Umbilical Cord Blood Mononuclear Cells Research Articles

The Potential of Ex-Vivo Created Human Di- Chimeric Cells for Migration and Engraftment in the Nude Rat Model.

Introduction: We introduce new cellular therapy based on the ex-vivo creation of donor-recipient chimeric cells as an alternative approach to bone marrow (BM) based therapies in support of solid organ and VCA transplants. The aim of this study was to evaluate the survival and migratory pathways of ex-vivo fused human di-chimeric cells (hDCC) delivered into the bone of the nude rat recipients. Methods: Twenty ex-vivo fusions of human umbilical cord blood (UCB) cells were performed. UCB mononuclear cells from 2 different donors were stained separately with PKH26 and PKH67 fluorescent membrane dyes. Fusion procedure was performed using polyethylene glycol (PEG) technique. Double PKH26 and PKH67 stained human (hDCC) were sorted and subjected to viability assessments by LIVE/DEAD Cell Stain staining as well as lymphocytotoxicity (LCT) test and STR-PCR for class I and II antigens. hDCC (4-9x106) were injected to the bone of nude rat recipients. The presence of hDCC was assessed at the following time points: Group 1 - 24 hours, Group 2 - 72 hours, Group 3 - 7 days, Group 4 - 21 days, Group 5 - 30 days and Group 6 - 60 days. At the given time points, blood, BM (at the injected and contralateral bone), lymph nodes, spleen, lung, liver, skin and brain samples were harvested. The presence of hDCC in the nude rat tissues was assessed using flow cytometry (FC) and immunofluorescent staining. Results: The creation of hDCC was successfully confirmed by FC and fluorescent microscopy using PKH staining. LCT and STR-PCR confirmed that the HLA class I and II specific for both UCB donors used for cell fusion was present on the surface of the hDCC. Viability of hDCC ranged between 94-99%. The presence of hDCC was detected by anti-human HLA class I ABC staining. FC analysis confirmed the presence of hDCC up to 60 days after cells implantation. Within 24 to 72 hours after implantation the hDCC migrated from the injected to the contralateral bone as well as to the lymphoid organs. Conclusion: This study confirmed feasibility of hDCC creation, and their migratory potential as assessed by hDCC engraftment to BM compartment and lymphoid tissues of the nude ratrecipients. Further characterization of migratory pathways of hDCC will contribute to the better understanding of their mechanism of action and tolerogenic properties.

Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice

IntroductionInsulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system.MethodsIn this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer.ResultsParadoxically, experiment results showed that the highest expansion of CD34+CD38−CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ−/−) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38−CD90+ and the in vivo competitive repopulating unit (CRU) functional assay.ConclusionIGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38−CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38−CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.

Human cord blood stem cell paracrine factors activate the survival protein kinase Akt and inhibit death protein kinases JNK and p38 in injured cardiomyocytes

Background aimsWe hypothesized that paracrine factors from human umbilical cord blood mononuclear cells (hUCBC) activate in injured cardiomyocytes the survival protein kinase Akt and limit activation of death protein kinases JNK and p38. MethodsWe treated hUCBC with H2O2 and measured growth factors and cytokines secreted by hUCBC. We then treated cardiomyocytes with H2O2 for 24 h and measured Akt, JNK and p38 activation by means of Western blots. We also measured myocyte viability and apoptosis with the use of fluorescence-activated cell-sorting cytometry. We then investigated myocytes treated for 24 h with H2O2 plus hUCBC and myocytes without hUCBC or H2O2. Four million hUCBC were placed in transwells permeable only to hUCBC paracrine factors, and the transwells were placed in flasks with H2O2+Dulbecco's modified Eagle's medium or in flasks with myocytes plus H2O2+Dulbecco's modified Eagle's medium. ResultshUCBC increased secretion during H2O2 of hepatocyte growth factor by 338%, insulin-like growth factor by 200%, interleukin-4 by 200%, vascular endothelial cell growth factor by 192%, placental growth factor by 150%, interleukin-10 by 150% and angiogenin by 121%. H2O2 increased myocyte JNK activation by 237% and p38 activation by 60%, decreased myocyte viability by 38% and increased necrosis by 34% (all P < 0.01). hUCBC paracrine factors increased in myocytes with H2O2 Akt activation by ≥25%, decreased JNK and p38 activation by >35%, increased viability by >22% and decreased apoptosis by >33% (all P < 0.05). Akt inhibitor API-1 prevented the effects of hUCBC and enhanced H2O2 decrease of myocyte viability. Addition of JNK inhibitor SP600125 or p38 inhibitor SB203580 to myocytes plus H2O2 prevented H2O2 decrease in viability and increased hUCBC beneficial effects. ConclusionsDuring free radical stress, hUCBC paracrine factors activate myocyte Akt, which increases myocyte viability by decreasing activation of death-promoting protein kinases JNK and p38.

