Comparison of Umbilical Cord Blood Derived Mononuclear Cells and Endothelial Generating Cells in Response to Ischemia in the Murine Hind-Limb Injury Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3973-3973
Author(s):  
Marcie R. Finney ◽  
Vincent J. Pompili ◽  
Nicholass G. Greco ◽  
Matthew J. Joseph ◽  
Daniel G. Winter ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Hind Limb ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Cell Populations ◽  
Surface Phenotype ◽  
Limb Injury

Abstract Recent reports have studied the use of cell populations from bone marrow, peripheral blood and umbilical cord blood (UCB) in mediating therapeutic angiogenesis in patients with coronary artery disease. We investigated the surface phenotype of UCB derived-mononuclear cells (MNC), and endothelial generating cells (EGC) by flow cytometry and in vitro functional migration studies. We used a murine hind-limb injury ischemia model to assess in vivo efficacy of the cell populations. METHODS: MNC were isolated by density centrifugation and CD133+ cells were isolated by magnetic separation (Miltenyi). EGC were derived by adherence of CD133− cells cultured 16h on fibronectin-coated tissue culture plates in EGM2 media (Clonetics). Cell characterization included surface phenotype determined by flow cytometry for monocyte markers CD14 and CD11b, stromal markers CD73 and CD105, KDR (VEGFR2), and the receptor for SDF-1, CXCR4. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or EGC towards SDF-1 (100ng/mL). Following a 3 hour incubation, the cells migrated to the bottom wells was counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences. NOD/SCID mice underwent right femoral artery ligation and were injected with cytokines (EGM2 media, n=10), MNC (n=7) or EGC (n=8), 1X 106 cells/mouse. Laser Doppler blood flow measurements were recorded weekly for four weeks. RESULTS: Enhanced expression of CXCR4, CD105, KDR, CD14 and CD11b was found in the EGC cells generated after 16h culture on fibronectin. Surface Phenotype of UCB MNC and EGC CD14 CD11b CD73 CD105 KDR CXCR4 MNC (n≥5) 8.1 ± 2.7 22.7 ± 6.6 5.1 ± 2.2 6.7 ± 2.1 7.1 ± 2.1 28.5 ± 5.8 EGC (n=5) 67.7 ± 9.5 80.0 ± 4.8 7.1 ± 4.0 33.8 ± 5.8 37.6 ± 6.4 64.6 ± 7.8 Functional assays showed increased migration of both MNC and EGC to SDF-1 compared to control media (4.9 ± 2.9, n=2 and 3.2 ± 0.6, n=3 fold increases respectively). With VEGF as a chemoattractant MNC exhibited a 1.5 fold increase over the negative control (n=1) and EGC showed a 1.4 ± 0.3 fold increase (n=2). In the murine hind-limb ischemia model the ratio of ischemic/non-ischemic limb blood flow was used to compare vasculogenic potential. There was improvement of blood flow 14 days after injection of the EGC cells (p=0.019). On days 21 and 28, blood flow ratio was higher than control but was not statistically significant (p=0.06). The difference between MNC and EGC was not significant at any time point (p> 0.05). Histological studies are ongoing. CONCLUSION: In summary, UCB derived EGC exhibited monocyte and stromal surface antigen expression, migrated to an SDF-1 gradient, and mediated improved vascular blood flow. Ongoing studies are focused on direct cell vs. paracrine effects underlying observed neovasculogenesis mediated by EGC.

