stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application:...

ELSEVIER Critical Reviews in Oncology/Hematology 22 (1996) 61-78

Critical Reviews in

ONCOLOGY/ HEMA TOLOG Y

Stem cells from bone marrow, umbilical cord blood and peripheral blood for clinical application: current status and future application

Li Lu*~*~, Rong-Nian Shenavcyd, Hal E. BroxmeyeraTbTd ‘Department of Medicine (Hematology/Oncology), Indiano University School of Medicine, Indianapolis, IN 46202-5321. USA

bDepartment of Microbiology/Immunology, Indiano University School of Medicine, Indianapolis, IN 46202-5121, USA ‘Department of Radbtion Oncology, Indiano University School of Medicine, Indianapolis, IN 46202-5121, USA

dThe Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202-5121. USA

Accepted 28 October 1994

Contents

I. 2.

3. 4.

5.

6. 7. 8.

Introduction .......................................................................... Hematopoietic stem cell purification and ex vivo expansion .................................. 2.1. Stem cell purification ............................................................. 2.2. Long-term culture and ex vivo expansion ........................................... Bone marrow stem cell transplantation .................................................... Umbilical cord blood stem cell transplantation ............................................. 4. I. Quantity and quality of immature hematopoietic stem cells from umbilical cord blood .... 4.2. Immune cells in cord blood .......................................................

4.2.1. Number and phenotype of cord blood T-lymphocytes ......................... 4.2.2. Function of the T-lymphocytes in cord blood ................................ 4.2.3. Maternal cell contamination in cord blood ...................................

Peripheral blood stem cell transplantation ................................................. 5.1. Blood stem cell mobilization and collection .......................................... 5.2. Kinetics of hemopoietic reconstitution after ABSCT .................................. 5.3. Contamination of harvested blood tumor cells ..................................... 5.4. Stability and sustainment of short- and long-term hemopoietic reconstitution after ABSC T Fetal liver stem cells (FLSC) transplantation ............................................ In utero hematopoietic stem cell transplants for inherited diseases .......................... Stem cell as target for gene therapy .................................................... 8.1. Increase transduction efficiency .................................................. 8.2. Long-term gene expression in hematopoietic cells in vivo ............................

9. Conclusion ............................................................................ Acknowledgements .............................................................................. Reviewer ....................................................................................... References ..................................................................................... Biographies .....................................................................................

62 62 62 63 64 65 65 65 66 66 66 66 67 68 68 68 69 69 69 70 70 71 71 71 71 78

Abbreviations: ABMT, autologous bone marrow transplantation; ABSCT, autologous blood stem cell transplantation; ADA, adenosine deaminase; ALL, acute. lymphocytic leukemia; allo, allogeneic; AML, acute myeloid leukemia; BM, bone marrow; BMT, bone marrow transplantation; CB, cord blood, CBSC, cord blood stem cells; CBSCT, cord blood stem cell transplantation; CML, chronic myeloid leukemia; CSF, colony stimulating factors; CT, cytotoxic therapy; FL, fetal liver; FLSC, fetal liver stem cells; GM-CSF, granulocyte macrophage-colony stimulating factor; GVHD, graft-versus-host disease; HGF, hematopoietic growth factors; HLA, human leukocyte antigens; HPP-CFC, high proliferative potential-colony forming cells; HSC, hematopoietic stem cells; IL, interleukin; LTC-IC, long-term culture-initiating cells; NK, natural killer; PB, peripheral blood; PBSC, peripheral blood stem cells; PCR, polymerase chain reaction; rhu, recombinant human; SCID, severe combined immunodeficiency disease; SLF, steel factor (also termed stem cell factor).

*Corresponding author, Indiana University School of Medicine, 975 W. Walnut Street, Room 501, Indianapolis, IN 46202-5 121, USA. Tel.: (3 17) 274-7560; fax: (317) 274-7592.

1040-8428/96/$32.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 1040-8428(95)0179-B

62 L. Lu et al. /Critical Reviews in Oncology/Hematology 22 (1996) 61-78

Bone marrow transplantation (BMT) has progressed rapidly during the past two decades to that of a treatment of choice as a therapeutically effective modality for the treatment of selected patients with malignant disease and non-malignant hematological disorders. However, its use is limited by availability of human leukocyte antigens (HLA)-matched donor cells, engraftment and graft-versus-host disease (GVHD). Prevention of GVHD, improvement in the speed and quality of marrow reconstitution, and screening of new im- munomodulating agents which improve engraftment and augment hemopoiesis are intense areas of investigation. To this end there has clearly been progress in puri- tication and characterization of human stem cells from different tissue sources. Discussed in this review are: (a) stem cell purification, characterization and ex vivo expansion; (b) bone marrow stem cell transplantation; (c) cord blood stem cell transplantation; (d) peripheral blood stem cell transplantation; (e) fetal liver stem cell transplantation; (f) in utero stem cell transplantation; and (g) evaluation of the capacity of stem cells to serve as targets for gene therapy.

1. Illtmduction

As early as 1949, Jacobson et al, [l] showed that shielding the spleen allowed mice to recover from lethal irradiation. Shortly thereafter Lorenz et al. [2] showed that irradiation protection could be achieved by an intravenous infusion of syngeneic marrow. Since that time, marrow grafting has been used for hematopoietic recovery, especially after myeloablative therapy using irradiation and/or chemotherapy. Thomas et al. [3] demonstrated that large quantities of marrow could be safely infused intravenously, but grafting was difficult. Mathe [4] reported an enduring allogeneic marrow graft in a patient with leukemia, and Gatti et al. [5] performed marrow transplantation from a matched sibling for an infant with an immunological deficiency disease. Thus, began the ‘modem’ era of bone marrow transplantation (BMT) [6]. BMT was initially developed as a treatment for severe combined immunodeficiency disease (SCID), aplastic anemia and acute leukemia. Based on pro- gressively improved results during the past two decades, this therapy has provided a means for curative correction of a large number of lethal congenital and acquired disorders of the hematopoietic and lymphoid systems. BMT has become the treatment of choice for aplastic anemia and has emerged as a highly promising approach for the treatment of high-risk forms of leukemia, par- ticularly when applied to patients in remission early in the course of their disease, and for a large variety of im- munodeticiencies, hemopathies and inborn errors of metabolism. Intensive chemotherapy and/or irradiation followed by BMT reconstitution is being increasingly

used in the treatment of malignancies and inherited diseases. BMT represents a viable therapeutic approach, but its use is limited by the availability of HLA-matched donor cells, engraftment problems and graft-versus-host disease (GVHD).

