Generation of dendritic cells from fresh and frozen cord blood CD34+ cells

Extracellular recordings obtained from the extrastriate cortex of the California ground squirrel, a diurnal sciurid, show that large receptive fields and a strong direction selectivity are present in the middle lateral area (ML) and the lateral area (L), located laterally to V2 and V3. Direction selectivity was tested by presenting stimuli of varying dimensions, shapes and speeds at different locations in the visual field. Most cells in ML and L (84%) were direction selective, with a preference for fast speeds, indicating that these areas share a role in motion processing. Areas ML and L may be homologous to area MT or may represent a case of homoplasia. A directional anisotropy for motion towards the vertical meridian was found in ML and L cells, suggesting that these areas may be involved in detecting predators and other moving objects coming from the periphery, rather than in processing flow fields caused by forward locomotion, for which a centrifugal bias might be expected.     

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.     

Interleukin-2-induces development of denditric cells from cord blood CD34+ cells

Fiducial markers are reference points used in the registration of image space(s) with physical (patient) space. As applied to interactive, image-guided surgery, the registration of image space with physical space allows the current location of a surgical tool to be indicated on a computer display of patient-specific preoperative images. This intrasurgical guidance information is particularly valuable in surgery within the brain, where visual feedback is limited. The accuracy of the mapping between physical and image space depends upon the accuracy with which the fiducial markers were located in each coordinate system. To effect accurate space registration for interactive, image-guided neurosurgery, the use of permanent fiducial markers implanted into the surface of the skull is proposed in this paper. These small cylindrical markers are composed of materials that make them visible in the image sets. The challenge lies in locating the subcutaneous markers in physical space. This paper presents an ultrasonic technique for transcutaneously detecting the location of these markers. The technique incorporates an algorithm based on detection of characteristic properties of the reflected A-mode ultrasonic waveform. The results demonstrate that ultrasound is an appropriate technique for accurate transcutaneous marker localization. The companion paper to this article describes an automatic, enhanced implementation of the marker-localization theory described in this article.     

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.     

CD34++ CD38- and CD34+ CD38+ human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source

CD34++ CD38- and CD34+ CD38+ hematopoietic progenitor cells (HPCs) from human fetal liver (FL), cord blood (CB), and adult bone marrow (ABM) were isolated and investigated for their growth characteristics, cytokine requirements and response to two modulators of early hematopoiesis, interferon (IFN)-gamma and macrophage inflammatory protein (MIP)-1alpha. We observed first that a significantly lower percentage of CD34++ cells were CD38- in ABM than in FL and CB. Second, the functional differences between CD34++ CD38- and CD34+ CD38+ cells were less pronounced in FL and CB than in their ABM counterparts. Third, an inverse correlation was found between growth factor response and the ontogenic age of HPCs, and a direct correlation was noted between cytokine requirements and the ontogenic age of HPCs. Fourth, spontaneous colony formation in a classic semisolid culture system was reproducibly obtained only in the ontogenically earliest cells, that is, in FL but not in CB and ABM, in which no such spontaneous colony formation was observed. Fifth, the modulatory effects of IFN-gamma and MIP-1alpha were qualitatively different depending on the ontogenic age of the progenitor source: whereas IFN-gamma was only a selective inhibitor of primitive CD34++ CD38- ABM progenitor cells, it inhibited both CD34++ CD38- and CD34+ CD38+ FL and CB cells to the same extent. In contrast to the effects of MIP-1alpha on ABM, we could not find any consistently stimulatory or inhibitory effects on FL and CB progenitors. In conclusion, important functional and biologic differences exist between FL, CB, and ABM progenitor cells, and these differences could have major implications for the use of these cell populations in preparative protocols of ex vivo expansion, transplantation strategies, or gene transfer experiments.     

