Division History Research Articles

Multi-type branching models to describe cell differentiation programs

Cell proliferation and differentiation is described by a multi-type branching process, a probability model that defines the inheritance of cell type. Cell type is defined by (i) a repression index related to the time required for S-phase entry and (ii) phenotype as determined by cell markers and division history. The inheritance of cell type is expressed as the expected number and type of progeny cells produced by a mother cell given her type. Expressions for the expected number and type of cells produced by a multi-cellular (bulk culture) system are derived from the general model by making the simplifying assumption that cell generation times are independent. The multi-type Smith–Martin model (MSM) makes the further assumption that cell generation times are lag-exponentially distributed with phenotype transitions occurring just before entry into S-phase. The inheritance-modified MSM (IMSM) model includes the influence of generation time memory so that mother and daughter generation times are correlated. The expansion of human cord blood CD34 + cells by haematopoietic growth factors was division tracked in bulk culture using carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE). The MSM model was fitted to division tracking data to indentify cell cycle length, and the rates of CD34 antigen down-regulation and apoptosis. The IMSM model was estimated for mouse granulocyte–macrophage progenitors using live cell imaging data. Multi-type branching models describe cell differentiation dynamics at both single- and multi-cell scales, providing a new paradigm for systematic analysis of stem and progenitor cell development.

Hematopoietic Stem Cell Cycling Dynamics In Steady State and Upon Hematopoietic Challenge

AbstractAbstract 572Life-long blood production is maintained by a small fraction of hematopoietic stem cells (HSCs). Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assays with 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic mouse models (Wilson et. al., 2008; Foudi et. al., 2009). While the former studies showed that all HSCs equally divide and likely contribute to blood formation (clonal maintenance), the latter suggested that some HSCs divide frequently and contribute to blood formation until cell death or full differentiation, while some HSCs are quiescent and then get activated to follow the same fate as frequently dividing ones (clonal succession). However, due to low resolution, none of the labeling techniques used were able to track single cell divisions. Furthermore, methods used might have direct influence on cycling activity of HSCs. Thus it remains to be determined a) if HSC divide continuously, sequentially or repetitively and contribute to steady-state hematopoiesis, b) what is a relationship between divisional history and repopulating ability, and c) how self-renewal and differentiation capacity of HSC is impacted by naturally-occurring severe hematopoietic challenges as infections. To address this directly, we set up a high resolution non-invasive in vivo HSC divisional tracking assay with CFSE (carboxyfluorescein diacetate succinimidyl ester). We here show that i.v. transfer of CFSE-labeled HSCs into non-conditioned congenic recipient mice allows evaluation of steady-state HSC cycling-dynamics as CFSE is equally distributed to daughter cells upon cellular division. Transfer of Lin-c-kit+Sca-1+ cells (LKS) into non-irradiated mice revealed non- and 1–7x divided LKS in recipient bone marrow over 20 weeks. To test in vivo limiting dilution and single cell HSC potential, non- or ≥5x divided cells were sorted based on divisional history from primary recipients at different weeks after transplantation, and transplanted into lethally irradiated secondary recipients. Single non-divided LKS at 3 weeks post primary transfer was able to multi-lineage repopulate 24% of recipients long-term, while 50 of ≥5x divided LKS did not engraft. Interestingly, both non- and ≥5x divided LKS at 7 or 12–14 weeks after primary transfer engrafted and showed fluctuating contribution to multi-lineage hematopoiesis over serial transplantation. Mathematical modeling based on limiting dilution transplantation, revealed no evidence for a dichotomy of biologically defined HSCs in different groups. Instead, steady-state serial transplantation with temporary fast-cycling cells revealed that they can slow down over time, suggesting dynamically changing cycling activity of HSC. We next tested the effects of hemato-immunological challenge on HSC proliferation. Mice transplanted with CFSE-labeled LKS cells were repetitively treated with LPS. Analysis 8 days after final LPS injection, i.e. three weeks after steady-state transplantation revealed that all LKS entered cell cycle and the number of ≥5x divided LKS was increased. Secondary transplantation showed that 2–4 time and ≥5x divided LKS from LPS-treated mice reconstituted multi-lineage hematopoiesis whereas both fractions from control mice failed to engraft. This data clearly indicate that HSCs are activated from quiescence upon LPS challenge and provide evidence, that naturally-occurring hemato-immunological challenges, such as gram-negative bacterial infection induces proliferation and self-renewal of HSCs. Our data suggest in contrast to previously proposed concepts, a novel “dynamic repetition” model for HSC cycling activity and blood formation where some HSCs participate in hematopoiesis for a while, subsequently enter a resting phase and get reactivated again to contribute to blood formation in repetitive cycles, leading to homogenous total divisional history of all HSCs at end of life. These findings might represent a biological principle that could hold true for other somatic stem cell-sustained organ-systems and might have developed during evolution to ensure equal distribution of work-load, efficient recruitment of stem cells during demand, and reduction of risk to acquire genetic alterations or fatal damage to the whole HSC population at any given time. Disclosures:No relevant conflicts of interest to declare.

