In vivo fate-tracing studies using the Scl stem cell enhancer: embryonic hematopoietic stem cells significantly contribute to adult hematopoiesis

Evidence for the lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner, we expressed the tamoxifen-inducible Cre-ER(T) recombinase under the control of the stem cell leukemia (Scl) stem-cell enhancer in transgenic mice (HSC-SCL-Cre-ER(T)). To determine functionality, HSC-SCL-Cre-ER(T) transgenics were bred with Cre reporter mice. Flow cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis 5 months later. In order to investigate whether the de novo HSC generation is completed during embryogenesis, HSC-SCL-Cre-ER(T)-marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC compartment was not different, implying that no further HSC generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between midgestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ER(T) mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs.     

Clonal diversity of the stem cell compartment

PURPOSE OF REVIEW: Hematopoietic stem cells are functionally heterogeneous even when isolated as phenotypically homogenous populations. How this heterogeneity is generated is incompletely understood. Several models have been formulated to explain the generation of diversity. All of these assume the existence of a single type of hematopoietic stem cell that generates heterogeneous daughter stem cells in response to extrinsic or intrinsic (stochastic) signals. This view has encouraged the idea that stem cells can be instructed to adapt their function. Newer data, however, challenge this concept. Here, we summarize these findings and discuss their implication for applications of stem cells. RECENT FINDINGS: Hematopoietic stem cells that differ in function have been documented during development and within the adult stem cell compartment. The differences in function are stably inherited to daughter stem cells when these cells proliferate to self-renew. Collectively, the data show that the adult stem cell compartment consists of a limited number of distinct classes of stem cells. SUMMARY: The most important stem cell functions, including self-renewal and differentiation capacity, are preprogrammed through epigenetic or genetic mechanisms. Thus, stem cells are much more predictable than previously thought. Changes in the stem cell compartment through disease or aging can be interpreted as shifts in its clonal composition, rather than a modification of individual hematopoietic stem cells.     

Low-dose L-arginine administration increases microperfusion of hindlimb muscle without affecting blood pressure in rats

The objective of this work was to evaluate the influence of exogenous L-arginine on the capillary blood flow of peripheral tissues of normotensive subjects. Rats were anesthetized with sodium pentobarbital, and the blood flow of femoral, dorsal, and ventral skin and gastrocnemius and soleus muscle was measured by laser Doppler flow and microsphere methods to compare the blood flow before and after the L-arginine infusion. L-arginine lowered the mean blood pressure in a dose-dependent manner, but a statistically significant reduction in mean blood pressure was detected only at a high dose of 500 mg/kg of body weight. The significant blood flow increment was detected after the L-arginine infusion at doses of 50 and 150 mg/kg without causing hypotension. Nicardipine, a calcium channel blocker, also increased the skin blood flow, but the blood flow increment and blood pressure fall were comparable. A significant increment in microperfusion was detected in gastrocnemius, soleus muscle, and ventral skin compared with control group by the microsphere method. No adverse effects were observed during L-arginine and microsphere infusion. The present work indicates that l-arginine infusion increases muscle capillary blood flow in rats that are not performing exercise. Supplementation with l-arginine might provide additional blood flow at rest and during exercise and result in the improvement of muscle performance and exercise capacity.     

Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients

The factors affecting T cell reconstitution post haematopoietic cell transplantation (HCT) are not well characterised. We carried out a longitudinal analysis of T cell reconstitution in 32 HCT recipients during the first 12 months post transplant. We analysed reconstitution of na?ve, memory and effector T cells, their diversity and monitored thymic output using TCR rearrangement excision circles (TRECs). Thymic-independent pathways were responsible for the rapid reconstitution of memory and effector T cells less than 6 months post HCT. Thymic-dependent pathways were activated between 6 and 12 months in the majority of patients with na?ve T cell numbers increasing in parallel with TREC levels. Increasing patient age, chronic GVHD and T cell depletion (with or without pretransplant Campath-1H) predicted low TREC levels and slow na?ve T cell recovery. Furthermore, increasing patient age also predicted high memory and effector T cell numbers. The effects of post HCT immunosuppression, total body irradiation, donor leucocyte infusions, T cell dose and post HCT infections on T cell recovery were also analysed. However, no effects of these single variables across a variety of different age, GVHD and T cell depletion groups were apparent. This study suggests that future analysis of the factors affecting T cell reconstitution and studies aimed at reactivating the thymus through therapeutic intervention should be analysed in age-, GVHD- and TCD-matched patient groups.     

