶质细胞的状况。碱性成纤维生长因子可明显地促进培养细胞的生长和分化。免疫组化细胞计数显示 ,培养第 3 d,特异烯醇化酶、胶质纤维酸性蛋白阳性细胞数均明显增加 ;培养第 5 d,特异烯醇化酶阳性细胞数是对照组的 1.9倍 ,胶质纤维酸性蛋白阳性细胞数为对照组的 1.6倍 ,前者表达增加明显 ;培养第 10 d,两者的阳性细胞数仍高于对照组 ,但增加不明显。不同培养时间的胞体最长突起长度也均高于对照组 ;胞体直径及表面积随培养时间延长而增大。说明 ,碱性成纤维生长因子既能促进胚胎神经干细胞的生长 ,也可促使其分化为神经元及神经胶质细胞 ,尤以神经元为明显     

Stretching the limits: Stem cells in regeneration science

The focus of regenerative medicine is rebuilding damaged tissues by cell transplantation or implantation of bioartificial tissues. In either case, therapies focus on adult stem cells (ASCs) and embryonic stem cells (ESCs) as cell sources. Here we review four topics based on these two cell sources. The first compares the current performance of ASCs and ESCs as cell transplant therapies and the drawbacks of each. The second explores somatic cell nuclear transfer (SCNT) as a method to derive ESCs that will not be immunorejected. The third topic explores how SCNT and ESC research has led to the ability to derive pluripotent ESCs by the dedifferentiation of adult somatic cells. Lastly, we discuss how research on activation of intrinsic adult stem cells and on somatic cell dedifferentiation can evolve regenerative medicine from a platform consisting of cell transplantation to one that includes the chemical induction of regeneration from the body's own cells at the site of injury. Developmental Dynamics 237:3648–3671, 2008. © 2008 Wiley‐Liss, Inc.     

DNA fingerprinting of sister blastomeres from human IVF embryos

BACKGROUND: Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. METHODS: Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). RESULTS: The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. CONCLUSIONS: A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.     

人胚胎干细胞系HUES4的培养和染色体核型分析

目的建立人胚胎干细胞无动物源性饲养层培养方法,同时对长时间体外培养的人胚胎干细胞核型变化进行分析。方法人胚胎干细胞系HUES4细胞分别培养于小鼠胚胎成纤维细胞和人包皮成纤维细胞饲养层,并对其干细胞特性进行鉴定;在培养传代过程中,收获P27、P34、P41和P44细胞进行染色体核型分析,P27细胞还进行DNA短串联重复序列多态性分析。结果生长于人包皮成纤维细胞饲养层的HUES4细胞碱性磷酸酶染色以及SSEA-4、TRA-1-60和TRA-1-81抗原阳性,SSEA-1抗原阴性。所检测的4代细胞中均见46,XY/46,XY,t（9;15）（q22;q26）核型嵌合现象,且异常核型百分比随传代次数增加有上升的趋势。结论培养人胚胎干细胞的饲养层细胞可由无动物源性的饲养层细胞替代;长期体外培养有增加细胞染色体核型异常的风险。     

Expression of voltage-dependent sodium and transient potassium currents in an identified sub-population of dorsal root ganglion cells acutely isolated from 12-day-old mouse embryos

The electrophysiological properties of a subset of dorsal root ganglion (DRG) neurons microdissected from 12-day-old (E12) mouse embryos and acutely isolated were analyzed as soon as 3 after their isolation. Two classes of neurons were defined according to their mean diameter. The larger diameter class was examined in this study. They display uniform cytoskeletal properties with co-expression of vimentin and neurofilament triplet proteins. Patch-clamp methods also revealed a homogeneous and limited repertoire of ionic channels that included (1) a TTX-sensitive Na⁺ current whose properties are similar to that reported in mature mammalian neurons, and (2) two types of K⁺ currents that can be compared with the delayed rectifier (I _k ) and the transient (I _a) potassium currents found in other mammalian preparations. It may be possible to use this in vitro model to examine the development of new types of currents, such as Ca²⁺ currents during neuronal growth and differentiation.     

Chromosomal integrity maintained in five human embryonic stem cell lines after prolonged in vitro culture

There have been recent reports of human embryonic stem cell (hESC) lines developing chromosomal aberrations after long-term culture, indicating an unstable genomic status due to the in vitro milieu. This raises concern, since it would limit their use in therapeutics. In this study the chromosomal status of five well-characterized hESC lines, SA002, SA002.5, AS034.1.1, SA121 and SA461, was monitored during long-term in vitro culture. The criteria of defined hESCs were met by all of the five hESC lines (four diploid and one trisomic for chromosome 13). The genomes were screened for chromosomal aberrations and rearrangements using comparative genomic hybridization (CGH), interphase fluorescence in situ hybridization (FISH) and traditional karyotyping on several occasions while in culture. The genomic integrity was shown to be maintained after repeated freeze-thaw procedures and continuous culture in vitro for up to 22 months (148 passages). We discuss the most common de novo chromosomal aberrations reported in hESCs, as well as their possible origin.     

Development of the retinal pathway to the pretectum of the cat

The development of retinal projections to the pretectal complex of prenatal and early postnatal cats has been examined using the anterograde transport of horseradish peroxidase and tritiated amino acids. As early as embryonic day 38, the entire dorsal pretectum is penetrated by retinal ganglion cell axons. At this stage the bilateral complement of retinal efferents appears to be dispersed uniformly within the pretectal anlage. A week later, on embryonic day 46, indistinct foci of peroxidase reaction product can be discerned within 2 of the primordial nuclei: the nucleus of the optic tract and the olivary nucleus. By embryonic day 56, five distinct bilateral fields of retinal fiber termination are apparent within the following regions:

(i) the nucleus of the optic tract;

(ii) the pretectal olivary nucleus;

(iii) the posterior pretectal nucleus;

(iv) the anterior pretectal nucleus; and

(v) the medial pretectal nucleus. Four days before birth, on embryonic day 61, crossed and uncrossed retinal arbors are partially segregated within the nucleus of the optic tract and the pretectal olivary nucleus.

The early postnatal retinal connection to the pretectum has an overall pattern virtually indistinguishable from that of the mature cat. The ontogeny of the retinal influx to the pretectum is similar to that of the retinocollicular projection.⁶¹ However, the development of retinal projections to the pretectum and superior colliculus appears to lag behind those to the dorsal lateral geniculate nucleus.⁴⁹ These differences may reflect temporal and spatial gradients in the maturation of three major classes of retinal ganglion cells.