Hematopoietic growth factors (HGFs) were initially identified by their ability to support stem/progenitor cell growth in culture assays [7,8]. During the past decade, a number of HGFs have been purified, cloned and recombinant forms produced. This has allowed clini- cians the opportunity to stimulate blood cell production in a relatively controlled fashion to achieve a therapeutic benefit. In clinical trials it has already been shown that granulocyte-macrophage (GM)colony stimulating factor (CSF) and G-CSF can accelerate marrow recovery after both allogeneic and autologous marrow graft with significant shortening of the time in the hospital [9,10]. The purification, characterization and biological function of hematopoietic stem cells responsible for reconstitution of marrow and blood and for indefinite cell renewal have been a targeted goal of experimental hematologists. During the last decade, much progress has been made in the purification of stem cells [ 1 l- 161. Gene transfer for the correction of genetic diseases of the marrow is an area of intense investigation. The use of retroviral vectors has increased the efficiency of gene transfer in the laboratory [17-211. Sustained expression of genes transferred into hematopoietic stem cells has been successful [ 181. Thus, stem cells can not only be used for marrow reconstitution, but can also serve as targets for gene transfer and subsequent gene therapy.

2. Hematopoietic stem cell purification and ex vivo expansion

2.1. Stem cell purijkation Hematopoietic stem cells (HSC) constitute a very

small pool of undifferentiated cells that are able to maintain their number by self-renewal and have the capability to differentiate into committed progenitor cells for all lymphoid and myeloid cell lineages. They can reconstitute the hematopoietic system of a lethally irradiated recipient [l l-151. The frequency of HSC among bone marrow (BM) cells has been variously estimated at between one per 10 000 and one per 100 000 cells. The yolk sac is the first site of hematopoiesis during ontogeny and thus logically might be the initial source of hematopoietic stem cells. Stem cells have been identified in the fetal liver (FL), thymus and bone marrow at later stages of embryogenesis [ 141. The origin of hematopoietic cells from HSC has been demonstrated using chromosomal markers [ 161, and more recently by retroviral integration markers [ 17-231. Four tissue sources of HSC are BM, umbilical cord blood (CB), FL and adult peripheral blood (PB).

L. Lu et al. /Critical Reviews in Oncology/Hematology 22 (19%) 61-78 63

In mouse, hematopoietic stem cells have been purified by a variety of methods, and a population of cells (Thy- I”, Lin-, Ly6A/E+) can be obtained which are approx- imately 1000 times enriched [24-261. These cells have been used for transplantation into lethally irradiated mouse recipients, and have resulted in reconstitution of myeloid and lymphoid lineages [27]. These cells have also been used in single-cell transplantation studies [25].

In humans, there have been numerous attempts to purify or enrich hematopoietic stern/progenitor cells using density gradient centrifugation, counterflow cen- trifugal elutriation, and cell sorting based on light scatter and antigenic determinant properties. A marker for human stem and progenitor cells, while not specific for these cells is the cell surface antigen CD34 which is present on a small fraction (< 1%) of unfractionated cells [28-571. We have enriched a population of colony forming cells with 47% purity from normal human bone marrow by fluorescence - activated cell sorting with monoclonal antibodies against CD34 and major histocompatibility class II (HLA-DR) antigens [48]. Our results have confirmed that the majority of normal human bone marrow colony forming cells express deter- minants recognized by highest densities of CD34 and lower densities of HLA-DR. We then demonstrated that recombinant human (rhu) growth cytokines can act directly on the proliferation of single hematopoietic progenitor cells in the absence of accessory cells and serum [Sl]. Efforts have been focused on purification of immature stem cells, and these cells can be further enriched using monoclonal antibodies against CD38 [32,33], CD33 [34-351, YB5.B8 (c-kit proto-oncogene product) [36], CD45 (a leukocyte common antigen isoform) [30,31], CD71 (the transferrin receptor) [43-451, HLA- DR [39,40], rhodamine 123 [41,42], CD15 [56] and LFA-1 [46].

2.2. Long-term culture and ex vivo expansion Although there is no definitive assay yet available for

human stem cells with the ability to reconstitute the hematopoietic system in vivo, in vitro assays have identified cells with some characteristics of stem cells. These include long-term culture-initiating cells (LTC-IC), high proliferative potential-colony forming cells (HPP-CFC), and colony forming unit-blast. We have enriched a population of HPP-CFC with extensive replating capacity by isolating cells with highest density of CD34 antigens (CD34+++) from human cord blood (CB) [52]. These HPP-CFC colonies came from single CD34+++ cells, and individual HPP-CFC colonies could be replated into secondary (24 dishes. Furthermore, the extensive proliferative self-renewal potential of these cells was measured by their capacity to be replated from 2’ to 3’, and 3’ to 4’ plates with maintenance of proliferative capacity. There is no evidence yet that HPP-CFC repre-

sent long-term marrow repopulating cells, but they probably belong to a stem cell compartment based on their replating capacity in vitro [52].

The hematopoietic microenvironment is the milieu provided by non-hematopoietic cells in the bone marrow that supports and regulates hematopoiesis. Marrow stroma is composed of tibroblasts, endothelial cells, macrophages and other cells which are responsible for the production of an extracellular matrix and hematopoietic growth factors [58]. Dexter-type long- term BM cultures, which are stromal cell-dependent long-term cultures, are thought to mimic the marrow microenvironment closely [58]. Verfaillie et al. have demonstrated that primitive progenitors can differentiate and also be maintained when cells are co-cultured with stromal layers, but separated from the stromal layers by an 0.4 pm microporous transwell membrane [39]. Cultivation of primitive CD34+/HLA-DR- progenitor cells in these cultures resulted in the recovery of significantly more LTC-IC and clonogenic cells than when cells were cultured in direct contact with the stroma in a classical Dexter-type culture system. Moreover, significantly more LTC-IC and clonogenetic cells could be recovered from stroma-noncontact cultures supplemented with rhu interleukin (IL)-3 than from stroma-free cultures supplemented with a combination of 4 cytokines. An alternative approach for the growth of early cells is to supply the cytokines required for proliferation and differentiation to a stroma-free culture system. Long-term cultures can be established with CD34+ cells in a stroma-free system when defined cytokines are repeatedly added [54-57,59,60]. Cyto- kines thought to be important in the induction of differentiation and/or proliferation of these primitive hematopoietic progenitors include recombinant human G-CSF, GN-CSF, IL-l, IL-3, IL-6, IL-11, and steel factor (SLF, also termed stem cell factor). Bernstein et al. [29] showed that the combination of IL-3, G-CSF and SLF increased the number of single isolated CD34+Lin- cells giving rise to colonies in vitro by lo-fold. The number of colony-forming cells derived from these primitive cells increased 20- to 40-fold in the presence of these cytokines. Our recent studies have demonstrated expansion of a single sorted CD34+++ cell from cord blood, and compared that with 5 000 CD34+++ cells [54]. In the presence of SLF, IL-lo and IL-3, the fold expansion of nucleated cells was greater in cultures initiated with 1 cell/well (> 5 000-fold) compared to that with 5 000 cells/well (791-fold), while the fold expansion of progenitor cells was greater when 5 000 cells were used to initiate suspension culture. This type of single cell assay may allow identification of early acting cytokines that are produced endogenously and which act in a feeder fashion when large numbers of cells are cultured together.