PR1-specific cytotoxic T lymphocytes expanded from cord blood are functional in vitro (VAC11P.1002)

Abstract Introduction Neutrophil elastase (NE) and proteinase 3 (P3) are serine proteases often found overexpressed in myeloid leukemic cells. PR1 is a nine amino acid, HLA-A2 restricted peptide derived from NE and P3. The frequency of PR1-specific T cells (PR1-CTL) is very low in healthy adult peripheral blood (0.0005 to 0.05% of CD8+ T cells), but much higher in umbilical cord blood (UCB) (0.007 to 0.345% of CD8+ T cells, N=57). We have previously shown sort-purified UCB PR1-CTL to be active against AML in a murine model. We now investigate the potential of UCB PR1-CTL to undergo expansion while maintaining the anti-leukemic properties of the cells. Methods HLA-A*0201 transfected K562 cells were loaded with PR1 peptide and irradiated with 3000 cGy. UCB mononuclear cells were stimulated with K562/A2/PR1 cells once a week for three weeks. CD8+ T cells were then negatively isolated from the expanded population by magnetic beads and underwent intracellular cytokine staining. Results Prior to expansion, the median percentage of PR1-CTL in the 9 UCB units tested was 0.211% (0.00%-0.348%) of CD8+ T cells. After stimulation this percentage increased to 0.958% (0.346%-2.1%). The expanded UCB PR1-CTL was of memory phenotype (CD45RA-/CCR7-). In addition, expanded UCB PR1-CTL produced both IFN-γ and TNF-α in response to PR1 peptide-loaded T2 cells. Conclusion PR1-CTL can be expanded from UCB and these expanded cells produce cytokines in a specific manner in response PR1-peptide loaded targets.

Effects of Bone Marrow Mesenchymal Stem Cells on Hematopoietic Recovery and Acute Graft-Versus-Host Disease in Murine Allogeneic Umbilical Cord Blood Transplantation Model

To investigate the effect of bone marrow mesenchymal stem cells (MSC) on hematopoietic recovery and acute graft-versus-host disease (GVHD) in a murine allogeneic umbilical cord blood transplantation (allo-UCBT) model. MSCs were obtained from C57/BL mouse bone marrow. The MSC phenotypes were identified by flow cytometry (FCM), and their ability to differentiate into osteoblasts and adipocytes was tested. Once murine allo-UCBT and aGVHD models were established, mice were divided into five groups: (1) total body irradiation (TBI) group, each mouse receiving 0.3 ml sterile saline infusion after TBI and used as control; (2) UCB group, receiving 2 × 10(6) umbilical cord blood mononuclear cells (UCB-MNC) after TBI; (3) UCB+MSC group, receiving 2 × 10(6) UCB-MNC and 2 × 10(7) MSC after TBI; (4) UCB+SC group, receiving 2 × 10(6) UCB-MNC and 2 × 10(6) spleen cells after TBI; and (5) UCB+SC+MSC group, receiving 2 × 10(6) UCB-MNC, 2 × 10(7) MSC and 2 × 10(6) spleen cells after TBI. To evaluate the engraftment of HSC, the white blood cells, red blood cells, and platelets counts were tested at different time points after transplantation, and the ratio of chimerism was identified by FCM. The acute GVHD clinical scores, recipient mice survival, and the histopathological analyses were used to evaluate the effect of MSC on acute GVHD. MSCs were successfully obtained in vitro and FCM analysis showed that these cells are highly positive for CD90.2, CD44, and negative for CD34, CD45, and they are capable to differentiate into osteoblasts and adipocytes after being induced. Compared to UCB group, the UCB+MSC mice had shorter duration of myelosuppression and higher percentage of donor-derived cells which was up to 22.87 ± 4.3 % and the white blood cell (WBC), red blood cell (RBC), and platelet counts started to increase by day 6 after transplantation. Moreover, the average survival time for UCB+MSC mice was 25.0 ± 10.55 days, while for the UCB group it was 15.5 ± 12.50 days. The UCB+SC mice showed fatigue, loss of appetite, weight loss, arched back, and hair ruffling on day 13 post transplantation. Approximately 50 % of mice showed skin ulcers, had diarrhea and other manifestations of acute GVHD, and all mice were died within 20 days. Histopathological analysis confirmed grade II-IV GVHD manifestation. In addition to transient weight loss, some UCB+SC+MSC mice developed arched back, hair ruffling, diarrhea and other manifestations of acute GVHD. The clinical scores in UCB+SC+MSC mice with acute GVHD (grade I-II or without GVHD) were lower than UCB+SC group (P < 0.05). Bone marrow MSCs can promote hematopoietic recovery and decrease the incidence of acute GVHD in murine allo-UCBT model.

