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Progress and prospects in hematopoietic stem cell expansion and transplantation
Editorial / Experimental Hematology 32 (2004) 692–697
Progress and prospects in hematopoietic stem cell expansion and transplantation The fact that mice and humans have similar hematolymphoid systems and respond similarly to myeloablation and hematopoietic cell transplantation (HCT) has allowed research on mouse separated hematopoietic stem and progenitor cells to inform our progress on human HCT. Long-term engrafting hematopoietic stem cells (LT-HSC) and their multipotent progeny (short-term engrafting HSC [ST-HSC] and MPP), the lineage-committed common lymphoid progenitors (CLP) and common myeloid progenitors (CMP), downstream progenitors such as granulocye-monocyte progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP), and unipotent megakaryocyte progenitors (MkP) were all first isolated in mice, and subsequently from human sources [1]. The direct transplantation of graded numbers of these mouse stem and/or progenitor cells in syngeneic hosts revealed many surprising and sometimes very useful findings, such as the fact that the content of HSC/MPP within a bone marrow (BM) transplant accounted entirely for the radioprotective capacity of the graft as well as the time to engraftment of neutrophils, platelets, and red blood cells [2]. In the context of bone marrow transplantation, therefore, the level of oligolineage progenitors (largely responsible for in vivo short-term colony-forming units (CFUs) and in vitro myeloerythroid colony-forming cells (CFCs) found in a minimally radioprotective dose of BM did not contribute measurably to restoration of blood cell counts or radioprotection [3]. In the mouse, transplantation of small numbers of HSC (as few as 1) gave rise to tens of thousands of HSC and the aforementioned progenitors, while the transplantation of any progenitors downstream of LT-HSC resulted in failed or minimal self-renewal. Thus, the only progenitors of mouse hematopoiesis that self-renew extensively in vivo are LT-HSC [4]. Similarly, the autologous transplantation of highly enriched human HSC resulted in more rapid engraftment of neutrophils and platelets than would be expected from their content in mobilized peripheral blood and were fully functional in protection from myeloablative doses of combination chemotherapy [5,6]. While it was hoped that self-renewing expansion of HSC in vitro could be accomplished with the set of cytokines cloned and generally available, such as steel factor (SLF), EPO, and interleukin (IL)-1, 3, 6, and 11, in fact, no single cytokine or combination of cytokines led to more than a modest expansion of HSC in mouse [6,7] or man [8], a sad fact that was verified by clinical transplantation experience [9–11]. Despite the massive expansion of hematopoietic and myeloerythroid cells that followed the optimal protocol combinations, the HSC content remained the same as the input HSC content [12]. It is not yet clear whether the number of
693
oligolineage progenitors produced from HSCs in these studies mirrored the number expected from non-self-renewing expansions or if some cytokine combinations could lead to some progenitor self-renewal in vitro [13]. Therefore, the search for factors and pathways that could expand HSCs in vitro as they were obviously expanded in vivo continued. That search became even more significant for several reasons: 1) the requirement for more HSCs to achieve robust engraftment in allogeneic transplants than in syngeneic transplants in mouse studies became apparent [14]; 2) the looming specter that very large numbers of HSCs of a variety of HLA haplotypes might be required to radioprotect human populations threatened with radioactive or chemical warfare agents; and 3) the unexpected activities of oligolineage progenitors in situational transplants in mice revealed important and lifesaving properties. Some of these properties include 1) the ability of CMP and MEP given in very large doses in lethally irradiated mice to provide transient radioprotection until the few surviving host HSC could regenerate the hematolymphoid system [15]; 2) the ability of very large numbers of CLP to protect HSCtransplanted mice from otherwise lethal infections with murine cytomegalovirus (CMV) in both syngeneic and MHC minor antigen mismatch donor-host pairing [16]; and 3) the ability of very large numbers of CMP/GMP to protect HSCtransplanted mice from otherwise lethal infections with spores of the fungus Aspergillus fumigatus (obtained from a lethal human infection) or gram-negative rod bacteria Pseudomonas aeruginosa in both syngeneic and allogeneic circumstances [17,18]. If these properties of oligolineage progenitors extrapolate to human therapies, there would be no absolute requirement for donor-host matching. Such therapeutic potential underscores the importance of expanding human HSC in order to obtain (with the appropriate cytokine combinations, if necessary) substantial numbers of human CLP, CMP, GMP, MEP, and perhaps MkP. The ex vivo expansion of hematopoietic stem cells is the next logical step in the field of hematopoietic cell transplantation. Despite remarkable progress in the field of HCT, current therapeutic limitations include the availability of donors, imprecision in immune reconstitution reflected by susceptibility to infectious pathogens or by graft-vs-host disease (GVHD), and relapse/persistent disease. Ex vivo expansion of hematopoietic stem and progenitor cells offers the potential for providing a graft that would more rapidly recapitulate the immune response and be free of malignant cells. Genetic engineering is also facilitated by ex vivo expansion of cells, offering the potential for enhanced immunotherapy or response to pathogens. Immediate scientific challenges to ex vivo expansion include 1) optimizing culture conditions, 2) identification of optimal human cell populations to expand, 3) identification and expansion of human long-term repopulating cells, and 4) concerns that expansion may lead to early senescence or decreased functional capacity after transfer, which may be related to cell cycle at a crucial time.
