Correlation analysis between COX-2 gene polymorphism and eclampsia.

OBJECTIVE: The aim of this study was to investigate the expression of cyclooxygenase-2 (COX-2) in eclampsia patients, and to explore the correlation between COX-2 polymorphism and incidence of eclampsia. PATIENTS AND METHODS: From January 2016 to January 2018, a total of 280 pregnant women diagnosed in the Obstetrics and Gynecology Department of our hospital were selected for this study. All patients were divided into two groups, including normal pregnancy control group (n=120) and eclampsia group (n=160). The expression of COX-2 in placenta and umbilical cord tissues of eclampsia group and normal group was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blotting and immunohistochemical staining. The single-nucleotide polymorphisms (rs1526172, rs1245231 and rs2198532) in the promoter region of the COX-2 gene were typed via conformation difference gel electrophoresis. Whether the distribution frequency of COX-2 genotypes met Hardy-Weinberg equilibrium law was detected via the Chi-square test. Meanwhile, the correlation between COX-2 alleles and gene polymorphisms and the incidence of eclampsia was analyzed. RESULTS: The messenger ribonucleic acid (mRNA) and protein expression levels of COX-2 in the eclampsia group were significantly higher than those of the normal group (p<0.05). According to the analysis, three polymorphisms of COX-2 gene were all in line with Hardy-Weinberg equilibrium distribution (p>0.05). Gene association analysis revealed that only polymorphisms (rs1526172 and rs1245231) and alleles were correlated with the incidence of eclampsia (p<0.05). However, polymorphism rs2198532 and alleles were not correlated with the incidence of eclampsia (p>0.05). CONCLUSIONS: Rs1526172 and rs1245231 in the promoter region of COX-2 are correlated with the incidence of eclampsia, while rs2198532 has no correlation with eclampsia.

Divergent changes in the content and activity of MMP-26 and TIMP-4 in human umbilical cord tissues associated with preeclampsia.

OBJECTIVES: Preeclampsia is the most common disorder associated with pregnancy. Our earlier findings revealed a substantial increase in the amount of matrix metalloproteinase-26 (matrilysin 2; MMP-26) in preeclamptic umbilical cord blood. The role of MMP-26 in preeclamptic umbilical cord tissue has not been fully elucidated. Some reports have indicated that the expression of matrilysin 2 and tissue inhibitor of matrix metalloproteinase 4 (TIMP-4) is coordinately regulated during progression of various diseases. STUDY DESIGN: Therefore, we decided to assess the expression and activity of MMP-26 and TIMP-4 in normal and preeclamptic umbilical cord tissues - umbilical cord arteries (UCA), vein (UCV) and Wharton's jelly (WJ). Tissues obtained from 10 normal (control material) and 10 preeclamptic umbilical cords were assessed using immunoenzymatic assay, Western immunoblotting, reverse transcriptase - polymerase chain reaction and fluorometric determination of the enzyme activity. RESULTS: All umbilical cord tissues, both control and preeclamptic, expressed MMP-26 and TIMP-4 in macromolecular complexes. Preeclampsia induced a significant increase in the content and actual activity of MMP-26 in UCV and WJ, as compared to control. The content of TIMP-4 in preeclamptic UCV and WJ was reduced. The content of MMP-26 mRNA was lower in UCA and UCV, whereas higher in WJ in preeclampsia. CONCLUSIONS: Divergent changes in MMP-26 mRNA and protein expression suggest a difference in the factors controlling the matrilysin synthesis in healthy and preeclamptic subjects. The decrease in TIMP-4 content in preeclamptic UCV might be the main reason for significantly higher actual activity of MMP-26 in that tissue. Only in preeclamptic Wharton's jelly the changes were compatible in terms of the content and activity of MMP-26 and TIMP-4. It cannot be excluded that similar alterations can be observed for the whole vascular system of newborns delivered by mothers with preeclampsia.

Evaluation of platelet lysate as a substitute for FBS in explant and enzymatic isolation methods of human umbilical cord MSCs.