64 L. Lu et al. /Critical Reviews in Oncology/Hematology 22 (19%) 61-78

There has recently been great interest in the ex vivo expansion of hematopoietic cells for a variety of applications [61]. However ex vivo maintenance and generation of functional hematopoietic stem/progenitor cells are complex processes and are poorly understood.

We have demonstrated that incubation of cord blood cells for 7 days with SLF resulted in 7.9-, 2.2- and 2.7- fold increases in numbers of CFU-GM, BFU-E and CFU-GEMM compared to starting numbers; addition of a CSF with SLF resulted in even greater expansion of those progenitors [60]. We also found that CB plasma synergized with SLF plus PIXY321, a GM-CSF/IL-3 fusion protein, to promote increases in cell numbers by days 14-21 and in CFU-GEMM by day 7 [62].

As briefly outlined above, recent advances in stem cell biology have resulted in the phenotypic and functional characterization of at least subsets of human hematopoietic stem cells, and techniques to obtain relatively pure populations of these cells and to expand these cells ex vivo. However, it is not yet clear if the earliest human stem cells with long-term marrow engrafting capacity are being expanded under these conditions, since, as we previously mentioned, there is not yet an assay for these human cells. With this cautionary note, the above technology may be used to design methods for autologous transplants with tumor-free stem cell grafts using positively selected hematopoietic stem cells. Autologous transplant protocols employing positively selected human stem cells are underway in patients with solid tumors.

3. Bone marrow stem cell transplantation

Allogeneic (allo) BMT is a powerful therapeutic modality for treatment of hematologic malignancies [63]. The total number of all BMTs performed worldwide is well over 40 000, and the annual figure is still increasing [64,65]. Allo-BMT is applied to a variety of diseases [66-681, including malignant hemopathies [69], aplastic myelopathies [70], homozygous hemoglo- binopathies, such as thalassemia [71], congenital im- munodeliciencies [72], and storage diseases. Two-thirds of patients requiring BMT lack HLA-matched family donors and many others therefore look for unrelated marrow donors. Although methods have improved for typing cells and more potential donors are being recruited into registries, the time for searches for unrelated marrow donors, and the possibility that no HLA-matched will be found limit to some extent such transplants. Moreover, unrelated marrow transplants are associated with high incidence of graft failure and GVHD, presumably because of donor-recipient non- identity undetectable with currently available screening procedures.

Until tumor-specific effective new agents can be

developed, increasing dose intensity may be the best way to improve therapy for patients with hematological malignancies and solid tumors who fail conventional treatment and do not have a related or unrelated HLA- matched donor. Alkylating agents, including radiation, are likely candidates for use in dose-intensive combinations [73]. The dose of most antineoplastic chemothera- peutic agents that may be administered is however limited largely by the toxicity to the normal marrow. Autologous bone marrow transplantation (ABMT) in- volves reinfusion of the patient’s own marrow as protection from the myeloablative effects of high-dose cytotoxic chemotherapy or chemo-radiotherapy. For many tumor types, ABMT offers higher response rates than standard approaches. For leukemias and lymphomas, response rates of 60-80% may be achieved with the potential for cure. With solid tumors, response rates range from 30% in gliomas, 50% in melanomas and colon cancer, and to more than 60% in lung cancer, and 80% in breast cancer [74]. Prolonged disease-free survival is possible for patients with lymphoma, Hodgkin’s disease, leukemia and breast cancer when high-dose cytotoxic therapy (CT) is followed by ABMT. Bone marrow and/or peripheral blood can be cryopreserved before high-dose CT and utilized for stem cell rescue. Clinical trials of patients with lymphoma, leukemia, Hodgkin’s disease and breast cancer have proven the importance of timing the induction and high-dose CT, and properly sequenced therapy has resulted in prolonged disease-free survival in these patients. ABMT has become accepted therapy in selected patients with leukemia [75], Hodgkin’s disease, non-Hodgkin’s lymphoma [76] and neuroblastoma [77]. More recently, ABMT has also been used with encouraging results in breast carcinoma [78], small cell carcinoma of the lung [79], and other disorders [80].

A major concern in ABMT is the possibility that clonogenic tumor cells might contaminate the harvested bone marrow resulting in recurrence of the malignant disease on infusing these cells into the host. The frequency of microscopically identifiable bone marrow contamination range from very high in leukemias and neuroblastoma [81] to lower in solid tumors such as breast cancer [82,83].

Recently, enriched CD34+ marrow cells have been used in combination with growth factor administration post-transplant in 21 patients with advanced breast cancer [84]. Neutrophil recovery was faster in patients receiving marrow plus G-CSF in comparison with patients receiving marrow plus GM-CSF or marrow only. Positively-selected enriched CD34+ marrow cells have also been used for transplantation following high dose chemotherapy f total body irradiation in 13 patients with metastatic breast cancer and 2 patients with neuroblastoma [85]. Engraftment of neutrophils was ap-


parent, but slower than that seen with whole marrow. The results suggest that highly enriched CD34+ cells are capable of hematopoietic reconstitution after myeloablative therapy [84,85] and it is possible that this procedure could be used as a tumor-purging technique for autologous as well as allogeneic transplantation.

