Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver

Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.     

In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential

Human hematopoietic stem cells are pluripotent, ie, capable of producing both lymphoid and myeloid progeny, and are therefore used for transplantation and gene therapy. An in vitro culture system was developed to study the multi-lineage developmental potential of a candidate human hematopoietic stem cell population, CD34+CD38- cells. CD34+CD38- cells cocultivated on the murine stromal line S17 generated predominantly CD19(+) B-cell progenitors. Transfer of cells from S17 stroma to myeloid-specific conditions ("switch culture") showed that a fraction of the immunophenotypically uncommitted CD19- cells generated on S17 stroma had myeloid potential (defined by expression of CD33 and generation of colony-forming unit-cells). Using the switch culture system, single CD34+CD38- cells were assessed for their lymphoid and myeloid potential. Nineteen of 50 (38%) clones generated from single CD34+CD38- cells possessed both B-lymphoid and myeloid potential. 94.7% of the CD34+CD38- cells with lympho-myeloid potential were late-proliferating (clonal appearance after 30 days), demonstrating that pluripotentiality is detected significantly more often in quiescent progenitors than in cytokine-responsive cells (P = .00002). The S17/switch culture system permits the in vitro assessment of the pluripotentiality of single human hematopoietic cells.     

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.     

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.     

CD164, a novel sialomucin on CD34(+) and erythroid subsets, is located on human chromosome 6q21

CD164 is a novel 80- to 90-kD mucin-like molecule expressed by human CD34(+) hematopoietic progenitor cells. Our previous results suggest that this receptor may play a key role in hematopoiesis by facilitating the adhesion of CD34(+) cells to bone marrow stroma and by negatively regulating CD34(+) hematopoietic progenitor cell growth. These functional effects are mediated by at least two spatially distinct epitopes, defined by the monoclonal antibodies (MoAbs), 103B2/9E10 and 105A5. In this report, we show that these MoAbs, together with two other CD164 MoAbs, N6B6 and 67D2, show distinct patterns of reactivity when analyzed on hematopoietic cells from normal human bone marrow, umbilical cord blood, and peripheral blood. Flow cytometric analyses revealed that, on average, 63% to 82% of human bone marrow and 55% to 93% of cord blood CD34(+) cells are CD164(+), with expression of the 105A5 epitope being more variable than that of the other identified epitopes. Extensive multiparameter flow cytometric analyses were performed on cells expressing the 103B2/9E10 functional epitope. These analyses showed that the majority (>90%) of CD34(+) human bone marrow and cord blood cells that were CD38(lo/-) or that coexpressed AC133, CD90(Thy-1), CD117(c-kit), or CD135(FLT-3) were CD164(103B2/9E10)+. This CD164 epitope was generally detected on a significant proportion of CD34(+)CD71(lo/-) or CD34(+)CD33(lo/-) cells. In accord with our previous in vitro progenitor assay data, these phenotypes suggest that the CD164(103B2/9E10) epitope is expressed by a very primitive hematopoietic progenitor cell subset. It is of particular interest to note that the CD34(+)CD164(103B2/9E10)lo/- cells in bone marrow are mainly CD19(+) B-cell precursors, with the CD164(103B2/9E10) epitope subsequently appearing on CD34(lo/-)CD19(+) and CD34(lo/-)CD20(+) B cells in bone marrow, but being virtually absent from B cells in the peripheral blood. Further analyses of the CD34(lo/-)CD164(103B2/9E10)+ subsets indicated that one of the most prominent populations consists of maturing erythroid cells. The expression of the CD164(103B2/9E10) epitope precedes the appearance of the glycophorin C, glycophorin A, and band III erythroid lineage markers but is lost on terminal differentiation of the erythroid cells. Expression of this CD164(103B2/9E10) epitope is also found on developing myelomonocytic cells in bone marrow, being downregulated on mature neutrophils but maintained on monocytes in the peripheral blood. We have extended these studies further by identifying Pl artificial chromosome (PAC) clones containing the CD164 gene and have used these to localize the CD164 gene specifically to human chromosome 6q21.     