694
Editorial / Experimental Hematology 32 (2004) 692–697
However, as stated above, the attempts at the ex vivo expansion of LT-HSC with the known and available hematopoietic cytokines in mouse and man have resulted in massive production of myeloerythroid cells but little or no expansion of HSC. Clinical investigation in patients or using human samples have both reinforced the great utility of the preclinical animal studies and identified the limitations in the translation. Three promising avenues have recently emerged in studies of mouse HSCs. The homeobox gene HoxB4 is not only expressed in populations of cells enriched for HSCs, but transfection of expressable HoxB4 into embryonic-stage hematopoietic cells has increased their numbers and improved their ability to engraft in adult irradiated hosts [19,20]. Unfortunately, gene transfection is currently dangerous in HSCs due to the few cases of gene transfer repair of X-linked SCID (mutant common interleukin receptor γ chain etiology) by the insertional activation of the Lmo 2 gene and concomitant progression to lymphoid leukemias. A second approach uses activation of HSC Notch 1 receptors with soluble jagged or delta ligands, but only one or a few lines have been reported to date [21]. We have recently fostered another approach: LT-HSC express members of the wnt signaling pathway, and purified wnt 3A causes proliferation and at least 10-fold expansion of transplantable mouse HSC [22]. Wnt activation operates through release of nonphophorylated β catenin from a cytoplasmic polyprotein destruction complex. The nonphosphorylated β catenin then translocates to the nucleus and stimulates lef/tcf-based gene transcription [23,24]. Transfection of the activated form of β catenin into purified mouse LT-HSC results in a 100to 1000-fold ex vivo expansion of transplantable HSC [22]. Native HSC carrying a lef/tcf reporter show in vivo evidence of β catenin–triggered gene expression, while other dividing progeny—notably CMP and GMP—are proliferating without lef/tcf measurable transcription [22]. Finally, inhibition of wnt pathway signaling blocks HSC proliferation and survival even in the presence of levels of SLF, IL-6, and TPO that cause about 90% of LT-HSC to divide [22]. Thus, the current task is to find a way to most efficiently activate the β catenin pathway without gene transfection protocols, to test the ability of human HSC to respond to this method, to show greatly expanded functionally engrafting HSCs from this method, to do so in GMP conditions, and to test quantitative and qualitative methods to make and store desired HSC and progenitor populations. In addition to the great potential of ex vivo expansion, other strategies using HSC and hematopoietic progenitor cells deserve investigation, especially as these strategies are more immediately amenable to further clinical study. The benefits of stem cells in hematopoietic cell transplantation have been demonstrated in both experimental and clinical studies. As stated above, highly purified CD34+Thy-1+ cells were used as the sole source of the autologous hematopoietic graft that was free of tumor cells and resulted in
rapid and sustained hematopoietic engraftment of neutrophils, platelets, and red blood cells. Additionally, based on preclinical studies, the potential clinical utility of infusions of progenitors is expected to be broad and includes bridging the period of anticipated marrow suppression regardless of the cause of the myelosuppression. Although the significant majority of invasive fungal infections following HCT do not occur in the setting of neutropenia, the risk for developing invasive aspergillosis following chemotherapy remains unchanged and increases geometrically in relation to duration of neutropenia. The risk for bacterial infections and fever during neutropenia from any cause, even in the setting of HCT of grafts enriched for CD34+ cells, remains unchanged over the past several decades. Despite a growing armamentarium of antimicrobial agents, the cost of these early infections, the rising incidence of invasive fungal infections, and the high mortality rate have all been documented extensively. Exogenous administration of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and infusions of granulocytes, even if mobilized by administration of G-CSF, have failed to consistently improve clearance of bacterial, viral, or fungal infections. Recent studies using mouse models demonstrate the enhanced immune reconstitution of hosts that received grafts containing high doses of CMP and GMP along with the HSC vs those that received grafts