Cell-Based Regenerative Strategies for Treatment of Diabetic Skin Wounds, a Comparative Study between Human Umbilical Cord Blood-Mononuclear Cells and Calves' Blood Haemodialysate

BackgroundDiabetes-related foot problems are bound to increase. However, medical therapies for wound care are limited; therefore, the need for development of new treatment modalities to improve wound healing in diabetic patients is essential and constitutes an emerging field of investigation.MethodsAnimals were randomly divided into 8 groups (I–VIII) (32 rats/group), all were streptozotocin (STZ)-induced diabetics except groups III and VIII were non-diabetic controls. The study comprised two experiments; the first included 3 groups. Group I injected with mononuclear cells (MNCs) derived from human umbilical cord blood (HUCB), group II a diabetic control group (PBS i.v). The second experiment included 5 groups, groups IV, V, and VI received topical HUCB-haemodialysate (HD), calves' blood HD, and solcoseryl, respectively. Group VII was the diabetic control group (topical saline). Standard circular wounds were created on the back of rats. A sample of each type of HD was analyzed using the high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) system. Wound area measurement and photography were carried out every 4 days. Plasma glucose, catalase (CAT), malondialdehyde (MDA), nitric oxide (NO) and platelets count were assessed. Wound samples were excised for hydroxyproline (HP) and histopathological study.ResultsTreatment with HUCB MNCs or HUCB-HD resulted in wound contraction, increased CAT, NO, platelets count, body weights, and HP content, and decreased MDA and glucose.ConclusionSystemic administration of HUCB MNCs and topical application of the newly prepared HUCB-HD or calves' blood HD significantly accelerated the rate of diabetic wound healing and would open the possibility of their future use in regenerative medicine.

Umbilical cord blood cells regulate the differentiation of endogenous neural stem cells in hypoxic ischemic neonatal rats via the hedgehog signaling pathway

Transplantation of umbilical cord blood mononuclear cells (UCBMC) promotes the proliferation of endogenous neural stem cells (NSCs), but it has been unclear whether the proliferating NSCs can differentiate into mature neural cells. Therefore, we explored the effects of UCBMC transplantation on the differentiation of endogenous NSCs and their underlying mechanisms. Seven-day-old Sprague-Dawley rats underwent left carotid ligation followed by hypoxic stress. UCBMC were transplanted 24h after hypoxia ischemia (HI). BrdU/β-tubulin/HNA/DAPI, BrdU/GFAP/HNA/DAPI, Ngn1/DAPI, and BMP4/DAPI were measured by immunofluorescence staining; Shh, Gli1, Ngn1, and BMP4 proteins were measured by western-blot analysis 28 days after transplantation. More newborn neurons and fewer astrocytes were observed in the HI+UCBMC group, its neuronal percentage was higher, and glial percentage was lower compared with the N+UCBMC (P<0.05) and HI+PBS groups (P<0.01), while fewer newborn neurons and more newborn astrocytes were found in the HI+cyclopamine (an antagonist of the hedgehog protein)+UCBMC group compared with the HI+UCBMC group (P<0.01). The expression of Shh, Gli1, and Ngn1 proteins was higher and BMP4 protein was lower in the HI+UCBMC compared with the HI+PBS group (P<0.01) and the HI+cyclopamine+UCBMC group (P<0.01). Linear regression analysis showed that the differentiation of NSCs correlated with expression of Ngn1 and BMP4 proteins (P<0.01). In conclusion, UCBMC promote neuronal differentiation and reduce glial differentiation in hypoxic ischemic neonatal rats via the hedgehog signaling pathway.

Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 10(6)-10(7)-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~10(68)-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine.

Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.

Monocytes are essential for the neuroprotective effect of human cord blood cells following middle cerebral artery occlusion in rat

Systemic administration of human umbilical cord blood (HUCB) mononuclear cells (MNC) following middle cerebral artery occlusion (MCAO) in the rat reduces infarct size and, more importantly, restores motor function. The HUCB cell preparation is composed of immature T-cells, B-cells, monocytes and stem cells. In this study we examined whether the beneficial effects of HUCB injection were attributable to one of these cell types. Male Sprague Dawley rats underwent permanent MCAO followed 48 h later by intravenous administration of HUCB MNC preparations depleted of either CD14(+) monocytes, CD133(+) stem cells, CD2(+) T-cells or CD19(+) B cells. Motor function was measured prior to MCAO and 30 days post-stroke. When CD14(+) monocytes were depleted from the HUCB MNC, activity and motor asymmetry were similar to the MCAO only treated animals. Monocyte depletion prevented HUCB cell treatment from reducing infarct size while monocyte enrichment was sufficient to reduce infarct size. Administration of monocyte-depleted HUCB cells did not suppress Iba1 labeling of microglia in the infarcted area relative to treatment with the whole HUCB preparation. These data demonstrate that the HUCB monocytes provide the majority of the efficacy in reducing infarct volume and promoting functional recovery.

Effects of intravenous administration of umbilical cord blood CD34+ cells in a mouse model of neonatal stroke

Neonatal stroke occurs in approximately 1/4000 live births and results in life-long neurological impairments: e.g., cerebral palsy. Currently, there is no evidence-based specific treatment for neonates with stroke. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment in rodent models of neonatal brain injury. However, all of the studies examined the effects of administering either the UCB mononuclear cell fraction or UCB-derived mesenchymal stem cells in neonatal rat models. The objective of this study was to examine the effects of human UCB CD34+ cells (hematopoietic stem cell/endothelial progenitor cells) in a mouse model of neonatal stroke, which we recently developed. On postnatal day 12, immunocompromized (SCID) mice underwent permanent occlusion of the left middle cerebral artery (MCAO). Forty-eight hours after MCAO, human UCB CD34+ cells (1×105cells) were injected intravenously into the mice. The area in which cerebral blood flow (CBF) was maintained was temporarily larger in the cell-treated group than in the phosphate-buffered saline (PBS)-treated group at 24h after treatment. With cell treatment, the percent loss of ipsilateral hemispheric volume was significantly ameliorated (21.5±1.9%) compared with the PBS group (25.6±5.1%) when assessed at 7weeks after MCAO. The cell-treated group did not exhibit significant differences from the PBS group in either rotarod (238±46s in the sham-surgery group, 175±49s in the PBS group, 203±54s in the cell-treated group) or open-field tests. The intravenous administration of human UCB CD34+ cells modestly reduced histological ischemic brain damage after neonatal stroke in mice, with a transient augmentation of CBF in the peri-infarct area.

Clinical safety in using unmatched allogeneic umbilical cord blood mononuclear cells transplantations in non-haematopoietic degenerative conditions.

