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Human Wharton’s Jelly MSCs (hWJ-MSCs) have a considerable advantage and potential in treating the central nervous system diseases and can be a new alternative treatment of Multiple Sclerosis (MS). Aim. To study the persistence and distribution of hWJ-MSCs along the neuraxis following transplantation in central nervous system of rats with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Methods. Isolation and cultivation of hWJ-MSCs in vitro. Immunological phenotyping by flow cytometry. EAE induction. Intrathecal (suboccipital) injection of MSCs into CNS of SCH-induced EAE rats. Persistence of hWJ-MSCs in the CNS of hWJ-MSCs -treated rats was detected through detection of the human alpha-satellite DNA in the tissue sections and the cerebrospinal fluid (CSF) by PCR at days 2, 3, 4 and 5 Results. PCR-assays for alpha-satellite sequences revealed that Human DNA was detected during 5 days following intrathecal injection at the peak of disease in the treated rats. It has been demonstrated that the human DNA was traced in CSF and various segments of a spinal cord. Conclusions. The data obtained suggest that intrathecally delivered hWJ-MSCs, with time, can migrate through the CSF from the injection site to various segments of CNS and persist therein during the first week of post transplantation, which was performed at the EAE disease peak in the xenogeneic setting without immunosuppression. hWJ-MSCs may be considered as a delivery cell source of therapeutic molecules for CNS inflammatory diseases. Мезенхімальні стовбурові клітини Вартонового студня пуповини людини (МСК-ВС) мають значну перевагу і потенціал у лікуванні захворювань центральної нервової системи (ЦНС) і можуть стати новою альтернативною терапією для розсіяного склерозу (РС). Мета. Вивчити виживання і розподіл МСК-ВС в ЦНС щурів з ЕAЕ після субокціпітальної трансплантації. Методи. Виділення і культивування МСК-ВС in vitro. Імунологічне фенотипування методом проточної цитофлюориметрії. Індукція ЕАЕ. Інтратекальне введення МСК в ЦНС щурів лінії Wistar з ЕАЕ, індукованим гомогенатом спинного мозку (ГСМ). Виживання МСК-ВС в ЦНС щурів оцінювали за допомогою виявлення людської альфа-сателітної ДНК у зразках тканин та спинномозковій рідині (СМР) на 2, 3, 4, та 5 дні за допомогою ПЛР. Результати. ПЛР-аналіз для альфа-сателітних послідовностей показав, що ДНК людини виявлялася протягом 5 днів після інтратекального введення, на піку симптомів. Було показано, що трансплантовані ксеногенні МСК-ВС мігрували через СМР в різні сегменти спинного мозку. Висновки. Отримані результати дозволяють припустити, що МСК-ВС, введені інтратекально, можуть мігрувати зі струмом СМР в різні відділи ЦНС і перебувати в них протягом першого тижня після трансплантації на піку захворювання ЕAЕ в ксеногенному варіанті без імуносупресії. МСК-ВС можна розглядати як джерело клітин для доставки терапевтичних молекул для лікування запальних захворювань ЦНС. Мезенхимальные стволовые клетки Вартонового студня пуповины человека (МСК-ВС) имеют значительное преимущество перед другими видами стволовых клеток и потенциал в лечении заболеваний центральной нервной системы (ЦНС) и могут стать новой альтернативной терапией для рассеянного склероза. Цель. Изучить выживание и распределение МСК-ВС в ЦНС крыс с ЭАЭ после субоксипитальной трансплантации. Методы. Выделение и культивирование МСК-ВС in vitro. Иммунологическое фенотипирование методом проточной цитофлюориметрии. Индукция ЭАЭ. Интратекальное (субокципитальное) введение МСК в ЦНС крыс линии Wistar с ЭАЭ, индуцированным гомогенатом спинного мозга (ГСМ). Выживание МСК-ВС в ЦНС крыс оценивали с помощью выявления человеческой альфа-сателлитной ДНК в образцах тканей и спинномозговой жидкости (СМЖ) на 2, 3, 4 и 5 дни с помощью ПЦР. Результаты. ПЦР-анализ для альфа-сателлитных последовательностей показал, что ДНК человека обнаруживалась в течение 5 дней после интратекального введения, на пике симптомов. Было показано, что трансплантированные ксеногенные МСК-ВС мигрировали через спинномозговую жидкость (СМЖ) в различные сегменты спинного мозга. Выводы. Полученные результаты позволяют предположить, что МСК-ВС, введенные интратекально, со временем¸могут мигрировать с током СМЖ в различные отделы ЦНС и находиться в них на протяжении первой недели после трансплантации на пике заболевания ЭAЭ в ксеногенном варианте без иммуносупрессии . МСК-ВС можно рассматривать как источник клеток для доставки терапевтических молекул для лечения воспалительных заболеваний ЦНС.

