25 years of epidermal stem cell research.

This is a chronicle of concepts in the field of epidermal stem cell biology and a historic look at their development over time. The past 25 years have seen the evolution of epidermal stem cell science, from first fundamental studies to a sophisticated science. The study of epithelial stem cell biology was aided by the ability to visualize the distribution of stem cells and their progeny through lineage analysis studies. The excellent progress we have made in understanding epidermal stem cell biology is discussed in this article. The challenges we still face in understanding epidermal stem cells include defining molecular markers for stem and progenitor sub-populations, determining the locations and contributions of the different stem cell niches, and mapping regulatory pathways of epidermal stem cell proliferation and differentiation. However, our rapidly evolving understanding of epidermal stem cells has many potential uses that promise to translate into improved patient therapy

Optimizing doxorubicin-G-CSF chemotherapy regimens for the treatment of triple-negative breast cancer

La chimiothérapie cytotoxique reste une option de traitement de première intention pour la majorité des cancers. Un effet secondaire majeur dans les schémas chimio-thérapeutiques est la neutropénie. La thérapie prophylactique avec le facteur de stimulation des colonies de granulocytes (G-CSF), une cytokine endogène responsable de la régulation de la production de neutrophiles, est administrée en concomitance. Le moment et la dose exacts pour administrer la chimiothérapie et le G-CSF représentent des éléments cruciaux pour obtenir les résultats souhaités du traitement. En nous appuyant sur des travaux antérieurs qui optimisaient les schémas thérapeutiques du G-CSF, nous sommes basés sur une approche de pharmacologie quantitative des systèmes (QSP) pour étudier la fréquence et l’intensité de la dose dans le but de maximiser les effets anti-tumoraux de la chimiothérapie tout en minimisant la neutropénie. Dans ce travail, nous avons effectué une optimisation sur une large gamme de longueurs de cycle et de valeurs des doses de chimiothérapie afin d’identifier les meilleurs schémas en combinaison avec le G-CSF. Nos résultats suggèrent que la doxorubicine 45mg/BSA tous les 14 jours a un impact positif sur le contrôle de la croissance tumorale, et qu’il est préfèrable de retarder l’administration du G-CSF au septième jour après la chimiothérapie et de donner moins de doses pour minimiser le risque de neutropénie et le fardeau de ce médicament. Cette étude suggère des pistes possibles pour des schémas optimaux de chimiothérapie, avec le soutien prophylactique du G-CSF spécifiquement dans le contexte du cancer du sein triple négatif.Cytotoxic chemotherapy continues to be a first-line treatment option for the majority of cancers. A major side effect in chemotherapy regimens is neutropenia. Prophylactic therapy with granulocyte colony stimulating factor (G-CSF), an endogenous cytokine responsible for regulating neutrophil production, is administered concomitantly; the exact timing of the combination chemotherapy and G-CSF is crucial for achieving treatment results. Leveraging on previous work that optimized treatment regimens based on G-CSF timing, we developed a quantitative systems pharmacology (QSP) framework to study dose frequency and intensity of chemotherapy in order to maximize anti-tumor effects while minimizing neutropenia. In this work, we performed an optimization across a wide range of cycle lengths and dose sizes to identify the best cytotoxic chemotherapy regimens with G-CSF support. Our results suggest that doxorubicin 45mg/BSA every 14 days, has a positive impact on tumour growth control, and that to minimize the risk of neutropenia and the burden to patients it is best to delay the administration of G-CSF to day seven after chemotherapy and give fewer doses . This study suggests possible avenues for optimal chemotherapy regimens with prophylactic support of G-CSF in the context of Triple Negative Breast Cancer

Hematopoietic Stem Cells Are the Major Source of Multilineage Hematopoiesis in Adult Animals

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses

Temporal stochasticity leads to nondeterministic chaos in a model for blood cell production

All types of blood cells are formed by differentiation from a small population of pluripotent stem cells in the bone marrow. This population should maintain the balance between self-renewal and differentiation, even under severe perturbations, e.g. the massive cell death caused by chemotherapy or irradiation. The authors constructed a cellular-automata model for bone marrow dynamics, which retrieves its homeostatic capabilities even under periodic perturbations with constant or random amplitude. However, temporally stochastic perturbations result in a chaotic-like behavior. Several methods of analysis failed to distinguish between the time series in this case and a chaotic time series, although the chaotic-like behavior has no deterministic source

