Benedikt L. Ziegler

Expression of MUC-1 Epitopes on Normal Bone Marrow: Implications for the Detection of Micrometastatic Tumor Cells

PURPOSE The expression of the carcinoma-associated mucin MUC-1 is thought to be restricted to epithelial cells and is used for micrometastatic tumor cell detection in patients with solid tumors, including those with breast cancer. Little is known, however, about the expression of MUC-1 epitopes in normal hematopoietic cells. MATERIALS AND METHODS MUC-1 expression was analyzed by flow cytometry and immunocytology on bone marrow (BM) mononuclear cells and purified CD34+ cells from healthy volunteers, using different anti-MUC-1-specific monoclonal antibodies. In addition, Western blotting of MUC-1 proteins was performed. RESULTS Surprisingly, 2% to 10% of normal human BM mononuclear cells expressed MUC-1, as defined by the anti-MUC-1 antibodies BM-2 (2E11), BM-7, 12H12, MAM-6, and HMFG-1. In contrast, two antibodies recognizing the BM-8 and the HMFG-2 epitopes of MUC-1 were not detected. MUC-1+ cells from normal BM consisted primarily of erythroblasts and normoblasts. In agreement with this, normal CD34+ cells cultured in vitro to differentiate into the erythroid lineage showed a strong MUC-1 expression on day 7 proerythroblasts. Western blotting of these cells confirmed that the reactive species is the known high molecular weight MUC-1 protein. CONCLUSION Our data demonstrate that some MUC-1 epitopes are expressed on normal BM cells and particularly on cells of the erythroid lineage. Hence the application of anti-MUC-1 antibodies for disseminated tumor cell detection in BM or peripheral blood progenitor cells may provide false-positive results, and only carefully evaluated anti-MUC-1 antibodies (eg, HMFG-2) might be selected. Furthermore, MUC-1-targeted immunotherapy in cancer patients might be hampered by the suppression of erythropoiesis.

Extrathymic T cell differentiation in vitro from human CD34+ stem cells

Although it is well established that T cells are derived from CD34+ stem cells in vivo, and that T cells can develop in the absence of a functioning thymus, it has not proven possible thus far to generate human T cells in vitro from CD34+ cells in the absence of any thymic influence. We now present a limiting dilution cloning culture system that supports the differentiation of highly purified human CD34+ cells to CD3+ T cells in vitro in the complete absence of any thymic components. The culture system features the use of a serum‐free medium supplemented with a cocktail of cytokines including flt‐3 ligand, interleukin‐3 (IL‐3), stem cell factor (SCF), and IL‐2. CD4+ T cell clones capable of mitogen‐stimulated proliferation and response to IL‐2, and expressing a varied TCR‐Vβ repertoire were obtained under these conditions. This culture system therefore supports human T lymphopoiesis in the absence of any thymic influence and may prove useful for the evaluation of extrathymic T cell differentiation in vitro. J. Leukoc. Biol. 64: 733–739; 1998.

Generation of infectious retrovirus aerosol through medical laser irradiation

A novel model system was used to investigate the spread of infectious particles and live cells through the application of lasers commonly used in clinical medicine. Supernatants from a cell line producing recombinant retroviruses carrying a marker gene (neoR) were exposed to Er:YAG‐laser beams. Aerosols were collected from various sites and distances from the point of laser impact and were analyzed by reverse transcription‐polymerase chain reaction (RT‐PCR) for neoR. In addition, a susceptible indicator cell line was used to investigate the presence of infectious virions in collected aerosols. To test the possibility of dissemination of viable cells, a cell line was laser irradiated, and the generated aerosols were analyzed for the presence of viable cells. The viral marker gene neoR could be detected in 16% (distance: 5.0–6.3 cm) to 59% (0.5–1.6 cm) of wells adjacent to the point of laser impact. The presence of infectious viruses in laser vapors conferring G418 resistance could be detected in 3% (distance 5.0–6.3 cm) to 20% (distance: 0.5–1.6 cm) of wells containing susceptible cells, and subsequent PCR analysis of isolated resistant clones revealed the presence of neoR‐RNA and ‐DNA. Viable cells were detected in 40% (distance 0.7–3.6 cm) to 3% (distance 10.7–11.8 cm) of wells adjacent to the point of laser impact. These results demonstrate that laser vapors can contain infectious viruses, viral genes, or viable cells and may promote the spread of infections or tumor cell dissemination. Lasers Surg. Med. 22:37–41, 1998.

Ex vivo manipulation of hematopoietic stem and progenitor cells

Approaches to manipulate peripheral blood progenitor cells (PBPC) ex vivo currently include the selection of CD34+ cells as a means to purge contaminating tumor cells from leukapheresis preparations or to provide a homogeneous starting population for the expansion of hematopoietic progenitor cells as well as the induction of postprogenitor cells of either the myeloid or megakaryocytic lineage. The latter cell populations might be used for an additional transplantation together with PBPC to possibly shorten the period of aplasia. In addition, ex vivo expansion of CD34+ cells can be used to generate autologous tumor-antigen-presenting dendritic cells for immunotherapeutic approaches aiming to treat minimal residual disease following high-dose chemotherapy.