Transplantation of human umbilical cord blood cells in macrophage-depleted SCID mice: evidence for accessory cell involvement in expansion of immature CD34+CD38- cells

In vivo expansion and multilineage outgrowth of human immature hematopoietic cell subsets from umbilical cord blood (UCB) were studied by transplantation into hereditary immunodeficient (SCID) mice. The mice were preconditioned with Cl2MDP-liposomes to deplete macrophages and 3.5 Gy total body irradiation (TBI). As measured by immunophenotyping, this procedure resulted in high levels of human CD45(+) cells in SCID mouse bone marrow (BM) 5 weeks after transplantation, similar to the levels of human cells observed in NOD/SCID mice preconditioned with TBI. Grafts containing approximately 10(7) unfractionated cells, approximately 10(5) purified CD34+ cells, or 5 x 10(3) purified CD34+CD38- cells yielded equivalent numbers of human CD45+ cells in the SCID mouse BM, which contained human CD34+ cells, monocytes, granulocytes, erythroid cells, and B lymphocytes at different stages of maturation. Low numbers of human GpA+ erythroid cells and CD41+ platelets were observed in the peripheral blood of engrafted mice. CD34+CD38+ cells (5 x 10(4)/mouse) failed to engraft, whereas CD34- cells (10(7)/mouse) displayed only low levels of chimerism, mainly due to mature T lymphocytes. Transplantation of graded numbers of UCB cells resulted in a proportional increase of the percentages of CD45+ and CD34+ cells produced in SCID mouse BM. In contrast, the number of immature, CD34+CD38- cells produced in vivo showed a second-order relation to CD34+ graft size, and mice engrafted with purified CD34+CD38- grafts produced 10-fold fewer CD34+ cells without detectable CD34+CD38- cells than mice transplanted with equivalent numbers of unfractionated or purified CD34+ cells. These results indicate that SCID repopulating CD34+CD38- cells require CD34+CD38+ accessory cell support for survival and expansion of immature cells, but not for production of mature multilineage progeny in SCID mouse BM. These accessory cells are present in the purified, nonrepopulating CD34+CD38+ subset as was directly proven by the ability of this fraction to restore the maintenance and expansion of immature CD34+CD38- cells in vivo when cotransplanted with purified CD34+CD38- grafts. The possibility to distinguish between maintenance and outgrowth of immature repopulating cells in SCID mice will facilitate further studies on the regulatory functions of accessory cells, growth factors, and other stimuli. Such information will be essential to design efficient stem cell expansion procedures for clinical use.     

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34-, CD10+, CD19+, and cytoplasmic mu chain (C mu)-negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 10(3) CD34+ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a C mu phenotype, and a small number of cells had immunoglobulin mu chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.     

Fluorescence in situ hybridization of progenitor cells obtained by fluorescence-activated cell sorting for the detection of cells affected by chromosome abnormality trisomy 8 in patients with myelodysplastic syndromes

Myelodysplastic syndrome (MDS) is believed to be a stem-cell disorder involving cytopenia and dysplastic changes in three hematopoietic lineages. However, the involvement of pluripotent stem cells and progenitor cells has not been clarified conclusively. To address this issue, we used fluorescence in situ hybridization (FISH) of blood and bone marrow (BM) smears for mature cells and FISH of cells sorted by fluorescence-activated cell sorting for progenitor cells. Seven patients with MDS associated with trisomy 8 were studied. FISH showed +8 in granulocytes, monocytes, and erythroblasts, but not in lymphocytes. Sorted cells of T (CD3(+)), B (CD19(+)), and NK cells (CD3(-)CD56(+)) from peripheral blood did not contain +8, nor did CD34(+) subpopulations from BM including B (CD34(+)CD19(+)), T/NK (CD34(+)CD7(+)) progenitors, and pluripotent stem cells (CD34(+)Thy1(+)). The +8 chromosome abnormality was identified in stem cells only at the level of colony-forming unit of granulocyte-erythrocyte-macrophage-megakaryocyte (CFU-GEMM; CD34(+)CD33(+)). It may thus be concluded that cells affected by trisomy 8 in the context of MDS are at the CFU-GEMM level and that cells of lymphoid lineage are not involved. These results provide new insights into the biology of MDS and suggest that intensive chemotherapy and autologous BM transplantation may become important therapeutic strategies.     

