International Journal of Cell Cloning 8:76-91 Suppl 1 (1990)
Human Umbilical Cord Blood: A Clinically Useful Source of Transplantable Hematopoietic Stem/Progenitor Cells Hal E. Broxmeyer4 Eliane Gluckman,' Arleen Auerbach', Gordon W Douglasd, Henry Friedman,' Scott Cooper4 Giao Hangoc", Joanne Kurtzberg: Judith Bard< Edward A . B o y d a Departments of Medicine (Hematology/Oncology), Microbiology and Immunology, and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA; bBone Marrow Transplant Unit, HBpital Saint-Louis, Paris, France; 'Laboratory for Investigative Dermatology, the Rockefeller University, New York, New York, USA; dDepartment of Obstetrics and Gynecology, New York University Medical Center, New York, New York, USA; 'Department of Pediatrics (Hematology/Oncology), Duke University Medical Center, Durham, North Carolina, USA; Department of Microbiology and Immunology, University of Arizona Health Sciences Center, lkcson, Arizona, USA
Key Words. Umbilical cord blood Hematopoietic reconstituting cells
Hematopoietic stem/progenitor cells
Abstract. This is a review and discussion of studies leading to the first use of human umbilical cord blood, material usually discarded, for the provision of stendprogenitor cells for clinical hematopoietic reconstitution [l, 21. This prospect arose as a result of extensive studies of the harvesting and cryopreservation of cord blood and of its numerical content of progenitor cells demonstrable in vim. A male patient with Fanconi anemia (FA) was conditioned with a modified regimen of cyclophosphamideand irradiation that accommodates the abnormally high sensitivity to these agents that is characteristic of FA. Cryopreserved cord blood had been retrieved at birth from a female sibling known from prenatal testing to be unaffected by FA and to be human leukocyte antigen (HLA)-compatible with the prospective sibling recipient. After conditioning and therapeutic infusion of thawed cord blood, successful hematopoietic reconstitution was indicated by the general health of the patient, who had previously required supportive transfusions, by satisfactory hematological criteria and by counts of hematopoietic progenitor cells of various types in the bone marrow. Complete engraftment of the myeloid system with donor cells was evident from cytogenetics, ABO typing, study of DNA polymorphism, and normal cellular resistance to cytotoxic agents that reveal the fragility of FA cells; the blood contained a residuum of host lymphocytes Correspondence: Hal E. Broxmeyer, Ph.D., Walther Oncology Center and Department of Medicine, Indiana University School of Medicine, Room 501, Medical Research and Library Building, 975 W. Walnut Street, Indianapolis, IN 46202-5121, USA. Received November 7, 1989; accepted for publication November 7, 1989. 0737-1454/90/$2.00/0 @AlphaMedPress
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exhibiting chromosomaldamage, but the trend has been towards eliminating these damaged cells. This implies that cord blood from a single individual should provide sufficient reconstituting cells for effective hematopoietic repopulation of an autologous or an HLAcompatible allogeneic recipient.
