Repopulation Kinetics Research Articles

Impaired Hepatocyte Regeneration in Acute Severe Hepatic Injury Enhances Effective Repopulation by Transplanted Hepatocytes

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 10(7)/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-alpha and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.

Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions

A new langerin(+) DC subset has recently been identified in murine dermis (langerin(+) dDC), but the lineage and functional relationships between these cells and langerin(+) epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin(+) dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin(+) dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin(+) dDC, in contrast to LC, did not require TGFbeta1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin(+) dDC and LC revealed that langerin(+) dDC were required for optimal production of beta-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin(+) dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.

Noninvasive Imaging of Liver Repopulation following Hepatocyte Transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix™ System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.

Characterization and quantification of clonal heterogeneity among hematopoietic stem cells: a model-based approach

AbstractHematopoietic stem cells (HSCs) show pronounced heterogeneity in self-renewal and differentiation behavior, which is reflected in their repopulation kinetics. Here, a single-cell–based mathematical model of HSC organization is used to examine the basis of HSC heterogeneity. Our modeling results, which are based on the analysis of limiting dilution competitive repopulation experiments in mice, demonstrate that small quantitative but clonally fixed differences of cellular properties are necessary and sufficient to account for the observed functional heterogeneity. The model predicts, and experimental data validate, that competitive pressures will amplify small clonal differences into large changes in the number of differentiated progeny. We further predict that the repertoire of HSC clones will evolve over time. Last, our results suggest that larger differences in cellular properties have to be assumed to account for genetically determined differences in HSC behavior as observed in different inbred mice strains. The model provides comprehensive systemic and quantitative insights into the clonal heterogeneity among HSCs with potential applications in predicting the behavior of malignant and/or genetically modified cells.

Differentiation Profiling of Marrow Stem Cells: A Megakaryocytic Hotspot and the Continuum Model of Hematopoiesis

Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. Standard hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum and believe that sensitivity to cytokines change as a stem/progenitor cells goes through cell cycle transit. We previously have shown that stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. We have demonstrated differentiation hotspots on a cell cycle continuum (Exp Heme 35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine LRH marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around giving an inductive signal after 32-hours in primary culture; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over half hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. Stem cell differentiation hotspots may eventually be utilized to alter repopulation kinetics after bone marrow transplantation improving recovery time of platelets and neutrophils, translating into improved outcomes.

Linear quadratic and tumour control probability modelling in external beam radiotherapy

The standard linear-quadratic (LQ) survival model for external beam radiotherapy is reviewed with particular emphasis on studying how different schedules of radiation treatment planning may be affected by different tumour repopulation kinetics. The LQ model is further examined in the context of tumour control probability (TCP) models. The application of the Zaider and Minerbo non-Poissonian TCP model incorporating the effect of cellular repopulation is reviewed. In particular the recent development of a cell cycle model within the original Zaider and Minerbo TCP formalism is highlighted. Application of this TCP cell-cycle model in clinical treatment plans is explored and analysed.

Chemokine-mobilized adult stem cells; defining a better hematopoietic graft

Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.

Initially Accelerated Fractionation of the R1H Tumor of the Rat Increases Local Tumor Control and Might Reduce Impact of Overall Treatment Time

To study the effect of treatment time prolongation following initial dose acceleration on the response of subcutaneously growing R1H tumor. Continuous standard fractionation (30 fractions/40 days) was compared to initially accelerated treatment (30 fractions/21 days) followed by five to two fractions per week yielding total treatment times from 40 to 72 days. Local tumor control was assessed as endpoint. Radiation dose to control 50% of the tumors (TCD50%) decreased statistically significant from 83.5 Gy (95% confidence interval [CI]: 78.6 .. 88.4) for standard fractionation to 74.1 Gy (95% CI: 72.7 .. 75.5) determined for all accelerated treatment arms (p = 0.003). Prolongation of treatment time after initial acceleration from 40 to 72 days led to a small but statistically not significant increase in TCD50% from 72.0 Gy (95% CI: 71.0 .. 72.9) to 76.2 Gy (95% CI: 69.9 .. 82.4) corresponding to a repopulated dose of 0.9 Gy per week. This time factor is considerably smaller than for conventional radiation treatment as determined in previous experiments. The results indicate that initially accelerated irradiation not only improves local tumor control but also minimizes the negative effect of treatment time prolongation. This might be due to changes in tumor cell repopulation kinetics.

