Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.     

Metallothionein in human immunomagnetically selected CD34+ haematopoietic progenitor cells

Human umbilical CD34⁺ immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34⁺ was cultured in serum‐free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34⁺ cells. CD34⁺ cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34⁺ cells by 5.12±2.47‐fold (mean±S.D., n=3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81±1.07%, whereas it was 69.39±10.37% in none‐expanded CD34⁺ cells (mean±S.D., n=3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine‐rich metal‐binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34⁺ cells and compared it with more mature CD34^? cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34^? cells was higher than in the immature CD34⁺ cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34^? cells, greater than in CD34⁺ cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.     

Properties of monocytes generated from haematopoietic CD34+ stem cells from bone marrow of colon cancer patients

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34⁺ stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34⁺ haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14⁺ monocytes were found. They consisted of two subpopulations: CD14⁺⁺CD16⁺ and CD14⁺CD16^?, at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14⁺ monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3⁺ T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu_369–377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8⁺ T lymphocytes (CTL). The CD14⁺⁺CD16⁺ subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34⁺ stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.     

Surface localization of wheat germ agglutinin and concanavalin A binding sites on normal human blood cells: an ultrastructural histochemical study

In order to determine the extent and variations in surface concavanalin A (CON A) and wheat germ agglutinin (WGA) labeling of different varieties of normal blood cells, gluraraldehyde-fixed human blood cells were exposed to CON A-gold labeled horseradish peroxidase (CON A-HRP-G) and WGA-gold labeled ovomucoid (WGA-OVO-G) histochemical methods. The resultant particulate reaction product permitted assessment of binding and number of gold particles per micrometer of cell surface. Particle counts and data were subjected to statistical analysis. Six subjects (three female and three male) were used and compared in this study. In spite of moderate variations in surface labeling of the various types of leukocytes, erythrocytes and platelets within a given subject, determinations of mean labeling values for similar cell varieties proved quite similar between subjects with the given lectin. WGA and CON A had substantially different labeling densities on the various hemic cells. WGA surface labeling of all types of hemic cells, with the exception of platelets, showed far more labeling than was found with CON A. WGA mean labeling of the grouped subjects was significantly higher for each variety of leukocyte than for either erythrocytes or platelets although this distinction was not always evident in an individual subject. With CON A, mean labeling density for each of hemic cell types showed significant differences between each of the hemic cell varieties. Erythrocytes had only minimal CON A binding while monocyte and platelet populations represented the most reactive of the hemic cells. No difference was noted between corresponding cell varieties in the female vs. male subjects.     

The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke

The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day) and 144 hrs (6th day) since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM) and erythroid (BFU-E)-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.     

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.     

Presence of cytochrome P450 enzymes in human CD34+ haematopoietic progenitor cells

Human CD34+ cells (haematopoietic stem and progenitor cells separated from peripheral blood) were shown to express CYP2E1 protein by Western blotting. For the first time, the specific CYP2E1 activity (chlorzoxazone 6-hydroxylation) was also detected. No CYP3A4 protein neither the CYP3A-specific nifedipine oxidase activity were detectable. The results obtained indicate differences in content of active CYP proteins in populations of stem and progenitor cells from different species as the CYP3A2 (a rat form similar to human CYP3A4) was shown to be expressed in bone marrow derived cells by RT-PCR (Avital et al. 2001).     

Specific high-affinity binding sites for a synthetic gliadin heptapeptide on human peripheral blood lymphocytes

The synthetic peptide containing residues 43-49 of alpha-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. We now demonstrate using radiolabeled alpha-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (KD) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of alpha-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of [125I]alpha-gliadin(43-49) than T-lymphocytes. The lymphocyte alpha-gliadin(43-49) receptor may play an important role in mediating the immunological response to alpha-gliadin.     