Introduction Hematopoiesis is sustained by a pool of hematopoietic stem and progenitor cells, whose ability to proliferate and differentiate are regulated by the production and action of a number of cytokines that can stimulate, enhance and/or suppress the above processes [3-111. The primary site of production of transplantable hematopoietic reconstituting cells in the adult is the bone marrow [5], which is the major reason why autologous or major histocompatibilitycomplex-matched allogeneic bone marrow transplantationis the usual therapeutic source for hemampoietic reconstituting cells. Hematopoietic stedprogenitor cells have been found in the circulating blood of adults, and this source has been used with some success for clinical stendprogenitor cell transplantation [l2-23]. However, hematopietic stendprogenitor cells in circulating adult blood are present in concentrations one-tenth to one-thousandthof that found in bone marrow [5], so that use of adult blood for stendprogenitor cells for transplantation purposes requires multiple leukopheresisand has been obtained and used primarily in an autologous situation in patients undergoing temporary rebound hematopoiesis resulting from intensive chemotherapy [Q]. Efforts to enhance numbers of circulating progenitor cells in adult blood for such use have utilized infusion of certain hematopoietic colony-stimulating factors (CSFs) , such as granulocytemacrophage (GM)-CSF, alone and in combination with intensive cliemotherapy [24]. There has been a concern however that adult peripheral blood stem cells may have only a f ~ t ecapacity for maintaining hematopoiesis [25]. Ontologically, hematopoietic stendprogenitorcells are found first in the yolk sac, later in the fetal liver, and then in the marrow of the fetus [5,26-291. Transplantation of fetal liver cells has been used in a limited setting in attempts to correct hematopoieticdeficiencies [30,31], but the use of such cells presents ethical dilemmas. Hematopoietic stemlprogenitor cells occur in fetal blood, and human umbilical cord blood is a source of “stem” (S)cells, as well as multipotential (CFU-gemm), erythroid (BFUe), granuloqte-macrophage(CFU-gm), and megakaryocyte (CFU-meg) progenitor cells [l, 32-44]. Moreover, hematopoietic progenitor cells from human umbilical cord blood can be maintained for many weeks in “long-term” culture systems [45,46], suggesting their production from more primitive cells. The high frequency of hematopoietic progenitor cells in umbilical cord blood at birth, which is apparently an expansion of these cells during the last months of pregnancy and the end phases of bone marrow colonization from other sites of hematopoiesis [33], does not last long; in fact, very shortly after birth, the blood of the newborn contains very rapidly diminishing numbers of these cells [33, 431.
Broxmeyer et al.
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The use of human umbilical cord blood for hematopoietic reconstitution was suggested by EdwardA. B q s e , who found that lethally irradiated mice could be rescued and reconstituted with neonatal mouse blood, in discussions with Hal E. Broxmtyer on the potential uses of this usually discarded cellular source. This discussion set into motion a multi-investigator, multi-institutional, national collaborative study to evaluate human umbilical cord blood as an alternative source for the provision of transplantable stedprogenitor cells for hematopoietic reconstitution [I]. The studies presented here include a review of those recently published investigations [l, 21.
Study Design The major problem with assessing the numbers of hematopoietic repopulating cells that could be isolated from umbilical cord blood was the lack of a quantitative assay for these cells in humans. It is not clear whether the S cell [39] falls into this category, and in all likelihood, the S cell is probably not the hematopoietic reconstituting cell. Moreover, the extremely low frequency of this cell in adult bone marrow and umbilical cord blood [39,47-49], even after extensive cell purification procedures to enrich this cell type and the need to retrospectively identify this cell by its replating potential in culture, makes it a difficult cell to quantitate. Although there is no direct quantitative assay for human hematopoietic reconstituting cells, the CFU-gm assay has been used successfully as an estimator of the engrafting capability of human bone marrow in a transplantation setting [50-531. We examined more than 100 collections of human umbilical cord blood after transport of the cells by overnight express mail service from distant obstetrical services to the Indiana University