Telomere length measurement by Q-FISH

Telomeres are essential functional elements of eukaryotic chromosomes involved in genome stability maintenance. The most important indicator of correct telomere function is telomere length maintenance within the range typical for each species. Telomere length can be estimated by the classical methodology based on Southern blot. However, this methodology is relatively crude and can provide estimate of average telomere length only. To overcome disadvantages of classical telomere length estimate, a new technique termed Q-FISH has been invented. Q-FISH provides estimate of telomere length in each individual chromosome with the resolution of 200 base pairs. In addition, Q-FISH may be used to estimate telomere length in species containing interstitial telomeric sites in their genomes. The classical methodology is non-informative in these cases. Finally, Q-FISH has been essential in estimating telomere length in the mouse, a species with ultra-long telomeres difficult to measure using classical methods. Principles of Q-FISH and its applications are briefly described in this article.     

Telomere length in subpopulations of human hematopoietic cells

In order to test the hypothesis that the telomere length in human hematopoietic cells correlates with their proliferative potential, we analyzed the telomere length in highly purified subpopulations of bone marrow cells. Cells were sorted on the basis of CD34 and CD38 cell surface markers, and two samples were additionally sorted on the basis of Hoechst 33342 dye efflux allowing isolation of side population (SP) cells. The telomere length in limiting numbers of sorted cells was analyzed using a newly developed fluorescence in situ hybridization (flow-FISH) method in which hybridization of telomere probe in cells of interest is measured relative to control cells in the same tube. In all seven bone marrow samples analyzed, the telomere length in CD34(+)CD38(-) cells was longer than in CD34(+)CD38(+) cells from the same donor (p < 0.02). Results with sorted SP cells were less clear: the telomere fluorescence in these cells was very heterogeneous, and a reproducible difference in telomere length relative to CD34(+)CD38(-) cells could not be observed. We conclude that the telomere length in subpopulations of hematopoietic cells does appear to be correlated with the known proliferative potential of such cells and that further characterization of cells on the basis of telomere length is warranted for enrichment of very rare precursors of hematopoietic and other tissues.     

Telomere length in white blood cells, buccal cells and brain tissue and its variation with ageing and Alzheimer's disease

Changes in telomere length have been associated with ageing and with certain age-related degenerative diseases. We report results using a quantitative RTm-PCR method to measure absolute telomere length (in kb per diploid genome) and show the age-related changes in white blood cells and buccal cell telomere length (in kb per diploid genome) in normal healthy individuals and Alzheimer's patients. We observed a significantly lower telomere length in white blood cells (P < 0.0001) and buccal cells (P < 0.01) in Alzheimer's patients relative to healthy age-matched controls (31.4% and 32.3%, respectively). However, there was a significantly greater telomere length in hippocampus cells of Alzheimer's brains (P = 0.01) compared to control samples (49.0%). We also observed that telomere length in buccal cells was 52.2-74.2% shorter than that observed in white blood cells (P < 0.0001). The odd's ratio of being diagnosed with Alzheimer's disease (AD) was 10.8 (95% CI 1.19-97.85) if white blood cell telomere length was less than 115 kb per diploid genome with a specificity of 46% and sensitivity of 92.9%. The odds ratio for AD diagnosis was 4.6 (95% CI 1.22-17.16) if buccal cell telomere length was less than 40 kb per diploid genome with a sensitivity of 72.7% and a specificity of 63.1%. These results suggest important differences in telomere maintenance in Alzheimer's cases compared to healthy controls depending on sampled tissue. These results need to be replicated in larger studies and in cohorts of other neurodegenerative disorders to determine specificity of changes to Alzheimer's patients.     

Telomere length and telomerase activity: effect of ageing on human NK cells

Telomeres are repeats of TTAGGG sequences located at the end of eukaryotic chromosomes. They are essential for stabilisation and protection of chromosomal ends and for the regulation of cell replicative capacity. Due to the end-replication defect of DNA polymerase, telomeres shorten progressively with each cell division and telomere length may be an indicator of the replicative history of a cell. Compensatory mechanisms for the telomere loss have been identified. The most widely studied one is mediated by telomerase a ribonuclear protein-enzyme complex that synthesise telomeric repeats. In this study we have investigated whether NK cells, derived from a group of old healthy subjects, underwent the modifications of telomere length and telomerase activity observed in other sub-populations of lymphocytes with advancing age. We demonstrated that: (a) telomere shortening occurred and telomerase activity decreased in human NK cells with ageing; (b) the rate of telomere loss was different under and over 80 years of age; (c) similarly to telomere shortening, the modification of telomerase activity was particularly evident in octogenarians; (d) subjects with the most evident modifications of telomeres and telomerase were the oldest and those with increased NK cell numbers.     