Multiarm High-Throughput Integration Site Detection: Limitations of LAM-PCR Technology and Optimization for Clonal Analysis

Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix. Using artificial mixtures of retrovirally marked, single-cell-derived clones, we demonstrate that LAM-PCR fails to detect 30-40% of the clones, even after exhaustive analysis. Furthermore, the relative abundance of specific clones within a mix is not accurately represented, deviating by as much as 60-fold from their true abundance. We describe an optimized, multiarm, high-throughput modification of LAM-PCR that improves the global detection capacity to greater than 90% with exhaustive sampling, facilitates accurate estimates of the total pool size from smaller samplings, and provides a rapid, cost-effective approach to the generation of large insertion-site data bases required for evaluation of vector integration preferences. The inability to estimate the abundance of individual clones within mixtures remains a serious limitation. Thus, although LAM-PCR is a powerful tool for identification of integration sites and for estimations of clonal complexity, it fails to provide the semiquantitative information necessary for direct, reliable tracking of individual clones in a chimeric background.

Kinetics and functional assay of liver repopulation after human cord blood transplantation

Background and aims To evaluate donor cell engraftment and the kinetics of cell repopulation in the injured mouse liver following human umbilical cord blood cell transplantation. Methods Nonobese diabetic/severe immunodeficient mice were treated with allyl alcohol to induce liver injury. Twenty-four hours later, umbilical cord blood derived mononuclear cells were transplanted by intra-splenic injection. Mice were sacrificed from 1 to 180 days after transplantation. Temporal changes in the ratio of human cells and fluorescence counts of human sex-determining region Y alleles in mouse liver were determined to evaluate the kinetics of cell repopulation. Mouse liver and sera were examined for the presence of human albumin. Results Human cell repopulation was extremely rapid in the first week following transplantation, with a doubling time of 1.16–1.39 days apparent. Thereafter cell doubling rate slowed significantly. Cells displaying characteristics of human hepatocytes were still evident at 180 days. Human albumin was detected in mouse liver and sera. Conclusion These findings confirm those from previous studies demonstrating that cells derived from human umbilical cord blood have the capacity to differentiate into cells with human hepatocyte characteristics in mouse liver following injury. Moreover, the detailed information collected regarding the kinetics of human cell repopulation in mouse liver will be of relevance to future studies examining the use of umbilical cord blood cells in liver transplantation therapy.

Investigation of various growth mechanisms of solid tumour growth within the linear-quadratic model for radiotherapy

The standard linear-quadratic survival model for radiotherapy is used to investigate different schedules of radiation treatment planning to study how these may be affected by different tumour repopulation kinetics between treatments. The laws for tumour cell repopulation include the logistic and Gompertz models and this extends the work of Wheldon et al (1977 Br. J. Radiol. 50 681), which was concerned with the case of exponential re-growth between treatments. Here we also consider the restricted exponential model. This has been successfully used by Panetta and Adam (1995 Math. Comput. Modelling 22 67) in the case of chemotherapy treatment planning.Treatment schedules investigated include standard fractionation of daily treatments, weekday treatments, accelerated fractionation, optimized uniform schedules and variation of the dosage and α/β ratio, where α and β are radiobiological parameters for the tumour tissue concerned. Parameters for these treatment strategies are extracted from the literature on advanced head and neck cancer, prostate cancer, as well as radiosensitive parameters. Standardized treatment protocols are also considered. Calculations based on the present analysis indicate that even with growth laws scaled to mimic initial growth, such that growth mechanisms are comparable, variation in survival fraction to orders of magnitude emerged. Calculations show that the logistic and exponential models yield similar results in tumour eradication. By comparison the Gompertz model calculations indicate that tumours described by this law result in a significantly poorer prognosis for tumour eradication than either the exponential or logistic models. The present study also shows that the faster the tumour growth rate and the higher the repair capacity of the cell line, the greater the variation in outcome of the survival fraction. Gaps in treatment, planned or unplanned, also accentuate the differences of the survival fraction given alternative growth dynamics.

