Isolation of Human Progenitor Populations
Umbilical cord blood (CB) was collected from normal deliveries, according to guidelines approved by the University of California Los Angeles Investigational Review Board. Enrichment of CD34+ cells was performed using the magnetic-activated cell sorting system (Miltenyi Biotec, Auburn, CA). For fluorescence-activated cell-sorting (FACS) sorting, CD34+ enriched cells were incubated with the following anti-human–specific monoclonal antibodies: CD34 PerCP-Cy5.5, CD38 PE-Cy7, CD123 (interleukin-3 receptor alpha) PE, CD45RA PE-Cy5, FITC-labeled lineage-specific antibodies: CD2, CD3, CD4, CD8, CD7, CD10, CD11b, CD14, CD19, CD56, and glycophorin A (Gly A); all from Becton Dickinson, San Jose, CA). An unstained (no antibody) control was used to define negative gates. The following, previously published immunophenotypic definitions were used to isolate myeloid progenitors from thawed CB CD34+ enriched cells by FACS: CD34+CD38-lin-CD45RA-CD123lo (CMP) 
, CD34+CD10+lin- CLP 
and CD34+CD38-lin- hematopoietic stem/progenitor cells (HSPC) 
. Sorting was performed on a FACSAria (Becton Dickinson) equipped with five lasers (355, 405, 488, 561, and 633 nm). Isolated populations were analyzed by FACS to assess post sort purity. For all FACS sorted populations ~ 95–99% purity was achieved based on re-analysis.
Cocultivation on the murine stromal line OP9 
was used to test for B lymphoid and myeloid differentiation. Freshly sorted CD34+ cord blood cells (500–1500 cells) were seeded onto established non-irradiated OP9 stromal cells (American Type Culture Collection, Manassas, VA) in 96-well or 48-well flat-bottomed plates. Cells were grown in a modified medium (DMEM/F12, Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Invitrogen, Carlsbad, CA) treated with charcoal to remove LPA, 50 µM 2-mercaptoethanol (Sigma-Aldrich), penicillin/streptomycin (Gemini Bio Products, Calabasas, CA), IL-7 (5 ng/mL, R&D Systems, Minneapolis, MN), Flt3 ligand (FL, 5 ng/mL, R&D), and thrombopoietin (TPO, 5 ng/mL, R&D). This cytokine combination is permissive for both lymphoid (B-cells) and myeloid (monocytic, granulocytic and megakaryocytic) lineages. Every 3 days thereafter, half the medium was replaced with fresh medium. Lysophosphatidic acid 18
1 Oleoyl-LPA (Tocris Bioscience, MA) was reconstituted in 70% ethanol and added to the fresh culture medium at final concentrations 0.1, 1 or 10 uM initially to determine optimal dose response. All subsequent experiments used a concentration of 1 µM LPA. Cells were cultured for 4 weeks followed by harvesting, immunostaning with fluorochrome labeled antibodies and immunophenotypic analysis of cultured cells. Sphingosine-1-Phosphate and Ki-16425 were purchased from Tocris Bioscience and reconstituted in 4% fatty acid free albumin (Sigma Aldrich, St Louis, MO) solution in phosphate buffered saline or 70% ethanol respectively following the manufacturer’s instructions.
Immunophenotypic Analysis of Cultured Cells
FACS analysis of cultured cells was performed on an LSR II instrument (Becton Dickinson) by direct immunofluorescence staining with human specific monoclonal antibodies after incubation in 1.2% human intravenous immunoglobulin (IVIG; Cutter, Berkley, CA). Lineage-specific differentiation was determined using the following antibodies: CD45-APC Cy7, CD34-PE Cy7, CD41a-PE Cy5, CD66b-PE, GlyA-APC or -PE, CD19-PE, APC or Percp-Cy5.5, and CD14 PE or FITC (all from Becton Dickinson). The following immunophenotypes were used to identify terminally differentiated lymphoid and myeloid cells from culture: monocytes (CD14+CD45+), granulocytes (CD66b+CD45+), megakaryocytes (CD41a+CD45-GLYA-) and B-cells (CD19+CD45+) (). For long term (4 week) culture experiments, the total number of cells per well in each condition was determined by trypan blue microscopy, and the number of differentiated cells was calculated based on % of each lineage phenotype by FACS multiplied to a total cell number in each well.