Visual Activity Regulates Neural Progenitor Cells in Developing Xenopus CNS through Musashi1

Regulation of progenitor cell fate determines the numbers of neurons in the developing brain. While proliferation of neural progenitors predominates during early central nervous system (CNS) development, progenitor cell fate shifts toward differentiation as CNS circuits develop, suggesting that signals from developing circuits may regulate proliferation and differentiation. We tested whether activity regulates neurogenesis invivo in the developing visual system of Xenopus tadpoles. Both cell proliferation and the number of musashi1-immunoreactive progenitors in the optic tectum decrease as visual system connections become stronger. Visual deprivation for 2days increased proliferation of musashi1-immunoreactive radial glial progenitors, while visual experience increased neuronal differentiation. Morpholino-mediated knockdown and overexpression of musashi1 indicate that musashi1 is necessary and sufficient for neural progenitor proliferation in the CNS. These data demonstrate a mechanism by which increased brain activity in developing circuits decreases cell proliferation and increases neuronal differentiation through the downregulation of musashi1 in response to circuit activity.

The load of short telomeres, estimated by a new method, Universal STELA, correlates with number of senescent cells

Short telomeres are thought to trigger senescence, most likely through a single - or a group of few - critically shortened telomeres. Such short telomeres are thought to result from a combination of gradual linear shortening resulting from the end replication problem, reflecting the division history of the cell, superimposed by a more stochastic mechanism, suddenly causing a significant shortening of a single telomere. Previously, studies that have tried to explore the role of critically shortened telomeres have been hampered by methodological problems. With the method presented here, Universal STELA, we have a tool that can directly investigate the relationship between senescence and the load of short telomeres. The method is a variant of the chromosome-specific STELA method but has the advantage that it can demonstrate short telomeres regardless of chromosome. With Universal STELA, we find a strong correlation between the load of short telomeres and cellular senescence. Further we show that the load of short telomeres is higher in senescent cells compared to proliferating cells at the same passage, offering an explanation of premature cell senescence. This new method, Universal STELA, offers some advantages compared to existing methods and can be used to explore many of the unanswered questions in telomere biology including the role that telomeres play in cancer and aging.

Leishmaniadonovani s.l.: Evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry

Leishmania infantum causes visceral leishmaniasis in all countries in the Mediterranean basin. It uses Phlebotomine sandflies as vectors where the promastigote stage develops, reproduces and becomes infective. Therefore the reproductive power of the promastigotes determines the inoculum size of the isolate. Ten Leishmania strains from Cyprus: two Leishmania donovani and eight L. infantum were used to study the proliferation capacity of the promastigotes. Population increase during a 6-day culture period was assessed quantitatively, by haematocytometer enumeration, and qualitatively by following the division history of each population during the same period by CFSE staining and flow cytometry. The strains exhibited different proliferation rates with L. infantum showing higher multiplication rates than L. donovani. These differences may represent their fitness capabilities and their ability to synchronize the multiplication activity of individual members in the population for the production of a sizeable inoculum in time for the vector’s blood meal.