Evaluation of safety in using unmatched human allogeneic umbilical cord blood cells for therapeutic use in individuals with non-haematopoietic degenerative conditions. The historical data and several recent immunological arguments suggest the therapeutic use of allogeneic Cord Blood Mononuclear Cells (CBMNCs), as these cells do not elicit immune response. Customarily, HLA matched cord blood MNCs are used along with prolonged immunosuppression in treatment of haematological conditions. Lately, unmatched CBMNCs are widely used in case of unavailability of HLA matched cord blood. There have been suggestions for using unmatched allogeneic cord blood MNCs for degenerative conditions without an immunoconditioning regimen. 49 patients with non-haematopoietic degenerative conditions were treated with HLA-unmatched allogeneic hUCB MNCs. Intrathecal/I.V injections (1-2 million cells/kg body weight) were given. Clinical, biochemical and haematological adverse events were evaluated. The haematological and biochemical parameters showed no major deviation from the normal. Clinically, no acute adverse effects or GVHD were observed with the used dosage. This study supports/suggests clinical safety in therapeutic medical use of unmatched allogeneic CBMNCs when used at low dosage in non-haematopoietic degenerative conditions.

IL-21 Enhances Anti-Leukemia Effect By Acting On Both CD3+CD56+ CIK Cells and Regulatory T Cells Derived From Umbilical Cord Blood In Vitro

Cytokine-induced killer (CIK) cells are the effective kind of immunocytes participated in biotherapy of tumors and CD3+CD56+ cells display a major role in cytolytic activity against tumors. Recombinant human interleukin-2 (rhIL-2) is one of the most necessary cytokines during induction of CIK cells, however, some extra kinds of cells eapecially regulatory T (Treg) cells may expand during the induction of CIK cells because of the presence of IL-2. As Treg cells may exert a negative effect on function of CIK cells through cell-to-cell contact and some cytokines secreted by Treg cells, so what we need to consider is how to reduce Treg cells in the culture system of CIK cells. Some studies have confirmed that IL-21, which is belonged to a subset of cytokines where the receptors share the common cytokine receptor γ chain, could express an inhibitory influence on proliferation in Treg cells and our previous study has also confirmed its enhancement on proliferation and function of CIK cells. Here we hypothesize that IL-21 not only promotes the proliferation and function of CD3+CD56+ CIK cells, but also depressed Treg cells in the culure system of CIK cells and then result in the enhanced anti-leukemia effect.In this study, we first detected whether Treg cells existed in the culture system of CIK cells. Firstly, CIK cells were obaind from umbilical cord blood mononuclear cells (CBMCs) by the sequential addition of interferon-γ, anti-CD3 antibody , and rh-IL-2. Then the immunophenotype of Treg cells was detected by the flow cytometry through CD4-FITC and Foxp3-PE antibodies double staining. It was found that a certain proportion of Treg cells at about (10.24±1.42)% consisted in the culture system of CIK cells.The second step in this study was to explore the effect of IL-21 acting on CD3+CD56+ CIK cells. Firstly, CD3+CD56+ CIK cells were separated from the culture cells mentioned above by positive selection using Fluorescence-activated Cell Sorter. Cells were then stimulated with IL-21 for a defined period of time and subjected to CCK-8 and LDH assays to measure cellular viability and cytotoxicity against to leukemia cell line—K562 cells, respectively. The CCK-8 assay results showed that OD values in the cells without IL-21 stimulation was 1.08 which was much lower than that in the cells with IL-21 stimulaiton at 1.45 (P<0.01), thus IL-21 could significantly enhance in vitro proliferation of CD3+CD56+ CIK cells. And after 72 hours of stimulation by IL-21, cytotoxicity of CD3+CD56+ CIK cells against K562 cells at an Effector:Target ratio of 20:1 was observed to be (52.99±1.26)%, while the rate in the cells without IL-21 stimulation was (29.31±0.58)% , which was much lower than the cells with IL-21 stimulation (P<0.01).Next, we investigated the effect of IL-21 on both Treg and CIK cells in the culture system. The culture system of CIK cells was established as mentioned above, and then part of these cells were stimulated with IL-21 for 72 hours. Immunophenotype of Treg and CIK cells was detected by flow cytometry. It was found that cells stimulated by IL-21 showed a decreased proportion of CD4+Foxp3+ Treg cells at (1.48±0.06)% compared with the cells without IL-21 stimulation at (10.24±1.42)% (P<0.01). The CD3+CD56+ cells occupied a certain proportion at (39.80±1.80)% in the cells stimulated with IL-21, which was much higher than that in the cells without IL-21 stimulation at (20.46±1.11)% ( P<0.01). Then cytotoxicity of CIK cells was detected by LDH assay and the results showed that IL-21 could extremely strengthen the cytotoxicity of CIK cells to K562 cells, and the cytotoxity index in the cells with IL-21 stimulation at (52.99±1.26)% , which was much higher than that in the cells without IL-21 stimulation at (29.31±0.58)% ( P<0.01).In conclusion, our findings indicate that IL-21 could promote the proliferative and cytotoxic activity of CD3+CD56+ CIK cells, meanwhile it also could suppress the viability of Treg cells in the CIK cells culture system. So a conclusion can be summarized that IL-21 could enhance anti-leukemia effect not only by promoting CD3+CD56+ CIK cells but also by depressing Treg cells derived from umbilical cord blood in vitro. Disclosures:No relevant conflicts of interest to declare.