Mesenchymal stem cells (MSCs) are ideal candidates for cell-based therapy aimed at tissue repair and immunomodulation. Aim. to study the survival of transplanted human MSCs from umbilical cord Wharton’s Jelly (hWJ-MSCs) in the animal model of experimental osteoarthritis (OA) in rats after injecting cells into a knee joint and to explore the effect of collagen scaffold on the cell survival in vivo. Methods. MSC isolation and cultivation in vitro. Immunological phenotyping of propagated hWJ-MSCs was performed by flow cytometry. The retention of transplanted cells was studied by the PCR revealing of human specific sequences in genomic DNA extracted from animal tissues. Results. hWJ-MSCs, both individual and grown on scaffold, were used and it was shown by PCR that human alpha-satellite DNA was detected on the first day in the immunocompetent OA animals inside the injured knee joint. In the collagen matrix (in the model of subcutaneous implantation) human alpha-satellite DNA was detected on the 5th day but was not detected on the 12th day. Conclusions. According to the PCR results, hWJ-MSCs survived in the OA animal model for a short period. Collagenic scaffold increased the residence time of donor cells in the recipients. hWJ-MSCs may be considered as a perspective cell source for the treatment of OA in human. Мезенхимальные стволовые клетки (МСК) являются идеальными кандидатами для восстановление тканей и иммуномодуляции при клеточной терапии. Цель. Изучить выживание трансплантированных МСК Варто­но­во­го студня пуповины человека (МСК-ВС) на модели экспериментального остеоартроза у крыс (введение клеток в коленный сустав), и исследовать влияние коллагенового матрикса на выживание МСК-ВС in vivo. Методы. Вы­де­ление и культивирование МСК-ВС in vitro. Имму­но­ло­ги­ческое фенотипирование мультиплицированных МСК-ВС про­ведено с помощью проточной цитофлуорометрии. Сохра­не­ние трансплантированных клеток изучали с помощью ПЦР, выявляющей специфические для человека последовательности в геномной ДНК, изолированной из тканей животных. Результы. Исследование индивидуальних и выращенные на матриксе МСК-ВС, с помощью ПЦР, показало, что альфа-сателлитная ДНК человека детектировалась в первые сутки у иммунокомпетентных животных в поврежденном суставе. В коллагеновом матриксе (в модели подкожной имплантации) ДНК человека была обнаружена на 5, но не детектировалась на 12 сутки. Вы­воды. По результатам ПЦР МСК-ВС выживали у жи­вотных с остеоартритом в течение короткого периода. Коллагеновый матрикс увеличивал продолжительность пре­бывания МСК-ВС у реципиентов. МСК-ВС могут рассматриваться как перспективный источник клеток для лечения остеоартрита у человека. Мезенхімальні стовбурові клітини (МСК) є ідеальними кандидатами для відновлення тканин та імуномодуляції при клітинній терапії. Мета. Вивчити виживання трансплантованих МСК Вартанового студню пуповини людини (МСК-ВС) на моделі експериментального остеоартрозу у щурів (введення клітин в колінний суглоб), та дослідити вплив коллагенового матриксу на виживання МСК-ВС in vivo. Ме­тоди. Виділення і культивування МСК-ВС in vitro. Імуно­ло­гічне фенотипування мультиплікованих МСК-ВС проведено за допомогою проточної цитофлуорометрії. Збере­жен­ня трансплантованих клітин вивчали за допомогою ПЛР, що виявляє специфічні для людини послідовності в геномній ДНК, ізольованій з тканин тварин. Результи. Використано індивідуальні та вирощені на матриксі МСК-ВС, ПЛР показано, що альфа-сателітна ДНК людини в пошкодженому суглобі у імунокомпетентних травмованих тварин виявлялась за добу. У колагеновому матриксі (в моделі підшкірної імплантації) ДНК людини була виявлена на 5, але не детектувалась на 12 добу. Висновки. За результатами ПЛР МСК-ВС виживали у тварин з остеоартрітом протягом нетривалого часу. Кола­ге­но­вий матрикс збільшував тривалість перебування МСК-ВС у реципієнтів. МСК-ВС можуть розглядатися як перспектив­не джерело клітин для лікування остеоартриту у людини.

