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Homing of transplanted hematopoietic stem cells to recipient bone marrow is a critical step in engraftment and initiation of marrow reconstitution. At present, only partial understanding of the cellular and molecular mechanisms governing homing exists. Likewise, only an incomplete list of adhesion molecules implicated in directing the trafficking of stem cells to the marrow microenvironment is available. Opposing hypotheses that attribute homing to an orderly and orchestrated cascade of events or to random migration of circulating cells find ample experimental support. Also unsettled is the fate of marrow-homed cells shortly after transplantation and the rapidity at which they begin to proliferate in their new marrow microenvironment. The limited number of studies in this field and disparities in their experimental design intensifies the confusion surrounding these critical aspects of stem cell biology. However, this area of research is moving forward rapidly and results capable of clarifying many of these issues are forthcoming. [ABSTRACT FROM AUTHOR]

The functional and phenotypic properties of normal human CD3+CD5- T cells which have a higher frequency of cytotoxic cells than CD3+CD5+ T lymphocytes have been described. Using three- and four-colour immunofluorescence flow cytometric cell sorting, the CD3+CD5- and CD3+CD5+ populations were subdivided into αβ or γδ T cell receptor positive cells. The four subsets were examined for the in vitro cytotoxic activity and were also stimulated with mitogens in limiting-dilution assays to measure the frequencies of proliferating and interleukin-2 (IL-2) producing cells. CD+CD5-αβ+, CD3+CD5-γδ+ and CD3+CD5+γδ+ cells had lower frequencies of proliferating and IL-2-producing cells than did CD3+CD5+αβ+ cells. However, the cytotoxic activity of the different phenotypes was higher in the CD3+CD5- subsets, especially when these cells were γδ+. Expression of γδ or lack of expression of CD5 appeared to be associated with the acquisition of cytolytic potentials. CD8 was expressed on 20% of fresh CD3+γδ+ cells. Cultured γδ+ cells retained the expression of γδ, but quickly lost that of CD8 and with time modulated the expression of CD5. The expression of CD5 was found to be higher on sorted CD3+CD5+γδ- than on CD3+CD5-γδ+ cells. These observations indicate that γδ is preferentially expressed on CD5-negative or weakly positive T lymphocytes and that CD3+CD5-γδ+ cells appear to constitute a discrete small subset of mature T lymphocytes which are cytotoxic in nature. However, the exact immunological function of these cells and their place in T cell ontogeny are yet to be elucidated. [ABSTRACT FROM AUTHOR]

A small T ceil subpopulation expressing the phenotype Leu-5 (CD2)+ Leu-4 (CD3) +, Leu-1 (CD5) - can be found in peripheral blood and bone marrow of normal individuals. When these cells were sorted out by three colour immunofluorescence cell sorting and tested in limiting dilution assays, they were found to have lower frequencies of proliferating (9.0 ± 5.6 times, n = 7) and of IL-2 producing cells (11.5 ± 5.0 times n = 5), and a higher frequency of cytotoxic cells (3.1 ±2.6 times. n = 2) than T lymphocytes expressing the three markers. In peripheral blood lymphocytes, 1/3 of the CD3+, CD5- cells were positive for Leu-2a (CD8) while virtually all were negative for Leu-3a (CD4>. Four colour flow cytometric analysis revealed a small subset of T cells positive for CD3 and negative for CD5, CD4 and CD8. Approximately 75% of the CD3+, CD5- cells were negative for Leu-7 and CD 16 simultaneously. These results shed a light on the phenotype of T cells that escape killing by CD5 and complement in T cell depleted bone marrow and may explain why fewer residual T cells in the depleted marrow are detected by limiting dilution assays than by flow cytometric analysis. [ABSTRACT FROM AUTHOR]

We have previously shown by reverse transcriptase-PCR (rtPCR) that CML CD34+ HLA-DR- cells are enriched for BCR/ABL(-) hematopoietic progenitor cells (HPC) while leukemic HPC reside predominately within CML CD34+ HLA-DR+ cells. We investigated whether the 30/35 kDa fragment of fibronectin (FN) could be used to enhance retroviral-mediated gene transfer (RMGT) in chronic phase CML marrow HPC. CML CD34+ HLA-DR- and CD34+ HLA-DR+ cells were transduced with vector supernate containing the neomycin resistance gene on plates coated with either FN or bovine serum albumin (BSA) as control, then assayed for transduced HPC in progenitor cell assays in the presence or absence of G418. Transduction efficiency of CML CD34+ HLA-DR- cells over BSA ranged from 0.09 to 7.2% (mean 3.3 +/- 1.5%), while that over FN plates ranged from 3.8 to 23% (mean 11.0 +/- 4.5%) (n = 4). Transduction efficiencies of CML CD34+ HLA-DR+ cells ranged from 0.4 to 9.8% (mean 3.7 +/- 1.7%) and 6.0 to 26% (mean 17.3 +/- 4.5%) (n = 5) over BSA and FN, respectively. rtPCR analysis for BCR/ABL mRNA of individual G418-resistant HPC generated from CD34+ HLA-DR- cells revealed that normal BCR/ABL(-) HPC were successfully transduced under these experimental conditions. These results demonstrate the feasibility of transducing normal CML primitive HPC, and illustrate the potential clinical use of FN in the setting of gene therapy for CML, as well as other diseases. [ABSTRACT FROM AUTHOR]

In the use of autologous PBPC transplantation in patients with multiple myeloma, contamination of PBPC with myeloma cells is commonly observed. Enrichment for CD34+ cells has been employed as a method of reducing this contamination. In this study the reduction of myeloma cells in PBPC was accomplished by the positive selection of CD34+ cells using immunomagnetic bead separation (Isolex 300 system). PBPC were mobilized from 18 patients using cyclophosphamide (4.5 g/m2) and G-CSF (10 μg/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 × 1010 with a recovery of 1.11 × 108 cells after selection. The median recovery of CD34+ cells was 48% (range 17–78) and purity was 90% (29–99). The median log depletion of CD19+ cells was 3.0. IgH rearrangement, assessed by PCR, was undetectable in 13 of 24 evaluable CD34+ enriched products. Patients received 200 mg/m2 of melphalan followed by the infusion of a median of 2.91 × 106/kg CD34+ cells (1.00–16.30). The median time to absolute neutrophil count >0.5 × 109/l was 11 days, and sustained platelet recovery of >20 × 109/l was 14 days. We conclude that immunomagnetic-based enrichment of CD34+ cells results in a marked reduction in myeloma cells without affecting engraftment kinetics. [ABSTRACT FROM AUTHOR]

