G Van Zant

Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span

Normal somatic cells undergo replicative senescence in vitro but the significance of this process in organismic aging remains controversial. We have shown previously that hemopoietic stem cells of common inbred strains of mice vary widely in cycling activity and that this parameter is inversely correlated with strain-dependent mean life span. To assess whether cell cycling and life span are causally related, we searched for quantitative trait loci (QTLs) that contributed to variation of these traits in BXH and BXD recombinant inbred mice. Two QTLs, mapping to exactly the same intervals on chromosomes 7 and 11, were identified that were associated with variation of both cell cycling and life span. The locus on chromosome 11 mapped to the cytokine cluster, a segment that shows synteny with human chromosome 5q, in which deletions are strongly associated with myelodysplastic syndrome. These data indicate that steady-state cell turn-over, here measured in hemopoietic progenitor cells, may have a significant effect on the mean life span of mammals.Normal somatic cells undergo replicative senescence in vitro but the significance of this process in organismic aging remains controversial. We have shown previously that hemopoietic stem cells of common inbred strains of mice vary widely in cycling activity and that this parameter is inversely correlated with strain‐dependent mean life span. To assess whether cell cycling and life span are causally related, we searched for quantitative trait loci (QTLs) that contributed to variation of these traits in BXH and BXD recombinant inbred mice. Two QTLs, mapping to exactly the same intervals on chromosomes 7 and 11, were identified that were associated with variation of both cell cycling and life span. The locus on chromosome 11 mapped to the cytokine cluster, a segment that shows synteny with human chromosome 5q, in which deletions are strongly associated with myelodysplastic syndrome. These data indicate that steady‐state cell turn‐over, here measured in hemopoietic progenitor cells, may have a significant effect on the mean life span of mammals. de Haan, G., Van Zant, G. Genetic analysis of hemopoietic cell cycling in mice suggests its involvement in organismal life span. FASEB J. 13, 707–713 (1999)

A putative gene causes variability in lifespan among genotypically identical mice

Ageing is a complex process during which damage to multiple organ systems accumulates1. The time at which impairment of critical bodily functions becomes incompatible with life determines organismal longevity and is usually measured in a population as mean or maximal lifespan. Our current understanding of the genes affecting longevity, particularly in mammalian species, is rudimentary at best2. Here we studied the range of lifespans among individual animals within genetically identical recombinant inbred (RI) strains of mice. We found that the time between the deaths of the first and last members of a given strain fell into two temporal groups, despite the fact that strains may have had similar mean or maximal lifespans. The segregation of RI strains into wide and narrow ranges that roughly corresponded to characteristic lifespan ranges of the progenitor strains, suggested a genetic component. We therefore mapped a putative gene accounting for more than 60% of the phenotype to a genomic segment near the centromere on chromosome 11. This locus causes variability in the rate at which genetically identical members of a population die and, from a practical standpoint, affects the slope of a death curve for a population.

Autologous CD34 + cell transplantation for patients with advanced lymphoma: effects of overnight storage on peripheral blood progenitor cell enrichment and engraftment

In order to demonstrate the feasibility of mobilization, enrichment and engraftment of autologous peripheral blood CD34+ cells in patients with relapsed lymphoma, 59 peripheral blood progenitor cell (PBPC) collections from 21 patients were enriched for CD34+ cells using CEPRATE SC (CellPro, Bothell, WA, USA) immunoaffinity column. Following high-dose chemotherapy, a mean of 17 × 108 (range, 3–34) nucleated cells/kg containing 8.7 × 106 (0.3–26) CD34+ cells/kg were re-infused. Blood cell recovery in these patients was compared to engraftment capacity of unenriched PBPCs in a cohort of lymphoma patients treated with an identical high-dose chemotherapy regimen. Neutrophil and platelet engraftment was rapid in both groups including five patients who received ⩽1 × 106 CD34+ cells/kg. After infusion of CD34+ enriched cells, neutrophils exceeded 0.5 × 109/l in 11 (8–14) days and platelets exceeded 20 × 109/l (untransfused) in 15 (9–39) days. In order to optimize the immunoaffinity column utilization we stored the first PBPC collections overnight at 4°C and combined them with the next day’s collection prior to the CD34+ enrichment procedure in 11 patients. This maneuver resulted in a significant decrease in the CD34+ cell recovery (resulting in reinfusion of a mean of 42% less CD34+ cells). Although overnight storage did not affect neutrophil engraftment, platelet engraftment was prolonged in this group of patients even when >2.0 × 106 CD34+ cells/kg were re-infused. The overnight storage procedure should be further evaluated for its effects on the CD34+ immunoaffinity enrichment procedure, megakaryocyte progenitors and platelet engraftment. We conclude that CD34+ cells enriched from peripheral blood result in rapid engraftment after high-dose chemotherapy in patients with advanced lymphoma that is comparable to that of patients receiving unenriched PBPCs.