School of Medicine [l]. The cord blood was assessed for content of nucleated cells, CFU-gm, BFU-e, and CFU-gem, in many cases both before and after cryopreservation [l]. It was first determined that these progenitor cells remained functionally viable in cord blood untreated except for the addition of anticoagulant for at least 3 days at 4°C or 25°C (room temperature), although not at 3TC, thus implying that these cells could be transported in a viable state at room temperature. These cord blood progenitors responded normally to stimulationby GM-CSF, granulocyte(G)-CSF, macrophage (M)-CSF, interleukin 3 (IL-3), and erythropoietin (Epo). The major findings, based on analysis of 101 cord blood collections was that the numbers of progenitor cells present in the low-density fraction after separation with Ficoll-Hypaque typically fell within the range (although the lower end of it) which has been reported to be associated with successful engraftment by bone marrow cells. However, it was also noted that large losses of progenitors occurred during the cell separations, suggesting that we had grossly underestimated the actual number of hematopoietic progenitor cells present in unseparated cord blood. Studies to maximize collection of the cord blood and assessment of the numbers of progenitors actually obtained from unseparated cord blood demonstrated that a single collection of
Umbilical Cord Blood Transplantable Stem Cells
79
umbilical cord blood contained numbers of progenitors clearly in the range, and sometimes in the high range, of that which has been associated with successful hematopoietic engraftment. Other observations of practical importance were that umbilical cord blood nucleated cells and progenitor cells could be cryopreserved and thawed in viable form, but washing of thawed cells entailed large losses of both total nucleated and progenitor cells. These studies [I] suggested to us that umbilical cord blood from a single individual was typically a sufficient source of cells for autologous (syngeneic)and for major histocompatibilitycomplex-matched allogeneic hematopoietic reconstitution, but that for optimal therapy in an actual transplant setting, the collected umbilical cord blood cells should be frozen in an unseparated state without even attempting to remove red cells, and infused after thawing without prior washing to remove hemolyzed red cells or the cryopreservant. Such situations have been reported previously in the setting of bone marrow transplantation. Based on the above-mentioned laboratory findings [l], a survey was made to identify a suitable and relevant clinical disease model that could serve as a setting for a first trial of hematopoietic engraftment, using umbilical cord blood cells from a single individual for use in an HLA-matched allogeneic situation for a sibling. At this point, the studies were enlarged to include investigators at other institutions, both nationally and internationally [2]. Fanconi anemia (FA) is an autosomal recessive disorder that entails progressive pancytopenia and predisposition to malignancy, together with nonhematopoietic developmental anomalies [54-561. The FA phenotype is variable so that diagnosis by clinical manifestations alone is difficult [57,58], but hypersensitivity to the clastogenic effect of DNA cross-linkingagents such as diepoxybutane (DEB) or nitrogen mustard, as previously reported by our collaborators, provides a diagnostic indicator of the FA genotype both prenatally and postnatally [58-631. Patients with FA often die young from complicationsof bone marrow aplasia such as hemorrhage or infection, or from leukemia. Bone marrow transplantation affords the prospect of a cure for the hematologic manifestations of the syndrome, but in FA it can be used with relative safety only if an HLA identical sibling donor is available [64]. It was noted by our co-investigators that FA patients could not tolerate the agents usually used for pretransplant conditioning (200 mg/kg cyclophosphamide), because of their hypersensitivity to alkylating agents which cross-link DNA [64-661 and to irradiation [67,68]. To lessen toxicity from conditioning in these patients, a new protocol consisting of administration of 20 mgkg of cyclophosphamide (total dose over 4 days) and 5 Grays (Gy) thoraco-abdominal irradiation was used [69-711. This regimen was similar to that used for transplantation in other patients with severe aplastic anemia, but with reduction of dosage. Twenty-three patients with FA had received bone marrow tran'splants according to this protocol; the long-term actuarial survival rate was 70%,with a median follow up of 4 years [71].
Broxmeyer et al.