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.     

BCR/ABL- CD34(+)HLA-DR- progenitor cells in early chronic phase, but not in more advanced phases, of chronic myelogenous leukemia are polyclonal

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph-) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph- progenitors are highly enriched in the CD34(+)HLA-DR- fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph- progenitor or stem cells, leaving the unanswered question whether Ph- CD34(+)HLA-DR- progenitors are normal. To show the clonal nature of Ph- CD34(+)HLA-DR- CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34(+)HLA-DR+ and CD34(+)HLA-DR- progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA-, CD34(+)HLA-DR- progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34(+)HLA-DR- derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph-, BCR/ABL mRNA- and polyclonal state in the CD34(+)HLA-DR- and CD34(+)HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34(+) cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph-, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34(+)HLA-DR- cells that are Ph- and BCR/ABL mRNA- are polyclonal and therefore benign. This population is suitable for autografting in CML.     

CD38 ligation in human B cell progenitors triggers tyrosine phosphorylation of CD19 and association of CD19 with lyn and phosphatidylinositol 3-kinase

CD38 is a 45-kDa transmembrane glycoprotein highly expressed in lymphoid progenitors. Ligation of CD38 with specific Abs inhibits growth and induces apoptosis in human immature B cells. CD38 ligation also triggers tyrosine phosphorylation of syk, c-cbl, and phospholipase C-gamma and activates phosphatidylinositol 3-kinase (PI3-K). In the present study, we investigated whether the cell surface membrane molecules used in B cell receptor-mediated signaling, such as Ig alpha, Ig beta, and CD19, could be involved in the CD38-mediated signaling cascade. In the B cell receptor-negative immature B cell lines RS4;11, 380, and REH, Ig alpha and Ig beta were expressed exclusively in the cytoplasm and were not tyrosine phosphorylated after CD38 ligation. By contrast, CD19 was markedly tyrosine phosphorylated and was associated with lyn and PI3-K. PI3-K activation appears to be directly linked to the growth-arresting effects of CD38 ligation, which are reduced by PI3-K inhibitors. Ligation of either CD38 or CD19 resulted in a similar pattern of protein tyrosine phosphorylation; both signaling pathways caused tyrosine phosphorylation of c-cbl. Levels of CD38 surface expression were not affected by prolonged incubation with anti-CD19 Ab, while CD19 expression markedly decreased. These results indicate that CD19 is a major component of the CD38 signaling cascade in B cell precursors, serving as a cell surface membrane docking site for cytoplasmic kinases. CD38 and CD19 are not physically linked, but activate an overlapping set of kinases in human immature B cells.     

Four-color flow cytometric investigation of terminal deoxynucleotidyl transferase-positive lymphoid precursors in pediatric bone marrow: CD79a expression precedes CD19 in early B-cell ontogeny

