ABSTRACT
Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). We investigated the role of human MSCs in NOD-SCID mice repopulation by unrelated human hematopoietic cells and studied the immune interactions between human MSCs and unrelated donor blood cells in vitro. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cell-free MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. These preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic.
Subject(s)
Hematopoietic Stem Cells/cytology , Lymphocyte Activation/immunology , Mesoderm/cytology , Stem Cell Transplantation/methods , T-Lymphocytes/immunology , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Flow Cytometry , Humans , Immunosuppression Therapy , Interferon-gamma/analysis , Mice , Mice, Inbred NOD , Mice, SCID , Transplantation, HeterologousABSTRACT
Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investigated for a potential therapeutic role as hematopoietic support cells following chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited evidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich marked hMSCs using chemotherapy. We have determined that hMSCs are markedly sensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pretreated with O(6)-benzylguanine (BG) resulting in a more than four-fold decrease in BCNU IC(90). The MFG retroviral vector encoding a bicistronic transcript for green fluorescent protein (GFP) and mutant (G156A)-methylguanine methyltransferase (G156A-MGMT), which encodes O(6)-alkylguanine-DNA alkyltransferase (AGT), conferring, BG plus BCNU resistance, transduced a high percentage of hMSCs. Transduced hMSCs had high expression of GFP and AGT and became significantly resistant to BG and BCNU. Furthermore, the proportion of GFP expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% following BG and BCNU treatment in vitro. Intravenously infused hMSCs were detected in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultured from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse could be recultured at a low frequency, but enriched by BG and BCNU treatment from 0.05 +/- 0.03% to 0.55 +/- 0.4 (p = 0.028, Welch t-test). Our results indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGMT is a powerful selectable gene for a second marker gene in hMSCs. Drug resistance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.
Subject(s)
Antineoplastic Agents/pharmacology , Carmustine/pharmacology , Guanine/analogs & derivatives , Guanine/pharmacology , Mesoderm/cytology , O(6)-Methylguanine-DNA Methyltransferase/genetics , Stem Cells/drug effects , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Brain/metabolism , Cell Survival/drug effects , Cell Transplantation/methods , Cells, Cultured , DNA/genetics , DNA/metabolism , Dose-Response Relationship, Drug , Drug Resistance, Multiple , Dystrophin/genetics , Flow Cytometry , Genetic Vectors/genetics , Green Fluorescent Proteins , Humans , Infusions, Intravenous , Liver/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Lung/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Mutation, Missense , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retroviridae/genetics , Spleen/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Tissue Distribution , TransfectionABSTRACT
The biologic factors that control the behavior of basal cell carcinoma are poorly understood. This study was undertaken to elucidate the mechanisms responsible for the altered protein levels of several basement membrane components found in basal cell carcinoma. RNA was isolated from papulonodular basal cell carcinoma, normal human epidermal keratinocytes, and normal human skin, reverse transcribed to cDNA and amplified by the polymerase chain reaction utilizing primers specific for the 230 kDa bullous pemphigoid antigen (BPAG1), the 180 kDa bullous pemphigoid antigen (BPAG2), the alpha6 and beta4 chains of the alpha6beta4 integrin complex, and the beta3 chain of laminin 5. Southern blots probed with internal oligonucleotides confirmed that each polymerase chain reaction was specific for the basement membrane component amplified. The mRNA expressions of basement membrane components were indistinguishable between normal human epidermal keratinocytes and normal human skin, and subsequent experiments used normal human epidermal keratinocytes as controls. Quantitation of polymerase chain reaction products indicated that all basement membrane specific mRNA were significantly decreased in basal cell carcinoma as compared with normal human epidermal keratinocytes. The mean polymerase chain reaction product intensities were significantly less in the basal cell carcinoma as compared with the normal human epidermal keratinocytes at the following levels: p < 0.001 for alpha6 and beta4 integrins and the beta3 chain of laminin 5; p < 0.01 for BPAG1; and p < 0.05 for BPAG2. Our results demonstrate that decreased protein levels of basement membrane components in basal cell carcinoma are due at least partially to a downregulation of basement membrane mRNA species. We speculate that these alterations may lead to a structurally incompetent basement membrane that facilitates the basal cell carcinoma ability to invade tissues.
Subject(s)
Basement Membrane/chemistry , Carcinoma, Basal Cell/genetics , Carrier Proteins , Collagen , Cytoskeletal Proteins , Nerve Tissue Proteins , Non-Fibrillar Collagens , RNA, Messenger/metabolism , Antigens, CD , Autoantigens/analysis , Blotting, Southern , Cell Adhesion Molecules , Culture Techniques , Desmosomes/chemistry , Desmosomes/metabolism , Dystonin , Humans , Integrin alpha6 , Integrin beta4 , Keratinocytes/chemistry , Pemphigoid, Bullous/immunology , Polymerase Chain Reaction , RNA-Directed DNA Polymerase , Skin/chemistry , Kalinin , Collagen Type XVIIABSTRACT
The professionalisation of the care and protection of children in the West has resulted from a complex of events that are particular to Europe, and that reflect Western cultural beliefs about the self, subjective experience and interpersonal connections. Attempts to universalise Western definitions of 'child abuse' fail to take into account the cultural and social realities of 'non-Western' children and families. Clinical material is presented from two South Asian families in Britain, and attributions of meaning by Western professionals and the South Asian family are discussed.
