Umbilical Cord Mononuclear Cells Research Articles

Cytokine Profiling of Human Umbilical Cord Plasma and Human Umbilical Cord Blood Mononuclear Cells

Introduction. Umbilical cord plasma (UCB-PL) was shown to be beneficial for treatment of ischemic brain injury, Gerotarget/Aging and Alzheimer's disease (Yoo et al., 2016; Castellano et al., 2017). Also, umbilical cord mononuclear cell (UCB-MC) demonstrated therapeutic effect in animal models of stroke, encephalopathy, amyotrophic lateral sclerosis and cerebral palsy (Islamov et al., 2015; Mukhamedyarov et al., 2013). Therapeutic effect of freshly isolated cells is believed t be due to transcriptional activation of numerous neurotrophic, antiapoptotic, proangiogenic pro- and anti-inflammatory factors. The main purpose of this investigation was to analyze the cytokine profile of hUCB-PL and hUCB-MC.Materials and Methods. Umbilical cord blood was collected into CPDA-1 blood bag and used for plasma (hUCB-PL) separation. Total proteins were normalized in hUCB-PL samples. UCB-MCs were isolated by density gradient sedimentation using Ficoll (1.077g/ml). Separated mononuclear cells were washed and cultured in RPMI-1640 medium (10% FBS, L-glutamine and penicillin-streptomycin antibiotic) at 37°C and 5% CO2. After incubation (72 h) hUCB-MCs conditioned media (CM) was harvested, centrifuged and stored at −80°C. hUCB-PL and hUCB-MC CM were used to analyze cytokines using Bio-Plex Pro™ Human Cytokine 27-plex and Human Chemokine 40-Plex panels. Data collected was analyzed using Luminex 100 or 200 analyzer with Bio-Plex Manager 5.0 software (Bio-Rad).Results. Many cytokines were detected in hUCB-PL: IL-1β, IL-6, IL-1ra, IL-4, IL-10, IL-13, IL-2, IL-7, IL-12, IL-17, IFN-g, TNF-a, IL-5, IL-9, IL-15, G-CSF, GM-CSF, PDGF-bb, FGF-2, VEGF, IL-8, Eotaxin, MCP-1, IP-10, MCP-1, MIP-1a, MIP-1b, RANTES, 6Cine, BCA-1, CTACK, ENA-78, Eotaxin2, Eotaxin-3, Fractalkine, GCP, Gro-a, Gro-b, I-309, I-TAC, MCP-2, MCP-3, MCP-4, MDC, MIF, MIG, MIP-3a, MIP3-b, MPIF-1, SCYB16, SDF-1ab, TARC, TERC. It should be mentioned that cytokine concentrations varied between individuals. When cytokines were analyzed in hUCB-MC CM samples, increased level of CTACK as compared to that in hUCB-PL. Also, cytokines IL-4, IL-5, IL-7, IL-9, IL-13, IL-15 were absent in hUCB-MC CM samples(Fig.1).Conclusions. Our data demonstrated numerous cytokines in hUCB-PL and hUCB-MC. Also, we observed individual variability in the level of those cytokines. Therefore, we suggest that the therapeutic effects of hUCB-PL transfusion and hUCB-MC transplantation could be associated with cytokine actions as well as factors which were not included in this analysis. We plan to conduct proteomic analysis of UCB-PL and hUCB-MC to identify molecules producing therapeutic effect. We also suggest that target secretome modulation could be achieved by modification of cells using different genetic constructs containing genes of growth factors and therapeutic molecules. Describes approach is expected to increase therapeutic potential of transplanted cells and annihilate disadvantages of human umbilical cord blood application.This work was supported by RFBR grant № 16-04-01567 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human umbilical cord mononuclear cells could ameliorate schistosomal hepatic fibrosis. An experimental study

Hepatic fibrosis is considered a worldwide public health problem. Liver transplantation is the treatment of choice. Rejection, post-operative complications and unavailability of donors are major obstacles. Recent treatment modalities include cell therapy but more researches are still needed. Human umbilical cord blood is a rich source for mononuclear cells which recently have shown promising anti-fibrotic results. The present study aimed to evaluate the potential effect of human umbilical cord blood mononuclear cells infusion on an experimental model of hepatic fibrosis caused by Schistosoma mansoni. Assessment of liver fibrosis was done by haematoxylin, eosin and Masson's trichrome staining. Homing to injured liver was assessed by staining of human specific nuclear antigen. Liver functions, collagen and hepatic stellate cells staining were done. Our results revealed marked improvement of liver fibrosis raising the possibility for the mononuclear cells clinical use in patients with hepatic fibrosis.