The evolving role of alemtuzumab (Campath-1H) for immunosuppressive therapy in organ transplantation

Alemtuzumab is a monoclonal anti-CD52 antibody, which has been used extensively off label in solid organ transplantation. Its primary use has been as an induction agent at the time of organ transplantation, although there is limited experience using it to treat steroid-resistant rejection. Prolonged lymphocyte depletion can be expected following alemtuzumab treatment even with one dose of 30 mg intravenously. The nature and kinetics of lymphocyte repopulation depend on the maintenance immunosuppression being administered. In comparison with Thymoglobulin, a polyclonal depleting antibody preparation, alemtuzumab offers significant practical benefits with lower cost, fewer side effects in administration, and no specific issues with i.v. access. The risks and benefits of depleting induction agents, such as alemtuzumab, are compared with nondepleting agents, such as anti-CD25 induction therapy. While the majority of experience in solid organ transplantation has been in kidney transplantation, there is more limited experience in liver, pancreas, islet, small bowel, and lung transplantation. We herein review some of the lessons learned from clinical experience to date in solid organ transplantation using alemtuzumab as an immunosuppressant.

The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets

AbstractHematopoietic stem cells (HSCs) display extensive heterogeneity in their behavior even when isolated as phenotypically homogeneous populations. It is not clear whether this heterogeneity reflects inherently diverse subsets of HSCs or a homogeneous population of HSCs diversified by their response to different external stimuli. To address this, we analyzed 97 individual HSCs in long-term transplantation assays. HSC clones were obtained from unseparated bone marrow (BM) through limiting dilution approaches. Following transplantation into individual hosts, donor-type cells in blood were measured bimonthly and the resulting repopulation kinetics were grouped according to overall shape. Only 16 types of repopulation kinetics were found among the HSC clones even though combinatorially 54 groups were possible. All HSC clones, regardless of their origin, could be assigned to this subset of groups, and the probability of finding new patterns is negligible. Thus, the full repertoire of repopulating HSCs was covered. These data indicate that the HSC compartment consists of a limited number of distinct HSC subsets, each with predictable behavior. Enrichment of HSCs (Lin–Rho–SP) changes the representation of HSC types by selecting for distinct subsets of HSCs. These data from the steady-state HSC repertoire could provide a basis for the diagnosis of perturbed patterns of HSCs potentially caused by disease or aging.

Scheduling of Radiation and Chemotherapy for Limited-Stage Small-Cell Lung Cancer: Repopulation As a Cause of Treatment Failure?

Themeta-analystcancastabroadnettogainstatisticalpower, possibly obtaining results clouded by the combining of heterogeneous trials, or can focus on more homogeneous data, thereby sacrificingpower.Inthisissue,DeRuysscheretal 1 havechosenthe latter approach to test the plausible hypothesis that initial chemotherapy or protracted radiotherapy can trigger repopulation of surviving cells in limited-stage small-cell lung cancer (LSCLC), thus decreasing the effectiveness of treatment. Repopulation of cells in critical normal tissues between individual dose fractions of either radiotherapy or chemotherapy is important for recovery or retention of normal organ function, thereby improving tolerance to treatment. Thus, a schedule of radiotherapy administered over 6 to 7 weeks leads to less normal tissuereactionthanasimilartotaldoseadministeredoverashorter time, and the kinetics of repopulation in the bone marrow allow repetitive scheduling of cycle-active chemotherapy at approximately 3-week intervals. However, repopulation of surviving tumor cells also occurs between dose fractions of either radiation or chemotherapy and effectively decreases tumor-cell kill (Fig 1). 2,3 There is evidence in several tumor types for an increase in the rate of repopulation during the latter part of a course of radiation treatment, which is an effect long recognized as a mechanism of clinicallysignificantresistancetotreatment. 4 Therearefewdataon the role of repopulation in the longer intervals between cycles of chemotherapy, but accelerating repopulation probably occurs and can lead to clinical resistance, even if there is no change in the inherent sensitivity of the cancer cells. 2

Tracking Differential Repopulation Kinetics of Human Hematopoietic Progenitor Cells Using MRI Detection of Nanoparticles.