Modeling Stem Cell Population Dynamics

Because of the stochastic nature of biological systems, mathematical and computational modeling approaches have become more acceptable to experimentalists and clinicians in recent years as contributing to new understandings of complicated cell mechanisms and tissue physiology. Indeed, even single cell or small tissue samples are complex dynamic systems that adapt to environmental challenges in space and time which is poorly understood. Mathematical models and computer simulations can explain and uncover unknown aspects of cell behavior and tissue functions. Models based on key biological mechanisms can give interesting insights and formulate predictions that cannot be derived from physical experiments or statistical data alone. Therefore; novel research approaches should incorporate interdisciplinary dialogues between Biology, Mathematics and Computational Sciences to validate experimental data and non-intuitive scenarios such as the stem cell hypothesis. The tissue of a higher organism such as a human being can be described as a set of a large number of cells with certain functions and morphology. However, most of the mature cells are deprived of the potential to replenish themselves. Such imperfection of mature cells is compensated by the presence of a population of stem cells which possesses the capability to self renew and to differentiate into various cell lineages. This process of continual cell replacement, is called homeostasis, is critical for the maintenance of adult tissues, and is maintained through the presence of different control mechanisms. The homeostatic replacement: of cells varies substantially among different. tissues. Unquestionably, the most important ability of a stern cell is to maintain the homeostasis by continuously supplying specialized cells. The decision for an individual stem cell to either renew or differentiate can be described as a stochastic process. Several research programs supported by hospitals and health institutes are trying to understand the underlying mechanism of how stem cells proliferate, differentiate, and maintain equilibrium with or without feedback. At this stage researchers are not able to answer key questions, for example the rate of proliferation, stem cell homeostasis and feedback that plays a. crucial role in tissue equilibrium. This dissertation work is within the realm of Bioinformatics where computer scientists have to face more algorithmic challenges because of the huge amount of data with exception, numerous rules and conditions. This thesis attempts to present stochastic models which can predict stem cell growth, understand stem cell homeostasis characteristics, and formalize mathematical relationships of tissue lineage homeostasis

Mathematical models of granulopoiesis

Haemopoiesis (blood cell production) is a process subject to active physiological regulation. It constitutes one example of a biological process controlled through a hierarchy of feedback loops acting at a range of levels from the molecular to the macroscopic. The thesis describes mathematical studies of the more macroscopic (physiological) levels of the control of haemopoiesis, with special emphasis on granulopoiesis. Following review of pertinent background material in cybernetics, physiology and pathology, attention is focussed on the mathematical representation of cellular proliferation and maturation, and a representation formulated in tennis of experimental observables is proposed. This leads to the study of a non-linear transcendental equation of the form with the unknown quantity. An iterative method of solution is proposed for this equation, which permits kinetic analysis of a certain class of non-steady-state processes. The method is used for the analysis of maturation kinetics of embryonic erythroid cells. Attention is then turned to the causal basis of the control of haemopoiesis. It is pointed out that certain features of haemopoietic regulation lead to the expectation of oscillatory phenomena and th.at observation of such phenomena can be revealing. With this in mind, a simple model is advanced for the control of granulocyte production. The model comprises two feedback loops, one regulating 'de novo' granulopoiesis in accordance with marrow granulocyte numbers and one regulating release from the marrow in accordance with blood granu1ocyte numbers. The model is described by the system of delay-differential equations where Gm , GB are (respectively) the marrow granulocyte number and blood granulocyte number, both at time, and to are parameters, chosen to give maxim.um physiological realism. Since 'de novo' granulopoiesis is believed to constitute a drain on primitive 'stem, cells', regulation of stem cell number appears necessary. A model for stem cell mitotic autoregulation based on a diffusible inhibitor concept is proposed. This theory leads to the study of a pair of differential equations which, utilizing probable order-of-magnitude differences in relaxation times, may be reduced to the single equation which admits of a closed analytic solution. This equation exhibits both self-limiting and non-self-limiting modes of behaviour, the biological implications of which are discussed. With parameters selected for stability, the stem cell mitotic autoreguation loop may be adjoined to the granulopoiesis control system, previously considered, to obtain a composite model. However, the unstable and capricious behaviour of this multi-loop combination renders it physiologically unacceptable, Modifications which restore stability seem to imply heterogeneity of the stem cell population, representation of which lies outside the scope of the study described. In the following chapter, consideration is given to the physical, basis of regulation of 'de novo' granulopoiesis with emphasis on 'in vitro' evidence relating to 'colony stimulating factor' and the possible role of positive feedback in regulating granulocyte numbers in infection. By mathematical formulation of a. recently proposed pcsitive- feedback model it is shown that positive feedback systems can be stable in the absence of overt negative feedback loops provided passive damping elements (e.g, cell death) exist and satisfy'' certain criteria. It is shown, however, that the criteria concerned are incompa.tib3.e with known featu.res of the regulation of granulopoiesis in infection and the existence of additional, negatively-acting, loops is deduced. Some possibilities in this direction are proposed. The model studies may illuminate the pathogenesis of some disorders of the control of granulopoiesis, notably cyclical neutropenia and myeloid leukaemia