EFFECTS OF DIRECT AND INDIRECT DELIVERY OF THE VEGF GENE BY CORD BLOOD CELLS IN LIMB ISCHEMIA AREA IN RATS

The effects of delivery of recombinant human VEGF165 gene under conditions of direct and cell-mediated gene therapy were studied in the model of posterior limb ischemia. Immediately after the creation of the ischemia model, a solution with the VEGF gene (Ad-VEGF group) or the mononuclear blood cells of the human umbilical cord (PCC) mononuclear cells (group CCP + Ad-VEGF) transduced by the same gene was injected into the distal part of the gastrocnemius muscle. At day 14, an increase in the number of CD31- and CD34-immunopositive cells in the ischemic gastrocnemius muscle was shown in both groups of animals. The number of CD31- and CD34-immunopositive cells in the Ad-VEGF group increased by 23% and by 2 times, respectively, and by 4 and 5 times, respectively, in the CKP + Ad-VEGF group. At this time, the number of CD31-immunopositive cells in the CKP + Ad-VEGF group was greater by a factor of 3, and the CD34-immunopositive cells by a factor of 2 compared with the Ad-VEGF group. A significantly larger increase in the number of endothelial cells, when delivered to the ischemia region of the VEGF gene by PPC compared to direct injection of the gene into the same region, indicates a significantly higher efficiency of angiogenesis under conditions of cell-mediated gene therapy.

Anti-fibrotic potential of human umbilical cord mononuclear cells and mouse bone marrow cells in CCl4- induced liver fibrosis in mice

Liver fibrosis is the consequence of hepatocyte injury that leads to the activation of hepatic stellate cells (HSC). The treatment of choice is Liver transplantation; however, it has many problems such as surgery-related complications, immunological rejection and high costs associated with the procedure. Stem cell-based therapy would be a potential alternative, so the aim of this study is to investigate the therapeutic potential of human umbilical cord mononuclear cells (MNC) and mouse bone marrow cells (BMC) against carbon tetrachloride (CCl4) induced liver fibrosis in mice and compare it with that of silymarin. In the present study, male albino mice (N=60) were divided into six groups (10 mice each), the first group served as the normal control group while the remaining five groups were rendered fibrotic by intraperitoneal injections of CCl4 and being left for 6 weeks to develop hepatic fibrosis. Thereafter, the mice were divided into CCl4 group, CCl4 group receiving MNC or BMC or silymarin or MNC and silymarin combination. After the specified treatment period, animals were then euthanized, blood and tissue samples were collected for measurement of alanine aminotransferase(ALT), aspartate aminotransferase(AST), malondialdehyde(MDA), reduced glutathione(GSH), collagen, Laminin, transforming growth factor β1(TGFβ1), tumor necrosis factor alpha(TNFα). MNC, BMC, and the combination therapy showed a significant decrease in ALT, AST, MDA, collagen, Laminin, TGFβ1, and TNFα and a significant increase in GSH. The data displayed a similar regression of fibrosis with the histological and immunohistological parameters. In conclusion, MNC, BMC and the combination therapy showed a potential therapeutic effect against liver fibrosis via reducing oxidative stress, inflammatory mediators, and fibrogenic markers.

A Cost-Effective Method to Assemble Biomimetic 3D Cell Culture Platforms.