Accumulation of DNA damage in the aged hematopoietic stem cell compartment

Aging is associated with loss of functional potential of multiple tissue systems, and there has been significant interest in understanding how tissue-specific cells contribute to this decline. DNA damage accumulation has been widely associated with aging in differentiated cell types. However, tissue-specific stem cells were once thought to be a geno-protected population, as damage accrued in a stem cell population has the potential to be inherited by differentiated progeny, as well as propagated within the stem cell compartment through self-renewal divisions. This review will discuss the evidence for DNA damage accumulation in the aged HSC compartment, potential drivers, and finally the consequences of the acquired damage.     

Analysis of parameters affecting engraftment in children undergoing autologous peripheral blood stem cell transplants

Eighty-three pediatric patients underwent autologous peripheral blood stem cell transplants at a single institution and were included in a study evaluating the correlations between five engraftment parameters and the time to both neutrophil and platelet recovery. The parameters included: the number of nucleated cells per kg (TNC/kg), the absolute CD34+ cell content per kg (CD34+/kg), the number of mononuclear cells per kg (MNC/kg), the number of BFU-E/kg, and the number of CFU-GM/kg. A two-tailed Mann-Whitney test (alpha = 0.05) was used to determine if there were significant differences between patients with neuroblastoma (n = 45) and patients with other diagnoses (n = 38). No statistically significant differences existed between neuroblastoma patients and patients with other diagnoses. Therefore, the two groups of patients were pooled together. Data were analyzed using both a univariate and multivariate correlation method and Student's t-test (alpha = 0.05). Two statistically significant logarithmic relationships were found. The first relationship was between MNC/kg and time to ANC reconstitution (P = 0.05). The second relationship was between CFU-GM/kg and time to platelet recovery (P = 0.01). Based on the statistical data, we conclude that there is no correlation between nucleated cell dose, CD34+ cell dose, and BFU-E content with either neutrophil or platelet recovery. Accordingly, in this study MNC cell dose per kilogram was the most important parameter predicting the length of time between graft infusion and neutrophil recovery while CFU-GM content per kilogram was the most important parameter predicting the length of time until platelet recovery.     

Cytogenetically aberrant cells in the stem cell compartment (CD34+lin-) in acute myeloid leukemia