4. Umbilical cord blood stem cell transplantation

Until recently, human umbilical cord blood was a usually discarded material. Cord blood is rich in hematopoietic progenitor cells, as measured in standard clonogenic assays [60,86-891. In 1989, we reported that the neonatal blood in the placenta might contain sufficient hematopoietic stem cells to serve as a potential source of therapeutically transplantable cells 1861. This led to the first successful experience with cord blood stem cell transplantation (CBSCT) in a patient with Fanconi anemia [90]. This young male patient was transplanted with HLA-matched cord blood from his sister and now is healthy and cured of the hematological disorder 7 years post transplantation. Engraftment was confirmed by sex chromosomal analysis of cells, ABO- antigens in erythrocytes and restriction fragment length polymorphism study of DNA. Subsequently Wagner et al. have reported an HLA-matched sibling cord blood transplantation in a child with juvenile chronic myelogenous leukemia [91]. Engraftment was confirmed by chromosomal analysis, restriction fragment length polymorphorism and polymerase chain reaction (PCR) analysis of colony forming cells from bone marrow of the recipient after transplantation. Thus, it was conclud- ed that umbilical cord blood could be considered an ef- ficacious source of sufficient stem cells for clinical hematopoietic reconstitution. Presently, over 150 children have received CBSCT for the treatment of a variety of diseases [89-951, including Fanconi anemia, leukemias such as. Ph chromosome-positive chronic myeloid leukemia (CML), AML, ALL, neuroblastoma, congenital immunodeficiency, aplastic anemia, Wiskott- Aldrich syndrome, and X-linked lymproliferative syndrome. These results led to attempts at setting up cord blood banks [96-981. Programs for the banking of unrelated CBSCs have already begun in the United States, France, United Kingdom, The Netherlands, Ger- many and Italy. While the application of CBSCs in the treatment of patients with potentially life-threatening disorders is underway, it is unlikely that individual centers will be able to completely address questions regarding the role of CBSCs in transplantation. For this reason an international cord blood transplantation registry was started [97]. CBSC can also serve as targets for gene transfer. This will be discussed in the gene therapy section.

Potential advantages of using cord blood relate to the high number and quality of hematopoietic stem and progenitor cells present in the circulation at birth and to the relative immune ‘immaturity’ of the newborn immune cells. These two issues are discussed below.

4.1. Quantity and quality of immature hematopoietic stem cells from umbilical cord blood

Single collections of cord blood can be used to engraft the hematopoietic system of children [89-991. Whether a single collection also contains sufficient cells to suc- cessfully engraft an adult remains an open question, although recent studies using sibling or unrelated cord blood suggests that this is possible. In vitro studies have suggested that single collections of cord blood probably do contain sufficient cells to engraft the hematopoietic system of an adult [60,88]. Engraftment will depend not only on the number of stem progenitors, but also on the quality of these cells for proliferation and self-renewal. These qualities of stem and progenitor cells appear to be high. It has been demonstrated that committed hematopoietic progenitor cells are at least as numerous in cord blood as in normal bone marrow [ 1001. The tin- ding of more erythroid and high proliferative megakaryocyte progenitors in cord blood than in normal bone marrow suggests that more primitive progenitor cells are present in the former, results consistent with ours [60]. We [52] have purified a population of CD34+++ cells and sorted them as a single cell/well, to demonstrate that there are 8-fold more HPP-CFC with extensive replating capacity in cord blood than in adult marrow [52]. Long-term cultures have suggested that cord blood contains more early cells than bone marrow with high proliferative potential [loll. Cord blood cells can be extensively expanded in suspension culture with different combinations of cytokines [62] and we have recently demonstrated that ex vivo expansion can also be established from single primitive cells [54]. Thus, immature hematopoietic stem cell and progenitor cells appear to be of good quality in cord blood.

4.2. Immune cells in cord blood There has been little or no GVHD detected in child-

ren receiving HLA-matched or one antigen mismatched sibling cord blood [90-951, and results using unrelated cord blood have been similar. There is evidence that immune cells from cord blood react differently than those in adults. Monocytes/macrophage and T- and B- lymphocytes are the major immune response cells. It has been reported that a low antigen-presenting ability found in CB monocytes/macrophage [102,103] is due to deficient HLA-DR antigen expression [102] and their intrinsic immaturity [103]. Newborn B cells, although, are capable of producing amounts of IgM antibody comparable with those of adult B cells, but their capaci-

66 L. Lu er 01. /Critical Reviews in Oncology/Hemarology 22 (19%) 61-78

ty to produce IgG or IgA is remarkably reduced [ 1041. Cord blood T cell functions, such as helper T cell activities [IOS], expression of cell surface antigens [IO61 and receptors [107], interferonr production [IO81 and killer cell activities [IO91 are decreased compared with those of adult T cells. In contrast, some other studies with T cell mitogens demonstrate that CB T cells possess mature functions compatible with adult T cells [IOS]. Thus, the functional maturity of cord blood T cells is still not completely understood.

In this context, there are two issues of interest. One is the maturity of cord blood lymphocytes, and the other one is the possibility of maternal cell contamination of collected cord blood cells.

4.2.1. Number and phenotype of cord blood T- lymphocytes. In cord blood, about 50% of low density mononuclear cells express CD3, compared to 70% in adult blood [89,113]. However, phenotypic analysis of T cell subsets (CD4, helper T cells, and CDS, suppressor T cells) revealed no significant differences between adult and cord blood. CD3positive T cells are a variable sub- population representing 44.8% of lymphocytes 11123. The majority of the CD3 cells are CD3+CD8+. Newborn T cells have lower levels of both IL-2 receptors and HLA-DR than do adult T cells. The CD4-positive T cells represent 31% of lymphocytes with a great preva- lence of the CD4+/CD45RA+ population.

4.2.2. Function of the T-lymphocytes in cord blood It is of interest that little cytotoxic T cell activity is generated by cord blood cells even after primary, secondary and tertiary stimulation by allogeneic cells [ 1 IO]. Moreover, even though the proliferative response to alloantigen is high in primary response, the secondary response is relatively blunted compared to that of adult T cells, suggesting a tolerant state was induced [ 1131. The nature of cytoplasmic signal transduction was examined in cord blood T cells by stimulating them with tumor promoter and calcium ionophore [I 141. The max- imal responses observed in CB T cells were much less than those of adult T cells, whereas the Con A-induced proliferative responses of these cells showed no signili- cant differences. The decreased responses of CB T cells were due to their lower efficiency in activating the cellular events in T cell activation and proliferation phase, because cord blood T cells have significantly less ability than adult T cells to produce IL-2 and express functional IL-2R complexes, especially high-affinity IL- 2R complexes, and HLA-DR. These functional characteristics may be related to the immaturity of cord T cells.

