Stability and utility of the unique human small cell carcinoma line SHP-77.

The human small cell (oat cell) carcinoma line, SHP-77, established by Fisher and Paulson in 1977 and originally described as a "large cell variant of oat cell cancer" has been evaluated by several different parameters and shown even after more than 200 passages to retain properties described for the original cell line. Karyotypic, histological, and biochemical features are retained, as well as tumorigenicity in nude mice. The original authors' suggestion that this is a propitious cell line for both in vitro and in vivo studies is supported by this report. Modulation of growth characteristics in vivo (in xenografts) emphasizes the plasticity of this unique line which serves as a valuable model for basic as well as therapeutic studies. SHP-77 can serve as an in vitro target in 51Cr and 111In release cytotoxicity assays as well as in in vivo nude mouse assays for evaluating immune reactivity of cells and serum from lung cancer patients. The potential histological variability of SHP-77, despite its biochemical stability, calls attention to the inadequacy of histological criteria for lung tumor classification.

Long-term expression of the glucocerebrosidase gene in mouse and human hematopoietic progenitors.

Gaucher disease (GD), one of the most common inherited metabolic disorders, is an excellent candidate for gene therapy using hematopoietic stem cells as targets. Animal models have demonstrated the feasibility of introducing the human glucocerebrosidase (GC) gene into hematopoietic progenitors with long term expression using a variety of retroviral vectors. We have previously demonstrated the expression and integration of the human GC gene in mouse hematopoietic progenitors and their progeny 4-8 months post transplant in primary recipients using the retroviral vector MFG-GC. We now demonstrate enzyme expression in peripheral blood lymphocytes of secondary recipients more than 12 months post transplantation. We also show a transduction efficiency of up to 95% in colony forming unit-granulocyte macrophage (CFU-GM) colonies generated from transduced CD34+ cells from a variety of sources, using a centrifugation promoted infection protocol. Transduction has also been documented in long term culture initiating cells (LTCIC) from the same transduced CD34+ cells. These data indicate efficient transduction of mouse hematopoietic progenitors as well as human CD34+ cells using the retroviral vector MFG-GC.

Long-term expression and secretion of human glucocerebrosidase by primary murine and human myoblasts and differentiated myotubes.

Gaucher disease (GD) is caused by a deficiency in glucocerebrosidase (GC). Enzyme replacement for GD disease is effective but expensive and requires life-long treatment. Development of alternative therapeutic strategies is therefore important. One approach is an enzyme delivery system which could supply GC into the circulation continuously. We have previously reported that human GC cDNA in a retroviral vector (MFG-GC) efficiently transduced a murine myoblast line (C2C12) and expressed GC intracellularly and extracellularly. Now we have demonstrated that primary murine and human myoblasts are transduced at very high efficiency by MFG-GC (five to ten copies of human GC gene per cell at a multiplicity of infection of 5-10), 100% of MFG-GC transduced cells expressed human GC. The transduced primary murine and human myoblasts had an intracellular GC activities about five to ten times above nontransduced controls. Furthermore, transduced primary myoblasts secreted human GC extracellularly for up to 35 weeks in vitro. The secreted human GC is specifically taken up by bone marrow derived macrophages, the cell type most important to the pathogenesis of GD. These data suggest that transduced primary myoblasts may be useful in supplying GC as an alternative approach to the treatment of GD.

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors.

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.

Cytokine mobilization of peripheral blood stem cells in patients with Gaucher disease with a view to gene therapy.

