Outgrowing Endothelial Cells That Are Derived from Human Umbilical Cord Blood Improve Neovascularization in Hind-Limb Ischemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3697-3697
Author(s):  
Eun-Sun Yoo ◽  
KiHwan Kwon ◽  
Jee-Young Ahn ◽  
Soo-Ah Oh ◽  
Hye-Jung Chang ◽  
...  
Keyword(s):  
Blood Flow ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Hind Limb ◽  
Limb Ischemia ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hind Limb Ischemia

Abstract Backgroud: Human umbilical cord blood (UCB) contains a high number of endothelial progenitor cells (EPCs) and may be useful for the treatment of ischemic disease. Recently, we have isolated EPCs from UCB having different biologic properties for angiogenic capabilities in vitro. In this present study, the aim is to examine the usefulness of OECs in hind-limb ischemia. Methods: Mononuclear cells from UCB cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Early spindle-shaped cells (early OECs), which were grown during the first week of culture and late cobblestone shaped cells (late OECs), which were in peak growth during the third week of culture were found. The hind-limb ischemia was established as follows: Athymic nude mice (BALB/C-nu) 18–22 g in weight were anesthetized with pentobarbital (60 mg/kg) and their left femoral arteries and main extension arteries were operatively resected. To examine the effect of the vasculogenesis of the two types of OECs, the mice were divided into three groups (PBS, early and late OECs). Twenty-four hours after operative excision 5 × 105 OECs in 200 μl and an equal volume of PBS were administered by intramuscular injection into the mice on hind-limb ischemia. To compare the effect of OECs on neovascularization in vivo, the analysis of blood flow of ischemic and healthy hind limbs was performed on days 1 and 21 after surgery using near-infrared (NIR) imaging with incocyanne Green (ICG). Results: Late OECs expressed a high level of mRNA on endothelial marker genes and formed capillary tubes in Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. We divided the mice into two groups according to the degree of perfusion; good (22.5–50%/min) and poor (0–22.5%) perfusion. OECs improved the blood flow of the ischemic hind-limb in the ’good’ perfusion group but not in the ’poor’ perfusion group. Early OECs led to a more significant improvement in blood flow than that of the late OECs. Conclusion: The different types of OECs from UCB have different biologic properties in vitro and different vasculogenic potential in vivo as well. The results might have potential application for the treatment of hind-limb ischemia.

Umbilical Cord Blood CD133+ Stem Cells Exhibit Vasculogenic Capacity In Vitro and Augment Neovasculogenesis In Vivo in a Murine Model of Vascular Ischemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1790-1790
Author(s):  
M.R. Finney ◽  
L.R. Fanning ◽  
P.J. Vincent ◽  
D.G. Winter ◽  
M.A. Hoffman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blood Flow ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Hind Limb ◽  
Capillary Density ◽  
Functional Assays