Alpha-satellite DNA (AS) is a main DNA component of primate centromeres, consisting of tandemly repeated units of ~170 bp. The AS of humans contains sequences organized into higher-order repeat (HOR) structures, in which a block of multiple repeat units forms a larger repeat unit and the larger units are repeated tandemly. The presence of HOR in AS is widely thought to be unique to hominids (family Hominidae; humans and great apes). Recently, we have identified an HOR-containing AS in the siamang, which is a small ape species belonging to the genus Symphalangus in the family Hylobatidae. This result supports the view that HOR in AS is an attribute of hominoids (superfamily Hominoidea) rather than hominids. A single example is, however, not sufficient for discussion of the evolutionary origin of HOR-containing AS. In the present study, we developed an efficient method for detecting signs of large-scale HOR and demonstrated HOR of AS in all the three other genera. Thus, AS organized into HOR occurs widely in hominoids. Our results indicate that (i) HOR-containing AS was present in the last common ancestor of hominoids or (ii) HOR-containing AS emerged independently in most or all basal branches of hominoids. We have also confirmed HOR occurrence in centromeric AS in the Hylobatidae family, which remained unclear in our previous study because of the existence of AS in subtelomeric regions, in addition to centromeres, of siamang chromosomes.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot. Hum Mol Genet

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

The evolutionary history of alpha-satellite DNA, the major component of primate centromeres, is hardly defined because of the difficulty in its sequence assembly and its rapid evolution when compared with most genomic sequences. By using several approaches, we have cloned, sequenced, and characterized alpha-satellite sequences from two species representing critical nodes in the primate phylogeny: the white-cheeked gibbon, a lesser ape, and marmoset, a New World monkey. Sequence analyses demonstrate that white-cheeked gibbon and marmoset alpha-satellite sequences are formed by units of approximately 171 and approximately 342 bp, respectively, and they both lack the high-order structure found in humans and great apes. Fluorescent in situ hybridization characterization shows a broad dispersal of alpha-satellite in the white-cheeked gibbon genome including centromeric, telomeric, and chromosomal interstitial localizations. On the other hand, centromeres in marmoset appear organized in highly divergent dimers roughly of 342 bp that show a similarity between monomers much lower than previously reported dimers, thus representing an ancient dimeric structure. All these data shed light on the evolution of the centromeric sequences in Primates. Our results suggest radical differences in the structure, organization, and evolution of alpha-satellite DNA among different primate species, supporting the notion that 1) all the centromeric sequence in Primates evolved by genomic amplification, unequal crossover, and sequence homogenization using a 171 bp monomer as the basic seeding unit and 2) centromeric function is linked to relatively short repeated elements, more than higher-order structure. Moreover, our data indicate that complex higher-order repeat structures are a peculiarity of the hominid lineage, showing the more complex organization in humans.

Satellite DNA, a major component of eukaryotic centromeric heterochromatin, is potentially associated with the processes ensuring the faithful segregation of the genetic material during cell division. Structural properties of alpha-satellite DNA (AS) from African green monkey (AGM) were studied. Atomic force microscopy imaging showed smaller end-to-end distances of AS fragments than would be expected for the persistence length of random sequence DNA. The apparent persistence length of the AS was determined as 35 nm. Gel-electrophoresis indicated only a weak contribution of intrinsic curvature to the DNA conformations suggesting an additional contribution of an elevated bending flexibility to the reduced end-to-end distances. Next, the force-extension behavior of the naked AS and in complex with nucleosomes was studied using optical tweezers. The naked AS showed a reduced overstretching transition force (−18% the value determined for random DNA) and higher forces required to straighten the DNA. Finally, reconstituted AS nucleosomes disrupted at significantly higher forces as compared with random DNA nucleosomes which is probably due to structural properties of the AS which stabilize the nucleosomes. The data support that the AS plays a role in the formation of centromeric heterochromatin due to specific structural properties and suggest that a relatively higher mechanical stability of nucleosomes is important in AGM–AS chromatin.

Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45–50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.

Background and Purpose: Highly repetitive DNA sequences localized in centromeric and telomeric regions of chromosomes have essential roles in cell division and aging, respectively. (Peri)centromeric and (sub)telomeric repetitive DNA sequences are poorly understood because they exhibit extreme complexity and polymorphism of sequence and organization at all levels and because they are not yet fully assembled. The objective of this work was to develop the model for computational visualization of universal features as well as the variability that characterize the architecture of these DNA regions directly from their primary sequence using a repeat-finding strategy combined with color-coding of sequence- relatedness. The main purpose was to provide detailed maps that would help in understanding the relationship between the primary sequence, organization, evolutionary history and function of repetitive DNA sequences in eukaryotic genomes. Materials and Methods: The proposed approach, named SitaRam, is based on application of BLAST (blastn and bl2seq), MapDraw, multicolor DotPlot, Clustal and Sfor methods. It was developed on AC017075 and AC019063 BAC clone sequences that encompass the centromere gap at the human chromosome 7. Results: SitaRam approach was found to be equally suitable for the visual analysis of »new« and »old« alpha-satellite DNA arrays. Provided here are graphic presentations of the discontinuous progressive pattern at the border of the centromeric D7Z1 array and presentations of the entire multimerically arranged D7Z2 array together with its monomerically arranged surrounding. Multicolor, multiscale and multiregister visualization of tandem repeat variants within satellite arrays in their sequential order revealed diverse hallmarks of unequal crossover as the process generating these arrays. By DotPlot merging, a difference in the frequency and distribution pattern of CENP-B boxes is revealed between alpha-satellite domains characterized by higher-order, progressive and irregular monomeric organization. Conclusions: The proposed representations are characterized by small space requirements and enable simultaneous visualization of diverse types of satellite and interspersed DNA sequences, their inversions, insertions, deletions and potential protein- binding sites. According to the appearance of repetitive patterns, satellite DNA arrays can be visually classified in respect to their evolutionary age, chromosomal location and function. Among the potentials of this model are the comparative analysis of repetitive DNA sequences originating from different eukaryotes and from diverse parts of their chromosomes, the evaluation of individual polymorphism, the detection of as yet unidentified sequences, information compression and simplification of education on repetitive DNAs.

CENP-C is a fundamental component of the centromere, highly conserved among species and necessary for the proper assembly of the kinetochore structure and for the metaphase-anaphase transition. Although CENP-C can bind DNA in vitro, the identification of the DNA sequences associated with it in vivo and the significance of such an interaction have been, until now, elusive. To address this problem we took. advantage of a chromatin-immunoprecipitation procedure and applied this technique to human HeLa cells. Through this approach we could establish that: (1) CENP-C binds the alpha-satellite DNA selectively; (2) the CENP-C region between amino acids 410 and 537, previously supposed to contain a DNA-binding domain, is indeed required to perform such a function in vivo; and (3) the profile of the alpha-satellite DNA associated with CENP-C is essentially identical to that recognized by CENP-B. However, further biochemical and ultrastructural characterization of CENP-B/DNA and CENP-C/DNA complexes, relative to their DNA components and specific spatial distribution in interphase nuclei, surprisingly reveals that CENP-C and CENP-B associate with the same types of alpha-satellite arrays but in distinct non-overlapping centromere domains. Our results, besides extending previous observations on the role of CENP-C in the formation of active centromeres, show, for the first time, that CENP-C can associate with the centromeric DNA sequences in vivo and, together with CENP-B, defines a highly structured organization of the alpha-satellite DNA within the human centromere.