Cord blood natural killer (NK) cells are CD3- CD56+ cells and express CD2, CD7, CDII,, CD18, CD38, CD45RA and the P75 IL-2 receptor [I IS]. Only small amounts of CD3YDl6’ cells exist in cord blood [ 1161. The majority of NK cells are in CD3CDl6+CD-

56+ and CD3’CDl6CD56+ cell populations in CB as in adult blood.

Cord blood NK cell activity is low or similar to that in adult blood [ 116,117] but lymphokine activated killer cell activity (LAK) is readily induced in cord blood by cytokines such as IL-2 or IL-12 [116,117].

4.2.3. Maternal cell contamination in cord blood. Con- tamination of cord blood with maternal cells was originally considered a theoretical concern that might lead to massive GVHD. Little or no maternal cell contamination had been detected [I 181 and in over 150 cord blood transplants there has been little or no GVHD [89-951. PCR analysis for amplification of 2 minisatellite sequences (33.6 and MS 51) were used to evaluate the possibility of contamination [I 191. Mater- nal cells were very rarely (2”/) present within cord blood collected at birth, since they were detected in only one of 47 cases, and this was at an even lower level in the lymphocyte cell fraction (0. 1 - 1 .O%), although more sen- sitive techniques have detected maternal cells in cord blood. It is not clear what, if anything, this low level of maternal cell contamination might mean in a clinical setting.

Thus, umbilical cord blood stem cells are an altema- tive source of transplantable cells that can be used to reconstitute hematopoiesis in children with malignant and non-malignant diseases after treatment with myefoablative doses of chemotherapy. The availability of cord blood banks [97,98,120] will allow assessment of the efficacy of using cord blood to transplant the hematopoietic system of adult recipients.

5. Peripheral blood stem cell transplantation

The presence in peripheral blood of circulating stem cells (PBSC) with potential marrow repopulating ability has been considered for a while [121]. The use of autologous PBSC for transplantation has permitted patients whose bone marrow is unsuitable for collection to receive high-dose radiation and/or chemotherapy for a variety of malignancies, and is considered an attractive alternative to bone marrow transplantation in those patients. Autologous PBSCT offers a number of possible advantages over autologous bone marrow transplantation. These include collection of blood-derived hematopoietic progenitors without the use of general anesthesia, more rapid hematopoietic recovery after marrow-ablative therapy with the potential for reduced tumor cell contamination, and the chance of performing PBSCT in patients with residual bone marrow disease or fibrosis. A large series of high-dose therapy and autologous peripheral blood stem cell transplantation for various malignancies has been reported 11221. The present status of blood stem cell transplantation has been summarized [123]. Mobilized peripheral blood

L. Lu ei al. /Critical Reviews in Oncology/Hematology 22 (19%) 61-78 61

stem cells appear to produce faster neutrophil and platelet recovery, fewer febrile days, lower blood product requirement and shorter periods of hospitalization, compared with bone marrow stem cells [ 124,125]. Many groups have reported on clinical use of peripheral blood progenitor cells for transplantation of patients with malignant diseases [ 1261. A number of recent reports docu- ment the success of this therapeutic approach in patients with lymphoid malignancies; -[ 1271, Hodgkin’s disease [128-1311, non-Hodgkin’s lymphoma [132-1361, multiple melanoma [ 137,138], leukemia [139-1411, solid tumor, such as neuroblastoma [142], multiple myeloma [143,144] and breast cancer [145], and other adtive cancer [146] in adults and children.

Since autologous blood stem cell transplantation (ABSCT), and more recently allogeneic blood stem cell transplantation, have been introduced as a treatment modality, clinical research has mostly focused on the following aspects: (a) blood stem cell mobilization and collection; (b) kinetics of hemopoietic reconstitution after transplant; (c) contamination of harvested blood mononuclear cells with clonogenic tumor cells; and (d) stability and sustainment of hemopoietic reconstitution after transplant.

5.1. Blood stem cell mobilization and collection Stem cell numbers in peripheral blood are very low

compared to those in the bone marrow. Although stem cells can be collected by apharesis, this requires the pro- cessing of large volumes of blood. Amplification of PBSC, however, facilitates collection by a limited number of aphareses and has several advantages since it renders autologous stem-cell rescue feasible, even in the case of bone-marrow hypocellularity or bone-marrow metastatic invasion, and avoids general anaesthesia necessary for bone marrow harvesting. Many runs of leukapheresis are required to collect sufficient numbers of circulating stem cells for complete hematopoietic reconstitution. Several manipulators have been em- ployed to increase circulating progenitor cell numbers. Mobilization of hematopoietic progenitors from bone marrow into peripheral blood can be achieved either by chemotherapy [129,130,133,135,138,149,150], hematopoietic growth factors [ 15 1 - 1561 or by a combination of myelosuppressive chemotherapy and growth factors [145,157-1621.

It is important to find better mobilizing techniques to provide more efficient harvesting and faster hematopoietic recovery. A large series of reports documented that PBSC can be mobilized by chemotherapy [147-1501. It has been demonstrated that even single high dose cyclophosphamide enables the collection of high numbers of hematopoietic stem cells from the peripheral blood [ 1501. Disadvantages with chemotherapy, which appear to require high doses of chemothera- peutic agents, include the frequent occurrence of fever,

infections and complications, and occasional mortality during pancytopenia after chemotherapy priming. Recently, recombinant human (rhu) growth factors, such as granulocyte (G)- or granulocyte-macrophage (GM)colony stimulating factor (CSF), have been used as stimulators of hematopoiesis in patients with neutro- penis resulting from cytoreductive therapy. Early studies demonstrated higher numbers of circulating progenitor cells in patients receiving G-CSF or GM-CSF to mobilize peripheral-blood progenitor cells [ 15 1 - 1621. Unseparated blood mobilized by G-CSF enhanced the engraftment of platelet in patients receiving high-dose chemotherapy (1521. Patients with non-myeloid malignant disorders receiving G-CSF had an increased number of blood granulocyte-macrophage (CFU-GM) and erythroid (CFU-E and BFU-E) progenitor cells, and a significantly shortened period of severe throm- bocytopenia. It was noted that PBSC obtained at a single leukapherasis during hematopoietic recovery after chemotherapy and G-CSF, was sufficient to restore bone marrow function [ 1321. Rapid recovery of neutrophils was found in all patients by the administration of G-CSF following transplantation. In other reports, it was observed that the combination of myelosuppressive chemotherapy and G-CSF mobilized more PBSC than chemotherapy alone [ 159,160]. More recently, it has been suggested that hematopoietic progenitor cells mobilized into the peripheral circulation by G-CSF can be considered equivalent to those in bone marrow in their capacity to repopulate hematopoietic tissue [ 1561. Administration of G-CSF appears to be safe with few and minor side-effects. The harvesting -of peripheral blood progenitors could be performed in a blood bank setting such that general anaesthesia would be avoided. For the recipient, a faster platelet recovery may be achieved with reductions in resultant morbidity, suppor- tive care and longer periods of hospitalization.