Telomere length and LINE1 methylation is associated with chromosomal aberrations in peripheral blood

The frequency of chromosomal aberrations in peripheral blood predicts a probable cancer risk. The individual telomere length and methylation of repetitive elements may be susceptibility factors for chromosomal aberrations. A cohort of healthy Norwegian men (N = 364) recruited during 1980–1999 were analyzed for chromosomal aberrations in phytohemagglutinin‐stimulated lymphocytes from peripheral blood. Chromosome‐type or chromatid‐type aberrations were scored. DNA was extracted from slides cytogenetically analyzed and relative average telomere length and methylation of LINE1 repeats were determined by quantitative polymerase chain reaction and bisulfite pyrosequencing, respectively. Information about individuals with malignant tumors (N = 49) diagnosed after chromosomal aberrations testing until end of 2008 was obtained and two matched controls per case were used in a nested case–control analysis. Shorter relative telomere length and higher methylation of LINE1 were associated with higher frequency of total chromosomal aberrations (β = ?0.76, P = 0.022; and β = 0.042, P = 0.048, respectively; age‐adjusted ordinal regression). The telomere length was stronger associated with chromosome‐type (β = ?1.00, P = 0.006) than with chromatid‐type aberrations (β = ?0.49, P = 0.115). The LINE1 methylation was stronger associated with chromatid‐type (β = 0.062, P = 0.003) than with chromosome‐type aberrations (β = 0.018, P = 0.41). Telomere length [individuals with short telomeres odds ratio (OR) = 0.87, 95% confidence interval (CI) 0.38–2.0], LINE1 (individuals with high methylation OR = 1.04, 95% CI 0.43–2.5) and chromosomal aberrations (individuals with high frequency OR = 1.6, 95% CI 0.63–3.9) at baseline did not predict cancer risk, but the conclusions were hampered by low statistical precision. The results suggest that shorter telomere length and higher LINE1 methylation in peripheral blood lymphocytes are predisposition factors for increased frequency of chromosomal aberrations. © 2012 Wiley Periodicals, Inc.     

Impaired T-lymphocyte colony formation by cord blood mononuclear cells

When compared to adult mononuclear cells, cord blood mononuclear cells demonstrated significantly decreased T-lymphocyte colony formation (1351±643 vs 592±862,P<0.01). This diminished colony-forming activity did not appear to be associated with impaired responsiveness to the stimulant phytohemagglutinin or with excessive suppressor-cell activity. Irradiation reduced the colony-forming capacity of cord blood mononuclear cells more than it did that of adult mononuclear cells. Depletion of adherent cells reduced cord blood mononuclear-cell colony-forming capacity by 40%, while similar treatment reduced adult colony formation by 10%. Lymphocyte proliferation in liquid culture of cord and adult cells was minimally affected by these procedures. The colony-forming capacity of cord blood could be enhanced by the addition of irradiated adult cells (284±72 vs 752±78,P<0.01). This enhancement was demonstrated to be due to a soluble factor produced by a population of irradiated adult cells depleted of the OKT8⁺ subpopulation of lymphocytes. These results indicate that the progenitor cells of T-lymphocyte colonies in cord blood have distinct biologic characteristics when compared to colony progenitors present in adult blood. This assay may prove to be useful in our efforts to understand the differentiation of T-cell function in man.     

乳凝集素对脐带血树突状细胞功能的调节作用

目的研究乳凝集素(lactadherin)对未成熟树突状细胞吞噬功能以及对成熟树突状细胞刺激初始T细胞增殖能力等方面的调节作用。方法 ficoll密度梯度离心法分离脐带血单个核细胞,加入细胞因子IL-4、GM-CSF、TNF-α(成熟组应用)诱导分化为树突状细胞(dendritic cell,DC),阳性对照组分别在第0天或第5天起加入lactadherin,各组均培养至第7天时收集细胞,流式细胞仪检测各组未成熟DC吞噬FITC-DEXTRAN的能力,MTT法检测各组成熟DC刺激初始T细胞增殖能力的变化。结果同时加入lactadherin诱导培养时,未成熟DC对抗原(FITC-DEXTRAN)的吞噬功能增强,在培养第0天时即加入lactadherin组培养完成后吞噬功能增强更为明显;成熟组DC刺激初始T细胞增殖的能力也较无lactadherin组明显增强(P<0.001),且以第5天加入组培养完成时刺激增殖作用更为显著。结论 lactadherin能够增强DC细胞的免疫功能,包括未成熟时的抗原吞噬摄取和成熟时刺激T细胞增殖,激活适应性免疫应答等。     