Terminal deoxynucleotidyl transferase (TdT)-positive cells in human bone marrow (BM) are a phenotypically inhomogeneous population of precursor cells. In their majority, these TdT+ cells are unambiguously committed to the B lineage, as evidenced by CD19 expression. However, TdT+ precursors that lack CD19 also exist and these may encompass a differentiation potential for the B as well as for other lineages. Because recent data suggested that CD19 expression is not the earliest differentiation event in B-cell ontogeny, we sought to reevaluate TdT+ lymphoid precursors in pediatric BM to define the phenotypic denominator of B-lineage affiliation upstream of CD19. Using four-color flow cytometry, we focused on the assessment of the CD79a antigen, which is highly B-cell specific and which may also be expressed very early in B-cell ontogeny. We found that a majority of TdT+ cells coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in all investigated samples, some TdT+ precursors lacked CD19 but expressed CD79a, which suggestively indicates also their B-lineage affiliation. In contrast to the CD19(+) precursors, which were usually CD10(hi) and CD79b+, these CD19(-)CD79a+ putative B-cell precursors preferentially expressed CD10 at low levels and were CD79b+ in only 41%. About 17% of these TdT+CD19(-)CD79a+ precursors also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or cytoplasmic CD3, which is discussed in the light of cellular activation rather than lineage promiscuity. Our data confirm that the earliest differentiation stages of B cells can be dissected upon expression of the lineage antigens CD79a and CD19 and imply that CD79a is earlier expressed than CD19. This raises the chance to follow the sequential events heralding B-cell commitment in the most immature precursors by correlating phenotypic and genetic differentiation markers.     

Flt3high and Flt3low CD34+ progenitor cells isolated from human bone marrow are functionally distinct

We generated monoclonal antibodies against the human Flt3 receptor and used them to study the characteristics of normal human bone marrow cells resolved based on Flt3 expression. Human CD34+ or CD34+lin- marrow cells were sorted into two populations: cells expressing high levels of Flt3 receptor (Flt3high) and cells with little or no expression of Flt3 receptor (Flt3low). Flt3 receptor was detected on a subset of CD34+CD38- marrow cells, as well as on CD34+CD19+ B lymphoid progenitors and CD34+CD14+CD64+ monocytic precursors. Flt3 receptor was also present on more mature CD34-CD14+ monocytes. In colony-forming assays, Flt3high cells gave rise mainly to colony-forming unit-granulocyte-macrophage (CFU-GM) colonies, whereas Flt3low cells produced mostly burst-forming unit-erythroid colonies. There was no difference in the number of multilineage CFU-Mix colonies between the two cell fractions. Cell cycle analysis showed that a large number of the Flt3low cells were in the G0 phase of the cell cycle, whereas Flt3high cells were predominantly in G1. Cell numbers in the suspension cultures initiated with Flt3high cells were maintained in the presence of Flt3 ligand (FL) alone, and increased in response to FL plus kit ligand (KL). In contrast, cell numbers in the suspension cultures started with Flt3low cells did not increase in the presence of FL, or FL plus KL. Upregulation of Flt3 receptor on Flt3low cells was not detected during suspension culture. CD14+ monocytes were the major cell type generated from CD34+lin-Flt3high cells in liquid suspension culture, whereas cells generated from CD34+lin-Flt3low cells were mainly CD71+GlycA+ erythroid cells. These results show clear functional differences between CD34+Flt3high and CD34+Flt3low cells and may have implications concerning the in vitro expansion of human hematopoietic progenitor cells.     

Nontransformed colony-derived stromal cell lines from normal human marrows. III. The maintenance of hematopoiesis from CD34+ cell populations

Nontransformed stromal colony-derived cell lines (CDCLs) consist of a pure stromal cell population that differentiates following a vascular smooth-muscle cell repertoire. Here we study the maintenance of hematopoiesis by this cell population. We show that CDCLs allow the generation for several weeks of stroma-adherent colonies (comprising a cobblestone area) from CD34+, CD34+/CD38+, and CD34+/CD38- cells. Stroma-adherent colony-forming cells (CFCs) from CD34+/CD38- cells reach a maximum at week 4 and limiting dilution analysis gives a frequency of 1 per 10 cells seeded; in contrast to this, CFCs from CD34+/CD38+ cells are optimal by week 2 and the frequency is then only 1 per 120 cells seeded. Stroma-adherent colonies comprise hematopoietic cells from all lineages except the T lymphocytic, with a majority of granulomonocytes. CDCLs also allow the amplification of granulomonocytic colony-forming units (CFU-GMs), since cumulative outputs of CFU-GMs by week 6 are 190 and 8 times that observed at culture inception for the CD34+/CD38- and CD34+/CD38+ cell populations, respectively. Our results suggest that stromal cells from CDCLs allow the maintenance of primitive hematopoietic precursors and induce their proliferation and differentiation. This study underscores the potential role of one of the microenvironmental cell populations, that of myoid cells, in the regulation of hematopoietic precursor behavior.     