In Vitro Characteristics of Umbilical Cord Blood Derived Mononuclear Cells, CD133+ Cells and Endothelial Generating Cells and Their Effectiveness In Vivo in Mediating Neovascularization in a Murine Model of Hind-Limb Ischemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3922-3922
Author(s):  
Marcie R. Finney ◽  
Matthew Joseph ◽  
Daniel G. Winter ◽  
Omar Masari ◽  
Margaret Kozik ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Flow ◽  
Hind Limb ◽  
Mononuclear Cells ◽  
Limb Ischemia ◽  
Histological Studies ◽  
Surface Phenotype ◽  
Migration Assays

Abstract Recent reports have studied the use of various cell populations from bone marrow, peripheral blood and umbilical cord blood (UCB) in mediating therapeutic angiogenesis. We sought to investigate the surface phenotype of UCB derived-mononuclear cells (MNC), CD133+ cells and endothelial generating cells (EGC) using flow cytometry and in vitro functional migration studies. A murine hind-limb injury ischemia model was used to assess in vivo efficacy of the different cell populations. METHODS: Mononuclear cells were isolated by density centrifugation. CD133+ cells were isolated from UCB MNC by magnetic separation (Miltenyi). EGC were derived by adherence of the CD133- cells overnight on fibronectin-coated tissue culture plates in EGM2 media (Clonetics). Surface phenotype was determined by flow cytometry for stem cell markers, CD133 and CD34, stromal markers CD73 and CD105, KDR (VEGFR2), and the receptor for SDF-1, CXCR4. Modified Boyden chambers (Neuroprobe) were used to observe chemotactic migration of MNC, CD133, EGC or the combination of CD133 and EGC towards SDF-1 (100ng/mL) compared to control wells containing media alone. In the in vivo studies, 40 NOD.SCID mice underwent right femoral artery ligation. Mice were randomized into five study groups: Cytokines (EGM2 media, n=5), MNC (n=5), CD133 (n=5), EGC (n=7), or CD133 and EGC (1:2 ratio, n=4). Laser Doppler blood flow measurements were recorded weekly for four weeks and the ratio of ischemic/non-ischemic leg was calculated. At day 28, tissue samples were harvested for histological studies. RESULTS: Surface phenotype by flow cytometry showed an average purity of 78.67% +/− 2.41% for the selection of CD133. In addition, an enhancement of the expression of CXCR4 was seen in the EGC population after overnight exposure to fibronectin and EGM2 media. Surface Phenotype of UCB MNC, CD133 and EGC MNC CD133 EGC CD34 4.03 87.42 3.40 CD133 3.19 78.67 5.06 CD73 5.07 N/A 7.15 CD105 6.74 22.74 33.78 KDR 7.14 3.59 37.65 CXCR4 28.54 8.66 64.57 The in vitro functional migration assays showed increased migration of MNC, EGC, and CD133 with EGC to SDF-1, where the CD133 cells alone showed no increased migration compared to control media. In the in vivo murine model of hind-limb ischemia, the blood flow ratio of ischemic/non-ischemic limb was used to estimate the rate of blood flow recovery. The rates of blood flow recovery were 0.0198 (CD133), 0.02 (CD133 and EGC), 0.0163 (MNC), 0.016 (EGC) and 0.0123 (cytokines). The rates were significantly different between CD133 and cytokines (p=0.011) and between CD133 and EGC compared to cytokines (p=0.011). The difference between MNC and cytokines (p=0.156) and between EGC and cytokines (p=0.176) was not significant. Histological studies are ongoing. CONCLUSION: Surface phenotype of UCB-derived MNC, CD133, and EGC were compared by flow cytometry. The in vitro functional chemotactic capacity toward SDF-1 of these cell types was determined by migration assays. The infusion of CD133+ or the combination of CD133 and EGC cells augmented the rate of blood flow recovery in the in vivo murine hind-limb model of ischemia compared to the crude MNC prep or the ECG alone.

