We have previously reported the ability of uncharacterized human bone marrow (BM) cells to engraft into preimmune fetal sheep, thereby creating sheep-human chimera suitable for in vivo examination of the properties of human hematopoietic stem cells (HSC). Adult human bone marrow CD34+ HLA-DR- cells have been extensively characterized in vitro and have been demonstrated to contain a number of primitive hematopoietic progenitor cells (PHPC). However, the capacity of such highly purified populations of human marrow CD34+ HLA-DR- cells to undergo in vivo self-renewal and multipotential lymphohematopoietic differentiation has not been previously demonstrated. To achieve that, human CD34+ HLA-DR- cells were transplanted in utero into immunoincompetent fetal sheep to investigate the BM-populating potential of these cells. Long-term chimerism, sustained human hematopoiesis, and expression of human cells belonging to all human blood cell lineages were demonstrated in two animals for more than 7 months' posttransplantation. Chimeric BM contained erythroid, granulocytic/monocytic, and megakaryocytic hematopoietic progenitor cells, as well as the primitive high proliferative potential colony- forming cell (HPP-CFC). Under a variety of in vitro experimental conditions, chimeric BM cells gave rise to human T cells expressing T- lymphocyte-specific markers, human natural killer (NK) cells, and human IgG-producing B cells. In vivo expansion and possibly self-renewal of transplanted PHPC was confirmed by the detection in chimeric BM 130 days' posttransplantation of CD34+ HLA-DR- cells, the phenotype of human cells constituting the stem-cell graft. These studies demonstrate not only the BM-populating capacity, multipotential differentiation, and most likely self-renewal capabilities of human CD34+ HLA-DR- cells, but also that this BM population contains human HSC. Furthermore, it appears that this animal model of xenogeneic stem-cell transplantation is extremely useful for in vivo examination of human hematopoiesis and the behavioral and functional characteristics of human HSC.

A mouse antihuman monoclonal IgG2a antibody, termed stem cell receptor- 1 (SR-1), specific for a determinant of the c-kit ligand receptor (KR), was used as an immunologic probe to analyze KR expression by human bone marrow hematopoietic progenitor cells. Monoclonal antibodies to CD34 and HLA-DR were used in a multicolor staining protocol in conjunction with SR-1 to further define the phenotypes of various classes of hematopoietic progenitor cells. Expression of KR (SR-1+) on hematopoietic progenitor cells identified subpopulations of cells expressing CD34 (CD34+). While one-half of the CD34- and HLA-DR- expressing cells (CD34+ HLA-DR+) expressed the KR (SR-1+), one-third of the CD34+ cells that lacked HLA-DR expression (CD34+ HLA-DR-) were SR- 1+. The CD34+ HLA-DR+ SR-1+ cell population contained the vast majority of the more differentiated progenitor cells, including the colony- forming unit (CFU) granulocyte-macrophage; burst-forming unit- erythrocyte; CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; and the CFU-megakaryocyte. The overall progenitor cell cloning efficiency of this subpopulation was greater than 31%. By contrast, the CD34+ HLA-DR- SR-1+ cell population contained fewer of these more differentiated progenitor cells but exclusively contained the more primitive progenitor cells, the BFU-megakaryocyte, high proliferative potential-colony-forming cell, and long-term bone marrow culture- initiating cell. The overall progenitor cell cloning efficiency of this subpopulation was greater than 7%. Both the CD34+ HLA-DR- and CD34+ HLA- DR+ cell subpopulations lacking KR expression contained few assayable hematopoietic progenitor cells. Long-term bone marrow cultures initiated with CD34+ HLA-DR- SR-1+ but not CD34+ HLA-DR- SR-1- cells, which were repeatedly supplemented with c-kit ligand (KL) and interleukin-3, generated assayable progenitor cells of at least 2 lineages for 10 weeks. These experiments demonstrate the expression of the KR throughout the hierarchy of human hematopoietic progenitor cell development. We conclude from our data that the KL and KR play a pivotal role in cytokine regulation of both the primitive and more differentiated human hematopoietic progenitor cells.

