Biologic and phenotypic analysis of early hematopoietic progenitor cells in umbilical cord blood

Umbilical cord blood (UCB) is an attractive potential alternative to bone marrow (BM) as a source of hematopoietic progenitor cells since the number of progenitors in UCB is similar or even greater than that in normal BM. It was the aim of the present study to analyze the degree of immaturity of UCB progenitor cells. UCB mononuclear (MNC) and/or CD34+ cells were tested for surface antigen phenotype, expression of cytokines receptor, effect of stem cell factor (SCF) on colony growth, resistance to mafosfamide and replating potential. We have found that 34.9 +/- 3.4% and 77.9 +/- 2.6% of UCB CD34+ cells did not express CD38 and CD45RA antigens, respectively, suggesting that UCB contains a high proportion of immature progenitor cells. By means of three-color analysis, the receptor for SCF was detected on the majority of the CD34+ HLA-DR+ subpopulation; in fact, 81.8% +/- 4.3% of CD34+ HLA-DR+ cells were defined as SCF(low) and 8.1 +/- 1.5% as SCF(high). Colony growth of MNC and CD34+ cells was enhanced by the addition of SCF to methylcellulose mixture, resulting in a statistically significant increase in CFU-GM and CFU-GEMM but not in BFU-E numbers. UCB progenitor cells showed a higher resistance to mafosfamide treatment, in comparison to BM; the addition of SCF to the culture medium resulted in a statistically significant increase in mafosfamide concentration required to inhibit 95% of colony growth (P < or = 0.05). Moreover, as shown by single colony transfer assays, the presence of SCF in primary cultures promoted a significantly higher replating potential for both untreated (42 +/- 3.3% vs 21 +/- 4.6%, P < or = 0.018) and mafosfamide-treated samples (62 +/- 5.6% vs 44 +/- 6.1%, P < or = 0.018). In conclusion, UCB is a source of progenitor cells with immature characteristics in terms of surface antigen expression, distribution of SCF receptor, resistance to mafosfamide and replating potential. Therefore, UCB progenitor cells represent an ideal candidate population for experimental programs involving gene transfer and ex vivo stem cell expansion.     

Analysis of myeloid and lymphoid markers on the surface and in the cytoplasm of mononuclear bone marrow cells in patients with myelodysplastic syndrome

Mononuclear cells of the bone marrow (BM) of patients in various subgroups of the myelodysplastic syndrome (MDS) were studied by flow cytometry for the expression of myeloid and lymphoid markers both on the surface and in the cytoplasm. A significantly higher percentage of the BM cells of MDS patients reacted with monoclonal antibodies (mAbs) to myeloid antigens (CD13, CD15 and CD33) by cytoplasmic staining as compared with cell surface staining. The percentage of BM cells expressing CD34 was markedly elevated in patients with RAEB-T. A distinct finding in MDS patients was the expression of myeloid antigens on mononuclear BM cells. The proportion of individuals whose mononuclear BM cells were positive for surface reactivity with anti-CD13 and anti-CD33 mAbs was highest among RAEB-T patients while none of the patients with RA expressed these surface antigens. Cytoplasmic staining significantly increased the percentage of CD13+ and CD33+ BM cells among RAEB and RAEB-T patients. The proportion of individuals whose BM cells possessed myeloid antigens was increased by cytoplasmic staining in all subgroups of MDS. The BM of a considerable proportion of RAEB-T and RAEB patients showed cells which coexpressed the CD7 and CD3 lymphoid markers along with the CD13 and CD33 myeloid antigens. The present study indicates the importance of comparative surface and cytoplasmic immunophenotyping with CD13 and CD33 mAbs for the diagnosis of subgroups of MDS. The coexpression of CD3 and CD7 with markers of the myeloid lineage may reflect derangement of the differentiation of pluripotent stem cells characteristic for MDS.     

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.     