Mesenchymal niche contribution to normal and malignant hematopoiesis

Normal hematopoiesis is tightly regulated by hematopoietic microenvironment/niche in bone marrow (BM) via direct hematopoietic cell-niche cell interaction and factors secreted by various types of cellular niches. The BM niche consists of cells of mesenchymal cell origin including mesenchymal stem cells (MSCs) and progenitor cells (MPCs). Accumulated evidence suggests the important role of BM mesenchymal cell niche for the maintenance of normal hematopoiesis and leukemogenesis. However, the exact role of different niche elements and the molecular mechanisms in leukemia development remain poorly defined. Knowledge is required for developing new therapeutic strategy to effectively treat the diseases. This thesis focuses on mesenchymal niche contribution to normal and malignant hematopoiesis, particularly, myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML). By using mouse models, multi-color flow cytometry, RNA-sequencing, transplantation and lineage tracing techniques, the thesis work has demonstrated the contribution of BM mesenchymal cell niche in the initiation of the myeloproliferative disease and progression of AML. The specific role of BM MSCs and laminin isoforms in AML progression and therapy response were studied. In addition, this thesis reports the features of native skin MSCs and their function in supporting normal hematopoietic and AML stem cells. In paper I, the instructive role of BM niches in MDS/MPN initiation is studied by using signalinduced proliferation associated 1 (Sipa1) gene deleted mice (Sipa1-/-) that develop agedependent MPN. The loss of Sipa1 induces BM niche alterations prior to the disease onset, including biased differentiation of Sipa1-/- MSCs towards adipocytes and upregulated expression of pro-inflammatory genes (Il-6 and TGF-β). Concomitantly, hematopoiesis maintenance gene (Cxcl12, Angptl1, Kitl) expressions were reduced in Sipa1-/- BM MSCs and MPCs. Transplantation of Sipa1+/+ hematopoietic cells to young Sipa1-/- mice resulted in MDS/MPN development, supporting the causative role of Sipa1 deficient niche for the development of MDS/MPN. The role of BM MSCs and MPCs during progression of AML is reported in paper II. By transplanting MLL-AF9+ AML cells to immunocompetent mice, we showed dynamic niche alterations induced by AML cells. During AML development, frequency of BM MSC & MPC were increased while hematopoiesis gene (Kitl, Cxcl12, Angptl1, Nov and Igf1) expression in BM MPCs were down regulated in correlation to AML engraftment in BM. Moreover, the expression of pro-inflammatory gene (Il-6) is elevated following the AML progression. Specifically, BM primitive subset of MSC (Ebf2+) is altered by AML cells to generate more progenies including Ebf2-MSC, MPC and CD44+ cells in the leukemic niche. The depletion of Ebf2+ cells accelerated AML development, demonstrating the suppressive role of Ebf2+ MSCs in AML progression possibly by maintaining normal hematopoiesis. In this study, upregulation of laminin 4 (Lama4) in both MSC and MPC was observed. To further investigate the functional consequence of Lama4 during AML development, the Lama4 (Lama4-/-) deficient mice were employed in paper III. We firstly studied the role of Lama4 in hematopoiesis regeneration following irradiationinduced stress and observed impaired recovery of erythropoiesis and megakaryopoiesis in Lama4-/- mice. On the contrary, AML progression and relapse were accelerated post transplantation of MLL-AF9+ AML cells. Furthermore, the Lama4-/- MSCs promoted AML cell growth and confer AML stem cell chemoresistance to cytarabine (Ara-C) via providing more metabolic support to the AML stem cells (LSCs). Taken together, paper III shows critical role of Lama4 in hematopoiesis recovery following irradiation and during AML development. Recent study has shown that AML LSCs infiltrate extramedullary organ. Meanwhile, skin has been reported to contain MSC-like population although the characteristics are not well defined. In paper IV, we employed Ebf2-gfp transgenic mice to prospectively characterize skin MSC phenotypically and functionally at bulk and single cell level. Skin Ebf2+ cells represent purified MSC while the Ebf2- fraction contained more differentiated MSCs that can be generated by the Ebf2+ cells, revealed by the in vivo lineage tracing of Ebf2+ MSCs. Both skin Ebf2+ cells and Ebf2-MSC displayed hematopoiesis supportive function, similar to their BM counterpart. Furthermore, co-culture of AML and AML CAFC on skin Ebf2+ and Ebf2-MSCs showed that skin MSCs also supported normal HSCs and provided chemoprotection for AML LSCs. In skin tissue of AML mice, infiltration of AML cells was observed and remained in skin tissue after Ara-C treatment, suggesting a possible contribution of skin MSCs to the persistence of AML cells. The skin Ebf2+ were found to be reduced in AML mice. However, the functional consequence of the skin MSCs remains to be investigated in the future. Altogether, paper IV reports skin harbors Ebf2+ and Ebf2-MSC with similar characteristics to BM MSC. Both skin Ebf2+ and Ebf2-MSCs support normal HSC and AML cells. Importantly, skin MSCs provide chemoprotection for AML LSC. In conclusion, the work in this thesis shows the role of BM niche for the initiation and progression of the myeloid malignancies using several transgenic mouse models. The work also provides evidence for critical role of Lama4 in hematopoiesis recovery following irradiation and AML progression. Furthermore, the biological features of skin MSCs and their function in supporting normal hematopoietic and AML cells. During Ara-C treatment, skin MSCs also displayed protective role for AML LSCs, indicating skin MSC possible role as a reservoir of chemoresistant AML LS

