Isolation of hematopoietic progenitors from placenta and chorionic membrane
This study was approved by the University of California at San Francisco Committee on Human Research. We used a method that our group devised for isolating human placental cells 8
that has been modified to achieve the maximum the recovery of hematopoietic cells 9
Isolation of hematopoietic progenitors from umbilical cord blood (UCB)
Samples of UCB were diluted 1:1 with PBS before centrifugation over Nycoprep 1.077 g/ml (Axis-Shield PLC, Oslo, Norway). The resulting light-density cell suspension of UCB was immediately used for phenotypic analyses, fluorescence in situ hybridization, or cell sorting experiments as described below for placental cells.
Isotype-matched control IgG1, IgG2a, IgG2b and IgG3 were purchased from BD Biosciences (San Jose, CA) or BioLegend (San Diego, CA), and IgM was obtained from Invitrogen Corporation (Carlsbad, CA). They were conjugated to fluorescein isothiocyanate (FITC), PE or APC as indicated in the figure legends. Propidium iodide (PI) was purchased from Sigma-Aldrich Chemical Co (St. Louis, MO) and used at a final concentration of 1 μg/ml. The antibodies that were used for cell separation and immunofluorescence included anti-CD34-PE (8G12, IgG1, Invitrogen Co.), anti-CD34-APC (581, IgG1, Beckman Coulter, Brea, CA), anti-CD45-FITC, -PE, and -APC (HI30, IgG1, Invitrogen Co.), anti-CD38-PE (HB-7, IgG1, BD Biosciences), anti-CD56-FITC (C5.9, IgG2b, Exalpha, Inc., Boston, MA), anti-HLA-DR-PE (L243, IgG1, BD Biosciences), anti-CD33-PE (P67.6, IgG1, BD Biosciences), anti-CD13-PE (L138, IgG1, BD Biosciences), anti-CD38-PE (HIT2, IgG1, BD Biosciences), anti-CD235a-FITC or -PE (11E4B7.6, IgG1, Beckman Coulter), anti-CD117-PE (YB5.B8, IgG1, BD Biosciences), anti-CD133-PE or -APC (AC133, IgG1, Milteny Biotec, Inc., Auburn, CA), anti-CD41-PE (HIP8, IgG1, BD Biosciences), anti-CD14-FITC, -PE, or -APC (MFP9, IgG2b, BD Biosciences), anti-CD15-FITC (MMA anti-Leu-M1, IgM, BD Biosciences), and anti-CD3-FITC, -PE, or -APC (SK7, anti-Leu-4, IgG1, BD Biosciences), anti-CD19-FITC (4G7, IgG1, BD Biosciences), anti-CD20-FITC (2H7, IgG2b, BD Biosciences).
Immunofluorescence and flow cytometry
Cell surface phenotypic analyses were performed as previously described 10
using a two-laser FACSCalibur or LSR II cytometer (BD Biosciences) for acquisition of samples and CellQuest (BD Biosciences) or FlowJo, version 9.2, (Tree Star, Inc., Ashland, OR) software for analysis.
Immunolocalization and fluorescence microscopy
Freshly isolated full-term placental tissue was fixed in 3% paraformaldehyde for 90 min, and processed as previously described 11
. Sections (8 μm) of the fixed/frozen tissue were stained with mAbs against CD34 (directly conjugated to FITC; clone 581, IgG1
, BD Pharmingen), and CD45 (clones 2B11 + PD7/26, IgG1
, Dako, Glostrup, Denmark). The binding of antibodies that were not direct conjugates was detected using the appropriate species-specific secondary antibody, a rhodamine-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA). For controls, some sections were stained with the secondary antibody alone. Staining with DAPI (Vectashield mounting medium with DAPI, Vector Laboratories, Burlingame, CA) was used to visualize nuclei. Slides were imaged on a Leica DM 5000 microscope equipped with a Leica 350DX camera (Leica Microsystems, Inc., Buffalo Grove, IL).
Human hematopoietic reconstitution of murine BM and spleen
/J (NOD-SCID) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Research was performed with approval of the Committee on Animal Research at the University of California San Francisco. Female mice were transplanted as adults (≥8 weeks old) by tail-vein injection using a 28g insulin syringe (BD Biosystems) several hours after 325 cGy γ-irradiation. Isolated placental cells were mixed with 2 × 107
irradiated (3000 cGy) human midgestation BM cells, isolated as previously described 12
, were used as carrier cells. Animals were maintained in a barrier facility in microisolator cages under specific-pathogen free conditions.
Analysis of engraftment
Femoral BM and splenocytes were harvested 11 weeks after transplantation. Spleens were passed through nylon sieves (BD Biosystems) to produce a single-cell suspension and light-density splenocytes were isolated by centrifugation over a layer of 1.077 g/ml Lymphoprep (Axis-Shield PLC) medium. Cells were suspended in PBS containing 5% normal mouse serum, 2 μg/ml anti-mouse CD16/CD32 (clone 93, BioLegend, San Diego, CA) and 0.01% NaN3 (Sigma Chemical Co., St. Louis, MO).
Mouse cells were identified by labeling with phycoerythrin (PE)-cyanine dye 7 (PE-Cy7) murine CD45 (clone 30-F11, BD Biosciences) and TER-119 (clone TER-119, BioLegend) and subsequent flow cytometrc analyses. Human leukocytes were identified by staining with allophycocyanin (APC)-H7 (clone 2D1, BD Biosciences). Human cells were further stained for the following fluorescein isothiocyanate (FITC)-labeled mAbs: CD15 (clone VIMC6, Invitrogen Co.), CD34 (clone 581, Invitrogen Co.), CD41 (clone VIP11, Invitrogen Co.), CD56 (clone C5.9, Exalpha Biologicals Inc.), and CD59 (clone MEM-43, Invitrogen Co.). Phycoerythrin (PE) mAbs used were: CD19 (clone SJ25-C1, Invitrogen Co.), CD33 (clone P67.6, BD Biosciences), CD38 (clone HB7, BD Biosciences), CD42b (clone MB45, Invitrogen Co.), and CD235a (clone 11E4B7.6, Beckman-Coulter Inc.). The following APC-labeled mAbs were used: CD14 (clone TüK4, Invitrogen Co.), CD34 (clone 581, Invitrogen Co.), CD71 (clone M-A712, BD Biosciences), and CD133 (clone AC133, Miltenyi Biotec).
After samples were stained with saturating levels of mAbs and washed, the cells were suspended in PBS containing 0.3% bovine serum albumin (Roche Diagnostic Corporation, Indianapolis, IN), 2 μg/ml PI to label dead cells and analyzed using an LSR II flow cytometer (BD Biosystems). Data were analyzed using FlowJo software, version 9.2 (Tree Star, Inc.).