Retrovirus-mediated gene transfer into CD4+ and CD8+ human T cell subsets derived from tumor-infiltrating lymphocytes and peripheral blood mononuclear cells

Summary Studies were undertaken to test the susceptibility of individual T cell subpopulations to retroviral-mediated gene transduction. Gene transfer into human tumor-infiltrating lymphocytes (TIL) or peripheral blood mononuclear cells (PBMC) was carried out by transduction with an amphotropic murine retroviral vector (LNL6 or N2) containing the bacterialneo ^R gene. The presence of theneo ^R gene in the TIL population was demonstrated by Southern blot analysis, detection of the enzymatic activity of the gene product and by the ability of transduced TIL to proliferate in high concentrations of G418, a neomycin analog that is toxic to eukaryotic cells. The presence of theneo ^R gene in TIL did not alter their proliferation or interleukin-2 dependence compared to nontransduced TIL. The differential susceptibility of CD4⁺ and CD8⁺ lymphoid cells to the retro-virus-mediated gene transfer was then tested. Transduction of heterogeneous TIL cultures containing both CD4⁺ and CD8⁺ cells resulted in gene insertion into both T cell subsets with no preferential transduction frequency into either CD4⁺ or CD8⁺ cells. In other experiments highly purified CD4⁺ and CD8⁺ T cell subpopulations from either TIL or PBMC could be successfully transduced with theneo ^R gene as demonstrated by Southern blot analysis and detection of the gene product neophosphotransferase activity. No such activity or vector DNA could be detected in controls of nontransduced cells. In these highly purified cell subsets the distinctive T cell phenotypic markers were continually expressed after transduction, G418 selection and long-term growth. Clinical trials have begun in patients with advanced cancer using heterogeneous populations of CD4⁺ and CD8⁺ gene-modified TIL. Current address: Bone Marrow Transplantation, Hadassah University Hospital, 91120 Jerusalem, Israel     

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34⁺ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34⁺ cells as well as peripheral blood CFU-GM was observed (P<0.01). Thus, the measurement of CD34⁺ cells is useful for predicting the number of circulating CFU-GM.     

Peripheral blood and BM CD34+ CD38- cells show better resistance to cryopreservation than CD34+ CD38+ cells in autologous stem cell transplantation

BACKGROUND: We and others have shown a critical role for CD34+ CD38- cells in hematopoietic recovery after autologous stem cell transplantation (ASCT), in particular for platelet reconstitution. Thus a routine assessment of CD34+ CD38- cells in freezing-thawing procedures for autografting could represent an important tool for predicting poor engraftment. METHODS: To compare the impact of cryopreservation on CD34+ CD38+ and CD34+ CD38- hematopoietic stem cell subsets, 193 autograft products collected in 84 patients with malignancies were assessed before controlled-rate cryopreservation in 10% DMSO and after thawing for autografting. RESULTS: Cell counts after thawing were significantly different from the pre-freezing counts for total CD34+ (P<0.0001) and CD34+ CD38+ (P<0.0001) cells, but not for CD34+ CD38- cells (P=0.252). Median losses for CD34+, CD34+ CD38+ and CD34+ CD38- cells were, respectively, 11.8%, 11.4% and 0.0%. The magnitude of fresh/post-thawing percentage cell variation was significantly different when comparing between the CD34+ CD38+ and CD34+ CD38- cell subsets (P<0.001). Moreover, CD34+ CD38- cells exhibited recovery values > or =100% in 85/160 graft products, compared with 51/193 in CD34+ CD38+ cells (P<0.0001). Also, recovery values > or =90% were significantly better in the CD34+ CD38- (98/160 grafts) than in the CD34+ CD38+ subsets (89/193 grafts) (P<0.01). DISCUSSION: In this work we have demonstrated that CD34+ cells that do not express the CD38 Ag show a significantly better resistance to cryopreservation. This could represent another example of the particular ability of less committed progenitor cells to overcome environmental injuries. Moreover, we consider routine assessment of CD34+ CD38- cells before freezing as clinically relevant, but post-thawing controls may be avoided because of their good resistance to freezing.     