A child case of CD34+, CD33-, HLA-DR-, CD7+, CD56+ stem cell leukemia with thymic involvement

It has been reported that the CD56+/CD7+/CD3- phenotype of natural killer (NK) cells develop from the CD34+/HLA-DR- bone marrow (BM) mononuclear cell population in long-term BM culture (LTBMC). An HLA-DR-/CD33+/CD56+/CD16- myeloid/natural killer cell acute leukemia has been described. We report here a 7-year-old boy who developed stem cell acute leukemia with superior vena cava syndrome secondary to thymic involvement. Surface marker analyses revealed that the leukemia cells showed CD34+/HLA-DR-/CD33-/CD7+/CD56+ phenotype. When stimulated with phorbol ester in vitro the leukemic cells morphologically differentiated to myeloid cells developing CD13, CD15 and CD56 antigens. Our results suggest that CD34+/HLA-DR-/CD7+/CD56+ stem cell leukemia may arise from transformation of a pluripotent precursor cell, which could differentiate to both myeloid and NK cell lineages.     

High-level expression of a novel epitope of CD59 identifies a subset of CD34+ bone marrow cells highly enriched for pluripotent stem cells

To further define the hierarchy of human hematopoietic progenitor cells, we have attempted to identify antibodies to cell-surface molecules expressed on CD34+ progenitor cell subsets. Herein we describe the utility of a new monoclonal antibody, HCC-1, which binds to a novel epitope of CD59 differentially expressed among CD34+ progenitor cells. HCC-1 subdivides the adult marrow CD34+ population into HCC-1high and HCC-1low/- fractions of approximately equal size. Cobblestone area-forming cells (CAFC) in long-term bone marrow culture were enriched 10-30-fold in CD34+HCC-1high cells compared with CD34+HCC1-low/- cells and two-fold compared with CD34+ cells. When injected into fetal human bone fragments implanted in SCID mice, the CD34+HCC-1high population showed potent engrafting activity leading to the production of myeloid, lymphoid, and erythroid elements, as well as the retention of progenitor cell phenotype. These studies demonstrate that the CD34+HCC-1high population contains primitive pluripotent hematopoietic stem cells. No hematopoietic engrafting activity was detected in the CD34+HCC-1low/- population. Consistent with this finding, simultaneous five-color flow cytometric analysis revealed that HCC-1high cells include virtually all CD34+Thy-1+Lin- cells, a cell population previously characterized as highly enriched for primitive pluripotent hematopoietic stem cells. The ability of CD34+ cells divided into subsets by HCC-1 to produce T cells was assessed by transplantation of sorted cells into human fetal thymus implanted into SCID mice. A higher frequency of thymus-engrafting activity was observed in the CD34+HCC-1high than in the CD34+HCC-1low/- population. Consistent with the limited ability to engraft in the SCID-hu thymus model, the CD34+HCC-1low/- population was shown to contain a low frequency of CD34+CD10+ lymphoid progenitor cells. We conclude that the HCC-1 epitope is expressed at high levels on a subset of CD34+ cells that contain virtually all primitive pluripotent hematopoietic stem cells and that the population of CD59 molecules expressed on CD34+ cells is not homogeneous.     

Colony formation of human fetal CD34+ hematopoietic cells

Manipulations to enhance engraftment of donated cells may be advantageous in transplantation of fetal hematopoietic cells (FHC). By assessing the formation of colonies, CD34+ enrichment was evaluated with and without cytokine stimulation (interleukins 3 and 6, stem cell factor, granulocyte-macrophage colony-stimulating factor). Cord blood cells and bone marrow cells served as controls. In FHC, cytokine stimulation and CD34+ enrichment always enhanced the formation of CFU-GM (colony-forming units--granulocytes, macrophages) and CFU-GEMM (colony-forming units-granulocytes, erythroid cells, macrophages, megakaryocytes). However, BFU-E (burst-forming units--erythroid cells) in FHC remained unchanged after cytokine stimulation and CD34+ enrichment. In FHC, the addition of cytokines and the enrichment of CD34+ cells usually contributed equally to enhance CFU-GM and CFU-GEMM colony formation. CD34-negative FHC produced the same number or more BFU-E and half the number of CFU-GM and CFU-GEMM as compared with crude cells. This CD34-negative cell population also responded to cytokine stimulation. Such findings may indicate that purification of CD34+ cells is not meaningful in fetal transplantation.     

Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium

Hematopoietic stem cells are capable of extensive self-renewal and expansion, particularly during embryonic growth. Although the molecular mechanisms involved with stem cell maintenance remain mysterious, it is now clear that an intraembryonic location, the aorta-gonad-mesonephros (AGM) region, is a site of residence and, potentially, amplification of the definitive hematopoietic stem cells that eventually seed the fetal liver and adult bone marrow. Because several studies suggested that morphologically defined hematopoietic stem/progenitor cells in the AGM region appeared to be attached in clusters to the ventrally located endothelium of the dorsal aorta, we derived cell lines from this intraembryonic site using an anti-CD34 antibody to select endothelial cells. Analysis of two different AGM-derived CD34(+) cell lines revealed that one, DAS 104-8, efficiently induced fetal-liver hematopoietic stem cells to differentiate down erythroid, myeloid, and B-lymphoid pathways, but it did not mediate self-renewal of these pluripotent cells. In contrast, a second cell line, DAS 104-4, was relatively inefficient at the induction of hematopoietic differentiation. Instead, this line provoked the expansion of early hematopoietic progenitor cells of the lin-CD34(+)Sca-1(+)c-Kit+ phenotype and was proficient at maintaining fetal liver-derived hematopoietic stem cells able to competitively repopulate the bone marrow of lethally irradiated mice. These data bolster the hypothesis that the endothelium of the AGM region acts to mediate the support and differentiation of hematopoietic stem cells in vivo.     

Culture system for extensive production of CD19+IgM+ cells by human cord blood CD34+ progenitors

We established a co-culture system with a monolayer of the murine bone marrow (BM) stroma cell line, MS-5, in which human cord blood CD34+ cells differentiated to CD19+ cells. The addition of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) highly enhanced the production of CD19+ cells. The expansion of the cell numbers was over 10(3)-fold. Furthermore, a significant proportion (<45%) of the cells expressed surface IgM (sIgM) after 5 weeks of co-culture. CD34+CD19- cells also showed a similar development of CD19+ cells and CD19+sigM+ cells. Filter separation of MS-5 cells and CD34+ cells did not inhibit the growth of CD19+ cells. However, when further purified CD34+CD19-CD13- CD33- cells were cultured in the presence of MS-5 cells with or without a separation filter, CD19+ cells did not appear in the non-contact setting. This result suggested that the highly purified CD34+CD19-CD13-CD33- progenitors require the cell-cell contact for the development of CD19+ cells, whereas other CD34+ fractions contain progenitors that do not require the contact. This co-culture system should be useful for the study of early human B-lymphopoiesis.     

Fas antigen (CD95) expression in peripheral blood progenitor cells from patients with leukaemia and lymphoma

Fas antigen (CD95) is a cell surface receptor belonging to the tumour necrosis factor/nerve growth factor superfamily and is able to induce apoptosis when triggered by its' natural ligand or an anti-Fas antibody. Fas expression is low on CD34+ bone marrow (BM) progenitor cells, but is increased by various cytokines in vitro. We investigated Fas expression on CD34+ cells from 39 peripheral blood progenitor cell (PBPC) harvests and from 5 normal BM harvests by dual colour flow cytometry to determine if Fas expression was altered during mobilisation. By including calibrated microbeads during flow cytometry, we quantified the number of Fas antigen molecules per cell. A low percentage of PBPC (22%) and normal BM (23%) CD34+ cells expressed Fas antigen. Fas expression varied on CD34+ cells from different diseases and the highest expression was found in ALL (52%). There was a significant three fold increase in the number of Fas molecules/cell expressed on CD34+ cells (PBPC 6,230 molecules/cell, BM 2,236; p = 0.0003). This level of expression was considerably less than that for CD3/CD19 lymphocytes (33,095 molecules/cell) and CD14 monocytes (47,467 molecules/cell) in the PBPC harvest. In conclusion, mobilisation including the use of growth of factors, has minimal effect on CD34 progenitor cell Fas expression.     

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.     

