Flowing cells through pulsed electric fields efficiently purges stem cell preparations of contaminating myeloma cells while preserving stem
http://www.100md.com
《血液学杂志》
Science Research Laboratory Inc, Somerville, MA
the Center for Regenerative Medicine and Technology, Massachusetts General Hospital
the Jerome Lipper Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.
Abstract
Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic–severe combined immunodeficient (NOD/SCID)/2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.
Introduction
Contaminant cancer cells in bone marrow (BM) or mobilized peripheral blood (PB) transfusions have been shown to correlate with disease relapse in the setting of some hematologic malignancies.1-4 In recent years, several technologies have been employed to purge tumor cells from autologous transfusions, including antibody-mediated selection for progenitor cells,2,5-8 depletion of tumor cells,9 genetic modification of tumor cells,10,11 selective chemical or physical means of tumor depletion,12,13 and in vitro expansion of hematopoietic cells.14 Clinical studies have employed tumor purging from stem cell preparations in non-Hodgkin lymphoma (NHL) with recent evidence of clinical benefit.1,15 Whereas no studies on a similar scale or with similar results exist for multiple myeloma (MM), we selected this hematologic disease as a test case for our purging technology since BM samples from patients with myeloma are consistently infiltrated with tumor, the tumor cells are readily quantified, and autologous transplantation is used to treat patients with this disorder. It should be emphasized however, that the data presented here simply use myeloma as an example and do not argue for or against the clinical utility of autologous transplantation with purging for MM.
The NHL studies that did use purging employed CD34 selection that is time-, cost-, and labor-intensive, and may not efficiently preserve hematopoietic stem cells. We sought to test a different method based on prior studies showing that defined electric field pulses applied to static, small volume samples selectively depleted breast cancer and megakaryocyte cell lines by 2 to 2.5 logs from mixtures with blood cells, and preserved small cells including lymphocytes and CD34+ cells.16 The principle behind selective pulsed electric field (PEF) purging is that a cell's cytosol is largely conductive, but the lipid cell membrane does not conduct electricity.17,18 The voltage developed across each cell is proportional to the cell's diameter. Under defined electric field conditions, larger cells are killed without altering the viability of smaller cells, including hematopoietic stem cells (HSCs). Whereas HSCs and resting lymphocytes are generally 6 μm to 8 μm in diameter,19,20 myeloma cells and other tumor cells are generally more than 10 μm in diameter.
We have now applied this technology in a modified format that permits continuous and rapid pulsing of clinically relevant numbers of cells (> 109 cells in 30 minutes) at controlled flow rates that negate the effects of cell concealment or cell settling (manuscript in preparation).22 Applying this technology to myeloma, we define tumor cell depletion by 3 to 6 orders of magnitude without sacrificing functional stem cells.
Study design
Preparation of primary cells and cell lines
PB was obtained from donors at Massachusetts General Hospital (MGH). BM was obtained from NDRI (Philadelphia, PA), and 2 mL to 5 mL of discard BM aspirates were obtained from patients with MM at the Dana-Farber/Harvard Cancer Center (institutional review board approval no. 1999-P008 401/4). Bone marrow or peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation. Cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA).
Flowing PEF apparatus
A prototype flowing PEF apparatus was designed and constructed by Science Research Laboratory (SRL). Components include the flowing treatment chamber, Cytopulse (Rockville, MD) electric pulse driver system, Tektronix oscilloscope (Beaverton, OR), PC-based control system, syringe pumps (Harvard Apparatus, Framingham, MA), and 4-way valve and outlet for purging air (manuscript in preparation).23
In vitro assays and staining
For tumor regrowth assays, cells were serially diluted in triplicate in conditioned medium/RPMI/fetal calf serum (FCS) in 96-well plates. After 2 weeks, plates were scored for colony formation (> 1 colony of > 4 viable tumor cells).
Colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays were performed as described.1
Engraftment of NOD/SCID/2m-/- mice
Six- to 8-week-old nonobese diabetic–severe combined immunodeficient (NOD/SCID)/2m-/- mice were obtained from the Jackson Laboratory (Bar Harbor, ME), handled, maintained, and underwent transplantation as described.21
Results and discussion
Selective PEF purging of carboxyfluoroscein diacetate succinimidyl ester (CFSE)–labeled myeloma cell lines (RPMI8226), mixed with PBMCs or bone marrow cells, was achieved at 1.4 kV/cm (Figure 1A-B). After PEF, viable cells were small and confined largely to the "lymphocyte gate" of a forward- and side-scatter plot, known to contain stem and progenitor cells, while the high forward- and side-scatter myeloma cells largely disappeared (Figure 1B, left). Purging of myeloma cells (CFDA-SE+) as well as monocytes (CD14+), with preservation of lymphocytes (CD3+/CD19+), was also evident.
Dose response to PEFs was evaluated. We noted purging of tumor cells (4 logs) with preservation of small lymphocytes (Figure 1C). While a representative experiment is shown, PEF consistently purged cancer cells by 3 to 4 logs, (4- to 5-log limit of detection) with preservation of small blood cells. Monocytes (CD14+), larger than lymphocytes and stem cells, were also purged by about 2 logs at 1.35 kV/cm. Experiments involving higher cell densities, up to 107 cell/mL, demonstrated similar tumor purging with preservation of small cells (data not shown), validating the utility of this system for large-scale tumor purging.
In order to quantify the purging of truly viable, proliferation-competent myeloma cells, tumor regrowth cultures were established (Figure 1D). Comparison between unpulsed and pulsed tumor cell regrowth showed 5 to 6 logs or greater purging (limit of detection for < 106 starting tumor cells). The mean log purging using RPMI8226 cells seeded into PBMCs, assessed by flow cytometry or tumor regrowth assay, is shown in Figure 1E (n = 6). These data suggest that flow cytometry enumeration, unable to distinguish between replication-competent cells and osmotically damaged but morphologically intact cells, underestimates the extent of tumor purging. Further, the lysing of cells by the PEF process releases large amounts of genomic DNA that cannot be effectively removed even with DNAse treatment. Therefore, DNA polymerase chain reaction for detection of MM cells after PEF purging could not be effectively applied.
After PEF purging, the capacity of human BM cells to engraft irradiated NOD/SCID/2m-/- BM was preserved, with more than 5% human CD45 cells after 7 weeks (Figure 1F). Preservation of engraftment capacity has also been demonstrated in NOD/SCID mice. Furthermore, CFC and LTC-IC frequency in vitro were similar or slightly enriched after PEF purging at 1.40 kV/cm, the upper limit and current standard field strength for most efficient tumor purging and progenitor cell preservation, and frequency decreased at and above 1.45 kV/cm (Figure 1G). These results demonstrate the functional and differentiation competence of the PEF-treated progenitor cells in vitro and in vivo.
Myeloma cells from the BM of patients with MM were killed at 1.40 kV/cm, demonstrated by trypan blue staining (Figure 2A) and flow cytometry (CD45dimCD138+CD38hi cells; Figure 2B). Percent survival of myeloma or small CD34+ cells after PEF treatment is summarized in Figure 2C (n = 4). In each case, the CD45dimCD38hiCD138+ population was purged at or near the limit of detection of 3 logs (due to small starting cell numbers); CD34+ cells were also affected at that pulse dose but with lower cell kill, preserving 50% ± 16%.
The data presented here confirm the principle of size selectivity using PEF technology, and demonstrate the utility of the flowing PEF technique for rapid purging of myeloma cells from progenitor and stem cell specimens. This method is appropriate for clinical sample volumes, and may provide a useful strategy for tumor cell purging from autologous BM or mobilized PB stem cell preparations. Overall purging efficacy, volume of specimen, and procedure time are significantly improved compared with our prior report.17 The patient sample data presented here emphasize the potential clinical utility of this approach.
Acknowledgements
We wish to thank Ai Hong Li at the Dana-Farber Cancer Institute and Aron Chiang and Meagan Kilbride at MGH for technical assistance.
Footnotes
Prepublished online as Blood First Edition Paper, August 3, 2004; DOI 10.1182/blood-2003-12-4399.
Supported by National Institutes of Health grant no. R44CA079364-02 and NASA grant no. NAS800009.
A.C. and Y.S. contributed equally to this work.