Analysis of cord blood CD34+ cells purified after cryopreservation

Many practical issues regarding processing blood samples for cord blood banking remain. After cryopreservation, a reduction in clonogenicity has been reported, although it is unknown whether this is associated with lower potential for long-term engraftment. CD34+ cell purification of cryopreserved cord blood (CB) may be important for the clinical application of in vitro expansion. We compared purity, yield, clonogenicity, and growth in long-term stromal-based culture of fresh and cryopreserved CD34+ purified cells (n = 12) using the miniMACS separation system. Mean purity of CD34+ cells was 93% when processed before and 73% when processed after cryopreservation. Fresh CD34+ cells had higher clonogenic potential than cryopreserved cells (45 vs 20%, p < 0.05) in CFU-Mix assays, indicating that progenitor cell loss during cryopreservation is due in part to reduced cloning efficiency of viable CD34+ cells. In long-term culture (LTC) on irradiated normal human bone marrow stroma (n = 7), CFU-GM production in the two groups was the same over 12 weeks, suggesting identical long-term culture-initiating cell (LTC-IC) numbers. We conclude that apparent clonogenic cell loss during cryopreservation is associated with relative sparing of the more primitive LTC-ICs. CFU-Mix assays may therefore underestimate the transplant potential of cryopreserved CB. Purification of CD34+ cells following cryopreservation gives sufficient purity for detailed evaluation of CD34+ cells and for stem cell expansion.     

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.     

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.     

Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.     

Cytokine mediated expansion of human umbilical cord blood CD34+ cells: comparison of the use of partially purified and pure CD34+ target cells

The study compares an occurrence rate of congenital malformations in newborn infants of mothers with insulin dependent diabetes (IDDM) and newborns of healthy mothers and mothers with pregnancy diabetes (GDM). This paper evaluates the influence of stage of advancement (a class) of diabetes in the mother and its control during early pregnancy on a rate of congenital malformations in the fetus. We have taken a group of 170 neonates of mothers with IDDM. The control group was 56 newborn infants of mothers with GDM and 26,368 newborn infants of healthy women. We found 11.2% of congenital malformations in newborn infants of mothers with IDDM, compared to 1.8% of ones in the newborn infant population of mothers with GDM and 2.2% in the population of healthy mothers. The occurrence rate of congenital malformations in offspring of diabetic mothers with IDDM was 5-times higher than in the general population of newborn infants of healthy and also mothers with GDM. A risk of major birth defect occurrence in the fetus was directly proportional to the grade of the blood glucose level control in mothers during the I trimester of pregnancy, but the presence of diabetic angiopathy (classes D-H) had a significant influence on the occurrence rate of major birth defects in the fetus only in metabolic imbalance cases.     

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.     

Ex vivo culture of peripheral blood CD34+ cells: effects of hematopoietic growth factors on production of neutrophilic precursors

A major potential application for ex vivo culture of hematopoietic progenitor cells is the treatment of cytopenia following high-dose chemotherapy and hematopoietic transplantation. We have previously postulated that infusion of a sufficient number of neutrophil postprogenitor cells generated by ex vivo culture of CD34+ cells may be able to abrogate neutropenia. In this article, we describe further development of an efficient stromal-free, cytokine-dependent, static culture system for generation of these cells. Our previous studies indicated that maximal production of nucleated cells and myeloid progenitor cells from PB CD34+ cells occurred with multiple hematopoietic growth factor (HGF), notably the 6-HGF combination of interleukin (IL)-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and stem cell factor (SCF). In the present study, we determine the contribution of each of these 6 HGF in generation of neutrophilic precursors. SCF, G-CSF, and IL-3 were found to be the most important HGF for production of neutrophilic cells. The 4-HGF combination of IL-3, IL-6, G-CSF, and SCF was optimized by performing dose-response experiments and shown to be as potent as 6 HGF for production of nascent CFU-GM and neutrophilic precursors.     

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.     

Extrathymic T cell differentiation in vitro from human CD34+ stem cells

Recent experiments have extended our understanding of how sensory information in premotor networks controlling motor output is processed during locomotion, and at what level the efficacy of specific sensory-motor pathways is determined. Phasic presynaptic inhibition of sensory transmission combined with postsynaptic alterations of excitatory and inhibitory synaptic transmission from interneurons of the premotor networks contribute to the modulation of reflex pathways and to the generation of reflex reversal. These mechanisms play an important role in adapting the operation of central networks to external demands and thus help optimize sensory-motor integration.     