In Vitro Characterization of Umbilical Cord Blood CD133+ and In Vivo Functionality in Response to Ischemia in a Murine Hind-Limb Injury Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3693-3693
Author(s):  
Marcie R. Finney ◽  
Nicholas G. Greco ◽  
Matthew E. Joseph ◽  
Daniel G. Winter ◽  
Shyam Bhakta ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hind Limb ◽  
Study Groups ◽  
Chemotactic Migration ◽  
Limb Injury ◽  
Injury Model

Abstract Previous reports have demonstrated efficacy of cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to determine the potential efficacy of adult umbilical cord blood (UCB) derived selected CD133+ cells in the murine hind limb ischemia model and to characterize these cells by surface phenotype and functionality prior to injection. Methods and Results: Mononuclear cells (MNC) from UCB were labeled with CD133+ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133+ cells was 0.51 ± 0.2% of MNC, with a purity of 78.7 ± 2.4% (n=30). Surface expression in the UCB CD133+ population was 3.59 ± 1.49% KDR(VEGFR2), 8.66 ± 3.79% CXCR4 and 22.74 ± 2.84% CD105 compared to 7.14 ± 2.15% KDR, 28.54 ± 5.81% CXCR4 and 6.74 ± 2.07% CD105 in the UCB MNC population. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or CD133+ cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. Following a 3 hour incubation, the cells migrating to the bottom wells were counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences). MNC and CD133+ cells migration to SDF showed a 4.9 ± 2.9 and 1.8 ± 0.7 fold increase over the negative control respectively. To test vasculogenic functionality of these selected cell populations, NOD/SCID mice underwent ligation of the right femoral artery and were randomized into 3 study groups: control (endothelial media with cytokines), non-selected MNC (1 x 106 cells/mouse) or CD133+ (0.5 x 106 cells/mouse) given via intracardiac injection immediately after injury. Doppler flow measurements were taken on both limbs each week for 4 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 CD133+ cells from UCB, 0.55 ± 0.07 (n=8) compared to cytokine controls 0.39 ± 0.02 (n=10, p=0.019). Mice receiving MNC did not show statistically significant improvement over control animals 0.42 ± 0.06 (n=7, p=0.27). Conclusion: Surface phenotyping was notable for increased expression of the receptor for SDF-1, CXCR4 on MNC when compared to CD133+ cells. In vitro functional assays showed that CD133+ and MNC exhibited increased chemotactic migration to SDF-1. In vivo studies showed that injection of UCB CD133+ cells improved blood flow compared with cytokines alone in the murine hind limb injury model, highlighting the vasculogenic potential of CD133+ cells from UCB.

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.

scholarly journals Impact of Intrauterine Growth Restriction Diseases on The Umbilical Cord Blood CD34+ Cell Counts

2020 ◽  
pp. 56-61
Author(s):  
Ekrem Ünal ◽  
Şerife Erdem ◽  
Ayten Biçer ◽  
Fatma Zehra Okuş ◽  
Alper Özcan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Gestational Hypertension ◽  
Healthy Controls ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Introduction: Different diseases in obstetrics and gynecology can affect the number of CD34+ cells in the umbilical cord blood. Objectives: This study aimed to evaluate the effect of Gestational Diabetes Mellitus (GDM), Gestational Hypertension (GHT) and Morbidly Adherent Placenta (MAP) on the content of CD34+ cells of umbilical cord blood and to compare the effectiveness of Sysmex XN20 analyzer to the flow cytometry method, which is the gold standard in CD34+ cells. Materials abd Methods: The umbilical cord blood (15 ml) was collected after the birth of the newborns. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus. The cells were stained with Procount ™ Progenitor Cell Count Kit with PE Labeled monoclonal anti-CD34+ antibody then analyzed with Flow Cytometry or Sysmex XN20 without staining, to identify CD34+ cells named as hematopoietic progenitor cells (HPC), respectively. Results: Flow cytometric evaluation revealed a significantly elevated (p<0.05) number of cord blood CD34+ cells in GDM and GHT groups compared with healthy controls. MAP patients had comparable CD34+ cells compared with healthy controls. A significant increase in lymphocyte counts was also observed in GDM and GHT groups compared with healthy controls. Sysmex analysis however only revealed an increase in lymphocyte numbers in GHT but picked no differences across groups in HPC. Correlation between Sysmex and flow cytometry results was weak in control, GHT, GDM and MAP groups r: 0570/p<0.01, r: 0.5727/p: 0.0708, r:0.2149/p: 0.4779, r: 0.111/p: 0.779, respectively. Conclusions: CD34+ cells were significantly higher in the GHT and GDM groups compared with healthy control cord blood. The correlations between Flow cytometry and Sysmex were not strong.