comprised of HSC alone, as measured by protection against lethal challenge with either Aspergillus fumigatus or Pseudomonas aeruginosa. It is striking that these doses of myeloid progenitors do not shorten the time of absolute peripheral neutropenia in these mice, but provide robust engraftment with myelomonocytic cells in marrow and spleen. Furthermore, the identification of human CMP/GMP makes clinical trials of progenitor infusions following chemotherapy or HCT an imminent reality. Enhancement of functional lymphoid reconstitution following HCT has also been demonstrated. Populations of lymphoid progenitors, both the common T lymphocyte progenitor (CTL) and the CLP, have been shown in mouse models to protect against lethal CMV infection following HCT. Moreover, CLP and CMP/GMP from donors with a minor or major mismatch in the MHC engrafted and enhanced the functional antiviral and antifungal immune response, respectively. Of great significance is that CLP-derived cells remained capable of distinguishing self and, thus, did not precipitate GVHD. Thus, infusions of progenitors should be studied in a clinical setting for 1) their potential to serve as a bridge in a variety of settings in which myelosuppression is expected as a strategy to reduce susceptibility to infections and 2) their efficacy as adjunctive treatment for opportunistic infections. Regarding the potential infusions of subpopulations of apheresed cells that are expanded ex vivo, the regulatory issues are relatively straightforward and precedent has already been established. These studies parallel the infusion of less well-defined whole blood or components of blood,
Editorial / Experimental Hematology 32 (2004) 692–697
most commonly from unrelated donors. That this practice is now considered to be routine is a remarkable achievement as the establishment of blood banks in the United States did not occur until the late 1940s. Standards for good manufacturing practices have also been established and are routinely relied upon for the processing of HCT grafts including the use of a variety of selection/purification techniques and ex vivo expansion. The establishment of cell therapy facilities should be a reality in the near future and, like blood banks, will likely be considered to be an essential service to hospitals. For cost containment reasons, one could envision centralized repositories to harvest, expand, and characterize the composition of grafts/infusions, as well as to perform HLA-typing and serologic and sterility testing. Both the national marrow donor program and the solid organ registries demonstrate that networks can be established to facilitate donor-host pairing. The dramatic advances made in the field of human hematopoietic cell biology have provided a foundation of both excellent preclinical studies and early clinical trials. It is clear that in order to make meaningful progress towards useful therapies, preclinical studies must continue to be performed in tandem with clinical trials. Clinical trials should proceed immediately on two general fronts. First, transplantation of highly purified HSC and highly purified progenitor cells without ex vivo expansion in both the autologous and allogeneic setting should be performed to confirm proof of concept and dose response curves. Second, studies of ex vivo expansion should continue to be actively pursued. Ideally, the establishment of consortia, each with a core facility for ex vivo expansion and a group of large clinical centers, would execute clinical studies of sufficient size and adequate design in parallel with laboratory investigation to determine correlates to clinical outcome. doi: 10.1016/j.exphem.2004.07.001
J.M. Brown and I.L.Weissman Stanford University, Stanford, Calif., USA
References 1. Manz MG, Miyamotot T, Akashi K, Weissman IL. Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A. 2002;99:11872–11877. 2. Uchida N, Aguila HL, Fleming WH, Jearbek L, Weissman IL. Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1loLin⫺Sca-1+ hematopoietic stem cells. Blood. 1994;83:3758–3779. 3. Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1loLin⫺Sca-1+ cells are the only stem cells in C57BL/ Ka-Thy-1.1 bone marrow. J Exp Med. 1992;175:175–184. 4. Uchida N, Jerabek L, Weissman IL. Searching for hematopoietic stem cells. II. The heterogeneity of Thy-1.1loLin⫺/loSca-1+ mouse hematopoietic stem cells separated by counterflow centrifugal elutriation. Exp Hematol. 1996;24:649–659. 5. Devine SM, Lazarus HM, Emerson SG. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects. Bone Marrow Transplant. 2003;31:241–252.