Mesenchymal stem cells (MSCs) have immense potential for cell-based therapy of acute and chronic pathological conditions. MSC transplantation for cell-based therapy requires a substantial number of cells in the range of 0.5-2.5 × 106 cells/kg body weight of an individual. A prolific source of MSCs followed by in vitro propagation is therefore an absolute prerequisite for clinical applications. Umbilical cord tissue (UCT) is an abundantly available prolific source of MSC that are fetal in nature and have higher potential for ex-vivo expansion. However, the ex-vivo expansion of MSCs using a xenogeneic supplement such as fetal bovine serum (FBS) carries the risk of transmission of zoonotic infections and immunological reactions. We used platelet lysate (PL) as a xeno-free, allogeneic replacement for FBS and compared the biological and functional characteristics of MSC processed and expanded with PL and FBS by explant and enzymatic method. UCT-MSCs expanded using PL displayed typical immunophenotype, plasticity, immunomodulatory property and chromosomal stability. PL supplementation also showed 2-fold increase in MSC yield from explant culture with improved immunomodulatory activity as compared to enzymatically dissociated cultures. In conclusion, PL from expired platelets is a viable alternative to FBS for generating clinically relevant numbers of MSC from explant cultures over enzymatic method.

Is there any role of prolidase enzyme activity in the etiology of preeclampsia?

OBJECTIVE: To evaluate a relationship between preeclampsia and prolidase enzyme activity. METHODS: A prospective cohort study of 41 pregnant women diagnosed with preeclampsia and 31 healthy pregnant women as control group was selected at Harran University Hospital Department of Obstetrics and Gynecology. The prolidase enzyme activity was analyzed in maternal and umbilical cord plasma, amniotic fluid and placental and umbilical cord tissues by Chinard method in addition to maternal serum levels of lactate dehydrogenase (LDH), serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT). RESULTS: A significant relationship was found between plasma prolidase activity (635 ± 83 U/L) (p = 0.007), umbilical cord plasma prolidase activity (610 ± 90 U/L) (p = 0.013), amniotic fluid prolidase activity (558 ± 100 U/L) (p = 0.001), umbilical cord tissue prolidase activity (4248 ± 1675 U/gr protein) (p = 0.013) and placental tissue prolidase activity (2116 ± 601 U/gr protein) (p = 0.001) in preeclamptic group when compared to healthy pregnant women. CONCLUSION: There is a strong correlation between prolidase enzyme activity and preeclampsia. Prolidase enzyme activity may play a role in preeclampsia.

Fingerprinting of gelatinase subtypes for different topographic regions on non-retaining placenta of Holstein cows.

The contribution of matrix metalloproteinases (MMP) to timely discharge of the placenta from bovine uterus at parturition is yet inconclusive, partly because of the presence of multiple MMP forms in situ. In the current study, the expression of different gelatinase subtypes on non-retaining placentas of Holstein cows was fingerprinted by using gelatin zymography. Different topographic regions on the placenta were measured separately, including the placentome-like structure and the fetal and maternal sides of interplacentomal placenta, all sampled from the central and peripheral areas of the placenta, respectively. The spontaneously ruptured umbilical cords were cross-sectioned as fetus end, middle and placenta end also for separate measurement. Body fluids including blood samples from the parturient cows, their neonatal calves and umbilical cord, as well as fetal fluids and the first colostrum were measured concomitantly. Results showed multiple forms of gelatinases subtypes in the placenta tissues and body fluids, including neutrophil gelatinase-associated lipocalin (NGAL)-MMP-9 complex, both the latent and active forms of MMP-2 and MMP-9; of them, the latent forms were much more abundantly and frequently expressed than the active forms. NGAL-MMP-9 complex was more prevalently present in the body fluids than in the placenta tissues. No distinguishable pattern of the expression of any gelatinase subtype was observed among the placentome-like structure, interplacentomal placenta and umbilical cord, or between fetal and maternal sides. Nonetheless, for interplacentomal placenta, proMMP-9 expression was higher in the central than in the peripheral area. In addition, proMMP-2 expression was higher in the rupture end (fetus end) than the placenta end of the umbilical cord. In conclusion, the current validated gelatin zymography detected a gradient proMMP-9 expression on the non-retaining placenta of cows in reverse to the proximity to the umbilical insertion point, and a gradient proMMP-2 expression on a section of the umbilical cord in reverse to the proximity to the rupture site, suggesting roles played by gelatinases in normal discharge of the placenta at term.

Osteogenesis of umbilical mesenchymal stem cells is enhanced in absence of DNA methyltransferase 3B (DNMT3B) through upregulating Runx2 expression.