Characterization of hematopoietic stem cells in the circulation

Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate into multiple cell lineages, giving rise to all blood components and immune cells, during the entire life of an individual. HSCs are localized in the bone marrow inside specialized compartments named “hematopoietic niches”. The niche contains stromal cells of mesenchymal origin, as is the case of adipocytes and osteoblasts as well as endothelial cells and cells of hematopoietic origin such as macrophages or megakaryocytes (1). All of these cells produce and deposit elements in the extracellular matrix but also secrete local hematopoietic cytokines that can induce or inhibit the proliferation and differentiation of progenitor cells. Early studies described that some of these HSCs are found travelling through the circulation of the organism (2). Additionally, the release of HSCs from the BM into peripheral blood follows circadian patterns, i.e. their numbers oscillate between day and night (3). In the present thesis we have analyzed whether HSCs in the circulation (named here circulating HSCs) have any physiological function and the mechanisms through which cHSCs are released into bloodstream. We have found that circulating HSC have a myeloid bias and are important for the repopulation of damaged niches. In addition, we found that multiple clones of these cHSCs enter the bloodstream and contribute to the regeneration of hematopoiesis in remote niches. We have found that the chemokine receptor CXCR2 is expressed in HSCs and is important for their homeostatic egress into the circulation. Genetic deficiency of Cxcr2 prevents the release of HSCs and the repopulation of remote damaged niches and gives rise to hematopoietic defects with age. Correspondingly, we have identified a population of perivascular cells inside the BM that express the chemokine ligand CXCL1 and could be key in the signaling of cHSC egress, and ultimately in preserving hematopoietic health through life.El estudio ha sido financiado por el proyecto SAF2015-65607-R otorgados al Dr. Andrés Hidalgo por el Ministerio de Economía, Industria y Competitividad (MEIC). Por su parte, Dª Itziar Cossío Cuartero ha sido beneficiaria de una beca FPI (SAF2015-65607-R). El CNIC recibe financiación del MEIC y la Fundación Pro-CNIC.N