Limited engraftment capacity of bone marrow–derived mesenchymal cells following T-cell–depleted hematopoietic stem cell transplantation

Abstract

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

Myeloablative doses of yttrium‐90‐ibritumomab tiuxetan and the risk of secondary myelodysplasia/acute myelogenous leukemia

“…The assay for committed colony-forming cells (CFCs), including multilineage colony-forming units (CFU-Mix), erythroid burst-forming units (BFU-E), and granulocytemacrophage CFUs (CFU-GM), was carried out as previously described. 37 Briefly, from 1 Â 10 4 to 5 Â 10 4 mononuclear cells were plated in 35-mm Petri dishes in cytokine-supplemented, methylcellulose-based medium (Methocult GF H4034; Stem Cell Technologies, Vancouver, British Columbia, Canada). Progenitor cell growth was evaluated according to previously published criteria.…”

“…Fibroblast CFUs (CFU-F) were assayed according to a previously described technique. 37 Briefly, mononuclear cells (5 Â 10 4 /mL resuspended in alpha-medium supplemented with fetal bovine serum; 12.5%, volume/volume), horse serum (12.5%, volume/volume), and freshly dissolved hydrocortisone (10 À6 M) were plated onto 60-mm Petri dishes. Fibroblastoid cell aggregates of >50 cells were scored as CFU-F after staining with crystal violet.…”

Interleukin-17A: A T-Cell-Derived Growth Factor for Murine and Human Mesenchymal Stem Cells

“…The contribution for the reconstitution of the HME following bone marrow transplantation (BMT) with stroma precursors remains controversial (donor vs. host) because some investigators were unable to detect donor-derived stroma cells, whereas others did [20 -24]. Although effects of chemoradiation are felt to play a major role in the impaired HME recovery following BMT, it has been hypothesized that T-cells might be involved in the process of stroma cell engraftment or host microenvironmental recovery or maintenance, although the mechanisms remain elusive [25]. Here, we have investigated the hypothesis that IL-17A might be one of the elusive T-cell-derived regulatory factors that exert a proliferative effect on MSC precursors.…”

Transformation by Retroviral Vectors of Bone Marrow-Derived Mesenchymal Cells Induces Mitochondria-Dependent cAMP-Sensitive Reactive Oxygen Species Production

“…MSCs were obtained from bone marrow aspirate from healthy subjects after informed consent was obtained, as described in [24]. Following centrifugation on a Ficoll-Hypaque density gradient (1.077 g/ml), marrow mononuclear cells were resuspended at a concentration of 2 ϫ 10 6 cells per milliliter in ␣ medium (Gibco-BRL, Grand Island, NY, http://www.gibcobrl.com) supplemented with 10% human serum, L-glutamine (2 mmol/l), 2-mercaptophenol (10 Ϫ4 mol/l), inositol (0.2 mmol/l), folic acid (20 mol/l), and hydrocortisone 10 Ϫ6 mol/l; cultured in 175-cm 2 flasks; and incubated at 37°C, 5% CO 2 in a humidified thermostat for 2-4 weeks until the confluent layer formed.…”

“…Puromycin selection was performed using puromycin at a concentration of 2.5 g/ml for 5-6 days. MSCs were phenotypically characterized by challenging against a panel of monoclonal antibodies recognizing CD45, CD14, CD90, CD105, and STRO-I and analyzed by cytofluorimetry as described in [24].…”