OBJECTIVE: Umbilical mesenchymal stem cells (UMSCs) is one of most popular regenerative medical source of bone replacement therapy in both clinical and scientific researches. However, it is still low effective to induce the osteogenesis of hUMSCs. In this study, we aimed to elucidate the roles of DNA methyltransferase 3B (DNMT3B) in the osteogenesis of hUMSCs. MATERIALS AND METHODS: Knockdown DNMT3B in hUMSCs was gained via RNA interference technology. After confirming the decrease of DNMT3B in mutant hUMSCs by immunostaining and qPCR, osteogenesis differentiation was carried out. To identify the phenotype of osteogenesis in both bone formation ability and function of bone, immunostaining, qPCR and functional test was performed, compared to wildtype hUMSCs. RESULTS: Real-time Quantitative PCR (qPCR) and immunostaining results indicated that lacking of DNTM3B the osteogenesis related genes were significantly downregulated. Meanwhile, the functional test was also consistent with the downregulated differentiation result. CONCLUSIONS: The osteogenesis differentiation of hUMSCs is impaired in the absence of DNMT3B.

Dramatic differences in activity of purines metabolizing ecto-enzymes between mesenchymal stem cells isolated from human umbilical cord blood and umbilical cord tissue.

The high quality human mesenchymal stem cells (MSCs) with remarkable expansion potential in culture are demonstrated to possess multifold clinical applications. However, their isolation and characterization are difficult and sometimes ambiguous. We exploited nucleotide metabolizing ecto-enzymes for more complete characterization of MSCs. Using standard methods of cell culturing and analyses, we detected significant differences in the activity of ecto-nucleotidases on the surface of MSCs isolated from umbilical cord tissue and MSC-like cells derived from umbilical cord blood. Interestingly, the proliferation rate and the immunophenotypic characteristics of mesenchymal stem cells also correspond to the activities of these enzymes. Compared with the CD90-, CD105-, and CD73-deficient and slowly proliferating UCB-MSC-like cells that had relatively higher ecto-NTPDases activity, the CD90-, CD105-, and CD73-positive and rapidly proliferating UC-MSCs rather had ecto-5'-nucleotidase activity and presented neither ecto-nucleotidases nor adenylate kinase activities. In summary, our results demonstrate for the first time that activity of purine nucleotide metabolizing ecto-enzymes differs significantly between mesenchymal stem cells drawn from different neonatal sources, corresponding with a distinct proliferative potential.

Human umbilical cord blood mononuclear cell-conditioned media inhibits hypoxic-induced apoptosis in human coronary artery endothelial cells and cardiac myocytes by activation of the survival protein Akt.

We have previously demonstrated in acute myocardial infarctions that human umbilical cord blood mononuclear cells (HUCBCs), which contain hematopoietic, endothelial, and mesenchymal stem cells, reduce acute myocardial infarction size by ≥50% and preserve LV contractility. We hypothesize that the beneficial effects of HUCBCs are due to secretion of biologically active factors that activate in cardiac endothelial cells and myocytes the cell survival protein Akt. We determined by protein microarrays the growth factors and anti-inflammatory cytokines secreted by HUCBCs into culture media during 12 h of hypoxia (1% O2). We then determined by Western blots the effects of cell-free media from hypoxic-conditioned HUCBCs (HUCM) on activation of the cell survival protein Akt in human coronary artery endothelial cells and cardiac myocytes in culture during 24 h of 1% O2. We also determined in separate experiments endothelial cell and myocyte apoptosis by caspase-3 and Annexin V. In the present experiments, HUCBCs secreted multiple growth factors, anti-inflammatory cytokines, and inhibitors of metalloproteinase during normoxia and hypoxia. Human cord blood cells increased the concentration in culture media of angiopoietin, hepatocyte growth factor, interleukin-4, insulin-like growth factor, placental growth factor, vascular endothelial cell growth factor, angiogenin, and stem cell factor by 100 to >10,000% during 12 h of 1% O2 (p<0.001). HUCM, which contained these biological factors, significantly increased Akt phosphorylation/activation in coronary artery endothelial cells and cardiac myocytes subjected to 24 h of 1% O2 by more than 60% (p<0.05) and increased the antiapoptotic protein Bcl-2 expression by 34-50% in comparison with endothelial cells and myocytes treated without HUCM in 1% O2(p<0.05). HUCM also significantly decreased caspase-3 activity and decreased hypoxic endothelial cell and cardiac myocyte apoptosis by more than 40% in comparison with cells cultured without HUCM (p<0.05). Inhibition of Akt activation in endothelial cells and myocytes by the sensitive and specific antagonist API-1 during 24 h of hypoxia nearly completely prevented the beneficial effects of HUCM on inhibiting caspase-3 activity and apoptosis. We conclude that HUCBCs secrete biologically active factors during hypoxia that activate survival proteins in endothelial cells and myocytes that significantly limit apoptosis.