Co‐culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34+CD38− fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34+, CD133+ and CD38−) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.

In Vivo divisional Tracking System Reveals Cycling Heterogeneity in Long-Term Hematopoietic Stem Cells.

Abstract 814Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and give rise to all mature cells of hemato-lymphoid system for the lifetime of an individual. To ensure this, HSCs are kept at homeostatic levels in adult bone marrow. Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assay using 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., PLoS ONE 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic model systems (Wilson et. al., Cell 2008; Foudi et. al., Nat. Biotech. 2008). Based on the latter, it was suggested that one HSC pool turns over faster than another, dormant pool with very limited divisions during a lifetime. However, the fast cycling HSCs did not have long-term multilineage reconstitution capacity in lethally irradiated animals in contrast to dormant HSCs (Wilson et. al., Cell 2008; Foudi et.al., Nat. Biotech. 2008). From these experiments remained unclear, whether the faster cycling HSC loose long-term repopulation potential according to divisional history, or whether they represent progenitors with limited self-renewal potential, sharing a long-term HSC phenotype. Therefore, the dynamics of steady-state long-term HSC homeostasis and blood production remains to be determined.To address this directly, we set up an in vivo HSC divisional tracking assay. Here we show i.v. transfer of CFSE (carboxyfluorescein diacetate succinimidyl ester) -labeled HSCs into non-conditioned CD45.1/2 congenic F1 recipient mice that allows evaluation of steady-state HSC dynamics as CFSE distributes equally to daughter cells upon each cellular division. Sorted naïve CD4+CD62L+ T cells were used as non-dividing control cell population to determine the zero division CFSE staining level over time. Upon transfer of Lin-c-kit+Sca-1+ cells (LKS) into sublethally irradiated mice, all donor derived Lin-c-kit+ cells had divided >5 times after 3 weeks. However, transfer of LKS cells into non-irradiated mice revealed non-divided LKS cells in recipient bone marrow over 20 weeks. FACS analysis with HSC or progenitor specific marker expression showed that most of 0-2 time-divided and few of >5x divided LKS cells maintained a long-term HSC phenotype (CD150+, c-mpl+, CD34-). In order to test HSC potential, non- or >5x divided cells were sorted based on divisional history from primary recipients at different time points after transplantation, and competitively transplanted into lethally irradiated secondary recipients. At 3 weeks post primary transfer, single non-divided LKS cell was able to multi-lineage repopulate recipients, while 50 of >5x divided LKS cells showed no engraftment. Interestingly, both non- and >5x divided LKS cells at 7 or 12-14 weeks after primary transfer had long-term multilineage repopulating potential. Limiting dilution transplantation experiments demonstrated that HSC with long-term multilineage capacity (LT-HSC) were maintained at constant numbers that fit the numbers of free bone marrow niche space, with non-divided LT-HSC decreasing and >5x divided LT-HSC increasing with a constant division rate. We next tested the effects of hemato-immunological challenge on HSC cycling dynamics. Upon i.p. LPS injection into mice, previously transplanted with CFSE-labeled LKS, almost all LT-HSCs entered cell cycle within one week after challenge.These findings directly demonstrate that some LT-HSCs are quiescent for up to one fifth of the life-time of a mouse, while other LT-HSCs divide more actively, thus proving asynchronous LT-HSC division and contribution to hematopoiesis in steady-state. In addition, the results demonstrate that quiescent LT-HSCs are driven into division in response to naturally-occurring hematopoietic challenges, such as systemic bacterial infection. The CFSE-tracking model established here now allows to directly test the role of intrinsic versus environmental cues on cycling-dynamics of HSCs as well as leukemia initiating cells in steady-state and upon challenge on multiple genetic and different species background. Disclosures:No relevant conflicts of interest to declare.