Bone marrow cells expressing the surface antigen CD34 comprise approximately 1% of harvested marrow and are highly enriched for marrow progenitor cells, including the cells believed to be responsible for long-term engraftment following bone marrow transplantation (BMT). Selection of CD34-expressing cells was applied in allogeneic BMT (alloBMT) to decrease the number of T lymphocytes in the infused marrow in an attempt to prevent severe graft-versus-host disease (GVHD). We report 14 patients who underwent HLA-identical sibling-matched alloBMT with marrow-enriched for CD34 cells using the Isolex 300 SA device. Patients received total body irradiation, thiotepa, cyclophosphamide, antithymocyte globulin and methylprednisolone prior to marrow infusion. No post-transplantation immunosuppressive therapy was given except for a 5-week course of steroids. The purity of the infused marrow was 64.9 ± 6.0% (mean ± s.e.m.) CD34-positive cells and patients received a mean of 1.24 ± 0.21 × 106 CD34 cells/kg. A mean of 9.4 ± 1.7 × 104 CD3 T cells/kg were present in the CD34-enriched product, representing a 2.7 ± 0.1 log depletion. There were no graft rejections and patients achieved a sustained absolute granulocyte count of >500 in a median of 10.5 days and a sustained platelet engraftment of >20000 untransfused in a median of 27 days. Patients were discharged a median of 21.5 days after marrow infusion. There were no instances of grade III or IV graft-versus-host disease (GVHD) and no unexpected adverse events during the transplant hospitalization. With a median follow-up of 12 months, the estimated 100 day survival is 86 ± 9%. CD34 selection in alloBMT permits rapid engraftment without unanticipated toxicities. [ABSTRACT FROM AUTHOR]

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1α was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1α (MIP-1α/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1α was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1α/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1α/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results... [ABSTRACT FROM AUTHOR]

It was hypothesized that during mammalian development, the extensive need for hematopoietic cells requires equal contribution to blood cell production from both quiescent and cycling hematopoietic stem cells (HSCs) while maintaining the stem cell pool. To investigate this hypothesis, the engraftment potential of umbilical cord blood (UCB) CD34(+) cells residing in either G(0) (G(0)CD34(+) cells) or G(1) (G(1)CD34(+) cells) phases of the cell cycle was assessed in nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice. Whereas the level of chimerism in mice transplanted with UCB G(0)CD34(+) cells was 69.9% +/- 24.0%, mice receiving equal numbers of G(1)CD34(+) cells harbored 46.7% +/- 21.3% human cells 8 weeks posttransplantation. Both groups of cells sustained multilineage differentiation and the production of CD34(+) cells in recipient animals. The relationship between the number of transplanted G(0)CD34(+) or G(1)CD34(+) cells and the level of chimerism was analyzed by a general linear models procedure. Although the initial level of chimerism following transplantation of G(0)CD34(+) cells was higher than that sustained by G(1)CD34(+) cells, the increment in the degree of chimerism obtained with each additional 10(3) cells of either phenotype was identical, suggesting that the reconstitution potential of these 2 types of cells was similar. Of interest is that human cells recovered from primary recipients of both G(0)CD34(+) and G(1)CD34(+) cells engrafted in secondary NOD/SCID recipients, albeit at a substantially lower level, confirming the primitive nature of UCB CD34(+) cells residing in G(1).

Engraftment potential of hematopoietic stem cells (HSCs) is likely to be dependent on several factors including expression of certain adhesion molecules (AMs) and degree of mitotic quiescence. The authors investigated the functional properties and engraftment potential of Sca-1(+)lin(-) cells subfractionated on the basis of expression, or lack thereof, of CD11a, CD43, CD49d, CD49e, or CD62L and correlated that expression with cell cycle status and proliferative potential of engrafting fractions. Donor-derived chimerism in mice receiving CD49e(+) or CD43(+) Sca-1(+)lin(-) cells was greater than that in mice receiving cells lacking these 2 markers, while Sca-1(+)lin(-) cells positive for CD11a and CD62L and bright for CD49d expression mediated minimal engraftment. AM phenotypes enriched for engraftment potential contained the majority of high proliferative potential-colony forming cells, low proliferative potential-colony forming cells, and cells providing rapid in vitro expansion. Cell cycle analysis of AM subpopulations revealed that, regardless of their bone marrow repopulating potential, Sca-1(+)lin(-) AM(-) cells contained a higher percentage of cells in G(0)/G(1) than their AM(+) counterparts. Interestingly, engrafting phenotypes, regardless of the status of their AM expression, were quicker to exit G(0)/G(1) following in vitro cytokine stimulation than their opposing phenotypes. When engrafting phenotypes of Sca-1(+)lin(-) AM(+) or AM(-) cells were further fractionated by Hoechst 33342 into G(0)/G(1) or S/G(2)+M, cells providing long-term engraftment were predominantly contained within the quiescent fraction. These results define a theoretical phenotype of a Sca-1(+)lin(-) engrafting cell as one that is mitotically quiescent, CD43(+), CD49e(+), CD11a(-), CD49d(dim), and CD62L(-). Furthermore, these data suggest that kinetics of in vitro proliferation may be a good predictor of engraftment potential of candidate populations of HSCs. (Blood. 2000;96:1380-1387)

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

We have investigated the expression and functional competence of folate receptor (FR) isoforms on human hematopoietic cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methodology, we find that a substantial fraction of low-density mononuclear and CD34(+) cells express both the beta and gamma isoforms of FR. The alpha isoform of FR (the form most commonly found on cancer cells) was surprisingly absent from all hematopoietic cells examined. Compared with KB cells (a human cell line known for its elevated expression of FR-alpha), the abundance of FR-beta on CD34(+) cell surfaces was relatively low (approximately 8% of KB cell levels). Because many antifolates and folic acid-linked chemotherapeutic agents enter malignant cells at least partially via FR endocytosis, it was important to evaluate the ability of FR on CD34(+) cells to bind folic acid (FA). Based on three FR binding assays, freshly isolated CD34(+) cells were found to display no affinity for FA. Thus, regardless of whether steps were taken to remove endogenous folates before receptor binding assays, FR on primitive hematopoietic cells failed to bind 3H-FA, fluorescein isothiocyanate (FITC)-linked FA, or FA-derivatized liposomes. In contrast, analogous studies on KB cells showed high levels of receptor binding for all three FR probes. These studies show that although multipotent hematopoietic progenitor cells express FR, the receptor does not transport significant amounts of FA. Consequently, antifolates and FA-linked chemotherapeutic agents that can be engineered to enter malignant cells exclusively through the FR should not harm progenitor/stem cell function.