Genetic Dissection of Age-Related Changes of Immune Function in Mice

Understanding of the genetic basis of normal and abnormal development of the immune response is an enormous undertaking. The immune response, at the most minimal level, involves interactions of antigen presenting cells (APCs), T and B cells. Each of these cells produce cell surface and soluble factors (cytokines) that affect both autocrine and paracrine functions. A second level of complexity needs to consider the development of the macrophage/monocyte lineage as well as the production of the common lymphoid precursor which undergoes distinct maturation steps in the thymus and periphery to form mature T cells as well as in BM (BM) and lymphoid organs to form mature B cells. A third level of complexity involves the immune response to infectious agents including viruses and also the response to tumour antigens. In addition, there are imbalances that predispose to decreased responses (immunodeficiencies) or increased responses (autoimmunity). A fourth level of complexity involves attempts to understand the differences in the immune response that occurs at a very young age, in adults, and at a very old age. This review will focus on the use of C57BL/6 J X DBA/2 J (BXD) recombinant inbred (RI) strains of mice to map genetic loci associated with the production of lymphoid precursors in the BM, development of T cells in the thymus, and T‐cell responses to stimulation in the peripheral lymphoid organs in adult and in aged mice. Strategies to improve the power and precision in which complex traits such as the age‐related immune response can be mapped is limited with the current set of 35 strains of BXD mice. Strategies to increase these strains by generating recombinant intercross (RIX) strains of mice are being developed to enable this large set of lines to detect quantitative trait loci (QTLs) with a much higher consistency and statistical power. More importantly, the resolution with which these QTLs can be mapped would be greatly improved and, in many cases, adequate to carry out direct identification of candidate genes. It is likely that, given the complexity of the immune system development, the number of cells involved in an immune response, and especially the changes in the immune system with ageing, mapping hundreds of genes will be required to fully understand age‐related changes in the immune response. This review outlines ongoing and future strategies that will enable the mapping and identification of these genes.

G-CSF Primed Autologous Marrow Harvest and Transplantation in Cytapheresis “Mobilization Failure” Patients: A Descriptive Analysis

Fifteen cancer patients, deemed blood HSC “mobilization failures” due to CD34+cell yields of < 0.5×106/kg from two consecutive daily cytaphereses, underwent G-CSF primed autologous bone marrow harvest in an attempt to obtain adequate hematopoietic support for subsequent autotransplantation. CD34 + cell yields from the primed marrow harvest were variable; however, some patients had > 5-fold increases in CD34 + cell yields in the marrow compared to cytapheresis, and 4 patients had CD34 + cell yields of > 1.0 (i.e., 1.2, 1.44, 1.61 and 2.45)×106/kg from the primed marrow harvest. None of the five patients previously exposed to stem cell toxins or fludarabine achieved > 0.85×106/kg CD34 +cells with the primed marrow harvest. A significant difference was noted between G-CSF primed blood and marrow for CD34 + cells but not for GM-CFU (p = 0.011 and p = 0.135, respectively, paired t-test). All evaluable patients engrafted; a median ANC > 0.5×109/L recovery was achieved on D + 12 (range+9 to+17) in 12 of 13 evaluable patients—one died on D + 9 without recovery. The last day of platelet transfusion occurred at a median D + 13 (range + 8 to > + 66); only one patient remained platelet transfusion-dependent beyond D + 34. As anticipated, patients with higher numbers of CD34 + cells transplanted had somewhat more rapid recoveries. Although stem cell damage is obviously a key factor in mobilization failure patients, these findings raise the possibility that poor mobilization, at least in some patients, results from a mechanism other than, or in addition to, simple stem cell damage. Moreover, they raise the issue of the minimum number of marrow CD34 +—or more arguably other—cells needed for adequate short- and long-term reconstitution. The role of G-CSF in this situation, especially regarding dose and/or schedule, is intriguing but remains to be clarified. G-CSF primed marrow harvest is a potential option in certain poor mobilizers but, as fully expected, is frequently inadequate. Whether such is preferable to “steady-state” marrow harvest, continued or repeated G-CSF primed cytapheresis (with or without chemotherapy), or primed marrow with G-CSF in other schedules—or with other cytokines—is unclear and will be the subject of further study.