Continuous Single Cell Observation Proves Lineage Instruction by Hematopoietic Cytokines.

Abstract 3607Poster Board III-543The significance of cytokines in hematopoiesis is irrevocable, but their contribution in lineage choice of multipotent progenitors is still controversial. Can late acting cytokines actively induce lineage decisions (instructive model), or do they only allow the survival and proliferation of already unilineage-restricted precursors, which have been differentiated by cell-autonomous mechanisms (selective/stochastic model)?A major reason for contradictory results is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. Both models can only be distinguished by constantly observing the fate of each cell and its progeny, but not by endpoint analyses of whole populations. Therefore, real-time tracking of individual cells in culture, tissues or whole organisms is an extremely powerful approach to fully understand the exact effect of cytokines on fate decisions.A computer aided culture and imaging system was developed to follow the fate of individual cells over long periods of time with high temporal and spatial resolution. New software modules were written, which help to record, display and analyze the divisional history, position, properties etc. of individual cells.Purified progenitor cells from mouse bone marrow of lineage-reporter mice were cultivated in defined media with the addition of selected cytokines. Video-time-lapse experiments enabled the tracking of individual cells and their progeny over many days. Pedigrees of hundreds of progenitor cells under each condition were generated and statistically analyzed. This novel single cell tracking technology revealed an unexpected instructive action of M-CSF and G-CSF directing lineage decisions of granulocyte-macrophage progenitors (GMPs) to differentiate into the monocytic or granulocytic lineage, respectively. Additionally, both late acting cytokines accelerated the differentiation process of GMPs. First results indicate an instructive in vivo action of these cytokines on progenitor lineage choice. Disclosures:No relevant conflicts of interest to declare.

Victory was Beyond Their Grasp: With the 272nd Volks-Grenadier Division from the Hürtgen Forest to the Heart of the Reich (review)

Reviewed by: Victory was Beyond Their Grasp: With the 272nd Volks-Grenadier Division from the Hürtgen Forest to the Heart of the Reich Steven R. Parker Victory was Beyond Their Grasp: With the 272nd Volks-Grenadier Division from the Hürtgen Forest to the Heart of the Reich. By Douglas E. Nash. Bedford, Penn.: Aberjona Press, 2008. ISBN 0-9777563-2-7. Guide to tactical symbols. Maps. Illustrations. Photographs. Appendixes. Notes. Bibliography. Index. Pp. xxxviii, 373. $24.95. In 1993 an American dealer in military documents journeyed to a small town in eastern Germany to purchase a collection of German military identity papers. Included in her purchase was a battered suitcase which, upon inspection, contained the almost complete files and documents for Füsilier Company 272 of the 272nd Volks-Grenadier Division, covering in detail the time from the unit's formation in September 1944 through its dissolution at the end of World War II. Fortunately [End Page 1004] for historians, the documents ended up in the possession of Douglas Nash, author of Hell's Gate: The Battle of the Cherkassy Pocket, January–February 1944. Nash used this treasure trove, along with other primary and secondary sources, as well as interviews with German and American veterans, to write a detailed and thorough history of this relatively unknown German division. Although it was in existence for less than a year, the division participated in several critical battles on the Western Front. Formed from the remnants of the 272nd Infanterie-Division that survived the intense fighting of the Normandy Campaign, the newly christened 272nd Volks-Grenadier Division had only six weeks of training before being deployed in the infamous Hürtgen Forest at the end of October, 1944. There it performed the critical mission of not only holding the current defensive positions, but also preventing the Americans from discovering the buildup of German troops in preparation for the Battle of the Bulge offensive. The division was successful in keeping American attacks in check, but at such a cost that it was judged too weak to participate in the ensuing offensive. After the failure of the Ardennes campaign, the division remained on the defensive along the Westwall, effectively delaying the Americans from capturing the Roer Dams for several weeks. Once pushed back across the Roer River, and suffering significantly from lack of supplies and support, the division endured a series of retreats, first to the Rhine, then to the Wied River, and finally most of the division was disbanded after being surrounded in the Ruhr Pocket. A few units managed to escape encirclement and make their way to the Harz Mountains before finally surrendering in late April, 1945. The amount of detail that Nash has been able to document is often quite remarkable, especially since during the last days of the Reich military records were often either nonexistent or deliberately destroyed. While ostensibly a divisional history, the narrative often describes events on a regimental, battalion, and even company level, often down to individual actions. Aiding the reader's understanding, Nash includes an excellent chapter discussing the organization and purpose of Volks-Grenadier Divisions. Often confused with Volkssturm units, which were usually made up of very young and very old conscripts, Volks-Grenadier Divisions were considered to be elite formations, often commanded by highly decorated officers. In the 272nd alone there were seven Knight's Cross holders, as well as eighteen soldiers decorated with the German Cross in Gold. The maps included in the book are very useful, although a few additional maps detailing the movements of the division's regiments and battalions would have been helpful, as the division often did not fight as a cohesive unit. The appendixes provide a detailed Order of Battle as well as well as a very helpful explanation of the numbering system used for creating the Volks-Grenadier divisions. Thanks to the efforts of an unknown German soldier in keeping the documents of his unit safe for almost forty years after the end of the war, Nash has written an excellent and highly readable history of an unheralded German division, and he has contributed significantly to our understanding of the final months of the Third Reich...