Developing effective stem cell based therapies requires the design of complex in vitro culture systems for more accurate representation of the stem cell niche. Attempts to improve conventional cell culture platforms include the use of biomaterial coated culture plates, sphere culture, microfluidic systems and bioreactors. Most of these platforms are not cost-effective, require industrial technical expertise to fabricate, and remain too simplistic compared to the physiological cell niche. The human amniotic membrane (hAM) has been used successfully in clinical grafting applications due to its unique biological composition and regenerative properties. In this study, we present a combinatorial platform that integrates the hAM with biomolecular, topographic and mechanical cues in one versatile model.MethodsWe utilized the hAM to provide the biological and the three dimensional (3D) topographic components of the prototype. The 3D nano-roughness of the hAM was characterized using surface electron microscopy and surface image analysis (ImageJ and SurfaceJ). We developed additional macro-scale and micro-scale versions of the platform which provided additional shear stress factors to simulate the fluid dynamics of the in vivo extracellular fluids.ResultsThree models of varying complexities of the prototype were assembled. A well-defined 3D surface modulation of the hAM in comparable to commercial 3D biomaterial culture substrates was achieved without complex fabrication and with significantly lower cost. Performance of the prototype was demonstrated through culture of primary human umbilical cord mononuclear blood cells (MNCs), human bone marrow mesenchymal stem cell line (hBMSC), and human breast cancer tissue.ConclusionThis study presents methods of assembling an integrated, flexible and low cost biomimetic cell culture platform for diverse cell culture applications.

Cell therapy for cerebral hemorrhage: Five year follow-up report.

The aim of the study was to examine treatment of cerebral hemorrhages with bone-marrow or human umbilical cord-derived mesenchymal stem cells (BMSCs or Hu-MSCs) and conventional surgical approaches, and determine and compare the effectiveness, feasibility, safety and reproducibility of each method. A retrospective analysis was performed on a cohort of cell-treated cerebral hemorrhage patients from October 1, 2007 to October 1, 2009. A total of 24 patients, all of whom received conventional surgical treatment, were classified as follows: i) The control group consisted of 8 patients who received only hematoma removal surgery, ii) the autologous group consisted of 7 patients who received additional autologous bone marrow mononuclear cell transplantation, and iii) the allograft group consisted of 9 patients who received additional umbilical cord mononuclear cell transplantation. After conventional hematoma removal surgery and X-ray supervision within 24 h and at 7 days, neurological disability and function tests were completed 3, 6, 12, 36 and 60 months later. The T-cell marker plasma levels were analyzed after 60 months. The results showed that, at approximately 3.5 months after graft the hematomas in all the groups were completely reabsorbed as observed on computed tomography scans. However, the functional outcomes in the cell-transplanted groups were better than in the control group after 5 years. While the National Institutes of Health Stroke Scale, modified Rankin score and modified Barthel index scores were simliar in the cell-transplanted groups, patients in the allograft group had better outcomes than those in the autologous graft group starting at 3 months and until the end of the follow-up period. The serum levels of T-cell markers CD4, CD56 and human leukocyte antigen-DR in the allograft group showed no signs of immunogenic graft complications and there were no significant differences in T-cell subtypes among the patient groups. The results of the present study suggest that, treatment of cerebral hemorrhage patients can be safely and effectively accomplished using Hu-MSC grafting and larger clinical trials should be considered in the future.

Maternal Obesity Affects Inflammatory and Iron Indices in Umbilical Cord Blood

To determine the impact of maternal obesity and gestational weight gain across pregnancy on fetal indices of inflammation and iron status. Eighty-five healthy term newborns delivered via elective cesarean were categorized by 2 maternal body mass index (BMI) thresholds; above or below 30kg/m(2) or above or below 35kg/m(2). Umbilical cord plasma levels of C-reactive protein, interleukin (IL)-6, tumor necrosis factor (TNF)-α, ferritin, and hepcidin were assayed. Cytokines released by phytohemagglutinin-stimulated umbilical cord mononuclear cells (MNCs) were assayed. Maternal class II obesity, defined as BMI of 35kg/m(2) and above, predicted higher C-reactive protein and TNF-α in umbilical cord plasma (P<.05 for both), and also proinflammatory cytokines (IL-1β, IL-6, and TNF-α) from stimulated MNC (P<.05 for all). The rise in plasma TNF-α and MNC TNF-α was not linear but occurred when the threshold of BMI 35kg/m(2) was reached (P<.005, P<.06). Poorer umbilical cord iron indices were associated with maternal obesity. When ferritin was low, IL-6 was higher (P<.04), but this relationship was present primarily when maternal BMI exceeded 35kg/m(2) (P<.03). Ferritin was correlated with hepcidin (P<.0001), but hepcidin was unrelated to either maternal BMI or inflammatory indices. Class II obesity and above during pregnancy is associated with fetal inflammation in a threshold fashion. Although maternal BMI negatively impacted fetal iron status, hepcidin, related to obesity in adults, was related to iron status and not obesity in fetuses. Pediatricians should be aware of these relationships.