Expansion of stem and progenitor cells.

An increasing interest exists in strategies to manipulate hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in vitro for clinical purposes by use of recombinant hematopoietic growth factors. A major goal of these ex vivo expansion strategies is to expand repopulating HSCs or to generate lineage-committed progenitors in the context of autologous or allogeneic stem cell transplantation. The ex vivo HSC/HPC expansion approach may have several clinical applications: 1) in the autologous setting, reduction of tumor cell contamination within the amplified transplantation product, 2) purging effects through possible growth advantage of HSCs/HPCs over contaminating tumor cells under appropriate in vitro conditions and purging effects of certain HGFs and antitumor agents during ex vivo culture, 3) the possibility of multiple transplants through repetitive use of cryopreserved HSC/HPC samples from one individual, 4) production of dendritic cells for immunotherapy, 5) production of mature blood cells for transfusion therapies including nadir rescue, and 6) gene therapy. Here, we summarize recent developments in the field of HSC/HPC expansion.

Surface Antigen Expression on CD34+ Cord Blood Cells: Comparative Analysis by Flow Cytometry and Limiting Dilution (LD) RT-PCR of Chymopapain-Treated or Untreated Cells

Expression of antigens coexpressed on cord blood (CB) CD34+ cells was evaluated by flow cytometry analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Antigen expression was also comparatively analyzed by flow cytometry and limiting dilution (LD) RT-PCR to investigate effects of chymopapain on epitopes of several cell surface markers: LD RT-PCR allows detection of the expression of antigens degraded by chymopapain which are not identified by flow cytometry. Monoclonal antibodies (MoAbs) that recognize chymopapain resistant epitopes on several coexpressed cell surface markers were identified: these included MoAbs directed against CD11a, CD13, CD18, CD38, CD45RO, CD51, HLA-DR, Thy-1, c-kit, flt-3 (STK-1), and mdr-1. Interestingly, chymopapain treatment caused enhanced staining with MoAbs against HLA-DR, Thy-1, flt-3, mdr-1, and CD51. The frequency (LD RT-PCR) of CD18, CD38, Thy-1, and c-kit RT-PCR signals on pure sorted CD34+ CD18-, CD34+ CD38-, CD34+ Thy-1-, and CD34+ c-kit- cells, respectively, was similar in corresponding subsets treated or not with chymopapain. In contrast, the frequency of CD33 RT-PCR signals on sorted CD34+ CD33- cells was higher in chymopapain-treated samples than in untreated samples and thus confirmed at the transcriptional level that the epitope recognized by anti-CD33 is chymopapain sensitive. Our findings extend data on the phenotypic profile of CB CD34+ cells and show that several key cell surface markers of hematopoietic progenitor cells are chymopapain resistant. In addition, the results of the present study demonstrate that the RT-PCR can be applied to the analysis of multiple RNA species in small numbers of hematopoietic progenitor cells and show that LD RT-PCR allows the identification and frequency determination of rare cells which are undetectable by flow cytometry.

Characterization of Purified and Ex Vivo Manipulated Human Hematopoietic Progenitor and Stem Cells in Xenograft Recipients

Abstract: Research on the biology, regulation, and transplantation of human hematopoietic stem cells requires test systems for the detection, monitoring, and quantitation of these cells. Xenografted animal models provide suitable stem cell assays, since they allow long‐term engraftment, multilineage differentiation, and serial transfer of human hematopoietic cells. Recent techniques for the separation of hematopoietic cells have provided highly purified cellular subsets selected on the basis of the surface marker phenotype. The stem cell content of these subsets, however, is still unclear. Also, innovative approaches for the induction of hematopoietic cell proliferation and differentiation have generated ex vivo manipulated cells whose biological properties and functions still remain to be assessed. This paper reports on the biological characterization of these cell populations by the use of xenograft models.

Ex Vivo Manipulation of Peripheral Blood CD34 + Cells

The success of autologous peripheral blood progenitor cell (PBPC) transplantation is challenged by relapse of malignant disease which might — at least in part — be mediated by graft contaminating tumor cells. Although the clinical role of tumor cell depletion still remains to be demonstrated in prospective, randomized trials, multiple purging strategies are currently pursued in the context of autologous stem cell transplantation. This report is discussing ex vivo manipulations of PBPC transplants with respect to purging of tumor cells, including the positive selection of CD34+ cells with or without negative depletion as well as ex vivo expansion techniques. In addition, adoptive immunotherapy strategies using ex vivo generated autologous dendritic cells for the treatment of minimal residual disease after stem cell transplantation will be discussed.