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The ability to perform HLA typing on fetal cells obtained during the first or second trimester of pregnancy [72] made it possible to ascertain whether a fetus was HLA identical to a sibling affected with FA. Three of 8 fetuses diagnosed as unaffected with FA were shown prenatally to be HLA identical to affected siblings [73]. This above information allowed us to evaluate the first instance of a hematopoietic stem/progenitorcell transplant using umbilical cord blood harvested at birth from an individual who was known, before birth, to be unaffected with FA, to be karyotypically normal, and to be HLA identical to her brother (who had FA). We were acutely aware that an umbilical cord blood hematopoietic stem/progenitor cell transplant was not a usual occurrence, and serious concern was given to ethical considerations inherent in such a procedure. This case was evaluatedand approved by the Institutional Review Board (IRB) for Clinical Investigationof the Duke University Medical Center from where the patient with FA was referred. They considered the following arguments in favor of the umbilical cord blood transplant: 1) the HLA-matched sibling donor, who was to be born and would be very young at the time of the transplant, would be spared the dangers of anesthesia and other complications potentially associated with bone marrow donation, 2) proof that umbilical cord blood could serve as an effective source of stem/progenitor cells would constitute a notable advance in the treatment of many diseases now being treated by autologous or HLA-matched allogeneic bone marrow transplantation, and 3) by the time of the transplant, marrow cells from the HLA-matched sibling could, if necessary, be used as a backup source of stem/progenitor cells. Written informed consent was obtained from the family for the collection of cord blood and again prior to referral of the patient to France. IRB approval from the Indiana University School of Medicine was given for the in vitro studies, for cryopreservation, for storage, and for transportation of the cord blood to the site of its eventual use. The case was also reviewed and approved by the Ethical Committee of the HBpital Saint-Louis in Paris. The parents of the recipient and the donor signed an informed consent for the transplant. It was explained that if the umbilical cord blood transplant failed, it would still be possible to use marrow cells from the HLA-identical sibling, who would be 6-months old at the time of the initial cord blood transplant. Regarding the regulatory issues, following discussions and correspondence, the Division of Biologics of the U.S. Food and Drug Administration informed us that it considered the cryostorage and subsequent use of cord blood stem and progenitor cells equivalent to bone marrow banks and other non-blood tissue banks, a category which is currently not subject to FDA regulation. Immediately upon uncomplicated vaginal delivery of the infant, the umbilical cord was doubly clamped 5 to 6 cm from the umbilicus and transected between the clamps. The infant was removed from the field. Blood was collected from the maternal (placental) end of the transected cord, while the placenta remained in situ, to take advantage of the enhanced blood flow generated by uterine
-
Umbilical Cord Blood Transplantable Stem Cells
81
contraction. Blood was also obtained from the removed placenta by needle aspiration of exposed, engorged vessels on the fetal surface. Collection was made into sterile wide-mouth glass bottles containing anticoagulant acid-citrate dextrose and penicillin-streptomycinand was sent at ambient temperature by overnight express service to the Indiana University School of Medicine for cellular analysis, cryopreservation and storage. A small sample of cord blood was also sent to the Rockefeller University for cytogenetic analysis. After removing a small sample of cells (
Human Umbilical Cord Blood: A Clinically Useful Source of Transplantable Hematopoietic Stem/Progenitor Cells Hal E. Broxmeyer4 Eliane Gluckman,' Arleen Auerbach', Gordon W Douglasd, Henry Friedman,' Scott Cooper4 Giao Hangoc", Joanne Kurtzberg: Judith Bard< Edward A . B o y d a Departments of Medicine (Hematology/Oncology), Microbiology and Immunology, and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA; bBone Marrow Transplant Unit, HBpital Saint-Louis, Paris, France; 'Laboratory for Investigative Dermatology, the Rockefeller University, New York, New York, USA; dDepartment of Obstetrics and Gynecology, New York University Medical Center, New York, New York, USA; 'Department of Pediatrics (Hematology/Oncology), Duke University Medical Center, Durham, North Carolina, USA; Department of Microbiology and Immunology, University of Arizona Health Sciences Center, lkcson, Arizona, USA
Key Words. Umbilical cord blood Hematopoietic reconstituting cells
Hematopoietic stem/progenitor cells
Abstract. This is a review and discussion of studies leading to the first use of human umbilical cord blood, material usually discarded, for the provision of stendprogenitor cells for clinical hematopoietic reconstitution [l, 21. This prospect arose as a result of extensive studies of the harvesting and cryopreservation of cord blood and of its numerical content of progenitor cells demonstrable in vim. A male patient with Fanconi anemia (FA) was conditioned with a modified regimen of cyclophosphamideand irradiation that accommodates the abnormally high sensitivity to these agents that is characteristic of FA. Cryopreserved cord blood had been retrieved at birth from a female sibling known from prenatal testing to be unaffected by FA and to be human leukocyte antigen (HLA)-compatible with the prospective sibling recipient. After conditioning and therapeutic infusion of thawed cord blood, successful hematopoietic reconstitution was indicated by the general health of the patient, who had previously required supportive transfusions, by satisfactory hematological criteria and by counts of hematopoietic progenitor cells of various types in the bone marrow. Complete engraftment of the myeloid system with donor cells was evident from cytogenetics, ABO typing, study of DNA polymorphism, and normal cellular resistance to cytotoxic agents that reveal the fragility of FA cells; the blood contained a residuum of host lymphocytes Correspondence: Hal E. Broxmeyer, Ph.D., Walther Oncology Center and Department of Medicine, Indiana University School of Medicine, Room 501, Medical Research and Library Building, 975 W. Walnut Street, Indianapolis, IN 46202-5121, USA. Received November 7, 1989; accepted for publication November 7, 1989. 0737-1454/90/$2.00/0 @AlphaMedPress
Umbilical Cord Blood Transplantable Stem Cells
77
exhibiting chromosomaldamage, but the trend has been towards eliminating these damaged cells. This implies that cord blood from a single individual should provide sufficient reconstituting cells for effective hematopoietic repopulation of an autologous or an HLAcompatible allogeneic recipient.