The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis

Mucin-like molecules represent an emerging family of cell surface glycoproteins expressed by cells of the hematopoietic system. We report the isolation of a cDNA clone that encodes a novel transmembrane isoform of the mucin-like glycoprotein MGC-24, expressed by both hematopoietic progenitor cells and elements of the bone marrow (BM) stroma. This molecule was clustered as CD164 at the recent workshop on human leukocyte differentiation antigens. CD164 was identified using a retroviral expression cloning strategy and two novel monoclonal antibody (MoAb) reagents, 103B2/9E10 and 105.A5. Both antibodies detected CD164/MGC-24v protein expression by BM stroma and subpopulations of the CD34(+) cells, which include the majority of clonogenic myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (blast-forming unit-erythroid [BFU-E]) progenitors and the hierarchically more primitive precursors (pre-CFU). Biochemical and functional characterization of CD164 showed that this protein represents a homodimeric molecule of approximately 160 kD. Functional studies demonstrate a role for CD164 in the adhesion of hematopoietic progenitor cells to BM stromal cells in vitro. Moreover, antibody ligation of CD164 on primitive hematopoietic progenitor cells characterized by the cell surface phenotype CD34(BRIGHT)CD38(-) results in the decreased recruitment of these cells into cell cycle, suggesting that CD164 represents a potent signaling molecule with the capacity to suppress hematopoietic cell proliferation.     

Effects of prior therapy on the in vitro proliferative potential of stem cell factor plus filgrastim-mobilized CD34-positive progenitor cells

The quantity of hematopoietic progenitors in an apheresis collection is defined by the number of CD34(+) cells or granulocyte macrophage colony-forming units present. These parameters are believed to give roughly equivalent information on graft quality. We here report that the in vitro proliferative potential of r-metHuSCF (stem cell factor) plus filgrastim (granulocyte colony-stimulating factor; r-metHuG-CSF) mobilized peripheral blood (PB) CD34(+) cells obtained from previously heavily treated non-Hodgkin's lymphoma patients inversely correlates with extent of prior therapy. CD34(+) cells were enriched using the CellPro Ceprate system and placed in liquid culture for 4 weeks in the presence of either r-metHuSCF, IL-3, IL-6, filgrastim (S36G), or S36G plus erythropoietin (S36GE) with a weekly exchange of media and cytokines with reestablishment of culture at the starting cell concentration (Delta assay) and enumeration of progenitors. Starting with 4 x 10(4) CD34(+) cells from apheresis samples from patients who had received <10 cycles of prior chemotherapy, progenitors were detectable in culture at 4 weeks 81% of the time as compared to 14% with CD34(+) cells from patients who had received >10 cycles and 5% for >10 cycles plus radiotherapy. The total number of progenitors generated over the duration of culture (area under the curve) was calculated using the trapezoidal rule as a novel measure of the proliferative potential of the enriched PB CD34(+) cell population. The median area under the curve of CD34(+) cells from patients receiving <10 cycles of prior chemotherapy was 7.4 and 5.7 (x10(5)) using S36G or S36GE, respectively, 1.8 and 1.9 if the patients received >10 cycles of prior chemotherapy, and 1.4 and 1.2 if the patients received >10 cycles of prior chemotherapy plus radiotherapy (P < 0.001). These data show that prior therapy impacts on the quality of PB CD34(+) cells as measured by their ability to generate committed progenitors over a number of weeks in liquid culture.