Several of the authors (J.A.M., A.C., A.L., and J.F.) are employed by, and 2 authors (H.M.E. and J.A.M.) have declared a financial interest in, Science Research Laboratory Inc, whose potential product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Van Besien K, Loberiza FR, Bajorunaite R, et al. Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood. 2003;102: 3521-3529.
Gertz MA, Witzig TE, Pineda AA, Greipp PR, Kyle RA, Litzow MR. Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation. Bone Marrow Transplant. 1997;19: 337-342.
Brenner MK, Rill DR, Moen RC, et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet. 1993:341: 85-86.
Deisseroth AB, Zu Z, Claxton D, et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood. 1994;83: 3068-3076.
Negrin RS, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant. 2000;6: 262-271.
Vescio R, Schiller G, Stewart AK, et al. Multicenter phase III trial to evaluate CD34(+) selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood. 1999;93: 1858-1868.
Hildebrandt M, Serke S, Meyer O, Ebell W, Salama A. Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield. Transfusion. 2000;40: 507-512.
Tricot G, Gazitt Y, Leemhuis T, et al. Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood. 1998;91: 4489-4495.
Anderson KC, Andersen J, Soiffer R, et al. Monoclonal antibody purged bone marrow transplantation therapy for multiple myeloma. Blood. 1993; 82: 2568-2576.
Thirukkumaran CM, Luider JM, Stewart DA, et al. Reovirus oncolysis as a novel purging strategy for autologous stem cell transplantation. Blood, 2003;102: 377-387.
Teoh G, Chen L, Urashima M, et al. Adenovirus vector-based purging of multiple myeloma cells. Blood. 1998;92: 4591-4601.
Soboloff J, Zhang Y, Minden M, Berger SA. Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging. Exp Hematol. 2002;30: 1219-1226.
Anderson GS, Tsujino I, Miyagi K, Sampson R, Sieber F. Preferential inactivation of paedriatic solid tumor cells by sequential exposure to Merocyanine 540-mediated photodynamic therapy and Edelfosine: implications for the ex vivo purging of autologous hematopoietic stem cell grafts. J Photochem Photobiol B. 2003;69: 87-95.
Lundell BI, Vredenburgh JJ, Tyer C, DeSombre K, Smith AK. Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor cell content through passive purging. Bone Marrow Transplant. 1998;22: 153-159.
Bierman PJ, Sweetenham JW, Loberiza Jr FR, et al. Syngeneic hematopoietic stem-cell transplantation for non-Hodgkin's lymphoma: a comparison with allogeneic and autologous transplantation— The Lymphoma Working Committee of the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. J Clin Oncol. 2003;21: 3744-3753.
Eppich HM, Foxall R, Gaynor K. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nat Biotechnol. 2000;18: 882-887.
Tsong TY. Electrically stimulated membrane breakdown. In: Lynch PT, Davey MR, eds. Electrical Manipulation of Cells. New York, NY: Chapman & Hall; 1996: 15-37.
Sale AJH, Hamilton WA, Sale AJ, Hamilton WA. Effects of high electric fields on micro-organisms. Biochim Biophys Acta. 1968;163: 37-43.
Radley JM, Ellis S, Palatsides M, Williams B, Bertoncello I. Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status. Exp Hematol. 1999;27: 365-369.
Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. Functional isolation and characterization of human hematopoietic stem cells. Science. 1995;267: 104-108.
Wang Z, Miura N, Bonelli A, et al. Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells. Blood. 2002;99: 2740-2747.(Abie Craiu, Yoriko Saito,)
the Center for Regenerative Medicine and Technology, Massachusetts General Hospital
the Jerome Lipper Myeloma Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.
Abstract
Autologous stem cell transplantation, in the setting of hematologic malignancies such as lymphoma, improves disease-free survival if the graft has undergone tumor purging. Here we show that flowing hematopoietic cells through pulsed electric fields (PEFs) effectively purges myeloma cells without sacrificing functional stem cells. Electric fields can induce irreversible cell membrane pores in direct relation to cell diameter, an effect we exploit in a flowing system appropriate for clinical scale. Multiple myeloma (MM) cell lines admixed with human bone marrow (BM) or peripheral blood (PB) cells were passed through PEFs at 1.35 kV/cm to 1.4 kV/cm, resulting in 3- to 4-log tumor cell depletion by flow cytometry and 4.5- to 6-log depletion by tumor regrowth cultures. Samples from patients with MM gave similar results by cytometry. Stem cell engraftment into nonobese diabetic–severe combined immunodeficient (NOD/SCID)/2m-/- mice was unperturbed by PEFs. Flowing cells through PEFs is a promising technology for rapid tumor cell purging of clinical progenitor cell preparations.