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6. Negrin RS, Atkinson K, Leemhuis I, et al. Transplantation of highly purified CD34+Thy-1+ hematopoietic stem cells in patients with metastatic breast cancer. Biol Blood Marrow Transplant. 2000;3:262–271. 7. Peters SO, Kittler EL, Ramshaw HS, Quesenberry PJ. Ex vivo expansion of murine marrow cells with interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor leads to impaired engraftment in irradiated hosts. Blood. 1996;87:30–37. 8. Traycoff CM, Cornetta K, Yoder MC, Davidson A, Srour EF. Ex vivo expansion of murine hematopoietic progenitor cells generates classes of expanded cells possessing different levels of bone marrow repopulating potential. Exp Hematol. 1996;24:299–306. 9. Devine SM, Lazarus HM, Emerson SG. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects. Bone Marrow Transplant. 2003;31:241–252. 10. Brugger W, Heimfeld S, Berenson RJ, Mertelsmann R, Kanz L. Reconstitution of hematopoiesis after high-dose chemotherapy by autologous progenitor cells generated ex vivo. N Engl J Med. 1995;333:283–287. 11. Holyoake TL, Alcorn MJ, Richmond L, et al. CD34+ PBPC expanded ex vivo may not provide durable engraftment following myeloablative chemoradiotherapy regimens. Bone Marrow Transplant. 1997;19:1095– 1101. 12. Alcorn MJ, Holyoake TL, Richmond L, et al. CD34+ cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity. J Clin Oncol. 1996;14:1839–1847. 13. Ikuta K, Ingolia DE, Friedman J, Heimfeld S, Weissman IL. Mouse hematopoietic stem cells and the interaction of c-kit receptor and steel factor. Int J Cell Cloning. 1991;9:451–460. 14. McKenna HJ, de Vries P, Brasel K, Lyman SD, Williams DE. Effect of flt3 ligand on the ex vivo expansion of human CD34+ hematopoietic progenitor cells. Blood. 1995;86:3413–3420. 15. Shizuru JA, Jerabek L, Edwards CT, Weissman IL. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol Blood Marrow Transplant. 1996;2:3–14. 16. Na Nakorn T, Traver D, Weissman IL, Akashi K. Myeloerythroidrestricted progenitors are sufficient to confer radioprotection and provide the majority of day 8 CFU-S. J Clin Invest. 2002;109:1579–1585. 17. Arber C, Bitmansour A, Brown JM. MHC-mismatched murine committed myeloid progenitors engraft and protect against invasive Aspergillosis. Blood. 2003;102:3504. 18. Bitmansour A, Burns SM, Traver D, et al. Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood. 2002;100: 4660–4667. 19. Arber C, BitMansour A, Sparer TE, et al. Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation. Blood. 2003; 102:421–428. 20. Sauvageau G, Thorsteindottir U, Eaves CJ, et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. 1995;9:1753– 1765. 21. Krosl J, Austin P, Beslu N, Kroom E, Humphries RK, Sauvageau G. In vitro expansion of hematopoietic stem cells by recombinant TATHOXB4 protein. Nat Med. 2003;9:1428–1432. 22. Varnum-Finney B, Xu L, Brasheim-Stein C, et al. Pluripotent, cytokinedependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med. 2000;6:1278–1281. 23. Reya T, Duncan AW, Ailes L, et al. A role for Wnt signalling in selfrenewal of haematopoietic stem cells. Nature. 2003;423:409–414. 24. Reya T, O’Riordan M, Okamura R, et al. Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity. 2000;13:15–24.