SIRT1 prevents replicative senescence of normal human umbilical cord fibroblast through potentiating the transcription of human telomerase reverse transcriptase gene.

SIRT1, the mammalian homolog of sirtuins, has emerged as a mediator of the beneficial effects of calorie restriction. Among them, we focused on the SIRT1-induced prevention of cellular senescence, and tried to reveal the molecular mechanisms that define the effects of SIRT1. Firstly in this study, we observed that overexpression of SIRT1 resulted in the prevention of cellular senescence of normal human umbilical cord fibroblast HUC-F2 cells. Here, we focused on the human telomerase reverse transcriptase (hTERT) gene as a target of the SIRT1-induced prevention of cellular senescence. Results showed that SIRT1, SIRT1 activator, resveratrol, and SIRT1 activating condition, starved condition, increased the transcription of hTERT in HUC-F2 cells. Next, we found that SIRT1 increased hTERT transcription in a c-MYC-dependent manner, triggered the transcription of the c-MYC gene and increased the amount of c-MYC recruited to the hTERT promoter. Further, SIRT1 increased the transcriptional activation ability of c-MYC and correspondingly increased the amount of acetylated H4 histone at the hTERT promoter. All of these results indicated that SIRT1 activates hTERT transcription through the involvement of c-MYC, and suggested that this SIRT1-induced augmentation of hTERT transcription resulted in the extension of the cellular life span of HUC-F2 cells.

Extracellular matrix remodeling of the umbilical cord in pre-eclampsia as a risk factor for fetal hypertension.

The human umbilical cord forms a connection between the placenta and the foetus. It is composed of two arteries and one vein surrounded by Wharton's jelly. Pre-eclampsia is accompanied by extensive remodeling of extracellular matrix of umbilical cord. Matrix metalloproteinases (MMPs) are engaged in degradation of extracellular matrix proteins and activation/inactivation of certain cytokines and enzymes. These enzymes will probably play a central role in the release of matrix-embedded cytokines and growth factors. MMP-2 (gelatinase A) is the main collagenolytic enzyme of both umbilical artery and vein. Other metalloproteinases are present in several times lower amounts. Reduced activity of collagen-degrading enzymes may be a factor, which enhances the accumulation of collagen and some other proteins in the pre-eclamptic umbilical cord tissues. It seems to be possible that similar alterations occur in other fetal blood vessels. It may result in an increase in peripheral resistance as well as an increase in the blood pressure in the fetal vascular system. Some observations suggest that the raised pressure may persist after birth. Pre-eclampsia may be a factor that evokes an initiation of hypertension in utero and its amplification through childhood and adulthood.

Immunohistochemical and transcriptional expression of matrix metalloproteinases in full-term human umbilical cord and human umbilical vein endothelial cells.

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodelling of extracellular matrix in physiological and pathological processes. MMPs also have a role on cell proliferation, migration, differentiation, angiogenesis and apoptosis. Umbilical cord is a special organ subjected to many changes during pre-natal life and whose cells can maintain a certain degree of plasticity also in post-natal period; for example recently they have been used as a source of stem cells. In this work we investigated the expression of MMPs in human umbilical cord and human umbilical vein endothelial cells (HUVEC) though immunohistochemistry, RT-PCR and gelatin zymography. MMP-2 protein is expressed in the amniotic epithelium of human umbilical cord and in few sub-epithelial fibroblasts, while MMP-3 and MMP-10 only in the umbilical epithelium. MMP-8, MMP-9 and MMP-13 immunoreactivity is localised in the epithelium and in Wharton's jelly mesenchymal cells. Immunocytochemistry also revealed protein expression for MMP-2, 3, 8, 9 and 10 in cultured HUVEC. In agreement with immunohistochemical data, RT-PCR analysis performed on samples of whole umbilical cord confirmed the transcriptional expression for the genes encoding all the six matrix metalloproteinases investigated, while in HUVEC only the expression of MMP-2, 3, 9, 10 and 13 mRNAs was detected. Gelatin zymography showed a clear MMP-2 and MMP-9 enzymatic activity in the conditioned medium of HUVEC at different culture passages, suggesting that HUVEC secrete gelatinases, that afterwards undergo extracellular activation, and this ability is not affected by passage number.