Abstract Recent reports have utilized a variety of cell types for cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to assess the vasculogeneic potential of selected CD133+ hematopoietic stem cells (HSC) from umbilical cord blood (UCB) utilizing in vitro functional assays and an in vivo murine hind-limb ischemia model. Methods & Results: Mononuclear cells (MNC) from UCB or bone marrow (BM) were incubated with CD133+ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133+ cells was 0.5±0.2% of UCB MNC and 0.7±0.3% of BM MNC, with a purity of 79±2% (UCB, n=30) and 84±5% (BM, n=12). Surface expression in the UCB CD133+ population was 3.6±1.5% KDR(VEGFR2), 8.7± 3.8% CXCR4 and 22.7±2.8% CD105 compared to 9.2±1.8% KDR, 14.4±1.3% CXCR4 and 23.7±2.3% CD105 in the BM CD133+ population. We measured chemotactic migration of cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. The fold increase over control was 4.9±2.9 UCB MNC, 1.8±0.7 UCB CD133+ and 8.3±1.7 BM CD133+ (n=3). Angiogenic protein assays of CD133+ cells demonstrated elevated levels of IL-8 production as compared to MNC (103+/−380 pg/mL greater in CD133+ than MNC from the same UCB unit) when cultured for 24h in basal media. NOD/SCID mice underwent ligation of the right femoral artery and were given cells or vehicle control via intracardiac injection immediately following injury. Mice were given 1 x 106 MNC or 0.5 x 106 CD133+ cells. Laser Doppler flow measurements were obtained from both limbs each week for 6 weeks and the ratio of perfusion in the ischemic/healthy limb was calculated. At 28 days, perfusion ratios were statistically higher in study groups receiving UCB CD133+ cells, 0.55±0.06 (n=9), BM CD133+ cells 0.47±0.07 (n=8), BM MNC 0.48±0.8 (n=6) compared to cytokine controls 0.37±0.03 (n=12, p<0.05). Mice receiving UCB MNC did not show statistically significant improvement in measured blood flow over control animals 0.42±0.05 (n=8, p=0.34). At sacrifice, bone marrow was harvested to assess engraftment of human cells by flow cytometric analysis. Mice injected with UCB CD133+ cells showed 19±4.9% positive huCD45 cells compared to 2.5±0.6% for UCB MNC, 1.6±0.4% for BM CD133+ cells and 2.3±0.3% for BM MNC (n=3). Histological studies from day 42 tissue samples of muscle distal to arterial ligation were evaluated for capillary density. Control animals had capillary density of 131±6.9 cells/mm2. Capillary density was statistically higher that controls in animals receiving UCB CD133+ (320±18; p<0.0001), BM CD133++ (183±9.3; p<0.0001), and UCB MNC (164±10.5; p=0.011). Mice treated with BM MNC (135±9.4) did not have a statistically significant increase in capillary density from controls (p=0.73). In addition, animals treated with either UCB or BM-derived CD133+ cells had statistically higher capillary density than unselected MNC (p=<0.0001 and p=0.0004, respectively). Conclusions: In vitro functional assays showed that UCB-derived CD133+ HSC demonstrate enhanced homing capability (migration) as well as the potential for cellular recruitment (via IL-8 production) for angiogenesis in response to ischemia. Furthermore, UCB derived CD133+ HSC mediate significantly improved blood flow in an in vivo murine hind-limb injury model of ischemia, indicating the greater vasculogenic potential of selected CD133+ cells from of this stem cell source.

Novel Evidence for the Presence of Potent, Paracrine, Pro-Angiopoietic Effects of Purified Human Umbilical Cord Blood-Derived CD133+ Cells - Implications for Adult Stem Cell Therapies in Regenerative Medicine

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4740-4740
Author(s):  
Kasia Mierzejewska ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adult Stem Cell ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Cell Therapies