Progress and prospects in hematopoietic stem cell expansion and transplantation The fact that mice and humans have similar hematolymphoid systems and respond similarly to myeloablation and hematopoietic cell transplantation (HCT) has allowed research on mouse separated hematopoietic stem and progenitor cells to inform our progress on human HCT. Long-term engrafting hematopoietic stem cells (LT-HSC) and their multipotent progeny (short-term engrafting HSC [ST-HSC] and MPP), the lineage-committed common lymphoid progenitors (CLP) and common myeloid progenitors (CMP), downstream progenitors such as granulocye-monocyte progenitors (GMP) and megakaryocyte-erythrocyte progenitors (MEP), and unipotent megakaryocyte progenitors (MkP) were all first isolated in mice, and subsequently from human sources [1]. The direct transplantation of graded numbers of these mouse stem and/or progenitor cells in syngeneic hosts revealed many surprising and sometimes very useful findings, such as the fact that the content of HSC/MPP within a bone marrow (BM) transplant accounted entirely for the radioprotective capacity of the graft as well as the time to engraftment of neutrophils, platelets, and red blood cells [2]. In the context of bone marrow transplantation, therefore, the level of oligolineage progenitors (largely responsible for in vivo short-term colony-forming units (CFUs) and in vitro myeloerythroid colony-forming cells (CFCs) found in a minimally radioprotective dose of BM did not contribute measurably to restoration of blood cell counts or radioprotection [3]. In the mouse, transplantation of small numbers of HSC (as few as 1) gave rise to tens of thousands of HSC and the aforementioned progenitors, while the transplantation of any progenitors downstream of LT-HSC resulted in failed or minimal self-renewal. Thus, the only progenitors of mouse hematopoiesis that self-renew extensively in vivo are LT-HSC [4]. Similarly, the autologous transplantation of highly enriched human HSC resulted in more rapid engraftment of neutrophils and platelets than would be expected from their content in mobilized peripheral blood and were fully functional in protection from myeloablative doses of combination chemotherapy [5,6]. While it was hoped that self-renewing expansion of HSC in vitro could be accomplished with the set of cytokines cloned and generally available, such as steel factor (SLF), EPO, and interleukin (IL)-1, 3, 6, and 11, in fact, no single cytokine or combination of cytokines led to more than a modest expansion of HSC in mouse [6,7] or man [8], a sad fact that was verified by clinical transplantation experience [9–11]. Despite the massive expansion of hematopoietic and myeloerythroid cells that followed the optimal protocol combinations, the HSC content remained the same as the input HSC content [12]. It is not yet clear whether the number of
693
oligolineage progenitors produced from HSCs in these studies mirrored the number expected from non-self-renewing expansions or if some cytokine combinations could lead to some progenitor self-renewal in vitro [13]. Therefore, the search for factors and pathways that could expand HSCs in vitro as they were obviously expanded in vivo continued. That search became even more significant for several reasons: 1) the requirement for more HSCs to achieve robust engraftment in allogeneic transplants than in syngeneic transplants in mouse studies became apparent [14]; 2) the looming specter that very large numbers of HSCs of a variety of HLA haplotypes might be required to radioprotect human populations threatened with radioactive or chemical warfare agents; and 3) the unexpected activities of oligolineage progenitors in situational transplants in mice revealed important and lifesaving properties. Some of these properties include 1) the ability of CMP and MEP given in very large doses in lethally irradiated mice to provide transient radioprotection until the few surviving host HSC could regenerate the hematolymphoid system [15]; 2) the ability of very large numbers of CLP to protect HSCtransplanted mice from otherwise lethal infections with murine cytomegalovirus (CMV) in both syngeneic and MHC minor antigen mismatch donor-host pairing [16]; and 3) the ability of very large numbers of CMP/GMP to protect HSCtransplanted mice from otherwise lethal infections with spores of the fungus Aspergillus fumigatus (obtained from a lethal human infection) or gram-negative rod bacteria Pseudomonas aeruginosa in both syngeneic and allogeneic circumstances [17,18]. If these properties of oligolineage progenitors extrapolate to human therapies, there would be no absolute requirement for donor-host matching. Such therapeutic potential underscores the importance of expanding human HSC in order to obtain (with the appropriate cytokine combinations, if necessary) substantial numbers of human CLP, CMP, GMP, MEP, and perhaps MkP. The ex vivo expansion of hematopoietic stem cells is the next logical step in the field of hematopoietic cell transplantation. Despite remarkable progress in the field of HCT, current therapeutic limitations include the availability of donors, imprecision in immune reconstitution reflected by susceptibility to infectious pathogens or by graft-vs-host disease (GVHD), and relapse/persistent disease. Ex vivo expansion of hematopoietic stem and progenitor cells offers the potential for providing a graft that would more rapidly recapitulate the immune response and be free of malignant cells. Genetic engineering is also facilitated by ex vivo expansion of cells, offering the potential for enhanced immunotherapy or response to pathogens. Immediate scientific challenges to ex vivo expansion include 1) optimizing culture conditions, 2) identification of optimal human cell populations to expand, 3) identification and expansion of human long-term repopulating cells, and 4) concerns that expansion may lead to early senescence or decreased functional capacity after transfer, which may be related to cell cycle at a crucial time.