Introduction Hematopoiesis is sustained by a pool of hematopoietic stem and progenitor cells, whose ability to proliferate and differentiate are regulated by the production and action of a number of cytokines that can stimulate, enhance and/or suppress the above processes [3-111. The primary site of production of transplantable hematopoietic reconstituting cells in the adult is the bone marrow [5], which is the major reason why autologous or major histocompatibilitycomplex-matched allogeneic bone marrow transplantationis the usual therapeutic source for hemampoietic reconstituting cells. Hematopoietic stedprogenitor cells have been found in the circulating blood of adults, and this source has been used with some success for clinical stendprogenitor cell transplantation [l2-23]. However, hematopietic stendprogenitor cells in circulating adult blood are present in concentrations one-tenth to one-thousandthof that found in bone marrow [5], so that use of adult blood for stendprogenitor cells for transplantation purposes requires multiple leukopheresisand has been obtained and used primarily in an autologous situation in patients undergoing temporary rebound hematopoiesis resulting from intensive chemotherapy [Q]. Efforts to enhance numbers of circulating progenitor cells in adult blood for such use have utilized infusion of certain hematopoietic colony-stimulating factors (CSFs) , such as granulocytemacrophage (GM)-CSF, alone and in combination with intensive cliemotherapy [24]. There has been a concern however that adult peripheral blood stem cells may have only a f ~ t ecapacity for maintaining hematopoiesis [25]. Ontologically, hematopoietic stendprogenitorcells are found first in the yolk sac, later in the fetal liver, and then in the marrow of the fetus [5,26-291. Transplantation of fetal liver cells has been used in a limited setting in attempts to correct hematopoieticdeficiencies [30,31], but the use of such cells presents ethical dilemmas. Hematopoietic stemlprogenitor cells occur in fetal blood, and human umbilical cord blood is a source of “stem” (S)cells, as well as multipotential (CFU-gemm), erythroid (BFUe), granuloqte-macrophage(CFU-gm), and megakaryocyte (CFU-meg) progenitor cells [l, 32-44]. Moreover, hematopoietic progenitor cells from human umbilical cord blood can be maintained for many weeks in “long-term” culture systems [45,46], suggesting their production from more primitive cells. The high frequency of hematopoietic progenitor cells in umbilical cord blood at birth, which is apparently an expansion of these cells during the last months of pregnancy and the end phases of bone marrow colonization from other sites of hematopoiesis [33], does not last long; in fact, very shortly after birth, the blood of the newborn contains very rapidly diminishing numbers of these cells [33, 431.
Broxmeyer et al.
78
The use of human umbilical cord blood for hematopoietic reconstitution was suggested by EdwardA. B q s e , who found that lethally irradiated mice could be rescued and reconstituted with neonatal mouse blood, in discussions with Hal E. Broxmtyer on the potential uses of this usually discarded cellular source. This discussion set into motion a multi-investigator, multi-institutional, national collaborative study to evaluate human umbilical cord blood as an alternative source for the provision of transplantable stedprogenitor cells for hematopoietic reconstitution [I]. The studies presented here include a review of those recently published investigations [l, 21.