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.

Ex vivo expansion of hematopoietic stem and progenitor cells is a very ambitious idea that would have major implications in the areas of stem cell transplantation and somatic gene therapy. However, successful ex vivo expansion has evaded and frustrated scientists for a number of years. The goal of ex vivo expansion is to induce cell division and proliferation of stem cells while maintaining their primary functional characteristic, namely, their ability to engraft and sustain long-term hematopoiesis. Only when a balance between these two requirements is reached can ex vivo expansion of stem cells be considered successful. Establishing such a balance has not been easy. However, many lessons have been learned along the way, and today we have a more profound understanding of the potential obstacles facing ex vivo expansion than we did only a few years ago. In this review, we discuss these obstacles and evaluate the current status of ex vivo expansion of stem and progenitor cells both from the perspective of basic stem cell biology and from the viewpoint of clinical utility of these cells in transplantation.

In the use of autologous PBPC transplantation in patients with multiple myeloma, contamination of PBPC with myeloma cells is commonly observed. Enrichment for CD34+ cells has been employed as a method of reducing this contamination. In this study the reduction of myeloma cells in PBPC was accomplished by the positive selection of CD34+ cells using immunomagnetic bead separation (Isolex 300 system). PBPC were mobilized from 18 patients using cyclophosphamide (4.5 g/m2) and G-CSF (10 microg/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 x 10(10) with a recovery of 1.11 x 10(8) cells after selection. The median recovery of CD34+ cells was 48% (range 17-78) and purity was 90% (29-99). The median log depletion of CD19+ cells was 3.0. IgH rearrangement, assessed by PCR, was undetectable in 13 of 24 evaluable CD34+ enriched products. Patients received 200 mg/m2 of melphalan followed by the infusion of a median of 2.91 x 10(6)/kg CD34+ cells (1.00-16.30). The median time to absolute neutrophil count >0.5 x 10(9)/l was 11 days, and sustained platelet recovery of >20 x 10(9)/l was 14 days. We conclude that immunomagnetic-based enrichment of CD34+ cells results in a marked reduction in myeloma cells without affecting engraftment kinetics.

Most primitive hematopoietic progenitor cells reside in vivo within the G0/G1 phase of the cell cycle. By simultaneous DNA/RNA staining it is possible to distinguish G0 and G1 states and to isolate cells in defined phases of the cell cycle. We report here the use of cell cycle fractionation to separate human mobilized peripheral blood (MPB) CD34(+) cells capable of repopulating the bone marrow (BM) of non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice. In freshly isolated MPB, repopulating cells were predominant within the G0 phase, because transplantation of CD34(+) cells residing in G0 (G0CD34(+)) resulted on average in a 16.6- +/- 3.2-fold higher BM chimerism than infusion of equal numbers of CD34(+) cells isolated in G1. We then investigated the effect of ex vivo cell cycle progression, in the absence of cell division, on engraftment capacity. Freshly isolated G0CD34(+) cells were activated by interleukin-3 (IL-3), stem cell factor (SCF), and flt3-ligand (FL) for a 36-hour incubation period during which a fraction of cells progressed from G0 into G1 but did not complete a cell cycle. The repopulating capacity of stimulated cells was markedly diminished compared with that of unmanipulated G0CD34(+) cells. Cells that remained in G0 during the 36-hour incubation period and those that traversed into G1 were sorted and assayed separately in NOD/SCID recipients. The repopulating ability of cells remaining in G0 was insignificantly reduced compared with that of unstimulated G0CD34(+) cells. On the contrary, CD34(+) cells traversing from G0 into G1 were largely depleted of repopulating capacity. Similar results were obtained when G0CD34(+) cells were activated by the combination of thrombopoietin-SCF-FL. These studies provide direct evidence of the quiescent nature of cells capable of repopulating the BM of NOD/SCID mice. Furthermore, these data also demonstrate that G0-G1 progression in vitro is associated with a decrease in engraftment capacity., (Copyright 1998 by The American Society of Hematology.)

Seventy-one patients with poor-prognosis breast cancer were enrolled after informed consent in a multicentre randomized study to evaluate the use of selected peripheral blood CD34+ cells to support haematopoietic recovery following high-dose chemotherapy. Patients who responded to conventional chemotherapy were mobilized with chemotherapy (mainly high-dose cyclophosphamide) and/or recombinant human granulocyte colony-stimulating factor (rhG-CSF). Patients who reached the threshold of 20 CD34+ cells per microl of peripheral blood underwent apheresis and were randomized at that time to receive either unmanipulated mobilized blood cells or selected CD34+ cells. For patients in the study arm, CD34+ cells were selected from aphereses using the Isolex300 device. Fifteen patients failed to mobilize peripheral blood progenitors and nine other patients were excluded for various reasons. Forty-seven eligible patients were randomized into two comparable groups. CD34+ cells were selected from aphereses in the study group. Haematopoietic recovery occurred at similar times in both groups. No side-effect related to the infusion of selected cells was observed. The frequency of epithelial tumour cells in aphereses was low (8 out of 42 evaluated patients), as determined by immunocytochemistry. We conclude that selected CD34+ cells safely support haematopoietic recovery following high-dose chemotherapy in patients with poor-prognosis breast cancer.

To investigate the mechanisms behind the leukaemic expansion of chronic myelogenous leukaemia (CML), we examined the cell cycle status and activation kinetics of purified subpopulations of CD34+ cells from normal and CML bone marrow (BM). Propidium iodide staining was used to assess cell cycle status of fresh cells or those stimulated with cytokines. Although the cell cycle status of fresh low-density cells from CML and normal BM was similar, a larger percentage of CML CD34+ cells were cycling than those from normal BM. The HLA-DR compartment of CML CD34+ cells, a fraction enriched for normal, non-leukaemic progenitors, contained a higher percentage of quiescent cells than the CD34+ HLA-DR+ fraction. When the activation of CD34+ cells was examined in response to SCF or IL-3 alone, or SCF+IL-3+IL-6, CML CD34+ cells exited GO/G1 more rapidly than normal CD34+ cells. Interestingly, although normal BM CD34+ cells failed to cycle in response to IL-6 alone, or in the absence of exogenous cytokines, 30% of CML cells cycled under these conditions. No differences in the degree of apoptosis were documented among CML and normal CD34+ cells in these cultures. These data suggest that enhanced cell cycle activation of CML CD34+ cells, by either autocrine stimuli or via enhanced sensitivity to exogenous stimuli, may be partially responsible for the pronounced cellular expansion characteristic of CML.