694
Editorial / Experimental Hematology 32 (2004) 692–697
However, as stated above, the attempts at the ex vivo expansion of LT-HSC with the known and available hematopoietic cytokines in mouse and man have resulted in massive production of myeloerythroid cells but little or no expansion of HSC. Clinical investigation in patients or using human samples have both reinforced the great utility of the preclinical animal studies and identified the limitations in the translation. Three promising avenues have recently emerged in studies of mouse HSCs. The homeobox gene HoxB4 is not only expressed in populations of cells enriched for HSCs, but transfection of expressable HoxB4 into embryonic-stage hematopoietic cells has increased their numbers and improved their ability to engraft in adult irradiated hosts [19,20]. Unfortunately, gene transfection is currently dangerous in HSCs due to the few cases of gene transfer repair of X-linked SCID (mutant common interleukin receptor γ chain etiology) by the insertional activation of the Lmo 2 gene and concomitant progression to lymphoid leukemias. A second approach uses activation of HSC Notch 1 receptors with soluble jagged or delta ligands, but only one or a few lines have been reported to date [21]. We have recently fostered another approach: LT-HSC express members of the wnt signaling pathway, and purified wnt 3A causes proliferation and at least 10-fold expansion of transplantable mouse HSC [22]. Wnt activation operates through release of nonphophorylated β catenin from a cytoplasmic polyprotein destruction complex. The nonphosphorylated β catenin then translocates to the nucleus and stimulates lef/tcf-based gene transcription [23,24]. Transfection of the activated form of β catenin into purified mouse LT-HSC results in a 100to 1000-fold ex vivo expansion of transplantable HSC [22]. Native HSC carrying a lef/tcf reporter show in vivo evidence of β catenin–triggered gene expression, while other dividing progeny—notably CMP and GMP—are proliferating without lef/tcf measurable transcription [22]. Finally, inhibition of wnt pathway signaling blocks HSC proliferation and survival even in the presence of levels of SLF, IL-6, and TPO that cause about 90% of LT-HSC to divide [22]. Thus, the current task is to find a way to most efficiently activate the β catenin pathway without gene transfection protocols, to test the ability of human HSC to respond to this method, to show greatly expanded functionally engrafting HSCs from this method, to do so in GMP conditions, and to test quantitative and qualitative methods to make and store desired HSC and progenitor populations. In addition to the great potential of ex vivo expansion, other strategies using HSC and hematopoietic progenitor cells deserve investigation, especially as these strategies are more immediately amenable to further clinical study. The benefits of stem cells in hematopoietic cell transplantation have been demonstrated in both experimental and clinical studies. As stated above, highly purified CD34+Thy-1+ cells were used as the sole source of the autologous hematopoietic graft that was free of tumor cells and resulted in
rapid and sustained hematopoietic engraftment of neutrophils, platelets, and red blood cells. Additionally, based on preclinical studies, the potential clinical utility of infusions of progenitors is expected to be broad and includes bridging the period of anticipated marrow suppression regardless of the cause of the myelosuppression. Although the significant majority of invasive fungal infections following HCT do not occur in the setting of neutropenia, the risk for developing invasive aspergillosis following chemotherapy remains unchanged and increases geometrically in relation to duration of neutropenia. The risk for bacterial infections and fever during neutropenia from any cause, even in the setting of HCT of grafts enriched for CD34+ cells, remains unchanged over the past several decades. Despite a growing armamentarium of antimicrobial agents, the cost of these early infections, the rising incidence of invasive fungal infections, and the high mortality rate have all been documented extensively. Exogenous administration of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) and infusions of granulocytes, even if mobilized by administration of G-CSF, have failed to consistently improve clearance of bacterial, viral, or fungal infections. Recent studies using mouse models demonstrate the enhanced immune reconstitution of hosts that received grafts containing high doses of CMP and GMP along with the HSC vs those that received grafts comprised of HSC alone, as measured by protection against lethal challenge with either