Induction of telomerase activity during development of human mast cells from peripheral blood CD34+ cells: comparisons with tumor mast-cell lines

To further characterize the development of mast cells from human hemopoietic pluripotent cells we have investigated the expression of telomerase activity in cultured human peripheral blood CD34+ cells, and CD34+ /CD117+ /CD13+ progenitor mast cells selected therefrom, with the idea that induction of telomerase is associated with clonal expansion of CD34+ /CD117+ /CD13+ cells. A rapid increase in telomerase activity preceded proliferation of both populations of cells in the presence of stem cell factor and either IL-3 or IL-6. The induction was transient, and telomerase activity declined to basal levels well before the appearance of mature mast cells. Studies with pharmacologic inhibitors suggested that this induction was initially dependent on the p38 mitogen-activated protein kinase and phosphatidylinositol 3'-kinase, but once cell replication was underway telomerase activity, but not cell replication, became resistant to the effects of inhibitors. Tumor mast cell lines, in contrast, expressed persistently high telomerase activity throughout the cell cycle, and this expression was unaffected by inhibitors of all known signaling pathways in mast cells even when cell proliferation was blocked for extended periods. These results suggest that the transient induction of telomerase activity in human progenitor mast cells was initially dependent on growth factor-mediated signals, whereas maintenance of high activity in tumor mast cell lines was not dependent on intracellular signals or cell replication.     

HPRT mutations in vivo in human CD 34+ hematopoietic stem cells

The HPRT mutations in T lymphocytes are widely utilized as biomarkers of environmental exposure and effect. The HPRT gene detects a wide variety of mutation types, many of which are similar at the molecular level to those found in oncogenes in cancers. However, it remains to be determined whether the assay for mutations in T lymphocytes is reflective of mutagenic events in tissues or cells which have high frequencies of malignancy in humans. We now demonstrate that the HPRT gene can be utilized to detect mutations in myeloid stem cells, which are frequent progenitor cells of leukemias. This myeloid stem cell assay shows an age related increase in mutation at HPRT and also detects increases in mutant frequency (M-MF) in patients who have undergone chemotherapy. The myeloid mutants are confirmed to have mutations in the HPRT gene by DNA sequence analysis. Increases in M-MF are seen as expected in the clonally unstable myeloid stem cells of patients with myelodysplastic syndromes; however, unexpectedly these patients also have elevated T-lymphocyte mutant frequencies (T-MF). A good correlation is shown between M-MFs and T-MFs in the same patients. Thus, it appears that the T-lymphocyte assay, which is technically much less demanding than the myeloid assay, appears to faithfully represent the frequency of mutagenic events in the myeloid lineage.     

CD4+CD25high regulatory cells in human peripheral blood

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD(+)CD25(+) T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4(+) population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4(+)CD25(+) regulatory cells isolated in mice. With TCR cross-linking, CD4(+)CD25(high) cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4(+)CD25(-) responder T cells in a contact-dependent manner. The CD4(+)CD25(high) regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RB(low) on murine CD4(+)CD25(+) regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4(+)CD25(high) regulatory cells and CD4(+)CD25(-) responder cells. Whereas higher ratios of CD4(+)CD25(high) T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.     

Antigen-dependent colonies of human peripheral blood lymphocytes: an immunomorphologic study

Human peripheral blood mononuclear cells (PBMC), stimulated by sheep red blood cells (SRBC), focally proliferate in agar and form colonies of anti-SRBC antibody-secreting cells surrounded by hemolytic areas. Two types of colonies develop: type I (diffuse type), which grows deeply into the agar, and type II (compact type), which grows above the former. Immunochemical and ultrastructural studies show that diffuse colonies contain differentiating lymphoid cells, from small lymphocytes to mature plasma cells. About 50% of cells stain positively in their cytoplasm for IgM and only 1-2% for IgG. Most colonies produce light chains of one class, whereas only a few produce both classes. Many cells resemble monocytes or T lymphocytes in their general morphology and lie in close contact with immunoglobulin-positive cells. Compact colonies contain cells not engaged in antibody production. The culture system described here is the first available antigen-dependent colony assay for human PBMC and may be useful for in vitro studies on the mechanism of human B-cell activation.     

Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34+ megakaryocytic progenitor cells from human placental and umbilical cord blood

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34(+) cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34(+) CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.