Exit of primitive hematopoietic progenitor cells (HPCs) from the G0 phase of the cell cycle in response to in vitro cytokine stimulation is a limiting step in successful ex vivo expansion. Simultaneous DNA/RNA staining with Hoechst 33342 and pyronin Y was used to separate human bone marrow CD34+ cells residing in G0 (G0CD34+) from those cycling in G1 and S/G2+M. Compared with CD34+ cells isolated in G1, G0CD34+ cells were characterized by a delayed response to cytokine stimulation and were enriched for long-term hematopoietic culture-initiating cells. We next compared the activation kinetics of individually sorted G0CD34+ cells stimulated with stem cell factor (SCF), flt3-ligand (FL), or interleukin-3 (IL-3) as single factors. In a novel clonal proliferation assay, the functional status of cells that had remained quiescent after an initial 7-day period and of those that had completed successive division cycles under each of these three factors was evaluated by assessment of subsequent proliferative capacity and maintenance of colony-forming cell precursor (pre-CFC) activity. All three cytokines were equally able to support the survival of primitive HPCs in the absence of cell division. Cells that did not respond to any cytokine stimulation for 7 days retained higher proliferative and pre-CFC activities than dividing cells. The hematopoietic function of cells that divided in response to SCF, FL, or IL-3 decreased after each division cycle. However, G0CD34+ cells displayed a heterogeneous response pattern to cytokine stimulation whereby SCF appeared to have a superior ability to promote the cycling of cells with high proliferative and pre-CFC activities. These results indicate that HPCs reside in opposing hierarchies of hematopoietic potential and responsiveness to cytokine stimulation. The data also begin to indicate relationships between cellular division in response to different stimuli and maintenance of hematopoietic function.

NK cells are lymphocytes which exhibit spontaneous cytotoxicity against a variety of target cells, including cancer cells. Mature NK and T cells may derive from a common precursor which differentiates into T or NK cells depending on the microenvironment. We evaluated the effect of human fetal thymic stroma on human CD34+Lin- progenitors. The culture medium was supplemented with human AB serum with or without interleukin-2 (IL2; 1000 U/ml) and interleukin-7 (IL7; 1000 U/ml). After 3 weeks of culture, CD45/56 cells were detected by flow cytometry and their activity was tested against K562 targets. In cultures with IL2 the percentage of CD56-positive cells was much higher in the Transwell cultures (60.8 +/- 12.5% from CD34+Lin-DR+ and 51% from CD34+Lin-progenitors) than in adherent cultures (25 +/- 21.9% from CD34+Lin-DR+ and 25.3 +/- 9.5% from CD34+Lin-progenitors) or suspension cultures (23 +/- 21.4% from CD34+Lin-DR+ progenitors and 43.1 +/- 14.2% from CD34+Lin-progenitors). Cytolytic activity as measured by K562 lysis was also higher in Transwell cultures with IL2. NK cells were also obtained in cultures without factors or supplemented with IL7, but in smaller numbers. These data indicate that NK cells can be obtained in vitro by coculture of immature hematopoietic progenitors with thymic stromal cells and that IL2 appears to strongly favor their development in the absence of stromal contact. This would indicate a direct inhibitory effect of the thymic stroma on NK progenitors.

Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21(ras) (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1-/- hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1-/- progenitors. Nf1-/- fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1-/- progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol using neoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of neoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

Murine fetal liver (FL) and adult bone marrow (BM) hematopoietic stem cells (HSCs) are characterized by cell surface expression of CD38 and c-kit. Because murine yolk sac (YS) HSC activity precedes the initiation of FL hematopoiesis, we investigated whether YS-derived HSCs also expressed c-kit and CD38. c-Kit+ CD38+ lineage- cells derived from day 9 YS as well as adult BM were found to be enriched in high proliferative potential colony-forming cells. c-Kit+ CD38+ lineage- YS or adult BM cells were capable of long-term reconstitution (>6 months) of busulfan-conditioned newborn or lethally irradiated adult mice, respectively. In contrast, c-kit+ CD38- lineage- populations from both tissues were enriched in lineage-committed progenitors and had no long-term HSC activity. We concluded that c-kit and CD38 are cell surface markers of HSCs expressed throughout murine ontogeny.

We examined the decline in hematopoietic potential observed when human CD34+ cells are cultured in vitro by evaluating the association between proliferation history and the fate of long-term hematopoietic culture-initiating cells (LTHC-ICs) as well as the onset of programmed cell death. The membrane dye PKH2 was used to track ex vivo expanded human CD34+ cells from bone marrow, cord blood, and mobilized peripheral blood, and to identify and isolate CD34+ cells that had divided once, twice, three, or four times or more, as well as cells that had remained cytokine nonresponsive and therefore failed to proliferate. These isolated groups of cells were assayed for their hematopoietic potential, cell cycle status, and percentage of apoptotic cells. A gradual decline in the content of LTHC-ICs, as well as in their ability to initiate and sustain in vitro hematopoiesis, was found to correlate with the number of in vitro cellular divisions, such that the hematopoietic potential of CD34+ cells dividing four or more times was nearly depleted. DNA analysis revealed that cells dividing more than three times resided predominantly in G0/G1 phases of the cell cycle. In addition, the percentage of CD34+ cells undergoing apoptosis was found to increase concomitantly with the number of in vitro cellular divisions; less than 10% of cells dividing once were apoptotic, whereas more than 25% of CD34+ cells dividing four or more times underwent programmed cell death. Together, these data suggest that a proliferation-associated, and possibly activation-induced, loss of hematopoietic potential among dividing CD34+ cells may result from an increase in programmed cell death among dividing primitive hematopoietic progenitor cells.

