Synergy of single-cell sequencing analyses and in vivo lineage-tracing approaches: A new opportunity for stem cell biology

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

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The future of mammary stem cell biology: the power of in vivo transplants

Breast Cancer Research ◽

10.1186/bcr1986 ◽

2008 ◽

Vol 10 (3) ◽

Cited By ~ 13

Author(s):

Geoffrey J Lindeman ◽

Jane E Visvader ◽

Matthew J Smalley ◽

Connie J Eaves

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Mammary Stem Cell ◽

Stem Cell Biology ◽

The Future ◽

Mammary Stem

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Preclinical Challenges and Clinical Target of Nanomaterials in Regenerative Medicine

Biomedical Engineering ◽

10.4018/978-1-5225-3158-6.ch059 ◽

2018 ◽

pp. 1402-1423

Author(s):

Martin Reinhardt ◽

Shibashish Giri ◽

Augustinus Bader

Keyword(s):

Tissue Engineering ◽

Stem Cell ◽

Cell Biology ◽

Stem Cell Biology ◽

Organ Engineering ◽

Cell Therapies ◽

Existing Problems ◽

Present Generation

Currently, practical application of nanotechnological approaches and stem cell therapies remains a challenge in both preclinical and clinical settings. Many existing problems in tissue engineering to organ engineering have been solved by the combined approaches of nanotechnology and stem cell biology, but significant barriers remain. Details about the role of various types of nanomaterial in preclinical and clinical research have been reviewed elsewhere, but scant information exists about the influence of nanomaterials on stem cell biology. Herein, the authors highlight the current advances of nanotechnological approaches for expansion, differentiations, harvesting, labeling, imagining, tissue engineering, and organ engineering of different types of stem cells. The preclinical outcome of in vitro and in vivo animal experimentations along with some examples of clinical outcomes of nanomaterials on stem cell research is the main focus of this chapter. This book chapter might be an impetus for the present generation of young scientists to revolutionize the coming generation of effective human healthcare.

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Live imaging of intracellular pH in planarians using the ratiometric fluorescent dye SNARF-5F-AM

Biology Methods and Protocols ◽

10.1093/biomethods/bpz005 ◽

2019 ◽

Vol 4 (1) ◽

Author(s):

Wendy Scott Beane ◽

Dany Spencer Adams ◽

Junji Morokuma ◽

Michael Levin

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Cell Activity ◽

Tissue Level ◽

Model System ◽

Resting Potential ◽

Stem Cell Biology ◽

Ph Gradients ◽

Fluorescent Reporter

Abstract Physiological parameters such as resting potential and pH are increasingly recognized as important regulators of cell activity and tissue-level events in regeneration, development, and cancer. The availability of fluorescent reporter dyes has greatly increased the ability to track these properties in vivo. The planarian flatworm is an important and highly tractable model system for regeneration, stem cell biology, and neuroscience; however, no protocols have been published for investigating pH in this system. Here, we report a simple and effective protocol for imaging pH gradients in living planaria suitable for intact and regenerating flatworms.

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Distinct Embryonic Origin and Injury Response of Resident Stem Cells in Craniofacial Muscles

Frontiers in Physiology ◽

10.3389/fphys.2021.690248 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xu Cheng ◽

Bing Shi ◽

Jingtao Li

Keyword(s):

Stem Cell ◽

Progenitor Cells ◽

Cell Biology ◽

Stem Cell Biology ◽

Injury Response ◽

Limb Muscles ◽

Resident Stem Cells ◽

Embryonic Origin

Craniofacial muscles emerge as a developmental novelty during the evolution from invertebrates to vertebrates, facilitating diversified modes of predation, feeding and communication. In contrast to the well-studied limb muscles, knowledge about craniofacial muscle stem cell biology has only recently starts to be gathered. Craniofacial muscles are distinct from their counterparts in other regions in terms of both their embryonic origin and their injury response. Compared with somite-derived limb muscles, pharyngeal arch-derived craniofacial muscles demonstrate delayed myofiber reconstitution and prolonged fibrosis during repair. The regeneration of muscle is orchestrated by a blended source of stem/progenitor cells, including myogenic muscle satellite cells (MuSCs), mesenchymal fibro-adipogenic progenitors (FAPs) and other interstitial progenitors. Limb muscles host MuSCs of the Pax3 lineage, and FAPs from the mesoderm, while craniofacial muscles have MuSCs of the Mesp1 lineage and FAPs from the ectoderm-derived neural crest. Both in vivo and in vitro data revealed distinct patterns of proliferation and differentiation in these craniofacial muscle stem/progenitor cells. Additionally, the proportion of cells of different embryonic origins changes throughout postnatal development in the craniofacial muscles, creating a more dynamic niche environment than in other muscles. In-depth comparative studies of the stem cell biology of craniofacial and limb muscles might inspire the development of novel therapeutics to improve the management of myopathic diseases. Based on the most up-to-date literature, we delineated the pivotal cell populations regulating craniofacial muscle repair and identified clues that might elucidate the distinct embryonic origin and injury response in craniofacial muscle cells.

