Preservation of immunological and colony-forming capacities of long-term (15 years) cryopreserved cord blood cells

BACKGROUND: Cryopreserved cord blood may be stored for decades before being used for allogeneic stem cell transplantation. Little is known about the effect of long-term cryopreservation in liquid nitrogen on the viability and function of cord blood cells. We examined the recovery, viability, clonogenic capacity, and T-cell reactivity to HLA alloantigens of cord blood samples cryopreserved up to 15 years. METHODS: Progenitor cell recoveries were studied by (colony-forming unit-granulocyte-macrophage) clonogenic assays from 18 cord blood samples short-term frozen for 2-8 weeks and from 8 samples cryopreserved for 15 years. Proliferative and cytotoxic responses against HLA antigens of thawed cord blood mononuclear cells after short-term or long-term cryopreservation were tested in standard mixed lymphocyte cultures and cell-mediated lympholysis assays. RESULTS: After thawing, the mononuclear cell recovery from long-term frozen cord blood low-density fractions averaged 80% (range, 64% to 92%). The presented data show that long-term frozen cord blood cells keep their clonogenic potential. No damaging effect was seen on the proliferative and cytotoxic capacities of long-term frozen cord blood T cells. CONCLUSIONS: The results support the possibility of long-term storage of progenitor cells from umbilical cord blood for future bone marrow reconstitution.     

High-dose granulocyte-colony stimulating factor (G-CSF) in vitro induces the growth of high proliferative potential colony forming cells (HPP-CFC) in patients undergoing blood stem cell mobilization

We evaluated the role of high-dose granulocyte colony stimulating factor (G-CSF) in vitro, in inducing the generation of high-proliferative potential colony forming cells (HPP-CFC), from either mononuclear cells or purified CD34+ cells. Both normal controls and patients undergoing peripheral blood stem cell (PBSC) mobilization and transplantation were studied. In serum-driven agar cultures, G-CSF stimulated the proliferation of HPP-CFC in a dose dependent manner (r = 0.92). The number of HPP-CFC was four-fold greater in mobilized patients than in normal controls. Purified CD34+ cells yielded 11-fold more colonies than mononuclear cells. HPP-CFC from mobilized patients showed replating capacity, giving rise to secondary colonies of more mature appearance. In serum-free cultures, the effect of G-CSF appeared to be mediated by synergistic interaction with stem cell factor. Our results suggest that G-CSF stimulates primitive hematopoietic cells that are detectable in increased amounts in patients receiving mobilization therapy. Therefore, determination of G-CSF induced HPP-CFC could be a useful tool in the evaluation of mobilization strategies.     

Endogenous hematopoietic reconstitution induced by human umbilical cord blood cells in immunocompromised mice: implications for adoptive therapy

Cross-linking the heterotrimeric (alpha beta gamma 2) IgE receptor, Fc epsilon RI, of mast cells activates two tyrosine kinases: Lyn, which phosphorylates beta and gamma subunit immunoreceptor tyrosine-based activation motifs, and Syk, which binds gamma-phospho-immunoreceptor tyrosine-based activation motifs and initiates cellular responses. We studied three Fc epsilon RI-dimerizing mAbs that maintain similar dispersed distributions over the surface of RBL-2H3 mast cells but elicit very different signaling responses. Specifically, mAb H10 receptor dimers induce very little inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, spreading, ruffling, and actin plaque assembly, whereas dimers generated with the other anti-Fc epsilon RI mAbs induce responses that are only modestly lower than that to multivalent Ag. H10 receptor dimers activate Lyn and support Fc epsilon RI beta and gamma subunit phosphorylation but are poor Syk activators compared with Ag and the other anti-Fc epsilon RI mAbs. H10 receptor dimers have two other distinguishing features. First, they induce stable complexes between activated Lyn and receptor subunits. Second, the predominant Lyn-binding phospho-beta isoform found in mAb H10-treated cells is a less tyrosine phosphorylated, more electrophoretically mobile species than the predominant isoform in Ag-treated cells that does not coprecipitate with Lyn. These studies implicate Lyn dissociation from highly phosphorylated receptor subunits as a new regulatory step in the Fc epsilon RI signaling cascade required for Syk activation and signal progression.     

Neutrophilic cell production by combination of stem cell factor and thrombopoietin from CD34(+) cord blood cells in long-term serum-deprived liquid culture