Using simultaneous Hoechst 33342 (Hst) and Pyronin Y (PY) staining for determination of DNA and RNA content, respectively, human CD34(+) cells were isolated in subcompartments of the G0 /G1 phase of the cell cycle by flow cytometric cell sorting. In both bone marrow (BM) and mobilized peripheral blood (MPB) CD34(+) cells, primitive long-term hematopoietic culture-initiating cell (LTHC-IC) activity was higher in CD34(+) cells isolated in G0 (G0CD34(+) cells) than in those residing in G1 (G1CD34(+) cells). However, as MPB CD34(+) cells displayed a more homogeneous cell-cycle status within the G0 /G1 phase and a relative absence of cells in late G1 , DNA/RNA fractionation was less effective in segregating LTHC-IC in MPB than in BM. BM CD34(+) cells belonging to four subcompartments of increasing RNA content within the G0 /G1 phase were evaluated in functional assays. The persistence of CD34 expression in suspension culture was inversely correlated with the initial RNA content of test cells. Multipotential progenitors were present in G0 or early G1 subcompartments, while lineage-restricted granulomonocytic progenitors were more abundant in late G1 . In vitro hematopoiesis was maintained for up to 6 weeks with G0CD34(+) cells, whereas production of clonogenic progenitors was more limited in cultures initiated with G1CD34(+) cells. To test the hypothesis that primitive LTHC-ICs would reenter a state of relative quiescence after in vitro division, BM CD34(+) cells proliferating in ex vivo cultures were identified from their quiescent counterparts by a relative loss of membrane intercalating dye PKH2, and were further fractionated with Hst and PY. The same functional hierarchy was documented within the PKH2(dim) population whereby LTHC-IC frequency was higher for CD34(+) cells reselected in G0 after in vitro division than for CD34(+) cells reisolated in G1 or in S/G2 + M. However, the highest LTHC-IC frequency was found in quiescent PKH2(bright) CD34(+) cells. Together, these results support the concept that cells with distinct hematopoietic capabilities follow different pathways during the G0 /G1 phase of the cell cycle both in vivo and during ex vivo culture.

We investigated the survival and chimeric engraftment characteristics of a large consecutive series of lambs that were transplanted with human hematopoietic cells in utero. Approximately 50% of the fetal sheep survived. Neither the transplantation of human cells into fetal sheep, nor the parity of the ewe was associated with increased mortality, as compared with the risk of surgery alone. However, a breed-associated mortality was noted. Sixty percent of surviving recipient lambs contained donor, human hematopoietic cells in blood and bone marrow (BM) cells. Chimerism ranged from 0.0001-1%. Human hematopoietic progenitors were identified in the BM in 8 of 12 chimeric sheep examined. Some lambs engrafted with human cells maintained a human chimerism for up to at least 2 years. Our data demonstrate that a large proportion of fetal sheep are capable of engrafting human cells, albeit at widely variable levels of engraftment.

Bone marrow (BM) CD34+ cells residing in the G0 phase of cell cycle may be the most suited candidates for the examination of cell cycle activation and proliferation of primitive hematopoietic progenitor cells (HPCs). We designed a double simultaneous labeling technique using both DNA and RNA staining with Hoechst 33342 and Pyronin Y, respectively, to isolate CD34+ cells residing in G0(G0CD34+). Using long-term BM cultures and limiting dilution analysis, G0CD34+ cells were found to be enriched for primitive HPCs. In vitro proliferation of G0CD34+ cells in response to sequential cytokine stimulation was examined in a two-step assay. In the first step, cells received a primary stimulation consisting of either stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), or IL-6 for 7 days. In the second step, cells from each group were washed and split into four or more groups, each of which was cultured again for another week with one of the four primary cytokines individually, or in combination. Tracking of progeny cells was accomplished by staining cells with PKH2 on day 0 and with PKH26 on day 7. Overall examination of proliferation patterns over 2 weeks showed that cells could progress into four phases of proliferation. Phase I contained cytokine nonresponsive cells that failed to proliferate. Phase II contained cells dividing up to three times within the first 7 days. Phases III and IV consisted of cells dividing up to five divisions and greater than six divisions, respectively, by the end of the 14-day period. Regardless of the cytokine used for primary stimulation, G0CD34+ cells moved only to phase II by day 7, whereas a substantial percentage of cells incubated with SCF or FL remained in phase I. Cells cultured in SCF or FL for the entire 14-day period did not progress beyond phase III but proliferated into phase IV (with <20% of cells remaining in phases I and II) if IL-3, but not IL-6, was substituted for either cytokine on day 7. G0CD34+ cells incubated with IL-3 for 14 days proliferated the most and progressed into phase IV; however, when SCF was substituted on day 7, cells failed to proliferate into phase IV. Most intriguing was a group of cells, many of which were CD34+, detected in cultures initially stimulated with IL-3, which remained as a distinct population, mostly in G0/G1, unable to progress out of phase II regardless of the nature of the second stimulus received on day 7. A small percentage of these cells expressed cyclin E, suggesting that their proliferation arrest may have been mediated by a cyclin-related disruption in cell cycle. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HPCs and that unscheduled stimulation of CD34+ cells residing in G0 may result in disruption of cell-cycle regulation.

It is believed that long-term cultures of CML marrow cells favor the outgrowth of BCR/ABL negative hematopoietic progenitor cells (HPC) and that this phenomenon may be enhanced with negative hematopoietic regulators which can maintain primitive HPC in a quiescent state. Proliferation of CML marrow CD34+ cells in primary short-term cultures, maintained in the presence or absence of macrophage inhibitory protein-1 alpha (MIP-1 alpha), was tracked with the membrane dye PKH2. After 7 to 10 days it was possible to distinguish between cytokine responsive (CR) CD34+ cells (cells which had divided thus becoming PKH2dim) and cytokine nonresponsive (CNR) CD34+ cells (cells which had not divided and had therefore remained PKH2bright). CR and CNR CD34+ cells were isolated by flow cytometric cell sorting, seeded in secondary long-term cultures, and their progeny cells assayed weekly for their clonogenic progenitor cell content and expression of BCR/ABL by reverse transcriptase polymerase chain reaction (RT-PCR). Whereas CNR cells isolated from control primary cultures (control/CNR) sustained in vitro hematopoiesis, similar cells from cultures treated with MIP-1 alpha (MIP-1 alpha/CNR) supported a higher and, in some patients, a more extended production of clonogenic HPC, indicating that MIP-1 alpha was able to maintain primitive HPC in a quiescent state. Predominance of BCR/ABL negative progenitors in vitro was more evident in secondary cultures initiated with CNR cells than in those initiated with CR cells, especially those established with MIP-1 alpha/CNR cells. Of interest is the observed decline in the percentage of BCR/ABL+ progenitors in these cultures with time. Whereas up to 100% of progenitors were BCR/ABL+ on day 0, by day 14, only 46% of progenitors in MIP-1 alpha/CNR secondary cultures were BCR/ABL+ and by day 28 and beyond, the percentage of BCR/ABL+ progenitors dropped to below 20%. These results suggest that the quiescent nature of normal HPC present in CML marrow may favor their identification via cell tracking and, subsequently, their isolation from the more actively cycling leukemic cells. These studies also confirm the feasibility of employing negative hematopoietic regulators to augment the sequestration of normal HPC among the cytokine nonresponsive fraction of CD34+ cells, an approach that may be clinically feasible for autotransplantation.