Cyclin-dependent kinase 5 regulates endothelial cell migration and angiogenesis.

Angiogenesis contributes to various pathological conditions. Due to the resistance against existing antiangiogenic therapy, an urgent need exists to understand the molecular basis of vessel growth and to identify new targets for antiangiogenic therapy. Here we show that cyclin-dependent kinase 5 (Cdk5), an important modulator of neuronal processes, regulates endothelial cell migration and angiogenesis, suggesting Cdk5 as a novel target for antiangiogenic therapy. Inhibition or knockdown of Cdk5 reduces endothelial cell motility and blocks angiogenesis in vitro and in vivo. We elucidate a specific signaling of Cdk5 in the endothelium; in contrast to neuronal cells, the motile defects upon inhibition of Cdk5 are not caused by an impaired function of focal adhesions or microtubules but by the reduced formation of lamellipodia. Inhibition or down-regulation of Cdk5 decreases the activity of the small GTPase Rac1 and results in a disorganized actin cytoskeleton. Constitutive active Rac1 compensates for the inhibiting effects of Cdk5 knockdown on migration, suggesting that Cdk5 exerts its effects in endothelial cell migration via Rac1. Our work elucidates Cdk5 as a pivotal new regulator of endothelial cell migration and angiogenesis. It suggests Cdk5 as a novel, pharmacologically accessible target for antiangiogenic therapy and provides the basis for a new therapeutic application of Cdk5 inhibitors as antiangiogenic agents.

Ouabain-induced apoptosis and Rho kinase: a novel caspase-2 cleavage site and fragment of Rock-2.

Ouabain, a specific Na+/K+-ATPase inhibitor, has recently been identified as a mammalian hormone. Its elevated concentrations in human plasma have also been associated with pathogenesis of several diseases. Recent studies have shown that ouabain induces aponecrotic cell death in a cell-type- and dose-dependent manner. However, the exact mechanism of ouabain-induced cell death is not fully understood. The Rho GTPase effectors Rho kinases-1 and -2 (Rock-1 and Rock-2) which play central roles in the organization of the actin cytoskeleton, involve in several models of apoptosis. In this study, we investigated the possible involvement of Rocks in ouabain-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Ouabain treatment resulted in loss of cell-cell and cell-substratum adhesion and apoptotic blebbing. Pretreatment of cells with Y-27632, a specific Rock inhibitor, resulted in the inhibition of cell-to-cell detachment and formation of membrane blebs. However, Y-27632 did not prevent ouabain-induced cell-substratum detachment. Instead, treatment with Y-27632 actually accelerated this process. Ouabain treatment induced cleavage of Rock-1 and Rock-2, which was prevented by caspase-3 and caspase-2 specific inhibitors z-DEVD-fmk and z-VDVAD-fmk, respectively. Ouabain-induced Rock-2 cleavage generated a fragment of approximately 140 kDa corresponding to the consensus sequence of caspase-2 on the carboxy terminus of Rock-2. Although it has been previously shown that Rock-2 was cleaved by caspase-2, we have identified here a novel cleavage site and fragment of Rock-2. Our data indicate that ouabain induces both Rock-1 and Rock-2 cleavage via caspase-dependent mechanisms and provide evidence that Rocks are involved in ouabain-induced cell-to-cell detachment and apoptosis.

Expression patterns of endothelial and inducible nitric oxide isoforms in the porcine umbilical cord.

Stable fetal-placental blood pressure and flow are extremely important in fetal growth and development. Uncontrolled and long-standing increased or decreased vascular blood pressure in the umbilical cord (UC) affects hyperaemia or ischaemia and consequently causes fetal death. Nitric oxide (NO) is one of the most active factors controlling blood flow through relaxation of the vascular smooth muscle. In this study, we investigated endothelial (eNOS) and inducible (iNOS) nitric oxide synthase expression and NADPH-diaphorase activity (NADPH-d) in the porcine UC at various stages of pregnancy. The UCs were collected from pigs on days 40, 60, 75 and 90 of pregnancy and postpartum. Western blot analysis as well as immunohistochemical staining revealed protein presence for eNOS and iNOS in the UC of the pig. The eNOS expression was maintained at a significantly higher level in all analysed days of pregnancy when compared with postnatal stage. Additionally, a significant protein increase for eNOS was observed in a periplacental part of UC on day 90. There were no obvious differences in iNOS protein level in UC samples derived from different stages of pregnancy. NADPH-diaphorase histochemical activity was correlated with NOS immunoreactivity during all analysed days of pregnancy. These results suggest that NOS isoforms are responsible for regulation of blood circulation in UC and immune responses.