GM-CSF has also been used for mobilization of PBSC and has resulted in efficient collections of PBSC, and has provided rapid and sustained restoration of hematopoietic function following high-dose chemotherapy [ 1551. GM-CSF in combination with chemotherapy can mobilize more PBSC than either chemotherapy or GM-CSF alone [145,160-1621. The efficacy of GM- CSF alone or in combination with peripheral blood- derived hematopoietic progenitor cells was compared as support for patients with metastatic breast carcinoma receiving high-dose chemotherapy [ 1451. The results showed that the use of autologous blood stem cell transplantation plus GM-CSF accelerated hematological recovery after high-dose chemotherapy with reduced morbidity and platelet-transfusion require- ments, compared with the use of GM-CSF alone [145]. In a recent report [ 1471 results of 33 patients undergoing PBSCT were compared to 17 concurrent patients undergoing autologous marrow transplantation. PBSC


were mobilized with high-dose cyclophosphamide, GM- CSF or with combinations of these. An important ob- servation was that PBSCT patients had fast engraftment and a IO-day shorter length of hospital stay than those undergoing autologous marrow transplantation. Al- though the authors did not show a cost analysis, it seems reasonable to assume that PBSCT offers a cost advantage over ABMT.

It has been suggested that it is too early to define a panel of clinical indications for blood being the preferred option over marrow for autologous transplantation [163]. A randomized controlled trial is the method to address this point. The most striking advantage of autologous blood over autologous marrow seems to be the shortening of pancytopenia immediately after myeloablative therapy and transplantation. In addition to mobilization of stem and progenitor cells with G-CSF and GM-CSF, trials with IL-3 have been reported [ 1641. Other growth factors such as IL- 1, IL- 11 or steel factor, alone or in combination with G-CSF or GM-CSF may be of value in mobilizing stem cells to the blood.

Of interest is a report which examined serum levels of G-CSF, M-CSF, GM-CSF, IL-3 and IL-6 in 10 children undergoing a total of 12 PBSCT procedures in which only sustained increases in serum G-CSF levels were seen early after ABSCT [ 1651. In an another report, a sequential coordinated pattern of hematopoietic growth factor release after myeloablative chemotherapy was seen providing indications of possible mechanisms mediating hematopoietic recovery after stem cell transplantation [ 1661.

The future of PBSCT likely includes allogeneic transplantation and manipulation of the graft produce to provide therapeutic benefit and improve immunologic recovery after marrow ablative therapy. However, it is emphasized that there are not definitive data that peripheral blood, even after mobilization techniques are used, contains long-term marrow repopulating cells.

5.2. Kinetics of hemopoietic reconstitution after ABSCT Bone marrow and circulating stem cell pools are in

dynamic equilibrium with 98% of all committed progenitor cells located in the marrow, but only 0.1% circulating at any given time [167]. There is no doubt that circulating blood cells can reconstitute short-term hematopoietic function after myeloablative therapy. Whether circulating blood cells can sustain hemopoiesis lifelong in recipients of truly myeloablative therapy has not yet been determined. Correlation of determined numbers of CFU-GM infused and granulocyte recovery has been used to predict hematopoietic recovery kinetics [ 156,160]. It was found that progenitors’ mobilization with G-CSF reduced the time to granulocyte recovery by 2-28 days, and that the number of mobilized CFU- GM infused into patients predicts the recovery of granulocyte and platelet production after ABSCT [ 1601. In

more recent studies, CD34+ cell content has been used to quantitate stem cell autografts. Correlations between CD34+ cells, CFU-GM and/or bone marrow recovery are imperfect. The CD34+ population of cells is heterogenous and many of these cells are not stem cells. CD34+ hematopoietic progenitor cells are present in the circulation at about l/10 that of the marrow and subpopulations of these cells differ in peripheral blood compared with bone marrow [168]. Immunophenotyp- ing studies have demonstrated a small portion of mobilized CD34+CD38- cells in the peripheral blood which suggests the possibility that circulating cells with long-term repopulating ability are present [ 164,169]. The CD34+38- population is immature [ 1701.

Although numbers of blood cells needed for successful short- and long-term engraftment are not yet known some have recommended 6 x lO’/mobilized mononuclear cells/kg b.w. are needed [128], whereas others suggest 8 x lo6 CD34+ blood cells/kg b.w. will produce rapid and sustained engraftment [171].

5.3. Contamination of harvested blood tumor cells It has been hypothesized that blood stem cells might

be less contaminated by residual clonogeneic tumor cells than bone marrow and therefore the risk of relapse after blood stem cell transplantation might be less [128,163]. There is however still no definite evidence to prove this hypothesis. In many cancers, malignant cells are detected in the blood, even in early disease, using sensi- tive molecular biological techniques such as cytogene- tics, PCR, FISH and long-term tumor cell cultures [ 1721. Most data are based on steady state conditions which may not be applicable during blood stem cell harvesting due to the multiple apheresis procedures used. In a recent report, it was shown that in patients with stage IV or high-risk stage II/III breast cancer, small cell or non-small cell lung cancer, and other advanced malignancies, an even higher number of circulating tumor cells was detected after chemotherapy followed by G-CSF [ 1731. Thus caution is needed in the use of mobilized peripheral blood from patients with tumors and the question of tumor-cell purging, even of peripheral blood cells, has to be considered [174].

5.4. Stability and sustainment of short- and long-term hemopoietic reconstitution after ABSCT

There are two issues of concern in hematopoietic recovery after ABSCT. One is immediate recovery and the other is long-term engraftment. The most conventional way to monitor the immediate recovery is granulocyte recovery. It is obvious that granulocyte recovery is more rapid with blood versus bone marrow autografts, and this is detected when blood cells are collected after mobilization with chemotherapy or hematopoietic growth factors. Whether blood stem cell transplants result in long-term engraftment remains unclear. The major


limitation of studying this question in humans is the lack of an assay for stem cells responsible for long-term engraftment. Gene marking will probably be a way to follow engraftment and to study the correlation between engraftment and bone marrow recovery [ 175).