The isoflavone genistein inhibits LPS-stimulated TNFα, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects

BACKGROUND Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Stemline™ Hematopoietic Stem Cell Expansion Medium, a Serum-Free Medium for the Expansion of CD34+ Hematopoietic Stem Cells and Progenitors

Hematopoietic stem cells (HSC) have the ability to repopulate the hematopoietic system by differentiating into all of the necessary erythroid, lymphoid, and myeloid lineages. Due to this rare ability, HSC are used as therapeutic agents in the treatment of malignant and benign diseases of the blood forming and immune systems. There have been many advances in the area of clinical HSC research, but the availability of suitable cells for transplantation still remains a major limiting factor. HSC can be isolated from three different sources: umbilical cord blood (CB), bone marrow, and mobilized peripheral blood. CB is currently the preferred source because it has been shown to have a lower risk of graft versus host disease (GVHD), presumably due to its immunological naivete. However, because the volume of CB is limited, each umbilical cord has only enough cells to successfully transplant a small child. In order to transplant an adult, the HSC from CB must be expanded ex vivo. The expansion must be performed in a manner to ensure that the HSC not only differentiate along appropriate hematopoietic lineages, but also self-renew, leaving undifferentiated stem cells in the expanded culture. In order to expand these very specific cell types, an optimized serum-free medium and cytokine cocktail are needed. To this end, Stemline Hematopoietic Stem Cell Expansion Media were developed for the expansion of HSC. They are serum-free media that allow for expansion of both differentiated and undifferentiated HSC. StemlineTM and StemlineTM II are both able to expand HSC from CB, bone marrow, and mobilized peripheral blood. In bench-scale and clinical-scale expansions, both media have

Autonomous regulation of stem cell frequency

Abstract Lineage restricted growth factors regulate peripheral blood cell homeostasis. However, mechanisms that affect the frequency of primitive stem cells with long term proliferative potential remain largely unknown. We have shown that stem cell numbers in DBA/2 (D2) mice are higher than in C57BL/6 (B6) mice. To assess whether this is a cell-extrinsic (e.g. due to variation of growth factor concentrations) or cell-autonomous trait we have developed a model in which D2 and B6 stem cells co-exist is a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 recipients with mixtures of D2 and B6 day-14 fetal liver cells. Donor leukocyte contribution was assessed using anti-H2K b/d antibodies. In agreement with our previous findings, repopulating ability of D2 fetal liver cells was ∼10 fold higher than B6 cells. In order to determine the actual frequency of D2 and B6 stem and progenitor cells in these chimeric animals, we performed several experiments. Firstly, mice in which D2 and B6 leukocyte contributions were equal, were injected with G-CSF. Peripheral blood CFU-GM were cultured and the genotype of each colony was assessed using SSLP-PCR. D2 CFU-GM were 4-8 fold more numerous than B6 progenitors. Marrow was harvested from these chimeric mice and D2 and B6 cell populations were separated by flow cytometry. CAFC analysis on sorted samples showed that the frequency of D2 late appearing CAFC subsets was ∼10 fold higher, indicating that chimerism of mobilized progenitors reflected marrow stem cell chimerism. To confirm these in vitro data we performed a secondary transplantation using unfractionated chimeric marrow, transplanted in limiting doses to F1 recipients. Comparison of the proportion of animals showing D2 and B6 leukocyte contributions revealed that the frequency of D2 LTRA was ∼ 10 fold higher than B6 LTRA numbers. Our data demonstrate that two genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotype/frequency. This indicates that stem cells maintain their population size autonomously.

Bi-phasic CD34+ cell mobilization of a syngeneic donor during prolonged G-CSF delivery.

BACKGROUND G-CSF administration over 10 days and neutrophil cytapheresis have been reported in the literature, but the kinetics of CD34+ cells in this situation is unclear. CASE A 42-year-old female underwent syngeneic transplantation for metastatic breast cancer. The recipient was in critical condition peri-transplant, therefore the donor received G-CSF for 13 days, during which eight cytaphereses for both PBPC and neutrophils were performed. Two peaks in CD34+ cells were noted; the first on Day 5 and the second on Days 10-13 of G-CSF administration; a total of 11.6 x 10(6)/kg CD34+ cells and 38.11 x 10(8)/kg neutrophils were infused. The recipients ANC exceeded 0.1 x 10(9)/L on Day +3. DISCUSSION To our knowledge, this is the longest reported cytapheresis of CD34+ cells from a normal donor The bi-phasic pattern in the cytapheresis product is also of interest. It is an unusual pattern that suggests a profound and complicated alteration in the marrow progenitor cell pool. If substantiated, this finding may offer an alternative cytapheresis schedule for donors.