An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells

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Human hematopoietic stem cells (HSCs) represent rare cell populations that are characterized by their unique ability to self-renew, differentiate into multiple lineages, and rescue myeloablated hosts.1-3 Important differences in the functional properties of different tissue sources of HSCs have been well documented.2,3 Some of these functional differences appear to be based on intrinsic, ontogenyrelated properties.4-7 In addition, stem cells that have been exposed ex vivo to cytokines possess an impaired ability to repopulate myeloablated hosts.8-12 These acquired defects have been attributed to a cytokine-induced reduction in stem cell self-renewal (replicative senescence) or an acquired, cell cycle-specific defect in homing to the marrow.8,13-15 Many of the functional properties characteristic of the various tissue sources of human HSCs are readily apparent with their use as grafts during clinical transplantation.16-23 Cord blood (CB) grafts are associated with delayed times to hematologic reconstitution when compared to marrow grafts, whereas mobilized stem cell grafts are associated with shortened periods required for hematologic reconstitution.16,17,20-23 These functional properties have been further characterized using a variety of in vitro and in vivo stem cell assays.24-33 These assays include in vitro long-term stem cell cultures and a variety of in vivo assays in which human stem cells engraft and differentiate in severe combined immunodeficient disease (SCID) mice or early gestation sheep fetuses, producing xenogenic chimerism that persists for months to years.24-33 A direct comparison of the functional properties of these various sources of human stem cell populations has not, however, previously been possible. Studies of the long-term functional capacity of various sources of murine stem cells, however, have been achieved with the use of competitive repopulation assays.34-40 Stem cell function of a donor with a particular genotype has been assayed by mixing its marrow with a constant number of marrow cells from a second donor with a distinguishable phenotypic marker and measuring the relative ability of each donor cell population to reconstitute stem celldepleted recipients.34 Such competitive repopulation assays have been useful for the study of the behavior of various sources of murine HSCs and their behavior after ex vivo expansion.34-40 In this report, we describe a new in vivo assay system that now permits the direct assessment of the relative marrow repopulating capacity and differentiative capacity of various sources of human HSCs. Materials and methods Cell collection and separation Samples of CB were obtained from normal full-term deliveries after gaining informed parental consent according to guidelines established by the University of Illinois at Chicago Institutional Review Board. CB samples were collected by drainage of blood into sterile polypropylene tubes containing preservative-free sodium heparin (ICN, Aurora, OH) at a final concentration of 20 U/mL. CB samples were diluted in Hank balanced salt solution (HBSS) (BioWhittaker, Walkersville, MD) supplemented with 2% heat-inactivated fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), 1 mmol/L HEPES, and 10 U/mL preservative-free sodium heparin. CB mononuclear cells (MNC) were isolated using Isoprep (1.077 g/mL) (Robbins Scientific, Sunnyvale, CA) density centrifugation. Human adult bone marrow (ABM) aspirates were obtained from the iliac crests of healthy normal adult volunteers after informed consent was provided, according to guidelines established by the University of Illinois at Chicago Institutional Review Board. Low-density MNC were separated from ABM by density centrifugation (Ficoll-Paque; Pharmacia LKB, Uppsala, Sweden). CB or ABM low-density MNC were washed twice in HBSS plus 2% heat-inactivated FBS and cryopreserved in Iscove modified Dulbecco medium (IMDM) (BioWhittaker) with 40% FBS and 10% dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO). Cryopreserved samples were stored in liquid nitrogen. A sample of each tissue was reserved from human lymphocyte antigen (HLA)–type determination. Isolation of cord blood and adult bone marrow CD341 cells Cryopreserved CB and ABM CD341 cells were rapidly thawed at 37°C and slowly diluted in IMDM containing 10% FBS and 0.1 mg/mL Dnase I (Boehringer Mannheim, Indianapolis, IN) before further purification. CD341 cells were immunomagnetically enriched from CB or ABM samples using the MACS CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Inc., Auburn, CA) according to the manufacturer’s instructions. Briefly, the MNC were washed and resuspended in Ca11-free and Mg11-free Dulbecco modified phosphate-buffered saline (dPBS) (BioWhittaker) supplemented with 0.5% bovine serum albumin (BSA) (Fraction V; Sigma) and 2 mmol/L EDTA. Cells were incubated with hapten-labeled anti-CD34 antibody (QBEND-10) in the presence of a blocking reagent and then with antihapten coupled to MACS microbeads. Labeled cells were filtered through a 30-mm nylon mesh and separated using a high-gradient magnetic separation column placed in a strong magnetic field. Magnetically retained cells were eluted and their purity was determined by flow cytometry to be more than 85%. Ex vivo expansion cultures of cord blood and adult bone marrow CD341 cells The porcine microvascular endothelial cell line (PMVEC) is a primary cell line derived from 4- to 6-month-old Yucatan minipig brains (Sus scrofa).41 The ability of PMVECs to support the proliferation of early human marrow cells has been previously reported by our group.11,41-43 PMVEC (passages 22 through 29) were maintained and used for hematopoietic cell expansions as previously described.11,41-43 Triplicate cultures of CB or ABM CD341 cells were seeded into 6-well tissue culture plates (Corning-Costar, Cambridge, MA) at 3 to 15 3 104 cells/well with pre-established PMVEC monolayers (PMVEC cocultures) or into plates without PMVEC monolayers (stroma cell-free cultures). All cultures were maintained in IMDM with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin, 1 mg/mL streptomycin (all from BioWhittaker) in a humidified incubator maintained at 37°C with 5% CO2. Cultures were supplemented with a combination of recombinant human cytokines including interleukin (IL)-3 at 10 ng/mL (R&D Systems, Minneapolis, MN), IL-6 at 10 ng/mL (R&D Systems), granulocyte-macrophage colony-stimulating factor (GM-CSF) at 10 ng/mL (R&D Systems), stem cell factor (SCF) at 100 ng/mL (R&D Systems), and FLT-3 ligand (FLT-3L) at 100 ng/mL (R&D Systems). Cultures were replenished twice weekly by replacing half of the medium and cytokines and expanded into additional culture wells with or without pre-established PMVEC monolayers as required. At days 7, 14, and 21, aliquots of cells were harvested for the performance of cell counts, phenotypic analysis, and in vivo assays. The trypan blue exclusion method was used to determine the total viable cell content of expansion cultures. The large size and distinct appearance of PMVECs permitted their exclusion during cell counting and flow cytometric analysis.