Repeated administrations of human umbilical cord blood cells improve disease outcomes in a mouse model of Sanfilippo syndrome type III B.

Sanfilippo syndrome type III B (MPS III B) is an inherited disorder characterized by a deficiency of α-N-acetylglucosaminidase (Naglu) enzyme leading to accumulation of heparan sulfate in lysosomes and severe neurological deficits. We have previously shown that a single administration of human umbilical cord mononuclear cells (hUCB MNCs) into Naglu knockout mice decreased behavioral abnormalities and tissue pathology. In this study, we tested whether repeated doses of hUCB MNCs would be more beneficial than a single dose of cells. Naglu mice at 3 months of age were randomly assigned to either a Media-only group or one of three hUCB MNC treatment groups--single low dose (3 × 10(6) cells), single high dose (1.8 × 10(7) cells), or multiple doses (3 × 10(6) cells monthly for 6 months) delivered intravenously; cyclosporine was injected intraperitoneally to immune suppress the mice for the duration of the study. An additional control group of wild-type mice was also used. We measured anxiety in an open field test and cognition in an active avoidance test prior to treatment and then at monthly intervals for 6 months. hUCB MNCs restored normal anxiety-like behavior in these mice (p < 0.001). The repeated cell administrations also restored hippocampal cytoarchitecture, protected the dendritic tree, decreased GM3 ganglioside accumulation, and decreased microglial activation, particularly in the hippocampus and cortex. These data suggest that the neuroprotective effect of hUCB MNCs can be enhanced by repeated cell administrations.

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells

Context: Mangiferin (2-C-β-d-gluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) is a well-known natural antioxidant distributed in various plants of the Anacardiaceae and Gentianaceae families. Mangiferin can inhibit carcinogen-induced lung or colon tumor formation in experimental animals. However, the molecular mechanisms of its chemopreventive activity remain unexplored.Objective: This study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-ARE signaling in hematopoietic cells.Materials and methods: Mononuclear cells (MNCs) were isolated from human umbilical cord blood (hUCB). DNA damage was evaluated by comet and micronucleus assays. The expression of Nrf2 and NQO1 was examined by immunofluorescence and western blotting. An electrophoretic mobility shift assay (EMSA) was used to detect the binding activity of Nrf2 with NQO1-ARE sequences.Results: We found that mangiferin treatment significantly reduced DNA damage in etoposide-treated MNCs, which was verified by decreased olive tail moment (OTM) and micronucleus (MN) frequency. Mangiferin treatment significantly promoted Nrf2 translocation into the nucleus and increased nuclear Nrf2 expression. Moreover, NQO1, an Nrf2 signaling target, was significantly upregulated by mangiferin treatment, and the binding activity of Nrf2 with NQO1-ARE sequences was elevated after mangiferin treatment.Discussion and conclusion: Mangiferin activated Nrf2 signaling, upregulated NQO1 expression, and significantly reduced etoposide-induced DNA damage. Thus, mangiferin is a potential cytoprotective agent for hematopoietic cells.

A comparison of umbilical cord blood-derived endothelial progenitor and mononuclear cell transplantation for the treatment of acute hindlimb ischemia