New Molecular Evidence That Oct-4 Is Truly Expressed In a Rare Population Of Developmental Early Stem Cells In Human Umbilical Cord Blood (UCB) and That Epigenetic Modification Of Imprinting At Igf2-H19 Locus Regulates Their Quiescent State – Potential Implications For Regenerative Medicine

BackgroundOne of the most intriguing questions in stem cell biology is whether human umbilical cord blood (UCB) contains early-development stem cells that express markers of pluripotency and thus could be employed in regenerative medicine. Several groups have reported mRNAs for genes regulating stem cell pluripotency, such as Oct-4A, Nanog, and SSEA-1, in UCB cells. However, detection of the Oct-4A transcript may be hampered by the presence of several pseudogenes and Oct-4B isoform, which is not related to stem cell pluripotency. Another important question is: why are these primitive stem cells that are present in UCB highly quiescent and relatively resistant to ex vivo expansion? We previously identified a population of Oct-4+ CD133+Lin–CD45– cells in human UCB (Leukemia 2007;21:297–303) that may become specified into long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011;25:1278) and mesenchymal stem cells (Stem Cell & Dev. 2013;22:622). These Oct-4+CD133+Lin–CD45– cells present in UCB correspond to a population of murine Oct-4+Sca-1+Lin+CD45– cells that remain quiescent in bone marrow because of epigenetic modification of parentally imprinted genes, including the Igf-2-H19 tandem gene (Leukemia 2009;23:2042). The quiescence of these cells has been explained by erasure of imprinting in the regulatory differentially methylated region (DMR) at the Igf2-H19 locus. In appropriate animal models, these small cells also give rise to LT-HSCs (Exp. Hematol2011;3:225), mesenchymal stem cells (Stem CellsDev 2010;19:1557), and lung epithelium (Stem Cells2013;doi: 10.1002/stem.1413). Moreover, as we demonstrated, the epigenetic reversal of the maternal type of imprinting to the somatic type in the DMR for the Igf2-H19 locus, which is necessary to maintain balanced expression between insulin-like growth factor 2 (Igf-2) and noncoding H19 RNA (precursor for several inhibitory miRNAs) from paternal and maternal chromosomes, respectively, is required for these cells to enter the cell cycle. The crucial role of Igf2-H19 imprinting in quiescence of the most-primitive stem cells in murine BM has been very recently confirmed by another group (Nature 2013, doi: 10.1038/nature12303). Aim of the studyTo address whether human UCB Oct-4± CD133±Lin–CD45– cells truly express genes regulating pluripotency, we examined the DNA methylation state of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) and of the DMR for Igf2-H19. Materials and MethodsUCB CD133+Lin–CD45– cells were isolated by multiparameter fluorescence-activated cell sorting (FACS) after intra-cellular staining for Oct4 protein in lineage-depleted human UCB mononuclear cells. Bisulfide modification of DNA followed by sequencing was employed to evaluate the methylation state of CpG islands in the promoters for Oct-4, Nanog, and Sall4 as well as in the DMR for the Igf2-H19 locus. Salient ResultsWe observed that Oct4, Nanog, and Sall4 promoters in UCB Oct-4+CD133+Lin–CD45– cells were demethylated to a similar degree as the human teratocarcinoma NTERA2, which is evidence for true expression of these genes. Furthermore, in human UCB we observed Oct-4+CD133+Lin–CD45– cells that, like their murine counterparts, erase the imprinting in the DMR at the Igf2-H19 locus, which demonstrates that genomic imprinting could be a key mechanism for maintaining the quiescence of these cells. This imprinting data was subsequently confirmed by RQ-PCR analysis of gene expression, showing downregulation of autocrine Igf-2 and upregulation of noncoding H19 RNA. ConclusionOur methylation studies of the promoters for pluripotency/germ-line genes (Oct4, Nanog, and Sall4) provide for the first time strong molecular evidence that UCB contains cells that truly express pluripotent stem cell markers. Moreover, molecular analysis of the methylation state in the DMR for the Igf2-H19 locus also explains for the first time how the quiescent state of these cells is regulated by changes in parental imprinting at the Igf2-H19 locus. Thus, elucidation of this mechanism that controls and modifies genomic imprinting in VSELs will be crucial for developing strategies to expand these cells and employ them more efficiently in regenerative medicine and we are currently working on this. [Display omitted] Disclosures:Ratajczak:Neostem Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Transplantation of Cryopreserved Human Umbilical Cord Blood Mononuclear Cells Does Not Induce Sustained Recovery after Experimental Stroke in Spontaneously Hypertensive Rats