Proliferation and differentiation of myelodysplastic CD34+ cells

Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders of hematopoiesis involving hyperproliferative and ineffective hematopoiesis associated with morphologic evidence of marrow cell dysplasia resulting in refractory cytopenia(s), and an increased risk of transformation into acute myeloblastic leukemia (AML). The administration of colony-stimulating factor(s) (CSFs) to patients with MDS increased blood neutrophil concentrations, in most patients, and was also expected to be beneficial and to prevent infections. However, the progression to AML during the treatment with CSFs was suspected in some patients. Therefore, extensive in vitro studies were expected to lead to the establishment of criteria for selection of patients who are likely to benefit from CSF's as well as to establish the overall value of the different types of CSFs therapy. For this purpose, in vitro colony assays provide an excellent tool for investigating the biologic characteristics of MDS progenitor cells. However, conditions of the culture must be such that each progenitor can express its full potential for proliferation and differentiation. Because of the above, MDS progenitor cells cannot be used because they carry an impairment in proliferation and differentiation. To address this problem, one needs to know how many cells are being handled and the maximum numbers of colonies and clusters expected. CD34, a stem cell phenotype, is at present one of the best markers of progenitor cells, and can be used for purposes of purification. Using a defined number of CD34+ cells, it was feasible to make direct investigations on MDS progenitor cells. In this review the properties of MDS progenitor cells are described, in association with proliferation and differentiation, with special emphasis on the phenotypic subpopulations of MDS CD34+ cells.     

Population dynamics of the monogenean Anoplodiscus cirrusspiralis on the snapper, Pagrus auratus

Mobilized peripheral blood progenitor cells (PBPC) have been shown to differ qualitatively from bone marrow (BM) progenitors. The released progenitor cells are predominantly in G0/G1 and show a relatively high percentage of rhodamine dull cells. Within the BM these last two features are characteristic of the more primitive progenitors. Although the mobilized PB cells can give rise to long-term repopulation and thus contain stem cells, the frequency of stem cells is not much higher if long-term initiating cell (LTC-IC) assays are used. To determine whether quiescent stem cells are selectively released or the low-cycle status of PB progenitors is related to the release from the BM microenvironment, the cell cycle status and rhodamine content in the PB and BM during mobilization were studied and compared with steady-state BM. More differentiated and more primitive progenitors were separated based on differentiation markers and cloned in single cell assay. In mobilized PB 54% of the CD34+ cells (n=5) were rhodamine dull compared to 22% in steady-state BM (P=0.014) [n=6]. The percentage of CD34+ cells in the S/G2M phases of the cell cycle was 2.1% in the mobilized PB (n=11), and 18% in steady-state BM (n=11) [P=0.002]. During mobilization the fraction of cells in the S/G2M phase of the cell cycle was 16% in BM (n=7), similar to steady-state BM (P=0.34). The released progenitors represented a selection of BM progenitors, with significantly more primitive progenitors (CD34+/13+/33dim) and less lymphoid precursors (CD34+/19+). Within the more differentiated CD34+113+/33bright, myelomonocytic precursors, both in PB as well as in BM, the percentage S/G2M was relatively higher than in the CD34+/13+/33dim subfraction: in normal BM: median 18% vs 8% (P=0.006) [n=8]; in mobilized PB 3% vs 2% (P=0.03) [n=10]; and in BM during mobilization 24% vs 7% (P=0.01) [n=6]. The cycle status of mobilized PB progenitors was low both in the primitive and more differentiated subfractions. During the mobilization period the BM progenitors are cycling as in steady-state BM. The low-cycle status of the mobilized PB progenitors may be related to the loss of contact with the micro-environment.     