Study Design The major problem with assessing the numbers of hematopoietic repopulating cells that could be isolated from umbilical cord blood was the lack of a quantitative assay for these cells in humans. It is not clear whether the S cell [39] falls into this category, and in all likelihood, the S cell is probably not the hematopoietic reconstituting cell. Moreover, the extremely low frequency of this cell in adult bone marrow and umbilical cord blood [39,47-49], even after extensive cell purification procedures to enrich this cell type and the need to retrospectively identify this cell by its replating potential in culture, makes it a difficult cell to quantitate. Although there is no direct quantitative assay for human hematopoietic reconstituting cells, the CFU-gm assay has been used successfully as an estimator of the engrafting capability of human bone marrow in a transplantation setting [50-531. We examined more than 100 collections of human umbilical cord blood after transport of the cells by overnight express mail service from distant obstetrical services to the Indiana University School of Medicine [l]. The cord blood was assessed for content of nucleated cells, CFU-gm, BFU-e, and CFU-gem, in many cases both before and after cryopreservation [l]. It was first determined that these progenitor cells remained functionally viable in cord blood untreated except for the addition of anticoagulant for at least 3 days at 4°C or 25°C (room temperature), although not at 3TC, thus implying that these cells could be transported in a viable state at room temperature. These cord blood progenitors responded normally to stimulationby GM-CSF, granulocyte(G)-CSF, macrophage (M)-CSF, interleukin 3 (IL-3), and erythropoietin (Epo). The major findings, based on analysis of 101 cord blood collections was that the numbers of progenitor cells present in the low-density fraction after separation with Ficoll-Hypaque typically fell within the range (although the lower end of it) which has been reported to be associated with successful engraftment by bone marrow cells. However, it was also noted that large losses of progenitors occurred during the cell separations, suggesting that we had grossly underestimated the actual number of hematopoietic progenitor cells present in unseparated cord blood. Studies to maximize collection of the cord blood and assessment of the numbers of progenitors actually obtained from unseparated cord blood demonstrated that a single collection of
Umbilical Cord Blood Transplantable Stem Cells
79
umbilical cord blood contained numbers of progenitors clearly in the range, and sometimes in the high range, of that which has been associated with successful hematopoietic engraftment. Other observations of practical importance were that umbilical cord blood nucleated cells and progenitor cells could be cryopreserved and thawed in viable form, but washing of thawed cells entailed large losses of both total nucleated and progenitor cells. These studies [I] suggested to us that umbilical cord blood from a single individual was typically a sufficient source of cells for autologous (syngeneic)and for major histocompatibilitycomplex-matched allogeneic hematopoietic reconstitution, but that for optimal therapy in an actual transplant setting, the collected umbilical cord blood cells should be frozen in an unseparated state without even attempting to remove red cells, and infused after thawing without prior washing to remove hemolyzed red cells or the cryopreservant. Such situations have been reported previously in the setting of bone marrow transplantation. Based on the above-mentioned laboratory findings [l], a survey was made to identify a suitable and relevant clinical disease model that could serve as a setting for a first trial of hematopoietic engraftment, using umbilical cord blood cells from a single individual for use in an HLA-matched allogeneic situation for a sibling. At this point, the studies were enlarged to include investigators at other institutions, both nationally and internationally [2]. Fanconi anemia (FA) is an autosomal recessive disorder that entails progressive pancytopenia and predisposition to malignancy, together with nonhematopoietic developmental anomalies [54-561. The FA phenotype is variable so that diagnosis by clinical manifestations alone is difficult [57,58], but hypersensitivity to the clastogenic effect of DNA cross-linkingagents such as diepoxybutane (DEB) or nitrogen mustard, as previously reported by our collaborators, provides a diagnostic indicator of the FA genotype both prenatally and postnatally [58-631. Patients with FA often die young from complicationsof bone marrow aplasia such as hemorrhage or infection, or from leukemia. Bone marrow transplantation affords the prospect of a cure for the hematologic manifestations of the syndrome, but in FA it can be used with relative safety only if an HLA identical sibling donor is available [64]. It was noted by our co-investigators that FA patients could not tolerate the agents usually used for pretransplant conditioning (200 mg/kg cyclophosphamide), because of their hypersensitivity to alkylating agents which cross-link DNA [64-661 and to irradiation [67,68]. To lessen toxicity from conditioning in these patients, a new protocol consisting of administration of 20 mgkg of cyclophosphamide (total dose over 4 days) and 5 Grays (Gy) thoraco-abdominal irradiation was used [69-711. This regimen was similar to that used for transplantation in other patients with severe aplastic anemia, but with reduction of dosage. Twenty-three patients with FA had received bone marrow tran'splants according to this protocol; the long-term actuarial survival rate was 70%,with a median follow up of 4 years [71].