Previous studies have highlighted the enormous potential of cell-based therapies for stroke not only to prevent ischemic brain damage, but also to amplify endogenous repair processes. Considering its widespread availability and low immunogenicity human umbilical cord blood (HUCB) is a particularly attractive stem cell source. Our goal was to investigate the neurorestorative potential of cryopreserved HUCB mononuclear cells (MNC) after permanent middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats (SHR). Human umbilical cord blood MNC or vehicle solution was administered intravenously 24 hours after MCAO. Experimental groups were as follows: (1) quantitative polymerase chain reaction (PCR) of host-derived growth factors up to 48 hours after stroke; (2) immunohistochemical analysis of astroglial scarring; (3) magnetic resonance imaging (MRI) and weekly behavioral tests for 2 months after stroke. Long-term functional outcome and lesion development on MRI were not beneficially influenced by HUCB MNC therapy. Furthermore, HUCB MNC treatment did not change local growth factor levels and glial scarring extent. In summary, we could not demonstrate neurorestorative properties of HUCB MNC after stroke in SHR. Our results advise caution regarding a prompt translation of cord blood therapy into clinical stroke trials as long as deepened knowledge about its precise modes of action is missing.

Intra-arterial transplantation of human umbilical cord blood mononuclear cells in neonatal hypoxic–ischemic rats

Based on preclinical findings, cellular therapy has become a promising therapeutic approach for neonatal hypoxia–ischemia (HI). However, before translation into the clinical setting, new and effective routes of cell delivery must be determined. Intra-arterial (IA) delivery is an attractive route of cellular administration but has never been used in neonatal HI rats. Aims: In this study, we investigated the feasibility of IA transplantation of human umbilical cord blood (HUCB) mononuclear cells for the treatment of long-term behavior dysfunction and brain lesion after neonatal HI. Main methods: Seven-day-old rats were subjected to a HI model and the animals received HUCB mononuclear cells into the left common carotid artery 24h after HI insult. Key findings: At 9weeks post-HI, intra-arterially transplanted HUCB mononuclear cells significantly improved learning and long-term spatial memory impairments when evaluated by the Morris water maze paradigm. There was no effect of neonatal HI insult or IA procedure on body weight and on motor coordination and balance when evaluated by the accelerating rotarod test. Cellular transplantation by the IA route did not restore neonatal HI-induced brain damage according to stereological volume assessment. Furthermore, HUCB mononuclear cells were tracked in the injured brain and peripheral organs of HI transplanted-rats by nested polymerase chain reaction analysis at different time points. Significance: Our findings contribute to the translational knowledge of cell based-therapy in neonatal HI and demonstrate for the first time that IA transplantation into rat pups is a feasible route for cellular delivery and prevents long-term cognitive deficits induced by experimental neonatal HI.