Abstract Abstract 4740 Background. As populations of CD34+, CD34+CXCR4+, or CD133+ cells that are enriched in stem cells, adult stem and progenitor cells purified from bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (UCB) are currently employed in the clinic to treat damaged organs (e.g., heart after myocardial infarction [AMI] or injured spinal cord or liver). The cell populations expressing these phenotypes are highly enriched primarily for hematopoietic stem/progenitor cells (HSPCs) and small numbers of endothelial progenitors, and for many years it has been wrongly supposed that they can trans-dedifferentiate into tissue-specific cells. However, even when improvement of organ function is observed after employing them in therapy, the lack of a convincing demonstration for the presence of donor-recipient chimerism in treated tissues in most of the studies performed so far indicates that mechanisms other than trans-dedifferentiation of the HSPCs delivered to the damaged organs into tissue-specific cells play a significant role in some positive clinical outcomes. In support of this conclusion, evidence has accumulated that stem cells secrete a variety of growth factors, cytokines, chemokines, and bioactive lipids that interact with the surrounding microenvironment and, when used in therapy, improve cell viability in damaged organs. In particular, more attention is currently being paid to microvesicles (MVs), which are shed from the cell surface or derived from the intracellular membrane compartment as mediators in cell-to-cell communication. Hypothesis. We hypothesized that these positive outcomes in adult stem cell therapies (e.g., by employing CD133+ cells) can be explained by the paracrine effects of these cells, involving both soluble factors as well as cell membrane-derived MVs. Experimental strategies. CD133+ cells were purified from UCB by employing immunomagnetic beads (> 95% purity as checked by FACS) and incubated for 24 hours in RPMI at 37°C in a small volume of medium supplemented with 0.5% albumin. Subsequently, we harvested conditioned media (CM) from these cells and isolated CD133+ cell-shed microvesicles (MVs) by high speed centrifugation. We employed sensitive ELISA assays to measure the concentration of important pro-angiopoietic and anti-apoptotic factors in CD133+ cell-derived CM and isolated mRNA from both CD133+ cells and CD133+ cell-derived MVs for RQ-PCR analysis of gene expression. Subsequently, the chemotactic activity of CD133+ cell-derived CM and MVs was tested against human umbilical cord blood endothelial cells (HUVECs), and, in parallel, we tested whether CD133+ cell-derived CM and MVs induce major signaling pathways in HUVECs. Finally, in in vitro functional assays, we tested the ability of CD133+ cell-derived CM and MVs to induce tube formation by HUVECs and the ability of in vivo Matrigel assay implants to induce angiogenesis. Results. We observed that highly purified UCB-derived CD133+ cells express mRNAs and secrete proteins for several pro-angiopoietic factors (e.g. VEGF, KL, FGF-2, and IGF-1) into CM and shed microvesicles (MVs) from the cell surface and endosomal compartment that are enriched for mRNAs encoding VEGF, KL, FGF-2, and IGF-1. Both CD133+ cell-derived CM and MVs possessed anti-apoptotic properties, increased the in vitro cell survival of endothelial cells, stimulated phosphorylation of MAPKp42/44 and AKT in HUVECs, induced chemotactic migration, proliferation and tube formation in vitro in HUVECs, as well as stimulated in vivo angiogenesis in Matrigel implants. Conclusions. These observations suggesting an important role for CD133+ cell-derived paracrine signals should be considered when evaluating clinical outcomes using purified CD133+ cells in regenerative medicine. Overall, these cell-derived paracrine signals may explain the therapeutic benefits of adult stem cells employed in regeneration of, for example, heart AMI. Finally, we will discuss several possibilities for enhancing secretion and modulating the composition of these paracrine signals that could be explored in the clinic. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Assessment of Neuroprotective Effects of Human Umbilical Cord Blood Mononuclear Cell Subpopulations In Vitro and in Vivo

2012 ◽  
Vol 21 (4) ◽  
pp. 723-737 ◽  
Author(s):  
Johannes Boltze ◽  
Doreen M. Reich ◽  
Susann Hau ◽  
Klaus G. Reymann ◽  
Maria Strassburger ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Neuroprotective Effects ◽  
Cord Blood Mononuclear Cell ◽  
Cell Subpopulations

scholarly journals Galectin-3 Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Reduces Aberrant Tau Phosphorylation in an Alzheimer Disease Model

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Hoon Lim ◽  
Dahm Lee ◽  
Wan Kyu Choi ◽  
Soo Jin Choi ◽  
Wonil Oh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Galectin 3 ◽  
5Xfad Mice

The formation of neurofibrillary tangles has been implicated as an important pathological marker for Alzheimer’s disease (AD). Studies have revealed that the inhibition of abnormal hyperphosphorylation and aggregation of tau in the AD brain might serve as an important drug target. Using in vitro and in vivo experimental models, such as the AD mouse model (5xFAD mice), we investigated the inhibition of hyperphosphorylation of tau using the human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Administration of hUCB-MSCs not only ameliorated the spatial learning and memory impairments but also mitigated the hyperphosphorylation of tau in 5xFAD mice. Furthermore, in vivo experiments in mice and in vitro ThT fluorescence assay validated galectin-3 (GAL-3) as an essential factor of hUCB-MSC. Moreover, GAL-3 was observed to be involved in the removal of aberrant forms of tau, by reducing hyperphosphorylation through decrements in the glycogen synthase kinase 3 beta (GSK-3β). Our results confirm that GAL-3, secreted by hUCB-MSC, regulates the abnormal accumulation of tau by protein-protein interactions. This study suggests that hUCB-MSCs mitigate hyperphosphorylation of tau through GAL-3 secretion. These findings highlight the potential role of hUCB-MSCs as a therapeutic agent for aberrant tau in AD.