Leukemia may be viewed as a clonal expansion of blast cells; however, the role of primitive cells and/or stem cells in disease etiology and progression is unclear. We investigated stem cell involvement in leukemia using fluorescence in situ hybridization (FISH), immunofluorescence labeling of hematopoietic subpopulations, and flow cytometric analysis/sorting to discriminate and quantify cytogenetically aberrant stem cells in 12 acute myeloid leukemia (AML) and three myelodysplastic (MDS) specimens. Flow cytometric analysis and sorting were used to discriminate and collect a primitive subpopulation enriched in stem cells expressing CD34+ and lacking CD33 and CD38 (CD34+lin-). A subpopulation containing progenitors and differentiating myeloid cells expressed CD34, CD33, and CD38 (CD34+lin+). Nine specimens contained less than 10% CD34+ cells and, thus, were considered to be CD34- leukemias. Mature lymphoid, myeloid, and erythroid subpopulations were sorted on the basis of antigen-linked immunofluorescence. Cytogenetically aberrant cells in sorted subpopulations were identified using FISH with enumerator probes selected on the basis of diagnosis karyotype. Cytogenetically aberrant CD34+lin- cells were present at frequencies between 9% and 99% in all specimens. CD34+lin- cytogenetically aberrant cells comprised between 0.05% and 11.9% of the marrow/blood specimens. Cytogenetically aberrant CD34+lin+ cells constituted 0.01% tp 56% of the marrow/blood population. These data demonstrate that aberrant cells are present in primitive CD34+ stem cell compartments, even in CD34- leukemias. Stem cell involvement was confirmed further by sorting lymphoid and erythroid subpopulations from eight specimens in which the predominant leukemic population lacked lymphoid/erythroid differentiation markers. In these specimens, as well as in multiple lineages, suggests involvement of a cell(s) with multilineage capabilities. The ability of aberrant CD34+lin- stem cells to contribute to clonal and compartment expansion within immunofluorescently defined subpopulations was evaluated to explore the functional phenotype of aberrant CD34+lin- cells. Analysis of compartment size and aberrant cell frequency suggests that frequency of cytogenetically aberrant stem cells is uncoupled from compartment size. These data suggest that cytogenetically aberrant cells in the primitive compartment show varying abilities to expand primitive compartments. Cytogenetically aberrant CD34+lin- cells precede the blast subpopulation in hierarchical maturation and may in some cases by considered preleukemic, requiring maturation or additional mutations before transformation (eg, compartmental expansion) occurs.     

Allogeneic peripheral blood stem cell transplantation results in less alteration of early T cell compartment homeostasis than bone marrow transplantation

Since low T cell counts evaluated 1 month after allogeneic bone marrow transplantation (BMT) are associated with an increased risk of leukemia relapse (Powles et al., Blood 1998; 91: 3481-3486), we compared, in a randomized multicentric clinical study, the peripheral blood cells obtained 30 days after allogeneic BMT vs allogeneic G-CSF-mobilized peripheral blood stem cell transplantation (BCT) in an HLA-identical setting. T cell counts were higher 30 days after BCT (718+/-142 cells/microl, n = 20) than after BMT (271+/-53 cells/microl, n = 26, P = 0.006). However, T cells were less activated after BCT than after BMT, as demonstrated by a lower expression level of CD25 and a lower percentage of HLA-DR+ and CD95+ T cells. Furthermore, CD4+, CD8+ and CD45RA+ post-BCT T cell counts correlated with the number of cells infused with the PBSC graft, while such a correlation was not observed between post-BMT counts and BM graft cell numbers, suggesting that the intensity of post-transplant peripheral lymphoid expansion and/or deletion differed between BCT and BMT. A comparison of the input of T cells expressing different CD45 isoforms with the post-transplant cell recovery further confirmed that, within the CD4+ T cell subset, post-transplant expansions occurred at a higher level after BMT than after BCT, affecting mainly the CD4+ CD45RO+ subset. Altogether, our data demonstrate for the first time in a randomized setting that homeostasis of the T cell pool is less altered early after BCT than after BMT. This may have a strong impact on the graft-versus-leukemia (GVL) effect and subsequent relapse rate.     

Ethanol increases the formation of NADP+ in rat hepatocytes

The acute effects of ethanol on total (bound + free) pyridine dinucleotides were determined in freshly isolated rat hepatocytes. Pyridine dinucleotides and adenine nucleotides were determined by high-performance liquid chromatography. Exposure of the hepatocytes to 8 mmol/L ethanol resulted in a decrease in NAD+ and an increase in NADP+ after 2 min incubation. There were no significant changes in NADH and NADPH. Ethanol decreased ATP and increased AMP after 2 min, whereas an increase in ADP was only apparent after 15 min of incubation. Ethanol 8 mmol/L and 100 mmol/L resulted in an increased incorporation of [32P] into NADP+ from [32P]-prelabeled NAD+ and ATP. Ethanol increased hepatocyte NAD+ kinase activity; this effect was blocked by 4-methylpyrazole but reproduced by 10 mumol acetaldehyde. These observations indicate that ethanol increases the synthesis of NADP+ and that this effect is most likely the result of increased NAD+ kinase activity. The ethanol-induced decrease of NAD+ may limit ADP ribosylation of nuclear proteins, whereas increases in NADP+ may stimulate the pentose phosphate cycle.     