Introduction
Contaminant cancer cells in bone marrow (BM) or mobilized peripheral blood (PB) transfusions have been shown to correlate with disease relapse in the setting of some hematologic malignancies.1-4 In recent years, several technologies have been employed to purge tumor cells from autologous transfusions, including antibody-mediated selection for progenitor cells,2,5-8 depletion of tumor cells,9 genetic modification of tumor cells,10,11 selective chemical or physical means of tumor depletion,12,13 and in vitro expansion of hematopoietic cells.14 Clinical studies have employed tumor purging from stem cell preparations in non-Hodgkin lymphoma (NHL) with recent evidence of clinical benefit.1,15 Whereas no studies on a similar scale or with similar results exist for multiple myeloma (MM), we selected this hematologic disease as a test case for our purging technology since BM samples from patients with myeloma are consistently infiltrated with tumor, the tumor cells are readily quantified, and autologous transplantation is used to treat patients with this disorder. It should be emphasized however, that the data presented here simply use myeloma as an example and do not argue for or against the clinical utility of autologous transplantation with purging for MM.
The NHL studies that did use purging employed CD34 selection that is time-, cost-, and labor-intensive, and may not efficiently preserve hematopoietic stem cells. We sought to test a different method based on prior studies showing that defined electric field pulses applied to static, small volume samples selectively depleted breast cancer and megakaryocyte cell lines by 2 to 2.5 logs from mixtures with blood cells, and preserved small cells including lymphocytes and CD34+ cells.16 The principle behind selective pulsed electric field (PEF) purging is that a cell's cytosol is largely conductive, but the lipid cell membrane does not conduct electricity.17,18 The voltage developed across each cell is proportional to the cell's diameter. Under defined electric field conditions, larger cells are killed without altering the viability of smaller cells, including hematopoietic stem cells (HSCs). Whereas HSCs and resting lymphocytes are generally 6 μm to 8 μm in diameter,19,20 myeloma cells and other tumor cells are generally more than 10 μm in diameter.
We have now applied this technology in a modified format that permits continuous and rapid pulsing of clinically relevant numbers of cells (> 109 cells in 30 minutes) at controlled flow rates that negate the effects of cell concealment or cell settling (manuscript in preparation).22 Applying this technology to myeloma, we define tumor cell depletion by 3 to 6 orders of magnitude without sacrificing functional stem cells.
Study design
Preparation of primary cells and cell lines
PB was obtained from donors at Massachusetts General Hospital (MGH). BM was obtained from NDRI (Philadelphia, PA), and 2 mL to 5 mL of discard BM aspirates were obtained from patients with MM at the Dana-Farber/Harvard Cancer Center (institutional review board approval no. 1999-P008 401/4). Bone marrow or peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation. Cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA).
Flowing PEF apparatus
A prototype flowing PEF apparatus was designed and constructed by Science Research Laboratory (SRL). Components include the flowing treatment chamber, Cytopulse (Rockville, MD) electric pulse driver system, Tektronix oscilloscope (Beaverton, OR), PC-based control system, syringe pumps (Harvard Apparatus, Framingham, MA), and 4-way valve and outlet for purging air (manuscript in preparation).23
In vitro assays and staining
For tumor regrowth assays, cells were serially diluted in triplicate in conditioned medium/RPMI/fetal calf serum (FCS) in 96-well plates. After 2 weeks, plates were scored for colony formation (> 1 colony of > 4 viable tumor cells).
Colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays were performed as described.1
Engraftment of NOD/SCID/2m-/- mice
Six- to 8-week-old nonobese diabetic–severe combined immunodeficient (NOD/SCID)/2m-/- mice were obtained from the Jackson Laboratory (Bar Harbor, ME), handled, maintained, and underwent transplantation as described.21
Results and discussion
Selective PEF purging of carboxyfluoroscein diacetate succinimidyl ester (CFSE)–labeled myeloma cell lines (RPMI8226), mixed with PBMCs or bone marrow cells, was achieved at 1.4 kV/cm (Figure 1A-B). After PEF, viable cells were small and confined largely to the "lymphocyte gate" of a forward- and side-scatter plot, known to contain stem and progenitor cells, while the high forward- and side-scatter myeloma cells largely disappeared (Figure 1B, left). Purging of myeloma cells (CFDA-SE+) as well as monocytes (CD14+), with preservation of lymphocytes (CD3+/CD19+), was also evident.
Dose response to PEFs was evaluated. We noted purging of tumor cells (4 logs) with preservation of small lymphocytes (Figure 1C). While a representative experiment is shown, PEF consistently purged cancer cells by 3 to 4 logs, (4- to 5-log limit of detection) with preservation of small blood cells. Monocytes (CD14+), larger than lymphocytes and stem cells, were also purged by about 2 logs at 1.35 kV/cm. Experiments involving higher cell densities, up to 107 cell/mL, demonstrated similar tumor purging with preservation of small cells (data not shown), validating the utility of this system for large-scale tumor purging.
In order to quantify the purging of truly viable, proliferation-competent myeloma cells, tumor regrowth cultures were established (Figure 1D). Comparison between unpulsed and pulsed tumor cell regrowth showed 5 to 6 logs or greater purging (limit of detection for < 106 starting tumor cells). The mean log purging using RPMI8226 cells seeded into PBMCs, assessed by flow cytometry or tumor regrowth assay, is shown in Figure 1E (n = 6). These data suggest that flow cytometry enumeration, unable to distinguish between replication-competent cells and osmotically damaged but morphologically intact cells, underestimates the extent of tumor purging. Further, the lysing of cells by the PEF process releases large amounts of genomic DNA that cannot be effectively removed even with DNAse treatment. Therefore, DNA polymerase chain reaction for detection of MM cells after PEF purging could not be effectively applied.
After PEF purging, the capacity of human BM cells to engraft irradiated NOD/SCID/2m-/- BM was preserved, with more than 5% human CD45 cells after 7 weeks (Figure 1F). Preservation of engraftment capacity has also been demonstrated in NOD/SCID mice. Furthermore, CFC and LTC-IC frequency in vitro were similar or slightly enriched after PEF purging at 1.40 kV/cm, the upper limit and current standard field strength for most efficient tumor purging and progenitor cell preservation, and frequency decreased at and above 1.45 kV/cm (Figure 1G). These results demonstrate the functional and differentiation competence of the PEF-treated progenitor cells in vitro and in vivo.
Myeloma cells from the BM of patients with MM were killed at 1.40 kV/cm, demonstrated by trypan blue staining (Figure 2A) and flow cytometry (CD45dimCD138+CD38hi cells; Figure 2B). Percent survival of myeloma or small CD34+ cells after PEF treatment is summarized in Figure 2C (n = 4). In each case, the CD45dimCD38hiCD138+ population was purged at or near the limit of detection of 3 logs (due to small starting cell numbers); CD34+ cells were also affected at that pulse dose but with lower cell kill, preserving 50% ± 16%.
The data presented here confirm the principle of size selectivity using PEF technology, and demonstrate the utility of the flowing PEF technique for rapid purging of myeloma cells from progenitor and stem cell specimens. This method is appropriate for clinical sample volumes, and may provide a useful strategy for tumor cell purging from autologous BM or mobilized PB stem cell preparations. Overall purging efficacy, volume of specimen, and procedure time are significantly improved compared with our prior report.17 The patient sample data presented here emphasize the potential clinical utility of this approach.
Acknowledgements
We wish to thank Ai Hong Li at the Dana-Farber Cancer Institute and Aron Chiang and Meagan Kilbride at MGH for technical assistance.
Footnotes
Prepublished online as Blood First Edition Paper, August 3, 2004; DOI 10.1182/blood-2003-12-4399.
Supported by National Institutes of Health grant no. R44CA079364-02 and NASA grant no. NAS800009.
A.C. and Y.S. contributed equally to this work.