SPRY1 Is a Negative Regulator of Long-Term In Vivo Engraftment and Ex Vivo Expansion of Primitive Human Umbilical Cord Blood Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1715-1715
Author(s):  
Craig E. Eckfeldt ◽  
Eric M. Mendenhall ◽  
Catherine M. Verfaillie
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

Abstract Hematopoietic stem cells (HSCs) are functionally defined by their capacity to home to the bone marrow microenvironment, proliferate and differentiate to restore normal hematopoiesis in a myeloablated recipient; however, the molecular determinants of these processes are not well understood. By comparing the gene expression profiles of highly purified human HSC-enriched and HSC-depleted cell populations, and subsequently validating the hematopoietic function of a subset of these differentially expressed genes using zebrafish (Danio rerio), we previously identified human Sprouty 1 (SPRY1), an evolutionarily conserved antagonist of RTK signaling, as a potential regulator of mammalian HSC development and/or cell-fate decisions. To directly assess the role of SPRY1 in mammalian HSC and hematopoietic progenitor cell (HPC) function, we constructed a dual-promoter lentiviral vector to co-express SPRY1 and green fluorescent protein (GFP) (SPRY1-LV) and a control lentiviral vector to express GFP alone (GFP-LV) in the CD34+ fraction of human umbilical cord blood (UCB). While the enforced expression of SPRY1 in CD34+ UCB cells had no effect on the frequency or morphology of colonies generated in short-term in vitro colony-forming cell (CFC) assays (SPRY1-LV = 27.6 ± 12.3% and GFP-LV = 28.4 ± 20.0%; n = 3), it profoundly inhibited the capacity of UCB CD34+ cells to engraft in the bone marrow NOD-SCID mice in vivo. The estimated frequency of week 11 SCID-repopulating cells (SRC) (± 1 standard error) for SPRY1-LV (n = 13 mice) and GFP-LV (n = 15 mice) cells was 1 in 12,678 cells (6,167 – 26,064) and 1 in 3,412 cells (2,272 – 5,124), respectively, as determined using limiting dilution conditions and Poisson statistics. Furthermore, in 14 day cultures designed for the ex vivo expansion and/or maintenance of primitive hematopoietic cells, ectopic expression of SPRY1 in CD34+ UCB cells dramatically inhibited the expansion of total nucleated cells (SPRY1-LV = 37.9 ± 10.1 fold; GFP-LV = 71.1 ± 5.8 fold; n = 3; p&lt;0.05) and CFCs (SPRY1-LV = 5.7 ± 1.2 fold; GFP-LV = 20.8 ± 14.9 fold; n = 3), although it had no effect on expansion of CD34+CD38− cells. We are currently investigating potential mechanisms for the observed affects of SPRY1 on primitive hematopoietic cells, paying particular attention to the possible effects of SPRY1 expression on “early-acting” hematopoietic cytokines and growth factors that activate RTKs - including FGF1, FGF2, VEGF, SCF, FLT3L, and ANGPT1. In conclusion, enforced expression of SPRY1 negatively regulates primitive hematopoietic cell engraftment in vivo and expansion in vitro, thereby presenting the first example of a role for a Sprouty family member, SPRY1, in primitive human hematopoietic cell function. Moreover, this data further validates the use of model organisms, such as zebrafish, for evaluating the functional roles of transcripts identified in large-scale gene expression profiling experiments in mice and humans.

In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

Transfusion ◽  
2008 ◽  
Vol 48 (10) ◽  
pp. 2235-2245 ◽  
Author(s):  
Eun Jung Baek ◽  
Han-Soo Kim ◽  
Sinyoung Kim ◽  
Honglien Jin ◽  
Tae-Yeal Choi ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Blood Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Clinical Grade ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model

2005 ◽  
Vol 289 (6) ◽  
pp. G1091-G1099 ◽  
Author(s):  
Kazunobu Nonome ◽  
Xiao-Kang Li ◽  
Terumi Takahara ◽  
Yusuke Kitazawa ◽  
Naoko Funeshima ◽  
...  
Keyword(s):  
Liver Injury ◽  
Umbilical Cord Blood ◽  
Fas Ligand ◽  
In Vitro Study ◽  
Cell Types ◽  
Human Umbilical Cord Blood ◽  
Specific Gene ◽  
Human Umbilical Cord

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34+/−cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34+, or CD34−cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of α-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34+and CD34−cells were not observed in human hepatocyte-specific expression.

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