In the present study, we investigated the effects of stem cell factor (SCF) and/or thrombopoietin (TPO) on the cell production by cord blood CD34(+) cells using a serum-deprived liquid culture system. Although SCF alone supported a modest production of neutrophilic cells and a remarkable generation of mast cells, the addition of TPO to the culture containing SCF caused an apparent generation of neutrophilic cells, identified by immunocytochemical staining and flow cytometric analysis. The significant production of neutrophilic cells by SCF and TPO was persistently observed from 2 weeks to 2 to 3 months of culture. The interaction between SCF and TPO on the neutrophilic cell generation was greater than the combined effects of SCF with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The addition of neutralizing antibody against G-CSF or GM-CSF did not influence the SCF + TPO-dependent neutrophilic cell production. A single-cell culture study showed that not only CD34(+)CD38(+) c-kit+ cells but also CD34(+)CD38(-)c-kit+ cells were responsible for the neutrophilic cell generation. In clonal cell cultures, GM progenitors as well as erythroid progenitors and multipotential progenitors expanded in the cultures supplemented with SCF and TPO. The neutrophilic cells grown by SCF + TPO were at myeloblast to band cell stages, and scarcely matured to segmented neutrophils. In addition, the cells generated by SCF + TPO were stained with monoclonal antibodies against myeloperoxidase, elastase, lactoferrin, and CD11b, but they had negligible levels of alkaline phosphatase (ALP) and CD35. The replating of the CD34(-)c-kit-/low CD15(+) cells grown by SCF + TPO into a culture containing SCF + G-CSF permitted both the terminal maturation into segmented cells and the appearance of ALP and CD35. These results indicate the existence of a G-CSF/GM-CSF-independent system of neutrophilic cell production.     

Effects of diheptyldiselenide (DDS) on human tumor cell lines and on peripheral blood mononuclear cells

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS (250 microM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h 51Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.     

Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture

The participation of apical membranes of uterine epithelial cells in the process of blastocyst adhesion makes them an interesting object in the study of changes occurring during early pregnancy. In the study of these changes alkaline phosphatase (AIP), a typical brush border enzyme, was chosen for demonstration with the scanning electron microscope (SEM) by means of a backscatter detector. Thus the temporal and spatial pattern of enzyme activity on the uterine luminal surface was made visible with lead salt procedures. AIP activity was shown to be located on apical membranes and microvilli of endometrial epithelial cells with high activity on day 2 of pregnancy decreasing to virtually no activity on day 5. This decrease in overall AIP activity was shown to be asymmetrical with respect to the uterine cavity. It begins on the antimesometrial half of the uterine lining on day 2. A distribution pattern demarcating a presumptive implantation site along the uterine horn was not found. However, on day 5 of pregnancy, a characteristic pattern of surface folds was found, dividing the uterine horn into 'implantation segments'. In addition, SEM investigation revealed a marked variation of AIP activity from one individual cell to the next on day 2 of pregnancy resulting in a mosaic-like pattern. This pattern is lost with the decrease of AIP activity on day 5. Thus heterogeneity of uterine epithelial cells in AIP activity is apparently a feature of nonreceptive epithelium in contrast to the homogeneous epithelium on day 5. It is proposed that epithelial cell homogeneity could be a marker for uterine receptivity.     

Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood

Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult "competitor" marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.     

A genetic model for undifferentiated cell tumor formation: similar tumors formed by different cell lines transformed by the E1A oncogene

The activation of oncogenes and the mutation/deletion of suppressor genes may be involved in tumor heterogeneity. In an attempt to study tumor heterogeneity, we transformed cell lines from epithelial (PAM 212), mesenchymal (NIH-3T3), or melanocytic origin (L-BIOBR) with the wild type E1a oncogene. To make the cell lines tumorigenic, cells were infected with Harvey sarcoma virus carrying the v-H-ras oncogene. The transformed cells were injected into nude mice and the tumors studied by optical and electron microscopy. The tumors formed by v-H-ras-transformed cells consisted of epitheliod melanomas, spindle cells sarcomas and poorly-differentiated carcinomas, depending on the cell of origin. In contrast, the tumors obtained from cells also carrying the E1a oncogene showed a predominant small and undifferentiated cell pattern regardless of the cell of origin. We conclude that the E1a oncogene products induce a negative control of differentiation, independent of the cell type, and that tumors formed by cells carrying the E1a oncogene display an undifferentiated cell pattern.     

Synchronization of cells in the S phase of the cell cycle by 3'-azido-3'-deoxythymidine: implications for cell cytotoxicity

The mechanism of synergy between 3'-azido-3'-deoxythymidine (AZT) and anticancer agents was investigated with emphasis on cell-cycle events. Exposure of exponentially growing WiDr human colon carcinoma cells to AZT resulted in synchronization of cells in the S phase of the cell cycle. Following treatment with AZT at 50 or 200 microM, 62% +/- 3% or 82% +/- 4% of the cells were in the S phase as compared with 36% +/- 2% in the control. Bromodeoxyuridine uptake studies revealed that the synchronized cells actively synthesized DNA. At concentrations of up to 200 microM, AZT produced a cytostatic rather than cytotoxic effect as indicated by viability and cell growth measurements. At 200 microM, AZT-induced synchronization was significant (P = < 0.001) after 12 h of drug exposure, reached a maximum at 24 h, and reversed to baseline levels by 72 h even in the continued presence of the drug. This indicates that AZT-induced cytostasis is a transient and reversible effect. The cell-cycle events seen with AZT in WiDr cells were also observed in eight of nine human tumor cell lines tested. Isobologram analysis of WiDr cells preexposed to AZT for 24 h and then exposed to either AZT-5-fluorouracil or AZT-methotrexate for a further 72 h revealed synergy between AZT and the anticancer agents, indicating that AZT-induced synchronization may have therapeutic benefits.     