Cord blood stem cell expansion is permissive to epigenetic regulation and environmental cues

Augmentation of the number of cord blood (CB) hematopoietic stem cells (HSC) present in a unit is required before it can be considered as an alternative graft for hematopoietic reconstitution for adult patients. In order to further optimize strategies to augment HSC numbers, we examined whether expansion of HSC mediated by epigenetic mechanisms remains permissive to external environmental cues. The chromatin-modifying agents 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) were used to ameliorate epigenetic alteration of CB cells during ex vivo culture by adding various cytokines. After culture, CD34(+)CD90(+) cell numbers, their division history, in vitro clonogenic potential, and in vivo hematopoietic reconstitution potential and frequency were determined. 5azaD/TSA-treated, CD34(+)CD90(+) cells were greatly influenced in terms of their degree of expansion, clonogenic potential, cell-division rate, and transplantability by the combination of cytokines used in culture. Furthermore, our current results verify that the sequential addition of 5azaD followed by TSA is crucial for expansion of HSC. We demonstrate that following 5azaD/TSA treatment, the rate of CD34(+)CD90(+) cell division is also dependent on the cytokine cocktail and that this is associated with functional changes, including alteration of in vitro clonogenic potential and in vivo reconstitution potential. Our studies indicate there are interactions between intrinsic factors influenced by epigenetic mechanisms and external environmental signals in the regulation of HSC expansion. Epigenetic influences on HSC can be accentuated by environmental factors. Regulation of the rate of divisions may be a critical determinant for the maintenance of HSC functional potency during ex vivo expansion.

A novel view on stem cell development: analysing the shape of cellular genealogies