Several of the authors (J.A.M., A.C., A.L., and J.F.) are employed by, and 2 authors (H.M.E. and J.A.M.) have declared a financial interest in, Science Research Laboratory Inc, whose potential product was studied in the present work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
References
Van Besien K, Loberiza FR, Bajorunaite R, et al. Comparison of autologous and allogeneic hematopoietic stem cell transplantation for follicular lymphoma. Blood. 2003;102: 3521-3529.
Gertz MA, Witzig TE, Pineda AA, Greipp PR, Kyle RA, Litzow MR. Monoclonal plasma cells in the blood stem cell harvest from patients with multiple myeloma are associated with shortened relapse-free survival after transplantation. Bone Marrow Transplant. 1997;19: 337-342.
Brenner MK, Rill DR, Moen RC, et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplantation. Lancet. 1993:341: 85-86.
Deisseroth AB, Zu Z, Claxton D, et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood. 1994;83: 3068-3076.
Negrin RS, Atkinson K, Leemhuis T, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant. 2000;6: 262-271.
Vescio R, Schiller G, Stewart AK, et al. Multicenter phase III trial to evaluate CD34(+) selected versus unselected autologous peripheral blood progenitor cell transplantation in multiple myeloma. Blood. 1999;93: 1858-1868.
Hildebrandt M, Serke S, Meyer O, Ebell W, Salama A. Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield. Transfusion. 2000;40: 507-512.
Tricot G, Gazitt Y, Leemhuis T, et al. Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood. 1998;91: 4489-4495.
Anderson KC, Andersen J, Soiffer R, et al. Monoclonal antibody purged bone marrow transplantation therapy for multiple myeloma. Blood. 1993; 82: 2568-2576.
Thirukkumaran CM, Luider JM, Stewart DA, et al. Reovirus oncolysis as a novel purging strategy for autologous stem cell transplantation. Blood, 2003;102: 377-387.
Teoh G, Chen L, Urashima M, et al. Adenovirus vector-based purging of multiple myeloma cells. Blood. 1998;92: 4591-4601.
Soboloff J, Zhang Y, Minden M, Berger SA. Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging. Exp Hematol. 2002;30: 1219-1226.
Anderson GS, Tsujino I, Miyagi K, Sampson R, Sieber F. Preferential inactivation of paedriatic solid tumor cells by sequential exposure to Merocyanine 540-mediated photodynamic therapy and Edelfosine: implications for the ex vivo purging of autologous hematopoietic stem cell grafts. J Photochem Photobiol B. 2003;69: 87-95.
Lundell BI, Vredenburgh JJ, Tyer C, DeSombre K, Smith AK. Ex vivo expansion of bone marrow from breast cancer patients: reduction in tumor cell content through passive purging. Bone Marrow Transplant. 1998;22: 153-159.
Bierman PJ, Sweetenham JW, Loberiza Jr FR, et al. Syngeneic hematopoietic stem-cell transplantation for non-Hodgkin's lymphoma: a comparison with allogeneic and autologous transplantation— The Lymphoma Working Committee of the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. J Clin Oncol. 2003;21: 3744-3753.
Eppich HM, Foxall R, Gaynor K. Pulsed electric fields for selection of hematopoietic cells and depletion of tumor cell contaminants. Nat Biotechnol. 2000;18: 882-887.
Tsong TY. Electrically stimulated membrane breakdown. In: Lynch PT, Davey MR, eds. Electrical Manipulation of Cells. New York, NY: Chapman & Hall; 1996: 15-37.
Sale AJH, Hamilton WA, Sale AJ, Hamilton WA. Effects of high electric fields on micro-organisms. Biochim Biophys Acta. 1968;163: 37-43.
Radley JM, Ellis S, Palatsides M, Williams B, Bertoncello I. Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status. Exp Hematol. 1999;27: 365-369.
Berardi AC, Wang A, Levine JD, Lopez P, Scadden DT. Functional isolation and characterization of human hematopoietic stem cells. Science. 1995;267: 104-108.
Wang Z, Miura N, Bonelli A, et al. Receptor tyrosine kinase, EphB4 (HTK), accelerates differentiation of select human hematopoietic cells. Blood. 2002;99: 2740-2747.(Abie Craiu, Yoriko Saito,)