Even within cell populations enriched for hematopoietic stem cells, there exists phenotypic and functional heterogeneity, with subsets of progenitors differentially contributing to the total character of the transplant. Here, we have used a dynamic in vivo imaging technology to track the differential homing kinetics of such subsets, within a well defined stem cell pool, and identified patterns of short term homing unique to CD31 versus CD34 cells.Human umbilical cord blood derived (hUCB) hematopoietic progenitor cells (HPC) were isolated on high expression of aldehyde dehydrogenase (ALDH) to yield a population of cells we have previously reported to be enriched for stem cell activity by phenotypic and functional parameters. Cells were plated 12 hours on fibronectin in X-Vivo15 supplemented with SCF, flt-3, and TPO. During incubation, cells were labeled with Bang's Laboratories 0.9um SPIO nanoparticles containing the Dragon Green fluorescent dye at a ratio of approximately 10:1 beads to cells. Positively loaded cells were immunomagnetically selected and transplanted into sublethally irradiated NOD/SCID/MPS7 mice. The MPS7 mouse model is highly permissive of human cell engraftment, and allows easy identification of donor cells by virtue of a genetic defect in beta-glucuronidase production. Post-transplant, animals underwent MRI every 30 minutes for 12 hours to develop a dynamic, real-time survey of donor migration between organs, and then sacrificed at optimal times for analysis by flow cytometry.The utility of this approach was validated by quantitative PCR for human beta-globin versus murine rapsyn to correlate loss of MR signal intensity to human cell engraftment. At 12 hours, MR signal intensity had decreased by 29% relative to time 0, with internal standardization against a water control. In contrast, repetitive imaging of a non-transplanted animal resulted in deviation of only +/− 4% from the mean. This loss of MR signal correlated to a spleen engraftment of as few as 10,000 cells (homing efficiency of ~2.5%) in some cohorts. This demonstrates the efficacy and resolution of this paradigm for real time analysis and manipulation of homing in vivo.We have noted a preferential affinity of CD31+ ALDH high HPC that highly co-express markers for CD117 and CD133 to the splenic vasculature as early as 1 hour post-transplant and peaking at 12 hours post-transplant. Alternatively, we observed initial marrow seeding to contain almost exclusively CD34+ cells with little co-expression of CD31 or even CD133. Additionally, we found that the spleen-homed fractions of cells also express classical markers of activation such as CD71 and CD38, whereas the marrow homed fraction does not. From a functional standpoint, we believe these two subsets comprise two facets of contribution to the total graft, providing short-term radioprotection and cytokine delivery from the spleen-homed compartment, and long term durable engraftment and self-renewal from the marrow-homed compartment.

New Fine Structure within the Adult Hematopoietic Stem Cell Compartment Revealed by Longterm Analysis of Mice Repopulated with Single Cells or Their In Vitro Generated Clonal Progeny.

Assessments of hematopoietic stem cell (HSC) repopulating activity in vivo have historically relied on calculated average longterm (12–16 wk) progeny outputs using non-purified transplants, thereby precluding definitive clonal assignments of donor-derived cells. Viral marking circumvents this problem, but has not been used for large scale surveys. Heterogeneity observed in the repopulation patterns has generally been inferred to reflect stochastic processes. We now report the in vivo repopulation kinetics of 89 individual longterm repopulating cells (LTRCs) before (n=49) and after (n=40) 4 days of clonal growth in vitro. LTRCs were defined here as cells whose WBC progeny could be detected at levels of ≥1% for at least 16 wk in sublethally irradiated Ly5-congenic W41/W41 hosts. Recipients were transplanted with either freshly isolated, single lin−Rho−SP LTRCs or 4-day clones generated from similar cells in serum-free cultures (+ 300 ng/ml SF, 20 ng/ml IL-11 & 1ng/ml Flt3-L). 4, 8, 12, 16, and 24 wk post-transplant, blood samples were stained for donor-derived B, T, and myeloid cells using a procedure that identifies donor/recipient doublets and Ly6g/Mac1low cells (which have features of lymphoid rather than myeloid WBCs) to exclude false-positive myeloid events. Four distinct patterns of repopulation were revealed. Type 1 showed a delayed production of predominantly myeloid WBCs (low or undetectable before 12 wk) that increased progressively (reaching 0.4–15% of all WBCs by 16 wk). Type 2 showed a robust multilineage repopulation that remained stable or increased over time (6–84% of all WBCs at 16 wk). Type 3 also showed an initially robust pattern of multilineage repopulation (peak numbers of WBCs at 8–12 wk and 1–51% at 16 wk), but the contribution of donor-derived myeloid cells was transient (<0.5% by 16 wk). Type 4 showed a lymphoid-restricted pattern (myeloid contribution <0.5% at all time points), with repopulation levels peaking at 8 wk and decreasing thereafter (1–22% at 16 wk). Persisting granulopoiesis, indicated by a high proportion of donor-derived cells in the Ly6g/Mac1+SSChi population at 16–24 wk, clearly distinguished the type 1 and 2 patterns from types 3 and 4 which showed near or complete cessation of donor-derived granulopoiesis beyond 12 wk. Preliminary secondary transplant experiments show that donor-derived LTRCs (with and without longterm granulopoietic activity) were exclusively generated in primary recipients with type 1 and 2 repopulation patterns. Amongst the freshly isolated LTRCs, 18% (9/49) were type 1, 41% (20/49) were type 2, 22% (11/49) were type 3, and 18% (9/49) were type 4. In contrast, 4-day clones derived from cells of the same phenotype and containing LTRC activity showed a marked decrease in type 1 and type 2 activity with a corresponding increase in type 3 and type 4 activity: type 1 = 5% (2/41), type 2 = 18% (7/40), type 3 = 28% (11/40) and type 4 = 50% (20/40). Collectively, these data identify a new hierarchy of four biologically discrete states within the compartment of cells currently defined as LTRCs. Proliferation of LTRCs either in vitro or in vivo appears to induce an irreversible transition from one state to another (from Type 1 to 2 to 3 to 4), suggesting the existence of intrinsic molecular correlates for each of these states and specific mechanisms that underlie their sequential appearance.