Feasibility and potential of in utero foetal membrane-derived cell transplantation

Cells isolated from foetal membranes of human term placenta display multiple properties, including some features of stem/progenitor cells, together with immunomodulatory actions and the ability to secrete bioactive soluble factors. Whilst such properties support the potential applicability of these cells in transplantation settings aimed at regenerating/repairing tissues in adults, theoretically, using these cells in prenatal treatment strategies may also be achievable. To assess the feasibility of a foetal membrane-derived cell-based therapeutic treatment during foetal development, we firstly addressed the question of whether in utero transplantation using these cells was possible. To this end, we assessed postnatal microchimerism after transplantation of amniotic membrane-derived cells (a mixture of both mesenchymal stromal/stem cells and epithelial cells) in foetal sheep. Transplantation was performed with or without human umbilical cord blood mononuclear cells and chorionic membrane-derived mesenchymal stromal/stem cells, and was followed by a postnatal booster cell injection. Lambs were euthanized 2-4 months postnatally and their organs/tissues were analysed for microchimerism through detection of human DNA. Human DNA was found in almost all tissues of all of the lambs, with the seemingly random appearance of human cells in some of the analysed tissues suggesting long-term human microchimerism and donor cell migration after in utero/postnatal booster xenotransplation. Differences in microchimerism tissue distribution between animals transplanted with different cell types are discussed. This pilot study adds to ongoing efforts by different investigators to explore the potential of in utero cellular transplantation, and warrants further investigation of using foetal membrane-derived cells for prenatal cell therapies.

Co-transplantation of mesenchymal stromal cells and cord blood cells in treatment of diabetes

Background aimsStem cells provide a promising source for treatment of type 1 diabetes, but the treatment strategy and mechanism remain unclear. The aims of this study were to investigate whether co-transplantation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and cord blood mononuclear cells (CB-MNCs) could reverse hyperglycemia in type 1 diabetic mice and to determine the appropriate ratio for co-transplantation. The treatment mechanism was also studied. MethodsA simple and efficient isolation method was developed to generate qualified UC-MSCs. UC-MSCs and CB-MNCs were then transplanted into type 1 diabetic mice at different ratios (UC-MSCs to CB-MNCs = 1:1, 1:4, 1:10) to observe the change in blood glucose concentration. Histology, immunohistochemistry, and human Alu polymerase chain reaction assay were performed to evaluate for the presence of donor-derived cells and the repair of endogenous islets. We also induced UC-MSCs into islet-like cells under specific culture conditions to determine their differentiate potential in vitro. ResultsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 effectively reversed hyperglycemia in diabetic mice. The detection of human Alu sequence indicated that the engraftment of donor-derived cells had homed into the recipient's pancreas and kidney. Although neither human insulin nor human nuclei antigen was detected in the regenerated pancreas, UC-MSCs could differentiate into insulin-secreted cells in vitro. ConclusionsCo-transplantation of UC-MSCs and CB-MNCs at a ratio of 1:4 could efficiently reverse hyperglycemia and repair pancreatic tissue.

Does early n.3 fatty acid exposure alter DNA methylation in the developing human immune system?

Evaluation of: Lee HS, Barraza‑Villarreal A, Hernandez‑Vargas H et al. Modulation of DNA methylation states and infant immune system by dietary supplementation with n‑3 PUFA during pregnancy in an intervention study. Am. J. Clin. Nutr. 98(2), 480–487 (2013). Long-lasting effects of n-3 fatty acids on the immune cells involved in allergic disease may be due to epigenetic alterations. Pregnant women consumed the n-3 fatty acid docosahexaenoic acid (DHA; 400 mg/day) or placebo from mid-gestation until delivery, and the methylation status of CpG loci within the promoter regions of several genes involved in immune function was measured in umbilical cord blood mononuclear cells (CBMCs) using pyrosequencing. There was no significant effect of DHA supplementation in pregnancy on methylation within the promoter regions of any of the cytokine or transcription factor genes measured in CBMCs. Methylation of the promoter region of the genome stabilizer LINE-1 was higher in CBMCs from infants of mothers who smoked in the DHA group compared with those in the placebo group, although this effect was lost following statistical correction for confounders, but did not differ between nonsmoking women in the two groups. Several factors limit interpretation of the findings. It remains unknown whether there is a role for epigenetic alterations as a mechanism for long-lasting immune and clinical effects of very early n-3 fatty acid exposure.

Umbilical Cord Blood Mononuclear Cell Transplantation for Treating Spinal Cord Injury

Umbilical Cord Blood Mononuclear Cell Transplantation for Treating Spinal Cord Injury