Evaluation of methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as reagents in flow cytometry of hematopoietic progenitor cells

BACKGROUND: Flow cytometry is used to quantitate CD34+ hematopoietic progenitor cells for transplantation. The present study evaluates methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as flow cytometry reagents. STUDY DESIGN AND METHODS: The human myeloid leukemic cell lines KG 1 a (CD34+) and K562 (CD34-) were grown in culture under standard conditions and then prepared on ficoll gradients of different densities to determine which gave the component that was most reproducible. After ficoll preparation, the cells were frozen in standard cryopreservation media and four methods of thawing were examined. Determination of the method that gave the cell component that was most reproducible was based on viability, percentage of cell recovery, and maintenance of CD34 antigenicity status. RESULTS: Ficoll gradient preparation improved the ease of flow cytometry analysis when original viability was low, and it produced a more uniform cell population. However, it resulted in significant cell loss for both cell lines. While the cell recovery for K562 cells was not significantly different with any of the densities of ficoll, recovery was significantly better for KG 1 a cells with ficoll at a specific gravity of 1.077. Of the thawing methods examined, all three that involved a rapid thaw at 37 degrees C were statistically equivalent to each other and were better than thawing at 4 degrees C. CONCLUSION: With a standardized method of preparing cell lines as reagents for quality control purposes, data comparison among cell processing laboratories may more readily be initiated. Such cell lines could also be useful as a teaching tool for flow cytometry and in proficiency testing.     

Frequency of fetal cells in sorted subpopulations of nucleated erythroid and CD34+ hematopoietic progenitor cells from maternal peripheral blood

Fetal cells that circulate in maternal peripheral blood (PB) during pregnancy offer a potential source of nucleated fetal material for noninvasive prenatal diagnosis. Fluorescence-activated cell sorting was used to target two populations of fetal cells: nucleated erythroid cells (NECs; CD71/glycophorin-A+ CD45(lo-int) CD34-) and hematopoietic progenitor cells (CD34+ cells; CD34++ CD71/glycophorin-A- CD45(int)). Fetal cells were detected by fluorescence in situ hybridization (FISH) using directly conjugated chromosome X and Y probes in 65% (13 of 20) of the maternal PBs (fetal karyotype 46,XY). The frequency of fetal cells isolated from the NEC and CD34+ fractions was, respectively, 0 to 14 and 0 to 7 cells per 2 x 10(7) previously frozen maternal cells (approximately 20 mL of blood). In nonfrozen samples, the yield and recovery of fetal cells was moderately improved. Culturing the CD34+ sorted fractions in serum-free media with cytokines improved the quality of the FISH preparations and resulted in a slight expansion in detectable fetal cells. The frequency of fetal cells isolated from cultured CD34+ fractions was 0 to 35 and 0 to 93 cells per 2 x 10(7) previously frozen and nonfrozen maternal PB cells, respectively. These results document the isolation, characterization, and enumeration of fetal cells from the maternal periphery that appear to be present in most, but not all, samples analyzed.     

Ex vivo generation of functional dendritic cells from mobilized CD34+ hematopoietic stem cells

Gene engineering to enhance tumour immunogenicity and elicit curative responses against established tumours and tumour recurrences has become an attractive prospect. Gene engineering enables new genes to be selectively inserted into the genome of a tumour cell, or the construction of new fusion plasmids coding tumour antigens and immunomodulatory molecules. The rationale behind current research is to enhance the immune recognition of tumour antigens through their association with the molecules on which immune recognition depends. The immunotherapy data obtained in many experimental tumour systems provide a realistic assessment of the potential and limits of this technological approach. Experimental vaccination of rodents has been shown to induce a significant immune memory, even against poorly immunogenic tumours, that can prevent tumour growth and cure initial metastases, but is poorly effective against established tumours. Its use in tumour prevention is a fresh dawning perspective.     

Engraftment and retroviral marking of CD34+ and CD34+CD38- human hematopoietic progenitors assessed in immune-deficient mice

Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+ progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38- cells. Comparable extents of human engraftment and lineage development were obtained from 5 x 10(5) CD34+ cells and 2,000 CD34+CD38- cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38- cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38- cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.