Kinetics of EGFR expression during fractionated irradiation varies between different human squamous cell carcinoma lines in nude mice

Background and purpose Preclinical and clinical data indicate that high pretherapeutic EGFR expression is associated with poor local tumour control, possibly caused by a high repopulation rate of clonogenic cells during radiotherapy in these tumours. Previous data reported from our laboratory showed a correlation between EGFR expression and acceleration of repopulation in poorly differentiated FaDu human squamous cell carcinoma (SCC) during fractionated irradiation. To test whether this is a general phenomenon, two further SCC were investigated in the present study. Patients and methods GL and UT-SCC-14, two moderately well differentiated and keratinising hSCC, were grown as xenografts in nude mice. Functional data on the repopulation kinetics during fractionated irradiation for these tumour models have been previously determined. The expression of EGFR during fractionation was analysed by immunohistochemistry. Endpoints were the membrane-staining score and the proportion of EGFR-positive cells (EGFR labelling index). Results Different kinetics of EGFR expression during fractionated RT were found. In UT-SCC-14, EGFR staining score and labelling index increased significantly during radiotherapy. In GL SCC, the EGFR expression was unchanged. Both tumours are characterized by a small but significant repopulation rate during radiotherapy. Conclusions The expression of EGFR may change significantly during fractionated irradiation. No clear correlation between EGFR expression and the repopulation kinetics of clonogenic tumour cells during fractionated irradiation was found. The changes in EGFR expression during irradiation warrant further investigation on their prognostic implications and on their importance for therapeutic interventions.

Flt3 Ligand Treatment Reverses Endotoxin Tolerance-Related Immunoparalysis

Endotoxin tolerance, the secondary blunting of a subset of microbial product-driven responses, is presumed to provide protection from pathological hyperactivation of the innate immune system during infection. However, endotoxin tolerance can itself be harmful. A significant percentage of sepsis survivors exhibit the phenotype of systemic endotoxin tolerance, a state termed immunoparalysis. Similar immune hyporeactivity, associated with an elevated risk of succumbing to bacterial superinfection, is also seen in the aftermath of major trauma, surgery, and burns. We recently demonstrated that in vivo endotoxin tolerance in murine models involves dendritic cell loss as well as alterations in the responsiveness of macrophages and remaining dendritic cells. Furthermore, the kinetics of recovery from immunoparalysis-associated inhibition of proinflammatory and immunoregulatory cytokine production directly parallels the kinetics of dendritic cell repopulation in these models. Given this, we examined whether recovery from immunoparalysis could be accelerated therapeutically with flt3 ligand, a growth factor that stimulates the differentiation and mobilization of dendritic cells. Notably, administration of flt3 ligand rapidly reverses immunoparalysis in vivo, accelerating and amplifying repopulation of tissues with proinflammatory and immunoregulatory cytokine-producing dendritic cells.