6. Fetal liver stem cells (FLSC) transplantation

The human fetus is immunologically immature until the sixteenth week of gestation [176]. This makes the very early fetus a potentially ideal transplant donor or recipient. The use of fetal tissue for transplantation has a number of theoretical advantages. The most important one is the potential immunologic ‘immaturity’ of fetal tissue. This means that the recognition of ‘self’ occurs during an early stage of fetal life and that tissues used before that could be immunologically privileged. During human embryogenesis, hematopoiesis is first detected in the yolk sac, then from the fifth week it is apparent in the fetal liver [ 1771, Transplants of hematopoietic stem cells derived from fetal liver could potentially reconstitute bone marrow function in various settings of bone marrow failure including various diseases and after cancer chemotherapy. In all species studied, including mice, rats, pigs, sheep, horses and monkeys, fetal liver transplants are associated with less GVHD than transplants of bone marrow derived stem cells. Experi- mental data indicates that T- and B-lymphocytes from second trimester fetal liver have reduced immune reac- tivity compared to mature lymphocytes. It has been found that fetal liver transplants can reconstitute long- term hematopoiesis [ 1781. Some success of fetal liver transplants in humans has been confined to children with immune deficiency disorders [194-1961. Trials in aplastic anemia and leukemia were unsuccessful but were performed without sufficiently intensive immune suppression. Therefore, the fetal liver may be used for transplantation to treat hematological disorders, immunodeficiencies or lysosomal storage disease [ 179- 1811.

Over 230 fetal tissue transplants (especially fetal liver transplants associated with syngeneic fetal thymus) have been performed to treat 62 patients with severe immunodeficiency diseases, severe aplastic anemia or inborn errors of metabolism [ 1821. It has been reported that in a patient suffering from a severe combined immunodeti- ciency disease, full immunological reconstitution was obtained after fetal liver and thymus transplantation [ 180,182]. HLA typing revealed that the T cells of the patient were of donor origin, while B cells and monocytes were of host origin. Immunoglobulin allotype determination showed that antibodies were synthesized by host B cells indicating that transplanted T cells and recipient cells cooperated despite an HLA mismatch.

There are however serious concerns with fetal liver [ 1831. Whether one can use partial mismatched fetal

liver in a non-immunodeliciency disease is not at all clear. Moreover, it is not clear yet if one can adequately freeze and store fetal liver stem cells. Additionally, there is the constant ethical conflict that occurs when fetal liver transplants are considered.

7. In utero bematopoietic stem cell transplants for inherited dkases

Recent advances in prenatal diagnosis now allow the diagnosis of a number of congenital hematopoietic and metabolic disorders during the first trimester of pregnancy [184]. The immunocompetence of the early gestation fetus can be compared to the child with SCID, who can often engraft with allogeneic bone marrow without the need for immunosuppressive conditioning. The goal of in utero hematopoietic stem cell (HSC) transplantation is to achieve successful engraftment using a ready source of HSC with minimal conditioning. This would result in minimal short- and long-term side effects to the recipient, increased availability of donors for children with disease that might benefit from BMT, and significantly decreased cost of medical care. The use of the pre- immune fetus of animals as the recipient has permitted transplantation across immunologic barriers and xeno- grafts have now been performed [ 184-1901. In utero studies have been done on monkeys [ 191,192] and sheep [193-1971. Four potential sources of HSC for in utero transplants include bone marrow, fetal liver, mobilized peripheral blood, and cord blood. An attempt at treatment of a 17-week gestation human fetus with Rh in- compatibility by transplantation of T cell depleted adult marrow has been reported [ 1981. The fetus survived but there was no documentation of chimerism or engraftment. In contrast, using fetal donor cells, human fetuses with bare lymphocyte syndrome, SCID and thalassemia have been treated [ 199-2021.

In utero human hematopoietic stem cell transplantation is now technically feasible. Three potential clinical applications of in utero transplantation are: (a) reconstitution of congenitally diagnosed hematopoietic disease; (b) gene therapy for enzyme or factor deficiency states; and (c) prenatal-specific tolerance induction for postnatal allogeneic and xenogeneic organ transplantation.

8. Stem cell as target for gene therapy

Retroviral mediated gene transfer has been shown by a number of investigators to be an efficient method for introducing genetic sequences into mammalian cells [203,204] and gene replacement therapy has been suggested for many single gene disorders [205-2071. Adeno-associated viruses (AAV) can also be used as a vehicle for gene transfer. In this article, we focus on retroviral gene transduction. Target cells for gene trans-


fer should be able to have self-renewal capacity and differentiate into progeny cells. Hepatocytes, endothelial cells, muscle cells, keratinocytes, tibroblasts and hematopoietic cells, including stem/progenitor cells and lymphocytes, have been suggested as target cells. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic stem/progenitor cells, because of their wide distribution and their well- characterized capacities for proliferation, differentiation and self-renewal. In vivo and in vitro experiments in human and animals have shown that the repopulating bone marrow stem cells are an ideal target for gene transfer [208,209]. Diseases in which gene therapy may be beneficial include Fanconi anemia [90], @-thalassemia [210], adenosine deaminase (ADA) deficiency, purine nucleoside phosphorylase deficiency [211], chronic granulomatous disease [212], leukocyte adhesion deficiency [213], Gaucher’s disease [214], Niemann- Pick type B [215] and metachromatic leukodystrophy [216] etc.

Children with ADA deficiency have recurrent infections as well as failure to thrive because of chronic diar- rhoea and malabsorption and a high incidence of lymphoma. BMT can be curative, but most of these individuals do not have a histocompatible bone marrow donor and die early in childhood. The first gene therapy was done in a Cyear-old girl with ADA deficiency in September 1990 [217]. At that time, lymphocytes isolated from the peripheral blood of the patient were used as target cells and a retroviral vector containing Neo and ADA cDNA were used for gene transfer. The patient received gene-treated lymphocytes monthly, because blood lymphocytes are mature and are contin- uously replaced from bone marrow stem/progenitor cells which are not treated. Thus, an effort at using hematopoietic stem cells as targets for gene transfer is increasing. For efficient transduction of pluripotent hematopoietic stem cells (PHSC), an amphotropic retrovirus carrying the human ADA gene has been generated [218,219]. During the past decade, in vitro experiments have indicated that hemopoietic progenitors from mice, rhesus monkeys and man can be transduced at high efficiency. As preclinical tests, a number of groups performed autologous bone marrow transplantation in lethally irradiated mice and rhesus monkeys using retrovirus-infected hematopoietic stem cells [220-2261. Stable long-term expression of human ADA in mice in all hematopoietic lineages following full hematopoietic reconstitution with transduced bone marrow has been demonstrated by several groups [222-2241. The levels of human ADA expression were compatible with endogenous murine ADA protein levels. Long-term expression of human ADA has been demonstrated in large animal models, such as rhesus monkeys [225,226]. Recently, autologous cord blood CD34+ cells transduced with human ADA gene were infused into three children with ADA deficiency [227].