The analysis of individual cell fates within a population of stem and progenitor cells is still a major experimental challenge in stem cell biology. However, new monitoring techniques, such as high-resolution time-lapse video microscopy, facilitate tracking and quantitative analysis of single cells and their progeny. Information on cellular development, divisional history and differentiation are naturally comprised into a pedigree-like structure, denoted as cellular genealogy. To extract reliable information concerning effecting variables and control mechanisms underlying cell fate decisions, it is necessary to analyse a large number of cellular genealogies. Here, we propose a set of statistical measures that are specifically tailored for the analysis of cellular genealogies. These measures address the degree and symmetry of cellular expansion, as well as occurrence and correlation of characteristic events such as cell death. Furthermore, we discuss two different methods for reconstruction of lineage fate decisions and show their impact on the interpretation of asymmetric developments. In order to illustrate these techniques, and to circumvent the present shortage of available experimental data, we obtain cellular genealogies from a single-cell-based mathematical model of haematopoietic stem cell organization. Based on statistical analysis of cellular genealogies, we conclude that effects of external variables, such as growth conditions, are imprinted in their topology. Moreover, we demonstrate that it is essential to analyse timing of cell fate-specific changes and of occurrence of cell death events in the divisional context in order to understand the mechanisms of lineage commitment.

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)-derived HSC expanded in a serum-free/stromal-based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB-cells reached the ninth generation, whereas BM-cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7-3.8% of BM CD34(+) cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 +/- 6.3% to 30.6 +/- 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34(+)CD38(-) cells present at the end of culture arose from proliferating CD34(+)CD38(+) cells that downregulated CD38 expression, while in CB, a CD34(+)CD38(-) population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft.

Algunos avances y proyecciones en el campo de la pedagogía rural

Recibido 18 de junio 2007 • Aprobado 15 de octubre 2007
 
 
 Este artículo explora la forma en que se ha hecho Pedagogía Rural en la División de Educación Rural (DER) del Centro de Investigación y Docencia en Educación (CIDE), de la Universidad Nacional. Para destacar los avances de los últimos tiempos, se organiza este accionar pedagógico en dos grandes momentos, cada uno de ellos con sus características, logros y debilidades. Aunque describe con cierto detalle la forma en que se ha hecho Pedagogía Rural en el camino recorrido por la División, lo importante es destacar cómo esto permite un salto cualitativo hacia nuevos horizontes en la Pedagogía Rural que hace la DER. El artículo finaliza con algunas reflexiones sobre los factores que posiblemente decidirán el destino de la Pedagogía Rural en los ámbitos nacional y centroamericano.

Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells

Hematopoietic stem cells (HSCs) are thought to divide infrequently based on their resistance to cytotoxic injury targeted at rapidly cycling cells1, 2 and have been presumed to retain labels such as the nucleotide analogue 5-bromodeoxyuridine (BrdU). However, recently it has been demonstrated that BrdU-retention is neither sensitive nor specific for HSCs3. Here we show that transient, transgenic expression of a Histone2B (H2B)-Green Fluorescent Protein (GFP) fusion protein in mice allows superior labeling of HSCs and permits improved analysis of their turnover in combination with other markers. Mathematical modeling of H2B-GFP dilution in HSCs, identified with a highly stringent marker combination (L−K+S+CD48−CD150+)4, revealed unexpected heterogeneity in their proliferation rates and suggests that ~ 20% of HSCs turn over at an extremely low rate (≤ 0.8–1.8% per day). Prospective isolation and transplantation of L−K+S+CD48−CD150+ HSCs with different H2B-GFP levels revealed that higher H2B-GFP label retention correlates with superior long-term repopulation potential.

Hematopoietic Stem Cells Reversibly Switch from Dormancy to Self-Renewal during Homeostasis and Repair

Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.

Advanced age in horses affects divisional history of T cells and inflammatory cytokine production

A number of model systems have been employed to investigate age-associated changes in immune function. The purpose of the current study was to characterize senescent T cells and to investigate the inflamm-aging phenomenon both in vitro and in vivo using the old horse as a model. We examined whether decreased T cell proliferation induced by Con A is caused by increased apoptosis. We also utilized intracellular CFSE to analyze changes within each round of cell proliferation, in particular cytokine production. Intracellular staining with flow cytometry, RT-PCR, and ELISA were used to measure pro-inflammatory cytokines both in vitro and in vivo. While lymphocytes from old horses exhibit decreased proliferation, this is not the result of increased apoptosis. Instead, a larger percentage of the T cells remain in the parent generation and produce significant amounts of IFNγ. Likewise, old horses have increased frequency of CD8 −IFNγ + T cells and TNFα producing cells. We also show that old horses have elevated levels of IL-1β, IL-15, IL-18 and TNFα gene expression in peripheral blood and significant levels of TNFα protein in serum, all characteristics of inflamm-aging.