Regulation of Sertoli cell alpha 2-macroglobulin and clusterin (SGP-2) secretion by peritubular myoid cells

alpha 2-Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular alpha 2-macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of alpha 2-macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10(-11)-10(-5) M) had no apparent effect on the secretion of alpha 2-macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate alpha 2-macroglobulin secretion by hepatocytes, did not affect the alpha 2-macroglobulin secretion. However, dexamethasone at 10(-7)-10(-5) M stimulated alpha 2-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated alpha 2-macroglobulin secretion. These findings suggest that the synthesis and/or secretion of alpha 2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly (ADP-ribose) polymerase, a potential target for drugs: Part II. Regulation of differentiation by the poly ADP-ribose system

It is shown that eukaryotic differentiation is specifically sensitive to pADPRT regulation in Trypanosomna, Leishmania and Mytilus models. There is powerful inhibition of early differentiation without cell toxicity by pADPRT ligands.     

Decreased interleukin-12 (IL-12) from activated cord versus adult peripheral blood mononuclear cells and upregulation of interferon-gamma, natural killer, and lymphokine-activated killer activity by IL-12 in cord blood mononuclear cells

Remnants of lipoproteins, intestinal chylomicrons, and very low density lipoprotein (VLDL), are rapidly cleared from plasma and enter hepatocytes. It has been suggested that remnant lipoproteins are initially captured in the space of Disse by heparan sulfate proteoglycans (HSPGs), and that their subsequent internalization into hepatocytes is mediated by members of the LDL-receptor gene family. Similarly to lipoprotein remnants, malaria sporozoites are removed from the blood circulation by the liver within minutes after injection by Anopheles mosquitoes. The sporozoite's surface is covered by the circumsporozoite protein (CS), and its region II-plus has been implicated in the binding of the parasites to glycosaminoglycan chains of hepatocyte HSPGs. Lactoferrin, a protein with antibacterial properties found in breast milk and neutrophil granules, is also rapidly cleared from the circulation by hepatocytes, and can inhibit the hepatic uptake of lipoprotein remnants. Here we provide evidence that sporozoites, lactoferrin, and remnant lipoproteins are cleared from the blood by similar mechanisms. CS, lactoferrin, and remnant lipoproteins compete in vitro and in vivo for binding sites on liver cells. The relevance of this binding event for sporozoite infectivity is highlighted by our demonstration that apoliprotein E-enriched beta-VLDI and lactoferrin inhibit sporozoite invasion of HepG2 cells. In addition, malaria sporozoites are less infective in LDL-receptor knockout (LDLR -/-) mice maintained on a high fat diet, as compared with littermates maintained on a normal diet. We conclude that the clearance of lipoprotein remnants and sporozoites from the blood is mediated by the same set of highly sulfated HSPGs on the hepatocyte plasma membrane.     

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.     

Poly(alpha-hydroxyacids) for application in the spinal cord: resorbability and biocompatibility with adult rat Schwann cells and spinal cord

Future surgical strategies to restore neurological function in the damaged human spinal cord may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. We have studied the in vitro and in vivo degradability of various aliphatic polyesters as well as their effects on rat Schwann cells in vitro and on spinal cord tissue in vivo. In vitro, cylinders made of poly(D,L-lactic-co-glycolic acid) 50:50 (PLA25GA50) started to degrade at 7 days, compared with 28 days for cylinders made of poly(D,L-lactic acid) (PLA50). This faster degradation of PLA25GA50 was reflected by a much higher absorption of water. In vivo, after implantation of PLA25GA50 or PLA50 cylinders between the stumps of a completely transected adult rat spinal cord, the decrease in molecular weight of both polymers was similar to that found in vitro. In vitro degradation of poly(L-lactic acid) (PLA100) mixed with increasing amounts of PLA100 oligomers also was determined. The degradation rate of PLA100 mixed with 30% oligomers was found to be similar to that of PLA50. In vitro, PLA25GA50 and the breakdown products had no adverse effect on the morphology, survival, and proliferation of cultured rat Schwann cells. In vivo, PLA25GA50 cylinders were integrated into the spinal tissue 2 weeks after implantation, unlike PLA50 cylinders. At all time points after surgery, the glial and inflammatory response near the lesion site was largely similar in both experimental and control animals. At time points later than 1 week, neurofilament-positive fibers were found within PLA25GA50 cylinders or the remains thereof. Growth-associated protein 43, which is indicative of regenerating axons, was observed in fibers in the vicinity of the injury site and in the remains of PLA25GA50 cylinders. The results suggest that poly(alpha-hydroxyacids) are likely candidates for application in spinal cord regeneration paradigms involving Schwann cells.