Cord Blood Plasma Enhances Migration of Hematopoietic Stem and Progenitor Cells (HSPC)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2959-2959
Author(s):  
James G Farmar ◽  
Alexander Wendling ◽  
Kevin Lynch ◽  
Jose Tomsig ◽  
Mariusz Z Ratajczak ◽  
...  
Keyword(s):  
Stress Response ◽  
Blood Plasma ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Potential Effect ◽  
Hematopoietic Stem

Abstract Abstract 2959 Background: A greater understanding of the mechanisms behind HSPC trafficking is vital to increase the efficacy of HSPC therapy. The composition of adult blood plasma (ABP) is well documented, in particular proteins and metabolites, but very little is known about umbilical cord blood plasma (CBP) which may contain a host of bioactive proteins and lipids released as a stress response during birth as opposed to ABP which is generally regarded as homeostatic. Physiologic stress response may result in altered concentration gradients of these factors thereby creating differing gradients between the marrow and peripheral blood compartments. We sought to investigate factors in CBP and ABP and their effects on HSPC migration. Methods/Results: Both ABP (Research Blood Components, Boston, MA) and CBP was filtered through 0.22 μm filters to remove debris and any cells remaining in the plasma. ABP contained 14% Citrate Phosphate Dextrose Anticoagulant (CPDA) by volume while CBP varied between 28–40% CPDA by volume. ABP samples used in migration assays were diluted to match the CBP concentration with PBS. We screened for the concentrations of 115 known proteins using a multiplexed ELISA assay and compared a pool of 10 CBPs against a pool of 10 ABPs. Umbilical cord blood (UCB) from research units not meeting clinical cell dose threshold was provided by the New York Blood Center (P. Rubinstein, MD). We found 43 proteins were elevated at least two-fold in CBP versus ABP, 16 of which were elevated 10-fold relative to ABP. Out of these 43 proteins, 6 have potential implications on HSPC mobilization: IL8, GCSF (CSF-2), VCAM, MCP1, MIP3, and CXCL10. The concentrations of the proteins in CBP are in pg/mL: IL8 546.85; GCSF 609.91; MCP1 1142.02; MIP3 80.80; VCAM 4, 016, 017.46; and CXCL10 218.27. The fold increase in CBP for these proteins are: IL8 19.39; GCSF 6.39; MCP1 4.12; MIP3 3.48; VCAM 4.20; and CCL10 2.00. The relative contribution of each protein to migration was measured by preparing aliquots of CBP and treating the aliquots with neutralizing antibodies toward each protein (Abcam, Cambridge, MA). Antibodies were incubated at a concentration of 1μg/mL in accordance with recommended concentrations from Abcam. In addition to these 6 proteins, S1P and C3a concentrations were also investigated due to their potential effect in HSPC mobilization. Migration experiments were conducted using Transwell plates (Corning Life Sciences, Lowell, MA). UCB was obtained 24–48 hrs following delivery, and CBP and mononuclear cells were isolated by centrifugation through a Ficoll-Paque density gradient. UCB CD34+ cells were selected by magnetic labeling and sorting using AutoMACS magnetic cell sorter (Miltenyi Biotec, Auburn, CA). UCB HSPCs were placed in upper transwells (8.0 μm pores; 1.5 × 105 cells/well) and the lower well contained CBP, ABP, or fresh RPMI basal media as a control. The cells were allowed to migrate towards the various solutions for 3 h. Cells that migrated were counted and immunophenotyped via hemocytometer and flow cytometry (BD FACS Calibur). In a comparison of migration towards CBP vs. ABP (n = 11 and 10), CBP exhibited an average increase in migration by 157.8 ± 44.1%. Migrations towards CBP depleted of one of the 6 proteins exhibited the following HSPC migrations compared to untreated CBP (100%) were: 48.9 ± 17% for IL8-neutralized (n=6); 90.2 ± 20.4% for GCSF-neutralized (n=5); 102 ± 18.0% for MCP-neutralized (n=5); 71.7 ± 19.8% for MIP3-neutralized (n=6); 35.4 ± 14.7% for VCAM-neutralized (n=4); and 51.7 ± 9.5% for CXCL10-neutralized CBP (n=4). All samples of CBP and ABP used in the migration studies were analyzed for S1P concentration by LC-MS/MS. S1P concentrations in CBP samples ranged from 0.95 to 2.27 (n = 6) times the concentration of S1P in ABP. Additionally, C3a in these CBPs and ABP was analyzed by an ELISA (BD Biosciences, San Jose CA). In CBP samples, C3a varied from 111–297 ng/mL (n = 13) compared to 661 ng/mL in ABP. Conclusion: An improved knowledge of the factors that influence mobilization may provide us with a better approach towards stem cell priming and graft HSPC engineering prior to transplantation. The proteins examined here and the effects of S1P and C3a on HSPC migration may provide novel insights into the factors that influence HSPC trafficking. Further understanding of HSPC migration to proteins released in stress response may be exploited to direct HSPC trafficking in the autologous and allogeneic setting. Disclosures: Lynch: SphynKx Therapeutics LLC: Equity Ownership.