Gaucher’s disease, characterized by a deficiency of glucocerebrosidase, is treated at present by either allogenic BMT or enzyme replacement. Neither treatment is ideal. BMT is limited to those patients with an HLA matched donor, which is more difficult to obtain, and is associated with significant morbidity and mortality. Enzyme replacement is expensive and needs to be lifelong. Transduction of the hematopoietic stem cells of the patient followed by autologous transplantation (gene therapy) could be an effective alternative treatment [228-2311. A retroviral vector (LG) containing the Gaucher gene has been developed and used to transduce murine hematopoietic stem cells with high efficiency. The vector has been used to infect hematopoietic cells from Gaucher patients and has been able to correct the enzyme deficiency [230].

In HIV-l infection, it may be possible to apply the technology of gene therapy to deliver anti-viral agents directly to infected cells and potentially benefit the infected individuals [232,233].

The problem of poor transduction efficiency of primitive hematopoietic progenitor cells still remains with human cells. Efforts have been taken to improve transduction efficiency in primitive hematopoietic stem/progenitor cells with long-term gene expression in hematopoietic cells in vivo.

8.1. Increase transduction efficiency An important requirement for increasing gene trans-

fer efficiency is that the target cells must be cycling. The murine ADA gene could be transduced into hematopoietic stem/progenitor cells from human bone marrow and umbilical cord blood and with stable expression in long-term cultures [234]. The majority of the primitive hematopoietic stem cells from bone marrow, cord blood and peripheral blood are quiescent [234,235] and are thus difficult targets for gene transfer. In the absence of growth factor preincubation of cells for prestimulation, gene transfer efficiency was only 3-10% [236-2381. Recently, many reports, including our own, have presented encouraging results on increased gene transfer efficiency by use of recombinant growth cytokines [234,235,239-2421. Enriched CD34+ cells have been used for marker gene transfer studies with high efficiency. Our recent results have shown that a neo gene could be transduced into a primitive population of CD34+++ cells from CB post prestimulation with Epo, steel factor (SLF), IL-3, GM-CSF and G-CSF with 49% transduction efficiency compared to 25% presorted cells [235]. The transduction efficiency was increased up to 80% when transduction was performed at the single cell level. Stable integration was evident even after four replatings of HPP-CFC colonies [241].

8.2. Long-term gene expression in hematopoietic cells in vivo

A requirement for successful gene therapy is long-

L. Lu et al. /Critical Reviews in OncologylHematology 22 (19%) 61-78 11

term expression of transferred genes. Since there is no

suitable human in vivo assay available for long-term gene expression, animal models have been used for this purpose. Long-term and stable expression of introduced genes in cells derived from transduced murine bone marrow has now been well established in several laborato- ries. The success rate for gene expression of some introduced gene sequences at 6 months reaches lOO?/ [243]. Stable long-term expression of human ADA in mice in all lineages following complete hematopoietic reconstitution with transduced bone marrow has also been demonstrated [222-2241. More recently, long-term expression has been further confirmed using larger animal model of rhesus monkey [225,226].

Progress in the use of retroviral vectors as well as other newer vector systems, to transduce hematopoietic stem cells will lead to future clinical trials in patients with genetic and oncological diseases.

9. conclusloo

During the past two decades, BMT has been beneficial and curative for patients with many hematologic malignancies and inherited diseases and some immunodeficiencies. The major problems that limit the success of BMT consist of recurrence of the malignancy, GVHD, infection and the paucity of matched donors for most patients. Recent interest has been focused on the transplant of stem cells from umbilical cord blood and mobilized peripheral blood. It has been suggested that the quantity and the quality of hematopoietic stem cells in cord blood are as good as, or even possibly better than, that in normal adult marrow. To establish a cryopreserved human CB bank of unrelated donors for clinical transplantation is important. The clinical obser- vations of possible lowered GVHD in the context of HLA-matched sibling cord blood suggest the possibility of crossing certain major HLA barriers. Stem cell transplantation has progressed from a highly experimental procedure to being accepted as a preferred form of treatment for a wide variety of disease. Research has resulted in a steady improvement in overcoming some of the problems listed above with a consequent improvement in long-term survival. The efficacy and applicabili- ty of transplant will increase when all of the major problems are resolved. Molecular biology and recombinant technology have made gene therapy possible. Stem cells will most likely be the desired targets for gene transfer and gene therapy. Although still in its infancy, human gene therapy holds considerable potential for the long-term treatment of recessive diseases, such as genetic inherited diseases, cancer and some immunodeficiencies.

Acknowledgements

Some of our studies reported in this review were sup-

ported by US Public Health Service grants R37 CA36464, ROl CA HL46549 and ROl HL 49202 to Hal E. Broxmeyer, and grants from the Phi Beta Psi Sorority to Li Lu and Rong-Nian Shen.

Reviewer

This manuscript was reviewed by Li. T. Chen, Ph.D. Department of Anatomy, University of South Florida, College of Medicine, 12901 Bruce B. Downs Blvd., MDCO06 Tampa, Florida 336124799, USA.

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Biographies

Li Lu, M.D. is a Full Scientist of Medicine and a Full Member of the Walther Oncology Center, Indiana Uni- versity School of Medicine, Indianapolis, Indiana, USA. She is presently Corresponding Deputy Editor (China) for the Journal, Experimental Hematology. Rang-Nian Shen, M.D. was a Full Scientist of Medicine and Radia- tion Biology and a Full Member of the Walther On- cology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA. He is now retired and resides in China. Hal E. Broxmeyer, Ph.D. is the Mary Margaret Walther Professor of Medicine, Professor of Microbiology and immunology and the Scientific Direc- tor and a Full Member of the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, USA. He is a former President of the Intema- tional Society for Experimental Hematology and served on numerous national and international committees and editorial boards.

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