Acute lower limb ischemia is a common peripheral artery disease whose treatment presents many difficulties. Stem cell transplantation is considered a novel and promising method of treating this disease. Umbili- cal cord blood (UCB) is rich in stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs). However, historically, banked umbilical cord blood has been used mainly to treat blood-related diseases. Therefore, this study compared the efficacy of umbilical cord blood- derived mononuclear cells (UCB-MNCs) with EPC transplantation for the treatment of acute hindlimb ischemia (ALI) in mouse models. MNCs were isolated from UCB by Ficoll gradient centrifugation, after which the EPCs were sorted based on CD34+ and CD133+ markers and cultured according to a previously published protocol. To induce ALI, mice were immuno-suppressed using busulfan (BU) and cyclophosphamide (CY), after which the femoral arteries were burned. Induction of ALI in the immune suppressed mice was confirmed by the grade of tissue damage, pedal frequency in water, tissue edema, changes in histology, total white blood cell count, and white blood cell composition. Model mice were injected with a dose of MNCs or EPCs and un-treated control mice were injected with phosphate buffered saline. The efficiency of treatment was evaluated by comparing the grade of tissue damage between the three groups of mice. Mice aged 6-12 months were suitable for ALI, with 100% of mice exhibiting ischemia from grade I 10%, grade III 50%, grade IV 40%. For all ALI mice, a gradual in- crease in pedal frequency in water, increased tissue edema, necrosis of muscle tissue, and loss of hindlimb func- tion were observed after 20 days. Transplanted MNCs and EPCs significantly improved hindlimb ischemia com- pared with control treatment. Moreover, EPC transplantation significantly improved hindlimb ischemia compared with MNC transplantation. Following EPC and MNC transplantation, 44.44% and 33.33% of the mice recovered fully (grade 0), respectively. Specifically, all recovered mice exhibited hindlimb activities similar to those of nor- mal mice. Transplantation of UCB-derived MNCs and EPCs are promising therapies for hindlimb ischemia.

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection

Umbilical cord blood has been used for a wide variety of immunologic investigations including assessments of developmental perturbations by antenatal exposures. Recent advances in multiparameter flow cytometry have allowed finer characterization of lymphocyte phenotype and function, revealing important differences between the fetal and adult immune systems. The degree of variability between human subjects confounds the ability to draw firm conclusions. Artifacts resulting from processing techniques exacerbate this variability. The unpredictable nature of deliveries, especially of premature infants, makes it difficult to control variables such as timing of umbilical cord mononuclear cell (UCMC) isolation and method of collection. Additionally, in multicenter studies dependent on central processing, delays are inevitable. However, little available literature describes systematic testing of the degree to which processing variations affect UCMC phenotype and function. Using multiparameter flow cytometry, we tested the effect of collection technique and length of time prior to UCMC isolation on T cell phenotype and function, with the goal of creating a standardized operating procedure for a multicenter investigation. The study also provides a benchmark data set including extensive surface and functional phenotyping of umbilical cord T cells. UCMC isolation delay of up to 24 h produced similar T cell phenotype and function as tested by in vitro SEB stimulation. There were few statistically significant differences between time points based on data medians. We conclude that, for the purpose of immunologic investigations, a 24-h time delay from sample collection to mononuclear cell isolation does not introduce a significant degree of variation in T cell phenotype and function when adhering to strict standard operating procedures.

Effect of delay from sample collection to umbilical cord mononuclear cell isolation on T cell phenotype and function. (174.20)

Abstract Background: The unpredictable nature of deliveries makes it difficult to control timing of umbilical cord mononuclear cell (UCMC) isolation. Our study tested the effect of time delay from umbilical cord collection to UCMC isolation on T cell phenotype and function. Methods: Cord blood was drawn from 15 healthy term deliveries using a standardized collection method. Each sample was divided and processed immediately, 24 hours and 48 hours after collection. Samples were analyzed using flow cytometry for phenotype and cytokine secretion, with and without in vitro stimulation. Comparisons were made to determine differences in variation and median cell frequencies over time. Results: Viability and recovery were &amp;gt;92% for all time-points. There was a decrease after 48 hours for CD56+ and CD3+ frequencies. CD4+ T cells showed differences after 24 hours in 4 of 23 subpopulations, and 8 subpopulations after 48 hours. No differences were seen with cytokine positive frequencies for IFNg, TNFa, IL17, or IL4/5/13 after 24 and 48 hours, but there was a decrease in IL2 and increase in MIP1β frequencies for CD4+ T cells. IL2 alone was increased for CD8+ T cells after 24 and 48 hours. Variation analysis showed high assay reproducibility, and subject-subject variation exceeded time delay variation for most populations. Conclusion: A 24-hour delay prior to UCMC isolation does not significantly change phenotype or function of most cord T cell subsets. A 48-hour delay may affect study results.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations

AbstractMicrochimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele–specific and Y-chromosome–directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

HLA-targeted cell sorting of microchimeric cells opens the way to phenotypical and functional characterization