Posttransplantation lymphoproliferative disorders (PT-LPDs) occurring in T-cell depleted (TCD) allogeneic bone marrow transplant recipients seem to be different from those that arise in solid organ recipients in their early development, the high incidence of extensive dissemination at presentation, and their aggressive course and high fatality rate. We report a series of 10 patients with PT-LPDs after TCD allogeneic bone marrow transplant. We studied the correlation between the morphology of the lesions; their clonality based on immunoglobulin (Ig) heavy chain gene rearrangement analysis and immunohistochemistry; their proliferative activity as measured by immunoperoxidase staining for the proliferating cell nuclear antigen (PCNA) and the presence of p53 gene product overexpression. Histologically, our cases corresponded to the two morphologic categories of polymorphic B-cell lymphoma (PBCL, seven cases) and malignant lymphoma immunoblastic (ML-IB, three cases). Ig light-chain staining showed monoclonality in a minority of the cases, whereas Ig gene rearrangement analysis by polymerase chain reaction revealed B-cell clonality in three of seven cases of PBCL and in all three cases of ML-IB. The Epstein-Barr virus (EBV) genome, the expression of EBV latent membrane protein or both were found in all 10 specimens. High proliferative activity (PCNA > or = 66%) was found in all cases, with a mean PCNA value of 56% in PBCL and 84% in ML-IB. Five specimens were p53+ (two of seven PBCL and three of three ML-IB). Two of four PBCL cases resolved with the administration of donor leukocytes. All of the remaining patients died of the PT-LPD within a short time from admission. Our results show that the PT-LPDs after TCD bone marrow transplantation are characterized by a high frequency of high-grade histologic subtypes, frequent monoclonality, high proliferative activity, frequent overexpression of p53 gene product, and poor prognosis. These characteristics observed in only a minority of cases of PT-LPDs occurring after solid organ transplantation may account for the less aggressive clinical behavior observed in those diseases.

Between October 1991 and May 1994, 42 patients were treated with cyclophosphamide, thiotepa, and total body irradiation followed by an allogeneic transplantation of marrow depleted of T cells with soybean agglutinin and E-rosetting. Patients included in this study had acute myelogenous leukemia (13), chronic myelogenous leukemia (12), acute lymphocytic leukemia (nine), Hodgkin's disease or non-Hodgkin's lymphoma (four), multiple myeloma (three), or myelodysplastic syndrome (one). The mean age was 34 (range 8 to 51 years). Nineteen patients had a matched sibling donor and 18 received marrow from 6/6 matched unrelated donors while five received transplants from unrelated donors disparate at one DR locus (5/6 match). Time to granulocyte engraftment (AGC > or = 500/mm3) occurred at a mean of 16.5 days for related and 11.4 days for unrelated transplant recipients, and was related to the increased use of G-CSF in the unrelated population. There was no correlation with number of mononuclear cells, T cells, or CD34-positive cells infused, the rate of engraftment or the incidence of transplant complications. Multivariate analysis determined that G-CSF administration and a diagnosis other than ALL were the only factors associated with a faster rate of engraftment. Patients receiving unrelated donor transplants, those with ALL, or those who had a low T cell number infused (< or = 8.0 x 10(3) cells/kg) experienced delayed hospital discharge. The regimen resulted in excellent rates of engraftment (95.2%) with only one failure to engraft and one graft rejection. The incidence of grade III-IV acute graft-versus-host disease was 0% with sibling and 26.1% with unrelated donors. There were no cases of veno-occlusive disease. Fifty percent of patients are alive with a mean follow-up of 26.4 months. We conclude that this regimen is well tolerated and results in excellent engraftment with a low incidence of severe graft-versus-host disease and few therapy-related toxicities.

In addition to possessing multilineage differentiation and self-renewal capabilities, pluripotent hematopoietic stem cells are believed to be mitotically quiescent and metabolically inactive. Fractions of human bone marrow (BM) CD34+ cells can be further enriched for primitive hematopoietic progenitor cells (HPC) by using a number of cell-surface markers. All of these fractions, however, contain cells that are still heterogeneous as far as their metabolic and mitotic activities are concerned. We therefore used Hoechst 33342 (Hst) to identify quiescent cells and Rhodamine 123 (Rh123) to identify metabolically inactive cells. CD34+HstdimRh123dim (CD34+d/d) and CD34+HstbrightRh123bright (CD34+b/b) cells were isolated by flow cytometry to examine the hematopoietic functions of mitotically and metabolically homogeneous progenitors. Cell-cycle status, progenitor cell content, maintenance of in vitro hematopoiesis, and long-term hematopoietic culture-initiating cell (LTHC-IC) content of CD34+d/d and CD34+b/b cells were compared with CD34+HLA-DR- cells, a well-defined phenotype of primitive HPC. Whereas 99.2 +/- 0.5% of freshly isolated CD34+d/d cells were in G0/G1 phase of the cell cycle, only 74.4 +/- 11.5% of CD34+b/b and 75.6 +/- 1.1% of CD34+HLA-DR- cells were in G0/G1. The number of multipotential progenitors (colony-forming units-granulocyte/erythroid/ macrophage/megakaryocyte [CFU-GEMM]) detected in CD34+d/d cells was twice that observed in CD34+HLA-DR- cells and eight times that in CD34+b/b cells. In stromal cell-free long-term cultures maintained for 10 weeks, production of assayable progenitors in cultures initiated with CD34+d/d cells exceeded that detected in CD34+HLA-DR- cultures by more than three-fold. Only in CD34+d/d cultures were high proliferative potential colony-forming cell (HPP-CFC)-derived colonies detected over a period of 6 weeks. Limiting dilution analysis revealed that the frequency of LTHC-IC was highest among CD34+d/d cells (7.2 +/- 3.3%), followed by a frequency of 4.5 +/- 4.8% for CD34+HLA-DR- cells and 2.2 +/- 3.5% for CD34+b/b cells. The primitive nature of HPC identified by CD34, Hst, and Rh123 was confirmed by the ability of as few as 200 murine marrow cells isolated by this technique to radioprotect and fully reconstitute lethally irradiated recipients. These results indicate that Hst and Rh123 staining can be used in combination with CD34 immunofluorescence to isolate a quiescent subpopulation of human primitive hematopoietic progenitor cells. Cells isolated by this technique appear to have functional properties associated with stem cells, suggesting that they may be ideal candidates for studies requiring primitive HPC, such as ex vivo expansion and somatic gene therapy.