High-dose therapy with peripheral blood stem cell transplantation results in a significant reduction of the haemopoietic progenitor cell compartment

In order to study the effect of high-dose therapy with peripheral blood stem cell transplantation (PBSCT) on the haemopoietic reserve in man, the number and composition of bone marrow (BM) and peripheral blood (PB)-derived progenitor cells were examined in 137 cancer patients. In 45 patients, paired samples from BM and PB were obtained before PBSC mobilization and 6-27 months after transplantation. Following PBSCT. the proportion of CD34+ cells was significantly smaller than before mobilization (BM 1.99 +/- 0.24 versus 0.8 +/- 0.09, P < 0.001), and no change was observed at several follow-up visits thereafter. The reduction was most pronounced for the primitive BM progenitor subsets such as the CD34+/DR- and CD34+/ Thy-1+ cells. The impairment of hematopoiesis was also reflected by a significant reduction in the plating efficiency of BM and PB samples. No relationship was found between the decrease in the proportion of CD34+ cells and any particular patient characteristics, kind of high-dose therapy or the CD34+ cell content in the autograft. In conclusion, high-dose therapy with PBSC transplantation is associated with a long-term impairment of the haemopoietic system. The reduction in the number of haemopoietic progenitor cells is not associated with a functional deficit, as peripheral blood counts post-transplantation were normal in the majority of patients.     

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.     

Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.     

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.     

CD34 positive blood cells for allogeneic progenitor and stem cell transplantation

The transplantation of allogeneic peripheral blood progenitor cells (PBPC) provides complete and sustained hematopoietic and lymphopoietic engraftment. In healthy donors, large amounts of PBPC can be mobilized with hematopoietic growth factors. However, the high content of immunocompetent T-cells in apheresis products may expose recipients of allogeneic PBPC to an elevated risk of acute and chronic graft-versus-host disease. Thus, the use of appropriate T-cell reduction, but not depletion might reduce this risk. The hazards of graft rejection and a higher relapse rate can be avoided by maintaining a portion of the T-cells in the graft. The positive selection of CD34+ cells from peripheral blood preparations simultaneously provides an approximately 1000-fold reduction of T-cells. These purified CD34+ cells containing committed and pluripotent stem cells are suitable for allogeneic transplantation and can be used in the following instances: 1. As hematopoietic stem and progenitor cell transplantation instead of bone marrow cells, from HLA-identical family donors; 2. for increasing the stem cell numbers from HLA-mismatched or three HLA-loci different family donors in order to reduce the incidence of rejection but without increasing the T-cell number; 3. boosting of poor marrow graft function with stem cells from the same family donors; 4. transplantation from volunteer matched unrelated donors; 5. split transplantation of CD34+ and T-cells; 6. addition of ex vivo expanded CD34+ cells to blood cell or bone marrow transplantation; 7. generation of antigen specific immune effector cells and antigen presenting cells for cell therapy.     

Immune reconstitution after autologous progenitor hemopoietic cell transplantation. A study comparing autologous bone marrow and autologous peripheral blood transplantation

BACKGROUND: In order to find out the effect of peripheral blood (PB) hematopoietic progenitor cells on immune reconstitution the present study compares, through a randomized trial, some lymphoid subsets after peripheral blood (PBT) or bone marrow (BMT) autologous transplantation. MATERIAL AND METHODS: Twelve patients suffering from malignant hematological disorders were included (6 BMT and 6 PBT). From these patients 14 lymphoid and natural killer (NK) subsets were sequentially analyzed using appropriate dual staining. NK activity was analyzed by measuring Cr51 release from the K562 cell line. Studies were done in days and -6, +10, +17, +24, +31, +38, +52, +66, +90, +120, +180 and +360 after transplantation. RESULTS: The CD8+ cell regeneration was produced mainly by activated cells (CD38+), and no differences were observed between BMT and PBT, but CD8+ HLADR+ cells were higher in the PBT group. During the first year after transplantation CD4+ lymphoid cells were never within normal range, and its recovery was due to the memory subset (CD4+/CD45RO+). The CD19+ lymphocytes began their regeneration after the first month and it was produced mainly by by the CD19+/CD5+ subset. NK cells recovered faster in patients who underwent PBT, but NK activity was similar in both subgroups of patients and it was within normal range from day +17 until the end of the study. CONCLUSION: T, B and NK lymphoid reconstitution do not differ significantly between patients that receive BM or PB as hemathopoietic rescue, but PB seems influence a faster reconstitution of cytotoxic subsets (CD8+/HLADR+ and NK lymphoid cells).