To test the hypothesis that the c-kit ligand plays an important role in the regulation of early events occurring during human hematopoiesis, we determined the effect of a recombinant form of c-kit ligand, termed mast cell growth factor (MGF), on the high-proliferative potential colony-forming cell (HPP-CFC) and the cell responsible for initiating long-term hematopoiesis in vitro (LTBMIC). MGF alone did not promote HPP-CFC colony formation by CD34+ DR- CD15- marrow cells, but synergistically augmented the ability of a combination of granulocyte- monocyte colony-stimulating factor (GM-CSF) interleukin (IL)-3 and a recombinant GM-CSF/IL-3 fusion protein (FP) to promote the formation of HPP-CFC-derived colonies. MGF had a similarly profound effect on in vitro long-term hematopoiesis. Repeated additions of IL-3, GM-CSF, or FP alone to CD34+ DR- CD15- marrow cells in a stromal cell-free culture system increased cell numbers 10(3)-fold by day 56 of long-term bone marrow culture (LTBMC), while combinations of MGF with IL-3 or FP yielded 10(4)- and 10(5)-fold expansion of cell numbers. Expansion of the number of assayable colony-forming unit-granulocyte-monocyte (CFU- GM) generated during LTBMC was also markedly enhanced when MGF was added in combination with IL-3 or FP. In addition, MGF, IL-3, and FP individually led to a twofold to threefold increase in HPP-CFC numbers after 14 to 21 days of LTBMC. Furthermore, the effects of these cytokines on HPP-CFC expansion during LTBMC were additive. Throughout the LTBMC, cells receiving MGF possessed a higher cloning efficiency than those receiving IL-3, GM-CSF, or FP alone. These data indicate that the c-kit ligand synergistically interacts with a number of cytokines to directly augment the proliferative capacity of primitive human hematopoietic progenitor cells.

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 marrowhomed 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.

Summary: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/m 2 ) and G-CSF (10 g/kg/day). A median of two leukaphereses and one selection was performed per patient. The median number of mononuclear cells processed was 3.50 × 10 10 with a recovery of 1.11 × 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/m 2 of melphalan followed by the infusion of a median of 2.91 × 10 6 /kg CD34 ؉ cells (1.00-16.30). The median time to absolute neutrophil count Ͼ0.5 × 10 9 /l was 11 days, and sustained platelet recovery of Ͼ20 × 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. Keywords: CD34 selection; multiple myeloma; immunomagnetic separation; transplantation The use of standard-dose chemotherapy in the management of multiple myeloma yields unsatisfactory outcomes in terms of both complete response rate and overall survival. In contrast, high-dose chemotherapy and combination chemo-radiotherapy have significantly improved both the disease-free and overall survival rates of patients suffering from multiple myeloma. [1][2][3] This improvement has been seen in both refractory and newly diagnosed patients treated with high-dose chemotherapy and autologous peripheral blood stem cell support. [4][5][6][7] Nonetheless, the high rate of relapse in the setting of autologous transplantation continues to be a major problem. It has been speculated that some of these relapses, particularly early ones, result from the transplantation of clonogenic myeloma cells. 8,9 Indeed, several investigators have shown that peripheral blood progenitor cell (PBPC) products contain significant numbers of circulating clonogenic myeloma cells. [10][11][12][13] In order to reduce the risk of relapse due to the transfer of myeloma cells, several techniques that remove these cells from the transplanted products are being investigated. 14-17 One such technique is the positive selection of CD34 + cells, shown to contain the necessary precursors for both short-and long-term hematopoietic reconstitution. Since it is believed that the CD34 antigen is not expressed on clonal myeloma cells, selection of CD34 + cells from autologous mobilized peripheral blood progenitor cells may result in the reduction of myeloma contamination of these cells by several orders of magnitude. 18,19 We report here ...