Fer saline (PBS, AccuGENEH Lonza Cat: 51226). The second protocol involved the use of density gradient centrifugation over Ficoll-PaqueTM PREMIUM (GE healthcare). Cord blood mononuclear Cells (CBMCs) were isolated from the interphase after gradient centrifugation (at 840g) over FicollPaqueTM PREMIUM and treated once with lysis buffer due to Title Loaded From File residual RBCs. The pellet was washed twice with PBS, resuspended, and centrifuged at 1000g to isolate the light-density mononuclear cells. A Human Neural Stem Cell (hNSC) line that was generated and expanded [K. Pong, Nicholas, A. P., Subramanian, V., Thompson, P. R., Ferretti, P. Peptidylarginine deiminase 3 – a novel early mediator of calcium-induced death in human neural stem cells. Unpublished data, submitted, 2013] as previously described [14] was used as a positive control for Nestin, CD133, and Sox2. A line of Mesenchymal Stem Cells (UC-MSC) from Wharton’s jelly generated as 16574785 described previously by Weiss et al [15] was used in some experiments as a control for gene expression analysis.Flow CytometryFlow cytometry analysis was carried out with a BD FACSCalibur TM. Data analysis was performed using FlowJo 6.4.7 software (Tree Start, Inc. 1997?006). The monoclonal antibodies used were: from BD Bioscience: Lineage Cocktail 1 (FITC; Cat: 340546), CD34 (APC; clone: 581), CXCR4 (also known as CD184 Cat: 555974, clone: 12G5), 7AAD (Cat: 51-68981E), CD41a (FITC and APC; clone: HIP8), Oct3/4 (PerCP-Cy TM 5.5; clone: 40/Oct-3), Anti-Sox2 (Alexa Flour 488; clone: 245610), SSEA-4 (FITC and PE; clone:MC813-70), and Nestin (PE; clone: 25NESTIN); from eBioscience, CD45 (biotin; clone:HI30), CD235a (biotin; clone: HIR2), Hematopoietic Lineage (FITC including CD2, CD3, CD14, CD16, CD19, CD56, and CD235a; Cat: 22-7778); from Miltenyi Biotec: CD45 (FITC, PE, PerCP, and APC clone: 5B1); CD133 (PE and APC clone: 293C3), andMaterials and Methods Cell SourcesWhole cord blood from non-clinical standard units was supplied by the Anthony Nolan Cell Therapy Centre (http://www. anthonynolan.org/Title Loaded From File Healthcare-professionals/Cord-blood-services. aspx) from consented mothers with healthy full-term pregnancies. Total nucleated cells (TNCs) were prepared using two different protocols. The first protocol involved lysis of red blood cells using BD Pharm Lyse (Cat: 555899). Briefly, hUCB was centrifuged at 1000g to remove the cord plasma as reported by Bhartiya, D., et al. [5]. The pellet containing red blood cells (RBCs) and TNCs was treated with lysing buffer (dilution 1:5) for 15 minutes at roomhUCB ELSc Are a Heterogeneous PopulationFigure 1. Recovery, viability, and clonogenic potential of the Lin2CD452 population. (A) The box plots show the percentage of cells recovered after magnetic isolation using lysing buffer (Ly) or Ficoll gradient centrifugation (F) (*p = 0.0306). (B) The box plots show the percentage of Lin2CD452 CXCR4+/CD34+/Nestin+ viable (7AAD negative) and dead (7AAD positive) cells using lysing buffer: note that the percentage of viable cells is significantly higher than that of dead cells (n = 5; *p = 0.0001); and the gate shows the 7AAD negative Lin2CD452CD34+ as an example (Staining for 7AAD and Nestin is not technically possible as Nestin is an intracellular staining). (C) The box plots show the number of colonies formed by cells isolated using lysis buffer after 14 days in culture (n = 5; *p = 0.0005). (D) Absolute count of both Lin2CD452CD34+ and Lin2CD45dimCD34+ cells plated in CFUs. CFUs = Colony Forming Un.Fer saline (PBS, AccuGENEH Lonza Cat: 51226). The second protocol involved the use of density gradient centrifugation over Ficoll-PaqueTM PREMIUM (GE healthcare). Cord blood mononuclear Cells (CBMCs) were isolated from the interphase after gradient centrifugation (at 840g) over FicollPaqueTM PREMIUM and treated once with lysis buffer due to residual RBCs. The pellet was washed twice with PBS, resuspended, and centrifuged at 1000g to isolate the light-density mononuclear cells. A Human Neural Stem Cell (hNSC) line that was generated and expanded [K. Pong, Nicholas, A. P., Subramanian, V., Thompson, P. R., Ferretti, P. Peptidylarginine deiminase 3 – a novel early mediator of calcium-induced death in human neural stem cells. Unpublished data, submitted, 2013] as previously described [14] was used as a positive control for Nestin, CD133, and Sox2. A line of Mesenchymal Stem Cells (UC-MSC) from Wharton’s jelly generated as 16574785 described previously by Weiss et al [15] was used in some experiments as a control for gene expression analysis.Flow CytometryFlow cytometry analysis was carried out with a BD FACSCalibur TM. Data analysis was performed using FlowJo 6.4.7 software (Tree Start, Inc. 1997?006). The monoclonal antibodies used were: from BD Bioscience: Lineage Cocktail 1 (FITC; Cat: 340546), CD34 (APC; clone: 581), CXCR4 (also known as CD184 Cat: 555974, clone: 12G5), 7AAD (Cat: 51-68981E), CD41a (FITC and APC; clone: HIP8), Oct3/4 (PerCP-Cy TM 5.5; clone: 40/Oct-3), Anti-Sox2 (Alexa Flour 488; clone: 245610), SSEA-4 (FITC and PE; clone:MC813-70), and Nestin (PE; clone: 25NESTIN); from eBioscience, CD45 (biotin; clone:HI30), CD235a (biotin; clone: HIR2), Hematopoietic Lineage (FITC including CD2, CD3, CD14, CD16, CD19, CD56, and CD235a; Cat: 22-7778); from Miltenyi Biotec: CD45 (FITC, PE, PerCP, and APC clone: 5B1); CD133 (PE and APC clone: 293C3), andMaterials and Methods Cell SourcesWhole cord blood from non-clinical standard units was supplied by the Anthony Nolan Cell Therapy Centre (http://www. anthonynolan.org/Healthcare-professionals/Cord-blood-services. aspx) from consented mothers with healthy full-term pregnancies. Total nucleated cells (TNCs) were prepared using two different protocols. The first protocol involved lysis of red blood cells using BD Pharm Lyse (Cat: 555899). Briefly, hUCB was centrifuged at 1000g to remove the cord plasma as reported by Bhartiya, D., et al. [5]. The pellet containing red blood cells (RBCs) and TNCs was treated with lysing buffer (dilution 1:5) for 15 minutes at roomhUCB ELSc Are a Heterogeneous PopulationFigure 1. Recovery, viability, and clonogenic potential of the Lin2CD452 population. (A) The box plots show the percentage of cells recovered after magnetic isolation using lysing buffer (Ly) or Ficoll gradient centrifugation (F) (*p = 0.0306). (B) The box plots show the percentage of Lin2CD452 CXCR4+/CD34+/Nestin+ viable (7AAD negative) and dead (7AAD positive) cells using lysing buffer: note that the percentage of viable cells is significantly higher than that of dead cells (n = 5; *p = 0.0001); and the gate shows the 7AAD negative Lin2CD452CD34+ as an example (Staining for 7AAD and Nestin is not technically possible as Nestin is an intracellular staining). (C) The box plots show the number of colonies formed by cells isolated using lysis buffer after 14 days in culture (n = 5; *p = 0.0005). (D) Absolute count of both Lin2CD452CD34+ and Lin2CD45dimCD34+ cells plated in CFUs. CFUs = Colony Forming Un.