Analysis Of The Lin- CD45- CD34/CD133+ Cell Population In G-CSF-Mobilized Peripheral Blood By Polychromatic Flow Cytometry and Micro RNA Profiling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4841-4841
Author(s):  
Yajuan Jiang ◽  
David Mallinson ◽  
Daria Olijnyk ◽  
Sarah Paterson ◽  
Susan Ridha ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Lineage ◽  
Equity Ownership ◽  
Lineage Markers ◽  
Mobilized Peripheral Blood

Abstract Introduction There has been controversy over the existence of rare somatic stem cells in mouse and human bone marrow and in human umbilical cord blood that have been described to be pluripotent. These cells have been observed to lack expression of CD45 and blood cell lineage markers (Lin-), to express markers associated with both hematopoietic (CD34, CD133) and pluripotent (Oct4, Nanog) stem cells, to be smaller than a blood lymphocyte, and have been associated with the ability to differentiate into cells and tissues of all three germ layers. They have been given a variety of names, including Very Small Embryonic/Epiblast-Like (VSEL) cells. Methods and Results To better characterize these cells, we fractionated G-CSF mobilized adult peripheral blood by elutriation, CD34/CD133 immunomagnetic selection (Miltenyi Biotec, Inc., STEMCELL Technologies, Inc.) and fluorescence-activated cell sorting (FACS) using a MoFlo XDP cell sorter (Beckman Coulter, Inc.). Cell fractions were analyzed on a Beckman Coulter Gallios flow cytometer using a 6-color cocktail of antibodies to CD45, CD34, CD133 and blood cell lineage markers, together with membrane-permeable (DRAQ5) and impermeable (7-AAD) nuclear dyes that distinguish live nucleated cells from dead cells, extracellular vesicles and cell debris. We observe that over 95% of Lin- CD45- flow cytometry events are extracellular vesicles rather than nucleated cells, and have isolated a population of Lin- CD45- CD34+ vesicles from the earliest elutriation fractions (<35 ml/min counterflow). Rare Lin- CD45- live nucleated cells are also clearly evident, many of which express CD34 but not CD133. This population of Lin- CD45- CD34+ CD133- cells makes up approximately 0.003% of the mononuclear cell population in G-CSF mobilized peripheral blood (1 in 300,000 mononuclear cells, or approximately 1 for every 1,000 CD34+ CD45+ hematopoietic stem/progenitor cells). Lin- CD45- CD133+ live nucleated cells are also identified but are considerably more rare (approximately 1 in 10 million mononuclear cells). Similar cell and vesicle populations are also found in umbilical cord blood, although with frequencies about 10-fold higher than in mobilized adult blood. To begin to characterize these cell and vesicle fractions, we isolated total RNA from FACS-sorted Lin- CD45- CD34/CD133+ (CD34+ and/or CD133+) cells for miRNA expression profiling (Agilent SurePrint G3 Human v16 microRNA 8x60K microarray, representing 1205 Human miRNAs, 1199 verified as real miRNAs in miRbase 18). Array data were processed using a proprietary normalization algorithm (Sistemic, Ltd.) to generate miRNA expression profiles which were analyzed by microRNA-based fingerprinting (SistemQC™). A robust miRNA profile was generated from the initial Lin- CD45- CD34/CD133+ cell sample, with 107 miRNAs reliably detected (a number consistent with other cellular profiles). The detected miRNAs showed a range of expression levels and were expressed well above the limit of detection for the arrays. Further samples will be analyzed to confirm these preliminary findings. Conclusion The Lin- CD45- population observed by flow cytometry analysis of human mobilized peripheral blood and umbilical cord blood is heterogeneous, and made up of distinct populations of vesicles and live nucleated cells that variably express CD34 and CD133. The ability to determine miRNA profiles from rare sorted populations such as Lin- CD45- CD34/CD133+ cells will enable the possible further understanding of the function of these cells, as well as the role of miRNAs in regulating their cellular processes. It is also hoped that the data will enhance the understanding of the potential clinical utility of such cells isolated from human blood products. Disclosures: Jiang: NeoStem, Inc.: Employment. Mallinson:Sistemic, Ltd.: Employment, Equity Ownership. Olijnyk:Sistemic, Ltd.: Employment. Paterson:Sistemic, Ltd.: Employment. Ridha:Sistemic, Ltd.: Employment. Tang:NeoStem, Inc.: Employment. O'Brien:Sistemic, Ltd.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Fong:NeoStem, Inc.: Employment. O'Neill:NeoStem, Inc.: Employment.