DNA Methylation in Benign Breast Epithelium in Relation to Age and Breast Cancer Risk

Many established breast cancer risk factors are related to the timing and duration of exposure to reproductive hormones, which are known to drive breast epithelial cell proliferation. The epigenetic molecular clock hypothesis suggests that CpG island methylation records the cell division history of benign epithelium. In proliferative epithelium, such as breast, this may provide an individualized cell-based measure of cancer risk. Methylation of cyclin D2, APC, HIN1, RASSF1A, and RAR-beta2 was measured by quantitative multiplex methylation-specific PCR in 290 benign and malignant breast epithelial cell samples obtained by palpation-directed fine-needle aspiration biopsy from 164 women. Univariate, multivariate, and unsupervised cluster analysis was used to establish the relationship between TSG methylation and a personal history of breast cancer, predicted breast cancer risk, and specific breast cancer risk factors. RASSF1A methylation was highly correlated with breast cancer risk [odds ratio (OR), 5.28; 95% confidence interval (95% CI), 1.95-14.32; P = 0.001], atypical cytology (OR, 4.11; 95% CI, 1.30-12.98; P = 0.016), and benign breast disease requiring biopsy (OR, 6.12; 95% CI, 1.41-26.51; P = 0.016). RASSF1A methylation increased linearly between ages 32 and 55. Increasing parity was associated with decreased APC methylation. TSG methylation increases in benign breast epithelium with increasing age. Because it is independently related to a personal history of benign or malignant breast disease and to predicted breast cancer risk, it may have value for breast cancer risk stratification and as a surrogate endpoint marker in prevention trials.

Prospective Identification and Characterization of Human Mesenchymal Stem Cell Subpopulations.

Mesenchymal stem cells (MSC) are multipotent cells that readily differentiate into a variety of cell types including osteoblasts, chondrocytes, and adipocytes. They support hematopoiesis (stroma function) and have potent immunomodulatory properties making them promising candidates for clinical use. Although MSC cultures are heterogeneous in morphology, no specific markers are available that would allow for the prospective isolation of distinct subpopulations. Therefore we investigated whether potential MSC subpopulation markers could be identified by gene expression profiling of MSC that were sorted based on functional parameters (i.e. proliferation characteristics). Human MSC were generated in standard cultures and stained with carboxyfluoresceine-succinimidylesther (CFSE) for cell division tracking. FACS analysis of CFSE-stained cells after 10 days culture showed that the majority of cells had undergone more than 3 cell divisions (51.2 ± 1.7%) whereas only 3.5 ± 0.9 % had not divided. Based on these results MSC were sorted by FACS according to their divisional history into non-proliferating cells (NP-MSC) and rapidly-proliferating cells (RP-MSC). Interestingly, the CFU-F frequencies of the NP-MSC were considerably (5-times) lower compared to RP-MSC. Furthermore, RP-MSC with low forward-/side-scatter properties had a higher CFU-F frequency than those with high scatter properties. Comparative oligonucleotide microarray analysis of FACS-sorted NP-MSC and RP-MSC populations identified a total of 112 differentially (≥4-fold) expressed genes. Fifty genes were significantly higher expressed in RP-MSC with 12 of them being cell cycle associated. Sixty-two genes showed a higher expression in NP-MSC and more than 20 of them were related to adhesion molecules, extracellular matrix proteins or cell senescence. Two of the differentially expressed genes (VCAM-1, FMOD) that corresponded to cell surface molecules were further tested for their potential to prospectively identify MSC subpopulations. Remarkably, FACS analysis using anti-VCAM-1 (CD106) and anti-fibromodulin (fmod) antibodies showed that approximately 40% and 3% of MSC were CD106neg/fmodneg and CD106pos/fmodpos, respectively. MSC were then sorted by FACS according to their expression of these markers and assayed for CFU-F frequency. Significant differences in CFU-F frequencies were observed between the double-negative and the double-positive MSC (12.3 ± 2.8 and 4.7 ± 2.1 colonies per 100 cells for CD106neg/fmodneg and CD106pos/fmodpos MSC, respectively). Experiments to test for differentiation potential differences are ongoing. Taken together, the data clearly demonstrate functional differences between MSC subpopulations. Furthermore, cell sorting based on proliferation characteristics and gene expression profiling enabled to identify surface markers that allowed for the first time to prospectively identify MSC subpopulations.