As clinical trials for gene therapy in Gaucher disease (GD) begin, questions regarding the biology of the hematopoietic stem cell in this disease remain unanswered. This study demonstrates the ability to mobilize and collect CD34+ cells in three patients with the disorder. Our RAC/FDA-approved clinical trial utilizes mobilized peripheral blood stem cells (PBSC) as the target cells for gene transfer. In this approach, a white blood cell fraction is collected by apheresis, enriched for CD34+ cells, and transduced with a retroviral vector carrying the glucocerebrosidase (GC) gene. Transduced cells from the patient with activity corrected to at least normal levels will be returned to the patient without myelosuppressive therapy. We report here the effect of cytokines in mobilizing PBSC in three patients with GD. Two (patients 1 and 2) were given granulocyte colony-stimulating factor (G-CSF) at a dose of 5 micrograms/kg/d and one (patient 3) was given 10 micrograms/kg/d for 10 days. Leukaphereses were done daily for 5 days and the products enriched for CD34+ cells using the clinical Ceprate (CellPro) column. The CD34+ cells in all fractions were monitored daily during mobilization and leukaphereses. Subset analysis for the expression of Thy-1, CD38, HLA-DR, and CD33 on the CD34+ cells was performed. An increase in CD34+ cells in the peripheral blood was noted from day 5 onward (up to a six-fold increase). Up to a 625-fold enrichment in CD34+ cells in the apheresis product was noted using the clinical Ceprate column. Totals of 1.2, 3.5, and 2.1 x 10(6) CD34+ cells/kg were collected in the three patients. A diminution in the percent of CD34+/Thy-1+ cells was noted with enrichment. In vitro retroviral transduction of the CD34-enriched cells using centrifugation promoted transduction protocol previously described (Bahnson AB et al., Centrifugal enhancement of retroviral-mediated gene transfer. Journal of Virology Methods 54:131, 1995) and modified for clinical use, demonstrated a mean transduction efficiency of 37% (range 8.3-87.1%) in clonogenic cells and up to 50% in long-term culture-initiating cells (LTC-IC) at week 6. Significantly, we have been able to achieve up to a 50-fold increase in the level of GC above deficient levels in the patients' CD34+ enriched cells when maintained in vitro in culture. The study demonstrates that up to a six-fold increase in CD34+ cells in the PB can be achieved with cytokines in patients with GD. CD34+ cells can be collected in numbers sufficient for conventional transplantation and transduced efficiently in vitro. In gene therapy trials for genetic disorders to date, myelosuppressive therapy is not advocated. The clinical trial will demonstrate whether this number of transduced CD34+ cells will be adequate for competitive engraftment of genetically corrected PBSC.

Long-term expression, systemic delivery, and macrophage uptake of recombinant human glucocerebrosidase in mice transplanted with genetically modified primary myoblasts.

A critical requirement for treatment of Gaucher disease via systemic delivery of recombinant GC is that secreted enzyme be in a form available for specific takeup by macrophages in vivo. In this article we investigated if transplanted primary myoblasts can sustain expression of human GC in vivo and if the secreted transgene product is taken up by macrophages. Transduced primary murine myoblasts were implanted into syngeneic C3H/HeJ mice. The results demonstrated that transplanted mice sustained long-term expression of transferred human GC gene in vivo. Furthermore, human GC is secreted into the circulation of mice transplanted with syngeneic primary myoblasts retrovirally transduced with human GC cDNA. The transplanted primary myoblasts differentiate and fuse with adjacent mature myofibers, and express the transgene product for up to 300 days. Human GC in the circulation reaches levels of 20-280 units/ml of plasma. Immunohistochemical studies of the target organs revealed that the secreted human GC is taken up by macrophages in liver and bone marrow. Immunochemical identification of reisolated myoblasts from transplanted mice showed that MFG-GC-transduced cells also survived as muscle stem cells in the implanted muscle. These results present in encouraging prospect for the treatment of Gaucher disease.

Centrifugal enhancement of retroviral mediated gene transfer.

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

Methods for Retrovirus-Mediated Gene Transfer to CD34(+) Enriched Cells.

Hematopoietic stem cells (HSC) provide for contmuous replenishment of the entire immune and hematopoietic systems, and also replenish themselves in a process termed self-renewal (1).The HSCs can be enriched from hematopoietic tissues using MAbs that bind to the CD34 antigen, a universally recognized marker for hematopoietic progenitors (2-4).Enriched HSC populations are being widely investigated for use in transplantation and gene therapy because they appear to provide rapid hematopoietic reconstitution in myeloablated patients (5-11), and they offer good targets for gene transfer (12-17).

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34 + lin hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well can receive a single hematopoietic stem cell candidate. Sequential movement of each cell-containing well to a microscopic imaging system, serially over a several-day to several-week experiment, is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Bio-box built on the microscope stage. New image analysis software facilitates in the tracking of cell movement, recording of the time of cell division, and immunophenotyping of each of multiple individual or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and also may be applicable to a wide range of other systems of interest in tissue engineering.

Expansion of hematopoietic stem cells in vitro as a model system for human tissue engineering.

The authors have taken a new approach to finding optimal conditions for stimulating conservative division of single isolated CD34(+)lin(-) hematopoietic stem cell candidates from human umbilical cord blood. The approach required the design and development of a novel multi-well single cell combinatorial culture system. This system incorporates the use of a multi-well tissue culture plate in which each well receives a single hematopoietic stem cell candidate. During an experiment lasting several days to weeks, each cell-containing well is moved sequentially and serially to a microscopic imaging system. This movement is facilitated by computer control of a motorized stage and stabilization of the experiment in an environmentally controlled Biobox built on the microscopic stage. New image analysis software facilitates tracking of cell movement, recording the time of cell division, and immunophenotyping of multiple, individual, or recently doubled cells in real time by a robotically controlled pipetting station. The principles of single cell culture should help solve many problems in human hematopoietic stem cell expansion and may be applicable to a wide range of other systems of interest in tissue engineering.