Pre-mobilization therapy blood CD34+ cell count predicts the likelihood of successful hematopoietic stem cell mobilization

We examined pre-mobilization blood CD34+ cell count to predict ability to mobilize adequate peripheral blood progenitor cells (PBPC) in 106 cancer patients and 36 allogeneic donors. Mean pre-mobilization therapy blood CD34+ cell count was 3.1 cells x 10(6)/l (s.d. = 3.9, r = 0.3-37) and mean CD34+ cells collected were 5.3 x 10(6) cells/kg/leukapheresis procedure (s.d. = 7.0, r = 0.03-53). Yields correlated with pre-mobilization CD34+ cells x 10(6)/l (r = 0.37, P-value < 0.0001); correlation was stronger in allogeneic donors (r = 0.56, P-value = 0.0004) and males (r = 0.46, P-value < 0.0001). Based on classification and regression tree multivariate analysis, the predictive value of pre-mobilization blood CD34+ cell count was confounded by other variables, including age, gender, mobilization regimen and malignancy type. We generated an algorithm to predict a minimum PBPC yield of 1 x 10(6) CD34+ cells/kg/leukapheresis procedure after mobilization. A threshold pre-mobilization blood CD34+ cell count of 2.65 cells x 10(6)/l was the most important decision point in predicting successful mobilization. Only 2% of subjects with pre-mobilization blood CD34+ cell counts > 2.65 cells x 10(6)/l did not achieve the minimum per apheresis, whereas 24% with pre-mobilization values below threshold had inadequate mobilization. Prospectively identifying individuals at risk for mobilization failure would allow for improved treatment planning, resource utilization and time saving.     

Early restriction of peripheral and proximal cell lineages during formation of the lung

To establish the timing of lineage restriction among endodermal derivatives, we developed a method to label permanently subsets of lung precursor cells at defined times during development by using Cre recombinase to activate floxed alkaline phosphatase or green fluorescent protein genes under control of doxycycline-dependent surfactant protein C promoter. Extensive or complete labeling of peripheral lung, thyroid, and thymic epithelia, but not trachea, bronchi, or gastrointestinal tract occurred when mice were exposed to doxycycline from embryonic day (E) 4.5 to E6.5. Nonoverlapping cell lineages of conducting airways (trachea and bronchi), as distinct from those of peripheral airways (bronchioles, acini, and alveoli), were established well before formation of the definitive lung buds at E9-9.5. At E11.5, the labeled precursors of peripheral lung were restricted to relatively few cells along the bronchial tubes and clusters in bronchial tips and lateral buds. Thereafter, these cells underwent marked expansion to form the entire gas-exchange region in the lung. This study demonstrates early restriction of endodermal progenitor cells forming peripheral as compared with proximal airways, identifies distinct cell lineages in conducting airways, and distinguishes neuroepithelial and tracheal-bronchial gland cell lineages from those lining peripheral regions of the lung. This system for conditional gene addition or deletion is useful for the study of lung morphogenesis and gene function in vivo, and identifies progenitor cells that may serve as useful targets for cell or gene replacement for pulmonary disorders.     

Infusion of hemolyzed red blood cells within peripheral blood stem cell grafts in patients with and without sickle cell disease

Peripheral blood stem cell (PBSC) infusions are associated with complications such as elevated blood pressure and decreased creatinine clearance. Patients with sickle cell disease experience similar manifestations, and some have postulated release of plasma-free hemoglobin with subsequent nitric oxide consumption as causative. We sought to evaluate whether the infusion of PBSC grafts containing lysed red blood cells (RBCs) leads to the toxicity observed in transplant subjects. We report a prospective cohort study of 60 subjects divided into 4 groups based on whether their infusions contained dimethyl sulfoxide (DMSO) and lysed RBCs, no DMSO and fresh RBCs, DMSO and no RBCs, or saline. Our primary end point, change in maximum blood pressure compared with baseline, was not significantly different among groups. Tricuspid regurgitant velocity and creatinine levels also did not differ significantly among groups. Our data do not support free hemoglobin as a significant contributor to toxicity associated with PBSC infusions. This study was registered at clinicaltrials.gov (NCT00631787).     