Microchimerism refers to the presence of less than 1% of non-host cells in a person. Our group developed a reliable method for separating viable microchimeric cells from the host environment. Optimal separation of microchimeric cells at proportions as low as 0.01% could be established with two monoclonal antibodies directed against different HLA antigens, one targeting the microchimeric cells and the other the host cells. Purity of separated cell populations was validated by HLA-allele-specific and Y-chromosome directed real-time qPCR assays. The methodology was used successfully to separate microchimeric maternal cells from child umbilical cord mononuclear cells after pregnancy. Cell sorting with HLA monoclonal antibodies targeting allelic differences enables reliable microchimeric cell detection and separation in blood specimens. With this approach, maximal enrichment of potentially viable microchimeric cells from a background cell population is reached, which opens the way to phenotypical and functional characterization of microchimeric cells.

Differentiating Human ES and Patient Derived IPS Cells to Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 2628The ability to generate hematopoietic stem cells (HSC's) from patient derived induced pluripotent stem (iPS) cells, would enable the generation of an unlimited supply of HLA matched transplantable cells for the treatment of both hematological disorders and malignancies. The goal of this project is to find the molecular determinants of human ES and iPS cell differentiation to transplantable HSC's. We have developed an advanced ES/iPS-to-blood differentiation protocol that mimics embryonic development towards the hematopoietic lineage. Current progress has resulted in up to 84% of human CD45+ hematopoietic cells (mean 41 ± 16%, n = 7) and with significant numbers of hematopoietic progenitor cells (CD45+CD34+, 29 ± 4%, n = 6) as reported by FACS analysis. Moreover, the numbers of hematopoietic progenitor cells generated (mean 20 ± 1 for a keratinocyte derived iPS (kiPS)), as measured by colony forming unit assays were comparable to numbers obtained from fresh whole blood umbilical cord mononuclear cell isolates (mean 35 ± 2) on a per 10.000 CD45+ input cells. We now plan to overexpress key factors predicted to support the generation and maturation of bona fide HSC's with repopulating ability. For this we have generated a tet-regulatable lentiviral based gene expression system to, out of a pool of over 30 genes, express up to 5 cDNA's of interest simultaneously. By clonal selection of lenti-transduced ES and iPS cells we have generated novel pluripotent lines that carry the tet-transactivator gene (m2rtTA), capable of efficient differentiation into blood. FACS analysis of these lines show blood generation of 30.7% and 11.8% of cells positive for CD45 for the human ES line Hues3 and the kiPS line respectively, with progenitor levels (CD45+CD34+) at 8.8% and 20.2%. These values are similar in efficiency to that of untransduced sister lines from which they were derived. These lines strongly express genes of interest following lenti-viral transduction and the addition of Doxycyclin (as reported by GFP). It is our goal to show successful reconstitution with in vitro generated HSC's, and ultimately pave the way for therapeutically relevant HSC's for treating hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Altered gene expression in umbilical cord mononuclear cells in preterm infants with periventricular leukomalacia

Aim Periventricular leukomalacia (PVL) is one of the most important causes of adverse outcome of preterm infants. We hypothesized that inflammatory or some other specific pathways will have been activated at birth in preterm infants who later develop PVL. The aim of this study is to examine the difference in mRNA expression in umbilical cord blood according to the presence or absence of PVL. Methods A total of 61 umbilical cord blood samples were collected from preterm infants with gestational age less than 33 weeks together with the patients' medical information during perinatal period. RNA expression patterns in the collected cord bloods were analyzed by microarray. On the basis of cranial ultrasonography and brain MRI examination, 3 infants (4.9%) were diagnosed as cystic PVL and selected as the subjects. Five patients who showed similar perinatal factors to those of infants with PVL but did not show PVL were selected as the normal control. Results Five of the 15 up-regulated genes are coding ribosomal proteins, and another encodes a translation elongation factor. Three of the 7 down-regulated genes encode proteins that may be related to immune response and/or inflammation. Conclusions Up-regulation of the ribosomal proteins may indicate an activation of lymphocytes during the fetal period.

Both Naïve and Memory T-Cell Subsets Participate in Alloresponses to HLA-Mismatched Targets – Implications for Adoptive Transfer of Viral Antigen-Specific T Cells.