In vitro system for differentiation of hematopoietic cells.
Cell Migration Experiments
Migration assays were carried out in 24 well Transwell plates from Costar with 6.5 mm diameter and 8.0 µm pore size. Freshly sorted CMP, CLP or HSPC (1,000 cells each) were seeded into the upper chamber in DMEM/F12 supplemented with 5% charcoal treated serum with no growth factors in the presence or absence of LPA (1 uM) in the lower chamber. Migration was assessed based on the number of total cells on the bottom of the lower chamber after 12 hours, determined separately for each cell type using bright field microscopy. Independent experiments were carried out using progenitor populations isolated from 3 different donors.
Cell Proliferation and Apoptosis Analyses
Freshly sorted CMP, CMP or HSPC were seeded into 48 well plates (5,000 cells per well) on OP9 cells, with DMEM/supplemented with 5% charcoal treated serum, with no growth factors in the presence or absence of LPA (1 uM) and cultured for 48 hours without medium change to measure the effect of LPA on proliferation and apoptosis. Prior to harvesting, cells were incubated with bromodeoxyuridine (BrdU) (10 uM) for 30 minutes. Harvested cells were fixed, permeabilized, and stained with FITC or APC conjugated antibody against BrdU. Unstained cells were used to set negative gates. Apoptosis rates in progenitor populations wasere assessed using FACS-based Annexin V assay (BD Bioscience). Equal numbers (3000 of CD45 gated hematopoietic cells recovered from culture) of each population was analyzed for BrdU incorporation or Annexin V binding using FACS analysis.
Specimens of adult sponge bone (3 individual specimens) were provided by the UCLA Translation Pathology Core Laboratory. All specimens were from patients with no hematopoietic disorders. Fetal bones (16–18 weeks of pregnancy) were obtained from Novogenix (Los Angeles, CA) fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Lois, MO, in PBS). Fixed tissues were embedded in paraffin, sectioned and subjected to histological immunohistochemical analyses. The murine stromal cell lines MS5 and OP9 (American Type Culture Collection (ATCC, Rockville, MD, USA) or primary human bone marrow derived mesenchymal stromal cells (passage 2–3) (AllCells Inc, Emeryville CA) were seeded into chamber slides (BD Bioscience) in DMEM/F12 (Invitrogen) medium supplemented with 20% fetal bovine serum. The next day, cells were fixed with 4% paraformaldehyde and subjected immunohistochemical analysis. Polyclonal antibodies against autotaxin, PPAP2a and CD146 were purchased from Abcam Inc. (Cambridge, MA) Secondary horse radish peroxidase (HRP) conjugated IMPRESS anti-rabbit and anti-mouse antibodies and 3, 3′-diaminobenzidine (DAB) substrate (Vector Labs) were used for the visualization of positively labeled regions. Images were acquired using the Zeiss Axiovision software version 4.8 Carl Zeiss Microscope (Carl Zeiss, Germany) equipped with ApoTome.2: Modules for Axio Imager.2 and Axio Observer with 40x (1.3 numerical aperture (NA)) and 63x (1.4 NA) oil-immersion objectives.
Quantitative Real-time PCR
Total RNA was extracted from cells (~5,000 sorted cells were used for each population) using the RNeasy Micro Kit, and converted to cDNA using the Omniscript RT Kit (kits were from Qiagen Sciences, Maryland, USA). Total RNA concentration for all samples was within 5–20 ng/ml range as determined by Nanodrop analysis. No template amplification was carried out prior to cDNA synthesis.
Next, SYBR Green RT-PCR amplification and detection was performed using an ABI Prism 7900 HT (Applied Biosystems) as previously described. The comparative Ct
method for relative quantification (2−ΔΔCt
) was used to quantitate gene expression according to Applied Biosystems’ recommendations [7900 HT Real-Time fast and SDS enterprise and database user guide
]. Expression of target genes was normalized to the level of the house-keeping gene RPL-7 and expressed relative to a calibrator (sample in each set with lowest expression). All primer sequences were obtained from Harvard University Primer Bank (http://pga.mgh.harvard.edu/primerbank
) Primer sequences used for QPCR are available on request.