Factors affecting volume reduction and red blood cell depletion of bone marrow on the COBE Spectra cell separator before haematopoietic stem cell transplantation

The COBE Spectra is used to volume/red blood cell (RBC) deplete BM before transplantation or cryopreservation. We have audited our results to identify the effect of transit time, refrigerated storage, age and cellular composition on mononuclear cells (MNC) and CD34+ cell recoveries, volume/RBC depletion and neutrophil engraftment. In total, 88 consecutive collections from autologous (n = 25) and allogeneic (n = 63) donors were included. The mean collection volume was 1250 +/- 398 ml with RBC content of 341 +/- 113 ml. The MNC and CD34+ cell recoveries were 83.3 +/- 18.5 and 88.1 +/- 18.9%, respectively, volume depletion was 88.2+/-4.4% and RBC depletion 98.3 +/- 1.8%. Neutrophil engraftment was achieved in 20.1 +/- 6.4 days. Factors affecting MNC and CD34+ cell recoveries were transit time (P = 0.0060), overall age (P < 0.0210) and MNC/CD34+ cell concentrations (P < 0.0313). The presence of crenated RBC also reduced CD34+ cell recovery (P = 0.0028). Refrigerated storage did not adversely affect cell recovery (P > 0.8161) or neutrophil engraftment (P = 0.8959). This study demonstrates that time in transit, overall age, MNC and CD34+ cell concentrations and RBC condition were important factors affecting processing. RBCs show artefacts soon after collection at ambient temperatures and these may interfere with the separation and collection of MNC/CD34+ cells. Refrigeration at 4-6 degrees C during transit and storage may reduce formation of RBC artefacts and maximize MNC and CD34+ cell recoveries.     

Impact of transplanted CD34+ cell dose in allogeneic unmanipulated peripheral blood stem cell transplantation

The impact of the CD34+ cell dose on chronic graft-versus-host disease (cGVHD) and the clinical outcome was analyzed in 41 consecutive adult patients submitted to allogeneic peripheral blood stem cell transplantation from HLA-identical siblings. The patients were classified into 'low' or 'high' CD34+ cell dose groups based on whether they received less or more than a median CD34+ cell dose of 10.5 x 10(6)/kg, respectively. There was a significant difference in the incidence of extensive cGVHD (low vs high group, 25.0 vs 66.7%, P=0.021) and relapse (47.6 vs 20.0%, P=0.049) between the two groups. With a median follow-up of 335 days, the 3-year survival estimate for the whole population was 47.9%, while that for the low and high groups was 29.9 and 67.8%, respectively (P=0.0434). An inverse relation was noted between the relapse rate and the incidence of extensive cGVHD (P=0.043). It would appear reasonable that the optimal dose of CD34+ cells should be determined based on the disease status or aggressiveness of the malignant cells in each patient. Yet, in the case of patients with a high risk of relapse, transplantation with a CD34+ cell dose of >10.5 x 10(6)/kg would seem to be acceptable to minimize the risk of relapse.     

CD34+-enriched-CD19+-depleted autologous peripheral blood stem cell transplantation for chronic lymphoproliferative disorders: high purging efficiency but increased risk of severe infections