Quantitative evaluation of long-term liver repopulation and the reconstitution of bile ductules after hepatocellular transplantation

The treatment of liver disease is severely limited by a shortage of donor livers. In trying to address this growing problem, hepatocellular transplantation (HTx) has received much attention as an alternative to whole organ transplant. However, the expansion of transplanted cells is at low level, and the reconstitution of functional liver tissue is limited by this cellular property. We set up an animal model to better understand cell dose effect and the kinetics of liver repopulation following HTx. Dipeptidyl peptidase IV (DPPIV)-deficient rats treated with retrorsine and subjected to partial hepatectomy were infused with DPPIV-positive hepatocytes. Rats were injected with varying numbers of donor hepatocytes down to 100 cells low, and liver repopulation was examined at different time points up to 20 mo long. Repopulation was assessed by computer-aided quantitative detection. Transplanted hepatocytes underwent multiple rounds of proliferation and stably repopulated the injured livers after 20 mo and at all cell doses. Transplanted cells divided 14 times within the 3-mo time period following infusion, and the liver repopulation reached a plateau between 3 and 20 mo. Approximately 90% replacement occurred. Donor-derived cells also reconstituted the bile ductules of the recipients. The ability of transplanted hepatocytes to fully reconstitute injured livers strongly supports further investigation into the clinical potential of HTx. Additionally, the observation that transplanted hepatocytes also form components of the biliary system suggests that these cells may have bi-potential property of the stem cells.

Differences in the Kinetics of Hematopoietic and Stem Cell (SC) Engraftment Following Murine Newborn/Cord Blood (NB) and Bone Marrow (BM) Transplantation.

Cord blood is being used at an increasing frequency as a source of stem and progenitor cells in human hematopoietic transplantation. However, very little is known about the kinetics of engraftment of cord blood relative to bone marrow derived stem cells. We have used a murine model of newborn/cord blood transplantation to address this question. Our studies demonstrated that murine NB can provide long-term engraftment in primary, secondary, and tertiary transplant recipients. Although NB engrafts more slowly than BM, at one year, engraftment was similar in both types of recipients. We compared the rate of recovery of blood counts, hematopoietic progenitor counts and putative stem cell [SC] counts (i.e. Sca-1+, c-kit+, Lin- cells) in recipients of NB and BM transplantation. Interestingly, the SC ratio in mononuclear cells from donor NB/donor BM was 3.25 ± 0.8 (range = 2.45 – 4.75, n=15). Similarly, donor NB mononuclear cells contained approximately 30% of the number of megakaryocytic progenitors, 12% of the myeloid progenitors and 5% of the erythroid progenitors of donor BM mononuclear cells. The repopulation kinetics in recipients of donor BM and NB transplantation were analyzed at regular intervals, up to 8 months after transplantation. During the first two weeks, NB recipients had lower hemoglobin, WBC and platelet counts than BM recipients. However, by 1 month, the hemoglobin and WBC counts were at similar levels in NB and BM recipients. In contrast, NB transplantation recipients required 2 to 3 months to achieve platelet counts similar to those in BM recipients. These results are reminiscent of the well-known delayed platelet recovery following human cord blood transplantation. Progenitor cell counts in the bone marrow of recipients paralleled the hematological recovery described above. At 2 weeks post-transplantation, progenitor counts of all lineages in NB recipients were 25 to 35% of those in BM recipients. By 1 month, erythroid and myeloid progenitor numbers were similar in NB and BM recipients. In contrast, the appearance of megakaryocytic progenitors was delayed following NB transplantation and did not reach the same level as BM recipients until about 2 months after transplantation. During recovery, the number of SC in the bone marrow of both types of recipients increased gradually over time. At one month after transplantation, the number of SC in BM recipients was significantly greater than that in NB recipients, with a ratio of 4.2 ± 0.2. This SC ratio decreased gradually during the next several months. At 2, 4, and 8 months following transplantation, the ratios of SC in BM recipients/NB recipients were 3.5 ± 0.4, 2.6 ± 0.5, and 2.2 ± 0.3, respectively (n=5). This gradual decrease in the ratio of BM/NB SC suggests that NB SC increase more rapidly than BM SC. In conclusion, these data demonstrate that the kinetics of hematopoietic and stem cell recovery following NB and BM transplantation are significantly different. Although hematopoietic recovery after NB transplantation is slow at first, final engraftment is similar following NB and BM transplantation. Furthermore, the number of SC in NB recipients increases at a faster rate than the number of SC in BM recipients. These differences in SC recovery may be a reflection of differences either in the homing capacity or in the functional maturity of NB relative to BM SC. Further investigation is required to distinguish between these two possibilities.