Abstract 2439Poster Board II-416It is generally assumed that T cell responses to HLA disparate targets arise from the naïve pool. However, the risk of graft-versus-host disease (GvHD) also increases if the donor has circulating T cells recognizing multiple persistent DNA viruses, suggesting that memory T cells may contribute to the overall alloresponse. We used a flow cytometric CFSE-based proliferation & activation (CD38 expression acquisition) assay to systematically assess the contribution of naïve and memory T cells to alloreactivity. In some experiments donor peripheral blood mononuclear cells (PBMC) were primed for 8 days with allogeneic, HLA mismatched PBMC (alloPBMC), after which the intracellular cytokine production (ICS) was examined upon rechallenge with autologous or allogeneic PBMC (alloPBMC). CFSE-labeled umbilical cord mononuclear cells (UCMC) from five donors were stimulated with an irradiated pool of HLA-disparate donor peripheral blood mononuclear cells (alloPBMC). By day 8, over 80% of the CD4+ and CD8+ T cells were CFSE[dim], i.e. had proliferated, and expressed the activation marker CD38, indicating that naïve T cells can mount an alloresponse. Next, PBMC from adult donors were separated into naïve (CD57-CD45RO-), memory (CD57-CD45RA- and CD57-CD62L- T cells), and effector (CD27-CD45RO-) T cells. We found that adult donor naïve T cells can also mount an alloresponse; importantly, both memory T cell subsets – and to a lesser extent, effector cells – were CFSE[dim]CD38+ at least to the same extent as naïve T cells, indicating their potential to respond to alloantigens in addition to their priming foreign antigen. These data were confirmed using a different approach: By first priming responder T cells with HLA-disparate donor PBMC for 8 days, followed by the enumeration of alloreactivity by ICS after stimulation with the original stimulator PBMC. To exclude reactivity of responder T cells with viral antigens present in PBMC (EBV; CMV), the experiment was repeated using activated T cells (T-APC) as antigen presenting cells, and yielded identical results. The direct ex vivo alloreactivity of T cell subsets was next tested by stimulating donor PBMC with HLA mismatched donor PBMC or activated T cells as APC in an 18 hour ICS. This experiment confirmed the rapid kinetics of alloreactivity and dominance of memory T cells in the alloresponse. Since a prominent role of memory T cells was apparent from our experiments we next tested T cells specific for DNA virus-derived antigens. EBV- and CMV-specific T cells, tested against a panel of 30 T-APC with a broad coverage of the most prominent HLA, displayed exquisite specificity for certain mismatched HLA alleles, indicating that DNA virus antigen-specific T cells may indeed cross-react with cellular antigens presented in the context of mismatched HLA class I and II proteins. Collectively our data demonstrate that both naïve and memory T cells mount an alloresponse, but that memory T cells are more rapidly and strongly recruited by alloantigen stimulation. These findings indicate that in man alloresponses are not confined to particular subsets of post-thymic T cells and that donor-derived viral antigen-specific T cells in an unrelated recipient may cross-react with peptide-MHC complexes against which they were not negatively selected. Disclosures:No relevant conflicts of interest to declare.

Introduction to Cellular Therapy

Cell therapy represents a new frontier in the development of therapeutics for stroke. Aside from physical rehabilitation, few options exist to enhance recovery from stroke. Prior studies in the 1990s provided precedence for considering a cellular approach to treat neurological disorders with the pioneering work on transplantation in Parkinson disease. These trials furnished preliminary evidence for a modest benefit in young patients with a neurodegenerative disorder who underwent neural transplantation. From those initial studies, pilot work emerged on the application of cellular replacement in Huntington disease and other neurological disorders. In an entirely different area of medicine, stem cell transplantation in oncology provided support for the idea that stem cells could be administered intravenously for a range of disorders including stroke. Over the past decade, investigators accordingly began showing that intravenously injected stem cells from the bone marrow and umbilical cord could promote recovery in animal stroke models. Thus began a new area of investigation in which adult stem cells from other sources, rather than the brain, could be tested for therapeutic applications in stroke. As transplantation studies have been growing, it has also now clearly been established that the adult brain undergoes neurogenesis. Elegant work from Lindvall and Parent and others have shown that …

Platelet-Derived Microparticels Stimulate Proliferation of Colony-Forming Unit Granulocyte-Macrophage of Umbilical Cord Blood Hematopoietic Stem Cells.