To evaluate whether marrow contributes to relapse after autologous bone marrow transplantation (AuBMT) for acute leukemia, transplanted marrow was marked with the G1N retroviral vector (Genetic Therapy Inc.) containing the neomycin phosphotransferase gene (neo). Between April 1992 and August 1993, 4 patients were transplanted for acute myeloid leukemia (AML) in second complete remission (CR) and 1 patient for acute lymphoid leukemia in first CR. An average of 12.4% (range 5-19%) of transplanted marrow mononuclear cells were exposed to G1N vector for 4 hr. In the vector-treated portion of the marrow, 4.9% of GM-CFU and 3.6% of erythroid burst-forming units (BFU-E) were resistant to G418 in vitro. In the 5 patients, the polymerase chain reaction (PCR) detected the neo sequence on only two occasions after AuBMT. Of 4 patients surviving 1 year after transplantation, only 1 had evidence of gene marked cells by PCR. Two AML patients have relapsed, one of whom had evidence of neo sequences in the bone marrow at day 100 but not at relapse 11 months after AuBMT. The second patient relapsed 18 months after AuBMT but never had PCR evidence of neo sequences before or after relapse. Our results indicate vector-transduced autologous bone marrow from heavily pretreated adults with acute leukemia mark with low efficiency, although vector sequences have been detected in bone marrow and peripheral blood up to 1 year after transplant. Of the 2 relapsed patients, no evidence of vector-marked leukemic blasts have been detected.

The objective of ex vivo expansion of primitive hematopoietic progenitor cells (HPC) is to increase the number of progeny cells possessing hematopoietic potential similar to the original HPC. In the context of bone marrow (BM) transplantation in mice, this implies that expanding a number of HPC sufficient for long-term rescue of one lethally irradiated animal should generate enough cells to rescue more than one lethally irradiated recipient. In the present study, Sca-1+Lin- cells from male C57Bl/6 mice were expanded in vitro with stem cell factor (SCF), interleukin-1alpha (IL-1alpha), IL-3, and IL-6 and used to transplant lethally irradiated syngeneic female recipients. Expanded cells were tracked in vitro with the fluorescent membrane dye PKH2, which becomes evenly distributed among dividing daughter cells, and fractionated on day 7 into Sca-1+ cells which did not divide (Sca-1+PKH2bright), those which had divided 1 to 2 times (Sca-1+PKH2moderate), or those which had divided four or more times (Sca-1+PKH2dim). Grafts of expanded cells consisted of either the same number of fresh cells proven to rescue lethally irradiated animals [3X10(3) cells; referred to as one repopulating dose (1 RD)] or the expansion equivalent (EE) of these cells. One EE of cells represented 3X10(3) multiplied by the fold increase in the number of cultured cells on day 7. All animals transplanted with 3X10(3) freshly isolated Sca-1+Lin- cells survived long-term. Only 53% of animals receiving 1 EE of all cultured day-7 cells survived. One RD from all three PKH2 fractions (bright, moderate, and dim) of day-7 cultured Sca-1+ cells failed to rescue more than 30% of lethally irradiated recipients. Comparable survival rates were obtained when 1 EE of Sca-1+PKH2dim or only 4 RD of Sca-1+PKH2bright cells were used as grafts, suggesting that a larger frequency of long-term repopulating cells may have been retained within the fraction of Sca-1+ cells undergoing minimal or no proliferation in culture. Engraftment of male ex vivo expanded cells in recipients was confirmed by polymerase chain reaction (PCR) analysis with Y chromosome-specific primers. When analyzed for their cell cycle status, Sca-1+PKH2bright cells were mostly quiescent, whereas a higher percentage of Sca-1+PKH2dim cells were in active phases of cell cycle. These data suggest that ex vivo expansion does not augment the number of BM repopulating HPC and that ex vivo expansion generates classes of progenitor cells with different BM repopulating potentials depending on their proliferative history. These studies also suggest that the cell cycle status of graft cells may affect the ability of these cells to engraft in myeloablated hosts.

Mobilized peripheral blood hematopoietic progenitor cells obtained from cancer patients treated with high-dose cyclophosphamide (7g/m2) followed by G-CSF, GM-CSF, IL-3, PIXY321, or combinations of these cytokines have been successfully used for autologous stem cell transplantation. We investigated the ability of hematopoietic progenitor cells (HPC) derived from mobilized peripheral blood (PB) to undergo ex vivo expansion in short term cultures by enumerating numbers of de novo generated CD34+ cells, assayable progenitor cells, and the frequency of long-term hematopoietic culture-initiating cells (LTHC-IC). These parameters were examined in CD34+ cells generated in culture through the use of cell tracking with the membrane dye PKH2. Fresh isolated mobilized CD34+ cells contained 0.49 +/- 0.36% LTHC-IC. However, due to the high number of total CD34+ cells in mobilized PB, the absolute number of LTHC-IC was higher than that contained in a bone marrow (BM) harvest. Mobilized CD34+ cells were stained with PKH2 and incubated with SCF, IL-3, and IL-6. After 5 to 6 days, numbers of total CD34+ cells and clonogenic progenitors increased 1.4- and 2.2-fold, respectively. Numbers of total progenitors continued to increase such that 10 to 12 days after the initiation of cultures a 6.4-fold increase was demonstrable. However, between days 5 and 7 of culture, the frequency of LTHC-IC in CD34+PKH2bright cells (cells which did not divide) was less than 50% of that determined for fresh cells, while the frequency among CD34+PKH2dim cells (cells that had divided) was very low or undetectable. However, moderately higher frequencies of LTHC-IC were detected following expansion for 48 hours only. In similar assays, both BM and cord blood cells were capable of generating LTHC-IC in CD34+PKH2dim cells but not to expand the overall number of these progenitors. These observations suggest that although mobilized PB CD34+ cells contain large numbers of LTHC-IC, these cells might not be capable of further ex vivo expansion and generation of additional LTHC-IC in vitro. Furthermore, these data indicate that mobilized PB CD34+ cells may have undergone maximal "in vivo expansion" such that additional ex vivo expansion of primitive progenitor cells may not be possible.