OBJECTIVE: The main objective of this work was to decrease the incidence of relapse after autologous stem cell transplantation with a "double purging" procedure. METHODS: We used a "positive" (CD34) and "negative" (CD19) double selection method to improve the efficacy of "single purging" of hematopoietic harvests in poor-prognosis lymphoproliferative disorders. All patients included in the study had a positive molecular marker of their disease. Minimal residual disease (MRD) was studied by flow cytometry and PCR techniques during the purging procedure and after transplantation. RESULTS: Twenty-six patients fulfilled entry criteria. Median age of patients was 50 years (range: 33-66); 17 were male and 9 female. Thirteen (50%) of the patients mobilized an adequate number of CD34+ cells (>or=3 x 10(6)/kg) to proceed with the double-selection protocol. Twelve of the 13 harvests became PCR negative after purging. Ten patients were grafted with the selected products and all but one engrafted without delay. After a median follow-up of 30 months, 2 of 10 patients suffered a molecular relapse at 7 and 19 months respectively. The earlier relapse was observed in the patient who received a MRD+ product. Only one patient experienced a clinical relapse. Three patients died due to obliterans bronchiolitis, pneumococcal sepsis, and septic shock of unknown origin, respectively, and three others presented life-threatening infections. CONCLUSION: Therefore, CD34+/CD19+ positive/negative selection is an effective purging approach in patients with chronic lymphoproliferative disorders. This favorable effect is, however, counterbalanced by the high frequency of life-threatening infections.     

rHuG-CSF increases the platelet-neutrophil complex formation and neutrophil adhesion molecule expression in volunteer granulocyte and stem cell apheresis donors

Several reports have shown that granulocyte colony-stimulating factor (G-CSF) administration induces a transient, mild hypercoagulable state, which might predispose certain donors to thrombotic complications. In the present study, changes in the expression of neutrophil adhesion molecules (CD11b/CD18, CD62L) and platelet-neutrophil complex formation following rHuG-CSF administration were investigated in normal granulocyte and stem cell donors. For granulocyte apheresis (N = 10), rHuG-CSF (5 microg/kg) was given subcutaneously every 12 h three times and apheresis was carried out two hours after the last dose. For stem cell apheresis (N = 8), rHuG-CSF (10 microg/kg/day) was given subcutaneously for 5 days and apheresis was carried out when peripheral CD34+ cell counts exceeded 20 cell/microL. Expression of neutrophil adhesion molecules (CD11b/CD18, CD62L) and platelet-neutrophil complex formation following rHuG-CSF administration were investigated in donors by a flow cytometric method. A significant increase in neutrophil counts (P < 0.001), and decreases in platelet counts (P < 0.01) and hemoglobin levels (P < 0.01) occurred following G-CSF administration. The expression of CD11b/CD18 significantly increased (P < 0.001) over pretreatment values with G-CSF administration and returned to baseline 1 week after stopping the drug. In contrast, CD62L expression was decreased (P < 0.01) with G-CSF and returned to normal after cessation of the drug. rHuG-CSF caused more than a two-fold increase (from 0.3 to 7.0 x 10(9)/L) in circulating platelet-neutrophil complexes (P < 0.01), which returned to normal after 1 week. Although clinical significance of these laboratory changes is not clear, the occurrence of neutrophil activation and increased platelet-neutrophil complex formation might predispose certain donors or patients to thrombotic complications following G-CSF administration.     

CD34+ selected cells in mismatched stem cell transplantation: a single centre experience of haploidentical peripheral blood stem cell transplantation

Over the past 3 years we have performed 10 haploidentical peripheral blood stem cell transplants in patients with incurable haematological malignancies and no prospect of a matched unrelated donor within an adequate time period. Conditioning consisted of ATG, TBI, thiotepa, cyclophosphamide and additional radioimmunotherapy in five patients. All patients received G-CSF mobilized peripheral blood stem cell grafts. GVHD prophylaxis consisted of T cell depletion by CD34+ selection; no post-transplant immunosuppression was given in nine patients. Stable engraftment was achieved in nine patients; one case of acute graft rejection was observed. Seven patients developed grade I acute GVHD, and six patients have developed chronic GVHD. Infections were the most significant clinical problem post transplant. Two patients have suffered a relapse of their disease and two further patients have died of transplant-related complications. After a median follow-up of 13 months (range 5-37 months) six patients are surviving in remission. We conclude that haploidentical PBSCT is a reasonable alternative to a MUD transplant.