scholarly journals Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Bucar ◽  
André Dargen de Matos Branco ◽  
Márcia F. Mata ◽  
João Coutinho Milhano ◽  
Íris Caramalho ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Cell Number ◽  
Alternative Source ◽  
Hematopoietic Stem ◽  
Promising Alternative

Abstract Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

scholarly journals Use of Acellular Umbilical Cord-Derived Tissues in Corneal and Ocular Surface Diseases

Medicines ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 12
Author(s):  
Arianna A. Tovar ◽  
Ian A. White ◽  
Alfonso L. Sabater
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ocular Surface ◽  
Cellular Proliferation ◽  
Mononuclear Cells ◽  
Autologous Serum ◽  
High Concentration ◽  
Ocular Surface Diseases ◽  
Natural Healing

Blood derived products have become a valuable source of tissue for the treatment of ocular surface diseases that are refractory to conventional treatments. These can be obtained from autologous or allogeneic sources (patient’s own blood or from healthy adult donors/umbilical cord blood, respectively). Allogeneic cord blood demonstrates practical advantages over alternatives and these advantages will be discussed herein. Umbilical cord blood (UCB) can be divided, generally speaking, into two distinct products: first, mononuclear cells, which can be used in regenerative ophthalmology, and second, the plasma/serum (an acellular fraction), which may be used in the form of eyedrops administered directly to the damaged ocular surface. The rationale for using umbilical cord serum (UCS) to treat ocular surface diseases such as severe dry eye syndrome (DES), persistent epithelial defects (PED), recurrent epithelial erosions, ocular chemical burns, graft versus host disease (GVHD), among others, is the considerably high concentration of growth factors and cytokines, mimicking the natural healing properties of human tears. Allogeneic serum also offers the opportunity for therapeutic treatment to patients who, due to poor heath, cannot provide autologous serum. The mechanism of action involves the stimulation of endogenous cellular proliferation, differentiation and maturation, which is highly efficient in promoting and enhancing corneal epithelial healing where other therapies have previously failed.

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