Umbilical cord blood mesenchymal stem cells

We studied umbilical cord blood mesenchymal stem cells and compared mesenchymal stem cells derived from umbilical cord blood, adipose tissue, and skin. Umbilical cord blood mesenchymal stem cells were characterized morphologically, cytofluorometrically, and by their differentiation potential. Umbilical cord blood mesenchymal stem cells did not differ from cells isolated from adipose tissue and skin by the main parameters (by morphology, expression of surface markers, and differentiation potential). A specific feature of umbilical cord blood mesenchymal stem cells is their low count per volume of the initial material and very low proliferative activity. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 1, pp. 16–20, January, 2007     

1型糖尿病、2型糖尿病及2型糖尿病伴动脉硬化患者外周血白细胞端粒长度的研究

目的:检测1型糖尿病(T1DM)、2型糖尿病(T2DM)、2型糖尿病伴动脉硬化(DAS)患者外周血白细胞端粒长度,分析糖尿病患者端粒长度变化的因素。方法:选择T1DM患者30例、T2DM患者60例、DAS患者40例和健康对照(NC组)40例,分别提取外周血白细胞,然后提取基因组DNA进行real-time PCR检测端粒长度。利用多元线性回归分析影响端粒长度变化的因素。结果:T1DM组、T2DM组和DAS组的端粒长度均小于NC组(P0.05);T1DM组端粒长度短于T2DM组和DAS组;DAS组较T2DM组更短。多元线性回归分析显示,T1DM组中,年龄与端粒长度呈负相关(P0.05);T2DM组中,年龄、体重指数(BMI)与端粒长度呈负相关(P0.05);DAS组中,患病时间、BMI与端粒长度呈负相关(P0.05)。结论:糖尿病患者的外周血白细胞端粒长度明显短于正常人,并且T1DM患者端粒长度短于T2DM;在2型糖尿病的对比中,DAS患者的端粒长度明显短于T2DM。患者的年龄、患病时间、BMI与端粒长度的缩短有密切的关系。     

脐带血树突状细胞的免疫功能研究

目的： 探讨脐带血树突状细胞(DC)的免疫功能状况。 方法： 以CD34-Lin- HLA-DR+细胞群设门4色荧光分析方法检测20例脐带血和20例外周血DC前体细胞亚群pDC1/pDC2（CD11c+CD123-/CD11c-CD123+）比值；酶联免疫吸附法检测其血浆相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4水平。 结果： 脐带血、成人外周血pDC1/pDC2比值无显著差异（P>0.05）；脐带血血浆IL-12p40水平显著高于成人外周血（P<0.01），两者IL-10、IFN-γ、IL-4水平无显著差异（P>0.05）。 结论： 脐带血DC功能发育比较完善。     

The effects of retinoic acid on immunoglobulin synthesis by human cord blood mononuclear cells

Derivatives of vitamin A have attracted considerable attention as agents which have immune potentiating properties and possibly tumor-suppressive effects. Recent investigations have shown that retinoic acid (RA) can augment immunoglobulin production of B-cell hybridomas from patients with immune deficiency. In this study we examined the ability of RA to modify the mitogen-induced polyclonal immunoglobulin synthesis of cord blood mononuclear cells (CBMC). RA in concentrations ranging from 10(-5) to 10(-7) M augmented IgM synthesis of CBMC in response to formalinized Cowans I strain Staphylococcus aureus (SAC) up to 45.6-fold which was greater at suboptimal responses to SAC. There were no changes in IgG or IgA synthesis and minimal effects on SAC-induced proliferative responses. RA did not produce similar changes in IgM synthesis of SAC-stimulated adult peripheral blood mononuclear cells (PBMC), and RA had no effect on the immunoglobulin synthesis of Epstein-Barr virus (EBV)-stimulated CBMC or adult PBMC. Time course studies showed that peak enhancement occurred when RA was added between 4 and 24 hr after culture initiation and required prior activation by SAC for augmentation of IgM synthesis. Cell separation experiments showed that prior incubation (18 hr) of an enriched T-cell fraction with RA enhanced the IgM synthesis of a T-cell-depleted B-cell fraction. These experiments and the findings that RA-induced augmentation of IgM production in response to SAC, but not to EBV suggest that the immunoregulatory effects of RA may be mediated by either T cells or T-cell products. Further studies will be necessary to understand the mechanism by which RA augments IgM synthesis of CBMC.     

Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes

Chondrocyte ex vivo expansion currently performed to replace damaged articular surfaces is associated with a loss of telomeric repeats similar to decades of aging in vivo. This might affect the incidence or time of onset of age-related disorders within transplanted cells or tissues. This study examined whether more immature progenitor cells, such as mesenchymal stem cells (MSC), which can be expanded and subsequently differentiated into chondrocytes is advantageous regarding telomere-length related limitations of expansion protocols. Primary chondrocytes and bone-marrow-derived MSC were isolated from 12 donors, expanded separately to 4 x 10(6) cells, and (re-)differentiated as three-dimensional chondrogenic spheroids. Cells were collected during expansion, after three-dimensional culturing and chondrogenic differentiation, and sequential analyses of telomere length and telomerase activity were performed. Surprisingly, telomeres of expanded MSC were significantly shorter than those from expanded chondrocytes from the same donor (11.4+/-2.5 vs. 13.4+/-2.2 kb) and tended to remain shorter after differentiation in chondrogenic spheroids (11.9+/-1.8 vs. 13.0+/- kb). While telomere lengths in native chondrocytes and MSC were not related to the age of the donor, significant negative correlations with age were observed in expanded (136 bp/year), three-dimensionally reconstituted (188 bp/year), and redifferentiated (229 bp/year) chondrocytes. Low levels of telomerase activity were found in MSC and chondrocytes during expansion and after (re-)differentiation to chondrogenic spheroids. In terms of replicative potential, as determined by telomere length, ex vivo expansion followed by chondrogenic differentiation of MSC did not provide a benefit compared to the expansion of adult chondrocytes. However, accelerated telomere shortening with age during expansion and redifferentiation argues for an "age phenotype" in chondrocytes as opposed to MSC and suggests an advantage for the use of MSC especially in older individuals and protocols requiring extensive expansion     

Engraftment capacity of umbilical cord blood cells processed by either whole blood preparation or filtration

Umbilical cord blood (UCB) preparation needs to be optimized in order to develop more simplified procedures for volume reduction, as well as to reduce the amount of contaminating cells within the final stem cell transplant. We evaluated a novel filter device (StemQuick((TM))E) and compared it with our routine buffy coat (BC) preparation procedure for the enrichment of hematopoietic progenitor cells (HPCs). Two groups of single or pooled UCB units were filtered (each n = 6), or equally divided in two halves and processed by filtration and BC preparation in parallel (n = 10). The engraftment capacity of UCB samples processed by whole blood (WB) preparation was compared with paired samples processed by filtration in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse animal model. Filtration of UCB units in the two groups with a mean volume of 87.8 and 120.7 ml, respectively, and nucleated cell (NC) content of 9.7 and 23.8 x 10(8) resulted in a sufficient mean cell recovery for mononucleated cells ([MNCs] 74.2%-77.5%), CD34(+) cells (76.3%-79.0%), and colony-forming cells (64.1%-86.3%). Moreover, we detected a relevant depletion of the transplants for RBCs (89.2%-90.0%) and platelets ([PLTs] 77.5%-86.1%). In contrast, the mean depletion rate using BC processing proved to be significantly different for PLTs (10%, p = 0.03) and RBCs (39.6%, p < 0.01). The NC composition showed a highly significant increase in MNCs and a decrease in granulocytes after filtration (p < 0.01), compared with a less significant MNC increase in the BC group (p < 0.05). For mice transplanted with WB-derived progenitors, we observed a mean of 15.3% +/- 15.5% of human CD45(+) cells within the BM compared with 19.9% +/- 16.8% for mice transplanted with filter samples (p = 0.03). The mean percentage of human CD34(+) cells was 4.2% +/- 3.1% for WB samples and 4.5% +/- 3.2% for filter samples (p = 0.68). As the data of NOD/SCID mice transplantation demonstrated a significant engraftment capacity of HPCs processed by filtration, no negative effect on the engraftment potential of filtered UCB cells versus non-volume-reduced cells from WB transplants was found. The StemQuick((TM))E filter devices proved to be a useful tool for Good Manufacturing Practices conform enrichment of HPCs and MNCs out of UCB. Filtration enables a quick and standardized preparation of a volume-reduced UCB transplant, including a partial depletion of granulocytes, RBCs, and PLTs without the need for centrifugation. Therefore, it seems very probable that filter-processed UCB transplants will also result in sufficient hematopoietic reconstitution in humans.     