Aspergillus fumigatus or Pseudomonas aeruginosa. It is striking that these doses of myeloid progenitors do not shorten the time of absolute peripheral neutropenia in these mice, but provide robust engraftment with myelomonocytic cells in marrow and spleen. Furthermore, the identification of human CMP/GMP makes clinical trials of progenitor infusions following chemotherapy or HCT an imminent reality. Enhancement of functional lymphoid reconstitution following HCT has also been demonstrated. Populations of lymphoid progenitors, both the common T lymphocyte progenitor (CTL) and the CLP, have been shown in mouse models to protect against lethal CMV infection following HCT. Moreover, CLP and CMP/GMP from donors with a minor or major mismatch in the MHC engrafted and enhanced the functional antiviral and antifungal immune response, respectively. Of great significance is that CLP-derived cells remained capable of distinguishing self and, thus, did not precipitate GVHD. Thus, infusions of progenitors should be studied in a clinical setting for 1) their potential to serve as a bridge in a variety of settings in which myelosuppression is expected as a strategy to reduce susceptibility to infections and 2) their efficacy as adjunctive treatment for opportunistic infections. Regarding the potential infusions of subpopulations of apheresed cells that are expanded ex vivo, the regulatory issues are relatively straightforward and precedent has already been established. These studies parallel the infusion of less well-defined whole blood or components of blood,
Editorial / Experimental Hematology 32 (2004) 692–697
most commonly from unrelated donors. That this practice is now considered to be routine is a remarkable achievement as the establishment of blood banks in the United States did not occur until the late 1940s. Standards for good manufacturing practices have also been established and are routinely relied upon for the processing of HCT grafts including the use of a variety of selection/purification techniques and ex vivo expansion. The establishment of cell therapy facilities should be a reality in the near future and, like blood banks, will likely be considered to be an essential service to hospitals. For cost containment reasons, one could envision centralized repositories to harvest, expand, and characterize the composition of grafts/infusions, as well as to perform HLA-typing and serologic and sterility testing. Both the national marrow donor program and the solid organ registries demonstrate that networks can be established to facilitate donor-host pairing. The dramatic advances made in the field of human hematopoietic cell biology have provided a foundation of both excellent preclinical studies and early clinical trials. It is clear that in order to make meaningful progress towards useful therapies, preclinical studies must continue to be performed in tandem with clinical trials. Clinical trials should proceed immediately on two general fronts. First, transplantation of highly purified HSC and highly purified progenitor cells without ex vivo expansion in both the autologous and allogeneic setting should be performed to confirm proof of concept and dose response curves. Second, studies of ex vivo expansion should continue to be actively pursued. Ideally, the establishment of consortia, each with a core facility for ex vivo expansion and a group of large clinical centers, would execute clinical studies of sufficient size and adequate design in parallel with laboratory investigation to determine correlates to clinical outcome. doi: 10.1016/j.exphem.2004.07.001
J.M. Brown and I.L.Weissman Stanford University, Stanford, Calif., USA
References 1. Manz MG, Miyamotot T, Akashi K, Weissman IL. Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A. 2002;99:11872–11877. 2. Uchida N, Aguila HL, Fleming WH, Jearbek L, Weissman IL. Rapid and sustained hematopoietic recovery in lethally irradiated mice transplanted with purified Thy-1.1loLin⫺Sca-1+ hematopoietic stem cells. Blood. 1994;83:3758–3779. 3. Uchida N, Weissman IL. Searching for hematopoietic stem cells: evidence that Thy-1.1loLin⫺Sca-1+ cells are the only stem cells in C57BL/ Ka-Thy-1.1 bone marrow. J Exp Med. 1992;175:175–184. 4. Uchida N, Jerabek L, Weissman IL. Searching for hematopoietic stem cells. II. The heterogeneity of Thy-1.1loLin⫺/loSca-1+ mouse hematopoietic stem cells separated by counterflow centrifugal elutriation. Exp Hematol. 1996;24:649–659. 5. Devine SM, Lazarus HM, Emerson SG. Clinical application of hematopoietic progenitor cell expansion: current status and future prospects. Bone Marrow Transplant. 2003;31:241–252.
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