Umbilical Cord blood has become an important source of hematopoietic stem-progenitor cells for transplantation, however hematopoietic recovery after transplantation with umbilical cord blood is slower than with bone marrow or mobilized peripheral blood. Adhesion molecules on hematopoietic cells are involved in hematopoietic cells' homing, which is considered the most important step of hematological recovery. Some articles indicated that expressions of adhesion molecules on CD34+ cells could predict the time to hematopoietic recovery after transplantation with bone marrow and peripheral blood of many adhesion molecules (such as CD62, CXCR4) are significantly lower on umbilical cord blood than on bone marrow. It is a possible reason for the difficulty in hematopoietic recovery after umbilical cord blood transplant. Platelet -derived microparticles (PMPs) are submicroscopic (<1 μm) membrane vesicles released from platelet if they are stimulated with agonists such as thrombin, collagen, or calcium ionophore A23187 or if exposed to high-stress shear forces. PMPs express several platelet-endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) and several other platelet relevant receptors such as CXCR4 and PAR-1. Some articles indicate that PMPs can affect the function of hematopoietic stem cells by increasing the adhesion of hematopoietic cells to fibrinogen, which suggests that PMP-transferred CD41 antigen plays an important role in this process. PMPs can also increase the survival of human hematopoietic cells including human CD34+ clonogenic progenitors. In our research, we observe the function of PMP to affect the cloning efficiency of colony-forming unit granulocyte-macrophage (CFU-GM).We adopt different concentrations of Thrombin (2U/ml, 1.5U/ml, 1.0U/ml and 0.5U/ml) to activate the platelet and acquire PMPs. Then PMPs were evaluated by using flow cytometry. Based on the result that stimulation of platelets by Thrombin (1U/ml) can acquire the best efficiency of PMPs, we used this concentration in all subsequent experiments. Umbilical cord mononuclear cells (MNCs) were obtained from healthy donors and isolate the MNCs by Ficoll-Hypaque density gradient centrifugation. Briefly, MNCs incubated with or without PMPs cultured in 2.7% methylcellulose. CFU-GM growth was stimulated with 30% umbilical cord serum, rhIL-3 and rh GM-CSF. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. Colonies were counted under an inverted microscope after 7 or 10 days. The research was divided into four groups: 1. control group; 2. PMPs(10μg/ml); 3. PMPs(50μg/ml); 4. PMPs(100μg/ml). The colony formation was enhanced with PMPs and is dependently stimulated with PMPs. The number of colonies in the group of PMPs(100μg/ml) is more than that of other groups. The number of colonies in control group, PMPs(10μg/ml), PMPs(50μg/ml) and PMPs(100μg/ml) are 57.4±3.2, 65.6±5.6, 77.1±1.7 and 87.8±5.0 per 1×105 respectively. These increases in different groups were statistically significant when compared with control group(p<0.05). To sum up, PMPs can affect the cloning efficiency of CFU-GM of umbilical cord hematopoietic stem cells and the efficiencies are depended on the concentration of PMPs.

Effects of EGF and bFGF on expression of microtubule-associated protein tau and MAP-2 mRNA in human umbilical cord mononuclear cells

The aim of this study was to explore the regulatory effects of cytokines, such as EGF and bFGF, on expression of the neural-specific molecules tau and MAP2 mRNA in mononuclear cells (MNCs) derived from human umbilical cord blood (UCB). Phenotypic changes were monitored by inverse phase-contrast microscopy. Tau and MAP2 mRNA were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Tau and MAP2-positive cells were determined by immunocytochemistry. The expression of tau mRNA was negative in uncultured cells, but MAP2 mRNA was positive; in cultured cells, tau protein mRNA expression was positive, MAP2 mRNA expression was upregulated by EGF+bFGF, EGF and bFGF compared to the control group (no cytokines). EGF+bFGF had a greater effect on MAP2 mRNA expression than EGF or bFGF alone. The same upregulatory tendency was noted for tau mRNA expression. It is concluded that MNCs derived from human UCB cells may express some neural specific molecules that can be upregulated by cytokines, especially EGF and bFGF together.