Human Mesenchymal Stromal Cells Regulate Initial Self-Renewing Divisions of Hematopoietic Progenitor Cells by a β1-Integrin-Dependent Mechanism

In previous reports, we have demonstrated that only direct cell-cell contact with stromal cells, such as the murine stromal cell line AFT024, was able to alter the cell division kinetics and self-renewing capacity of hematopoietic progenitor cells (HPC). Because beta(1)-integrins were shown to be crucial for the interaction of HPC with the bone marrow microenvironment, we have studied the role of beta(1)-integrins in the regulation of self-renewing cell divisions. For this purpose, we used primary human mesenchymal stromal (MS) cells as in vitro surrogate niche and monitored the division history and subsequent functional fate of individually plated CD34(+)133(+) cells in the absence or presence of an anti-beta(1)-integrin blocking antibody by time-lapse microscopy and subsequent long-term culture-initiating cell (LTC-IC) assays. beta(1)-Integrin-mediated contact with MS cells significantly increased the proportion of asymmetrically dividing cells and led to a substantial increase of LTC-IC. Provided that beta(1)-integrin-mediated contact was available within the first 72 hours, human MS cells were able to recruit HPC into cell cycle and accelerate their division kinetics without loss of stem cell function. Activation of beta(1)-integrins by ligands alone (e.g., fibronectin and vascular cell adhesion molecule-1) was not sufficient to alter the cell division symmetry and promote self-renewal of HPC, thus indicating an indirect effect. These results have provided evidence that primary human MS cells are able to induce self-renewing divisions of HPC by a beta(1)-integrin-dependent mechanism.

Differential cell division history between neutrophils and macrophages in their development from granulocyte–macrophage progenitors

The appearance of monocytes before neutrophils in the blood during haematopoietic recovery in myelosuppressive patients is commonly observed, thus suggesting a difference in the cell division history between these two lineages in the differentiation from granulocyte-macrophage (GM) progenitors. We investigated the cell division histories of murine GM progenitors. When analysed by the dye dilution method, GM progenitors gave rise to Gr-1+Fms+ and Gr-1+Fms- cells that passed through similar rounds of cell division during initial 5 d of culture. The Gr-1+Fms+ cells showed morphological features of monocytes, while Gr-1+Fms- cells exhibited an immature morphology of neutrophils. In the subsequent culture, a decline in the number of Gr-1+Fms+ cells was observed, while Gr-1+Fms- cells increased. The proliferation of Gr-1+Fms- cells and no cell division of Gr-1+Fms+ cells were confirmed by DNA staining, Ki-67 expression, membrane dye staining and bromodeoxyuridine incorporation. These Gr-1+Fms- cells acquired mature neutrophil morphology, whereas Gr-1+Fms+ cells became macrophages. These results demonstrate that GM progenitors generate postmitotic monocytes earlier than mature neutrophils. Our data may also offer one explanation for the rapid recovery of monocytes in comparison with neutrophils in the early phase of haematopoietic regeneration.