Cytoplasmic DNA synthesis by cord blood cells of premature and full-term infants

Summary Cord blood cells from 8 full-term and 12 premature infants as well as blood leukocytes from 10 adults were incubated with³H-thymidine and were subsequently exposed to autoradiographic emulsion for prolonged periods of 45 and 90 days. Cytoplasmic label — which we interpreted as evidence of mitochondrial DNA synthesis — was found in 1.7 per cent of lymphoid cells from adult blood, in 2.9 per cent and 4.5 per cent of lymphoid cells from full-term and premature infants, respectively. Statistical analysis of the data shows that the number of labelled cells is significantly larger in the blood of premature infants than in adults (p<0.01).On leave from the Department of Pediatrics, University of Göttingen, West-Germany, under the auspices of the Committee for Scientific Co-operation between German Institutions and the Weizmann Institute     

Direct chromosome analysis from neonatal cord blood

Direct chromosome preparations of neonatal cord blood provides the unique opportunity for rapid chromosome analysis (turnaround time; 6 hr), without the necessity of bone marrow aspiration. Based on 42 samples we confirm the finding of Garnham and Sutherland [1987] for suitability of cord blood for direct chromosome preparation. Procedural modifications are provided for higher yield of cells for chromosome analysis. The procedure may well be of major significance for rapid diagnosis of neonates who suffer from aneusomy.     

新生儿脐血单核细胞在脂多糖刺激下分泌IL-6及出现IL-6mRNA表达的实验研究

目的 :通过观察LPS对新生儿脐血单个核细胞 (MC)分泌IL 6及表达IL 6mRNA基因的影响 ,探讨严重细菌感染时新生儿机体防御反应机制。方法 :取肝素抗凝剂脐血 ,用密度离心分离法分离MNC ,以RPMI16 40培养液调整细胞浓度为1× 10 6 ml- 1 ,将细胞悬液铺于 2 4孔培养板上 ,依次加入不同浓度脂多糖 (LPS)培养 36h或同一浓度LPS(1μg ml)培养不同时间 ,收集培养上清液及细胞 ,分别用ELISA和RT PCR方法测定IL 6及IL 6mRNA表达情况。结果 :①脐血MNC在LPS刺激 3、6、12、18、2 4、36h后IL 6分泌水平逐步增高 ,6h以后增加尤为明显 ,与其他各时间点比较有非常显著的差异 (P <0 0 0 1)。LPS刺激组与无LPS对照组相同时间点比较 ,6h内IL 6变化水平无差异 ,6h以上各点有显著差异 (P <0 0 1)。RT PCR方法检测显示LPS刺激后 3h即可见IL 6mRNA基因表达。②脐血MNC受不同浓度LPS刺激时 ,IL 6分泌水平随LPS浓度递增。③全部脐血MNC均检测到IL 6mRNA基因表达。结论 :LPS能诱导新生儿脐血MNCIL 6mRNA基因转录 ,从而促使IL 6合成、分泌 ,该作用呈时间、剂量依赖性变化。     

人脐血干细胞体外诱导为肥大细胞的研究进展

Mast cells (MCs) play a key role in the pathogenesis of allergic diseases. Tissue MCs are originated from hematopoiefic stem cells in bone marrow. In recent years, it was reported that human mast cells could be differentiated from stem cells of umbilical cord blood. In this review, we summarize the development in this novel area.     

Telomere shortening in peripheral blood cells was related with aging but not with white blood cell count

Summary Telomeres in somatic cells are progressively shortened with aging. We investigated the relationship between the telomere length and other factors which may affect the frequency of cell divisions, in peripheral blood cells. Shortening of telomeric repeats was correlated with aging (p<0.0001), but not with white blood cell count, neutrophil count, and smoking habit. Not only the number of cell divisions, but also some other factors, such as upregulation level of telomerase activity concomitant with the cell division in hematopoietic progenitor cells, might affect the length of telomeric repeats in blood cells.     

Human cord blood cells can differentiate into retinal nerve cells

Retinal degeneration and dystrophy are the major causes of blindness in the developed world. It has been reported that human cord blood cells (HCBCs) can differentiate into neuron-like cells in vitro. We have recently demonstrated that bone marrow cells (BMCs) of both mice and rats can differentiate into retinal nerve cells (RNCs). In the present study, we show the differentiation capacity of HCBCs into RNCs in vivo. We transplanted lineage-negative HCBCs into the subretinal space of severe combined immunodeficiency (SCID) mice. Two weeks after the transplantation, some of the transplanted cells expressed human nestin, human MAP2, human neuron specific enolase (NSE), beta-III tubulin and also rhodopsin. These results indicate that HCBCs can differentiate into RNCs and suggest that our new strategy could be used for the regeneration of retinal nerve cells in degenerative or dystrophic diseases.