Broxmeyer et al.
80
The ability to perform HLA typing on fetal cells obtained during the first or second trimester of pregnancy [72] made it possible to ascertain whether a fetus was HLA identical to a sibling affected with FA. Three of 8 fetuses diagnosed as unaffected with FA were shown prenatally to be HLA identical to affected siblings [73]. This above information allowed us to evaluate the first instance of a hematopoietic stem/progenitorcell transplant using umbilical cord blood harvested at birth from an individual who was known, before birth, to be unaffected with FA, to be karyotypically normal, and to be HLA identical to her brother (who had FA). We were acutely aware that an umbilical cord blood hematopoietic stem/progenitor cell transplant was not a usual occurrence, and serious concern was given to ethical considerations inherent in such a procedure. This case was evaluatedand approved by the Institutional Review Board (IRB) for Clinical Investigationof the Duke University Medical Center from where the patient with FA was referred. They considered the following arguments in favor of the umbilical cord blood transplant: 1) the HLA-matched sibling donor, who was to be born and would be very young at the time of the transplant, would be spared the dangers of anesthesia and other complications potentially associated with bone marrow donation, 2) proof that umbilical cord blood could serve as an effective source of stem/progenitor cells would constitute a notable advance in the treatment of many diseases now being treated by autologous or HLA-matched allogeneic bone marrow transplantation, and 3) by the time of the transplant, marrow cells from the HLA-matched sibling could, if necessary, be used as a backup source of stem/progenitor cells. Written informed consent was obtained from the family for the collection of cord blood and again prior to referral of the patient to France. IRB approval from the Indiana University School of Medicine was given for the in vitro studies, for cryopreservation, for storage, and for transportation of the cord blood to the site of its eventual use. The case was also reviewed and approved by the Ethical Committee of the HBpital Saint-Louis in Paris. The parents of the recipient and the donor signed an informed consent for the transplant. It was explained that if the umbilical cord blood transplant failed, it would still be possible to use marrow cells from the HLA-identical sibling, who would be 6-months old at the time of the initial cord blood transplant. Regarding the regulatory issues, following discussions and correspondence, the Division of Biologics of the U.S. Food and Drug Administration informed us that it considered the cryostorage and subsequent use of cord blood stem and progenitor cells equivalent to bone marrow banks and other non-blood tissue banks, a category which is currently not subject to FDA regulation. Immediately upon uncomplicated vaginal delivery of the infant, the umbilical cord was doubly clamped 5 to 6 cm from the umbilicus and transected between the clamps. The infant was removed from the field. Blood was collected from the maternal (placental) end of the transected cord, while the placenta remained in situ, to take advantage of the enhanced blood flow generated by uterine
-
Umbilical Cord Blood Transplantable Stem Cells
81
contraction. Blood was also obtained from the removed placenta by needle aspiration of exposed, engorged vessels on the fetal surface. Collection was made into sterile wide-mouth glass bottles containing anticoagulant acid-citrate dextrose and penicillin-streptomycinand was sent at ambient temperature by overnight express service to the Indiana University School of Medicine for cellular analysis, cryopreservation and storage. A small sample of cord blood was also sent to the Rockefeller University for cytogenetic analysis. After removing a small sample of cells (