Endothelial Src kinase regulates membrane recycling from the lateral border recycling compartment during leukocyte transendothelial migration.

When leukocytes cross endothelial cells during the inflammatory response, membrane from the recently described lateral border recycling compartment (LBRC) is selectively targeted around diapedesing leukocytes. This "targeted recycling" is critical for leukocyte transendothelial migration. Blocking homophilic PECAM interactions between leukocytes and endothelial cells blocks targeted recycling from the LBRC and blocks diapedesis. However, the cellular signaling pathways that trigger targeted recycling are not known. We show that targeted recycling from the LBRC is dependent on Src kinase. The selective Src kinase inhibitor PP2 blocked targeted recycling and blocked diapedesis by over 70%. However, Src kinase inhibition did not affect the structure or normal constitutive recycling of membrane from the LBRC in the absence of leukocytes. PECAM, a Src kinase substrate, traffics between the LBRC and the endothelial surface at the cell border. However, virtually all of the PECAM in the cell that was phosphorylated on tyrosine residues was found in the LBRC. These findings demonstrate that Src kinase activity is critical for the targeted recycling of membrane from the LBRC to the site of transendothelial migration and that the PECAM in the LBRC is qualitatively different from the PECAM on the surface of endothelial cells.

Angiotensin II induces endothelial cell senescence via the activation of mitogen-activated protein kinases.

Vascular endothelial cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related cardiovascular disorders, for example, atherosclerosis. Angiotensin II (Ang II), a principal effector of the renin-angiotensin system (RAS), an important signaling molecule involved in atherogenic stimuli, is known to promote aging and cellular senescence. In the present study, induction of Ang II promoted a growth arrest with phenotypic characteristics of cell senescence, such as enlarged cell shapes, increased senescence-associated beta-galactosidase (SA-beta-gal) positive staining cells, and depressed cell proliferation. Ang II drastically decreased the expression level of Bcl-2, in part via the activation of extracellular signal-regulated kinase (ERK). Our results suggest that Ang II can induce HUVEC senescence; one of its molecular mechanisms is a probability that the mitogen-activated protein kinase (MAPK) signal pathway is involved in the process of pathological and physiological senescence of endothelial cells as well as vascular aging.

Glycogen synthase kinase-3beta inhibition preserves hematopoietic stem cell activity and inhibits leukemic cell growth.

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use.

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.

Transient down-regulation of beta1 integrin subtypes on kidney carcinoma cells is induced by mechanical contact with endothelial cell membranes.

Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.

Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys.

Cells in the umbilical cord stroma have gained attention in recent years; however, differentiation to certain lineages in humans has been demonstrated in few studies. Unlike bone marrow MSCs, human umbilical cord stroma cells (HUCSCs) are far from being well characterized. This study attempts to describe proliferation, structural, and differentiation properties of these cells to account for their exceptional nature in many aspects. Cellular dynamics, cellular structure, and the degree of transformations during expansion and differentiation into mesenchymal and neuronal lineages were examined in vitro over a 10-month period. Comparisons with human bone marrow MSCs regarding differentiation were performed. HUCSCs in culture revealed two distinct cell populations, type 1 and type 2 cells, that possessed differential vimentin and cytokeratin filaments. Corresponding cells were encountered in cord sections displaying region-specific localization. alpha-Smooth muscle actin and desmin filaments, which were evident in cord sections, diminished through passages. No difference was noted regarding type 1 and type 2 cells in differentiation to chondrogenic, adipogenic, and osteogenic lineages, whereas a preferential differentiation was noted in neuronal lineage. Relative success was achieved by production of chondrocytic spheres and osteogenic monolayers, whereas adipocytes were immature compared with bone marrow MSCs. The presence of neuronal markers suggests that they transform into a certain state of maturity under neurogenic induction. Conclusively, HUCSCs retain their original phenotype in culture without spontaneous differentiation, have a limited lifespan, and bear multipotent stem cell characteristics. Given these characteristics, they may be generally considered progenitor cells if manipulated under appropriate conditions and deserve further study to be potentially used in cell-based therapies.