Cord blood (CB) has been used recently for stem cell transplantation. We have investigated two different approaches to deplete CB samples of T cells capable of mounting graft-vs.-host disease (GVHD). The methods used were selection of CD34+ cells using avidin-biotin columns (CellPro) and T cell immunodepletion with T10B9 monoclonal antibody (mAb) plus complement. Using the avidin-biotin columns, 10.3% of the original CD34+ cells were recovered. Although this technique yielded a population containing 60 +/- 5.5% CD34+ cells, about 1 log of CFU-GM progenitors were lost. In contrast, after the T10B9 mAb and complement immunodepletion, 75 +/- 19% and 62 +/- 7% of the CD34+ cells and CFU-GM were recovered, respectively. T cell depletion was 3.6 logs using the CellPro columns and 2.2 logs after immunodepletion. To investigate whether cell losses following T cell depletion could be overcome by ex vivo expansion, cells were cultured in the presence of recombinant human interleukin-3 (rhIL-3) and recombinant human c-kit ligand (stem cell factor [rhSCF]) for 7 days. There were 14- and six-fold expansions in the number of progenitors recovered after CellPro and immunodepletion, respectively. To asses the engraftment potential of expanded cells, we used a murine transplantation model in which the presence of human cells was identified by the anti-CD45 mAb. Cells expanded in vitro engrafted in irradiated BNXid mice as efficiently as nonexpanded cells, suggesting that expansion did not affect their transplantability. This study shows that both techniques resulted in significant T cell depletion of CB. Furthermore, in vitro expansion could overcome cell losses sustained during the separation techniques without impairing the engraftment potential of the expanded cells.

We have previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human adult marrow CD34+ HLA-DR- cells in sheep after transplantation in utero. The persistence in these animals of CD34+ HLA-DR- cells, the phenotype of the original graft, and the presence of assayable high proliferative potential colony-forming cells (HPP-CFCs), a primitive progenitor cell that has self-renewal capacity and multilineage differentiative capacity, suggested that these chimeric animals continued to contain human pluripotent hematopoietic stem cells (HSCs). To test this hypothesis, CD45+ cells from the bone marrow of such an animal were isolated by panning at 19 and 24 months after birth, were pooled, and were transplanted into 4 preimmune sheep fetuses (0.6 x 10(6) viable cells per fetus). Three of the sheep were born alive. Bone marrow from one of these secondary recipients (lamb 58) exhibited significant donor (human) cell presence at 3, 9, 12, and 15 months (last evaluation period) after transplant. The engraftment was sustained along with multilineage expression involving lymphoid, erythroid, and myeloid cells of human origin. These results indicated that human CD34+ HLA-DR- cells are capable of long-term marrow repopulation and that this cell population is enriched for human long-term repopulating HSCs.

In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)

Human cord blood (CB) can be utilized as a source of transplantable stem cells. In the clinical setting, the numbers of clonogenic progenitors and nucleated cells are used as indirect measures of the stem cell content of CB samples. In an attempt to define other parameters of engraftment potential, we have determined the numbers of CD34+ cells, and of CD34+ cells co-expressing CD38 and/or HLA-DR in 17 CB samples. At the same time, clonogenic progenitors were assayed in the presence of erythropoietin and different combinations of rhIL-3, rhG-CSF, rhGM-CSF, rh-Steel Factor and medium conditioned by the PU34 primate cell line. Unseparated CB contained a mean of 14.8 x 10(4) CD34+ cells/mL. CD34+CD38-, CD34+DR+ and CD34+DR+ CD38- cells represented 12.2%, 91% and 10% of all CD34+ cells, respectively. Linear regression analysis demonstrated that the total number of CD34+ cells, as well as the different subsets, correlated positively with the numbers of total colonies and day 14 CFU-GM. Furthermore, the proportion of CD34+CD38- cells was significantly higher than has been reported for adult marrow. Cord blood CD34+CD38- and CD34+DR+ cells have a high proliferative potential and are enriched for primitive hematopoietic progenitors. Thus, we conclude that a single collection of CB could be sufficient to engraft an adult recipient, and that quantitation of CD34+ cells and their subsets may be useful in determining the engraftment potential of CB samples.

Autologous bone marrow transplantation has become a very popular and successful treatment for many patients with lymphomas and other malignancies. The current indications, pretreatment regimes, and laboratory manipulations are discussed as well as the application of gene transfer to eliminate selected genetic diseases and detect disease relapse.

Currently, the most commonly used grafts of progenitor and stem cells for patients undergoing bone marrow transplantation (BMT) are derived from large collections of autologous or allogeneic adult human bone marrow (BM). The feasibility of using human umbilical cord blood (HUCB), normal peripheral blood (PB), and smaller collections of BM as sources of hematopoietic stem cell grafts for adult patients remains questionable. We investigated the ex vivo proliferative potential of HUCB CD34+ cells as a means of expanding HUCB grafts, thereby making them more acceptable for clinical transplantation. HUCB-derived CD34+HLA-DR+ cells, maintained for 5 days in suspension cultures supplemented with 10% HUCB plasma and a combination of stem cell factor (SCF) and interleukin-3 (IL-3), displayed a 10-fold increase in the total number of CD34+ cells. In contrast, only a four-fold increase was observed in identical cultures initiated with BM-derived CD34+HLA-DR+ cells. Whereas BM CD34+ cells failed to proliferate in response to SCF alone, HUCB CD34+ cells expanded 5.6-fold by day 5, thus demonstrating an enhanced response to SCF. When the effects of SCF on the exit of HUCB cells from G0/G1 phases of cell cycle were investigated, we found that although HUCB CD34+HLA-DR+ cells were more quiescent than BM CD34+HLA-DR+ and BM CD34+HLA-DR- cells (97.5% of HUCB CD34+HLA-DR+ in G0/G1 vs. 88.6% of BM CD34+HLA-DR+ and 92.0% of BM CD34+HLA-DR- [p < 0.005]), HUCB CD34+HLA-DR+ cells exited from dormancy more rapidly than BM cells, such that by 36 to 48 hours following exposure to SCF, only 55% remained in G0/G1. Furthermore, an 8.4-fold increase in the number of HUCB CD34+ cells still residing in G0/G1 was observed on day 5 in cultures supplemented with SCF and IL-3, suggesting the generation of large numbers of primitive hematopoietic progenitor cells (HPC) in vitro. When the contribution of HUCB plasma to the exist of HUCB CD34+HLA-DR+ cells from G0/G1 phases of cell cycle was investigated, it was found that in serum-free media supplemented with only SCF or IL-3, HUCB cells did not exist G0/G1 as rapidly as when HUCB plasma or SCF plus IL-3 was present. In contrast, when HUCB plasma was added to any cytokine combination, it did not enhance the exist of BM CD34+HLA-DR+ cells from G0/G1 phases of cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)