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Novel Pituitary Organoid Model as Powerful Tool to Unravel Pituitary Stem Cell Biology Across Ages and Disease

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.1330 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A652-A652

Author(s):

Hugo E J Vankelecom ◽

Emma Laporte ◽

Florian Hermans ◽

Charlotte Nys ◽

Annelies Vennekens

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Biology ◽

Cell Activation ◽

Stem Cell Biology ◽

Cell Compartment ◽

Organoid Culture ◽

Stem Cell Compartment

Abstract The pituitary gland harbors a population of stem cells. However, role and regulation of these cells remain poorly understood. We recently established organoids from mouse pituitary as a novel research tool to explore pituitary stem cell biology (Cox et al., J. Endocrinol. 2019; 240:287-308). In general, organoids represent 3D in vitro cell configurations that develop and self-organize from (single) tissue stem cells under well-defined culture conditions that typically mirror the stem cell niche and/or embryogenic processes. Organoids reliably recapitulate key aspects of the original organ, including of its stem cell compartment. Moreover, organoids are long-term expandable while retaining these properties. We demonstrated that pituitary organoids originate from the resident (SOX2+) stem cells, largely phenocopy these cells and retain the stemness phenotype during expansive culture. Interestingly, the organoids show confident in vivo translatability and, when developed from transgenically damaged gland, recapitulate the activation status of the stem cells as observed in situ following injury. Now, we found that the organoids also mirror the stem cells’ phenotype and biology in physiological conditions in which the stem cell compartment is either activated or compromised. Organoids from the neonatal maturing pituitary reproduce phenotypical and functional aspects of its activated stem cells, whereas organoids from aging gland mimic the declined functional state of the stem cells in old pituitary. Interestingly, this functional decay was found to be reverted during organoid culture, indicating that the old pituitary stem cells retain intrinsic functionality but are in vivo restrained by an obstructive microenvironment, not present in the organoid culture. Indeed, using single-cell transcriptomics and in vivo analysis, we found that the aging pituitary suffers from a prevailing inflammatory state (inflammaging) which appears to raise the threshold for stem cell activation. Interestingly, comparison of young and old pituitary led us to the discovery of pituitary stem cell activators. Finally, we found that activated parameters of organoid formation are also observed when tumorigenesis takes place in the gland, again mimicking the in situ stem cell activation that is occurring in this perturbed, pathological condition. Taken together, we identified, and applied, our new pituitary organoid model as advanced and powerful tool to gain profound insight into pituitary stem cell behavior across life and disease, which is expected to eventually translate into restorative and rejuvenative tactics when pituitary function is compromised by damage or age. In this context, our single-cell transcriptome database has strong potential to unveil appealing targets.

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The Role of Hyaluronic Acid in Normal and Perturbed Hemopoietic Stem Cell Biology.

Blood ◽

10.1182/blood.v108.11.708.708 ◽

2006 ◽

Vol 108 (11) ◽

pp. 708-708 ◽

Cited By ~ 1

Author(s):

Sarah E. Shaw ◽

David N. Haylock ◽

Hayley M. Johnston ◽

Richard Lock ◽

Susie K. Nilsson

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Stem Cell Niche ◽

Fold Increase ◽

Hemopoietic Stem Cell ◽

Stem Cell Biology ◽

Hemopoietic Microenvironment ◽

Cell Niche

Abstract Considerable evidence supports the proposal that the localisation of hemopoiesis to the bone marrow (BM) involves developmentally regulated adhesive interactions between primitive hemopoietic stem cells (HSC) and the hemopoietic microenvironment of the marrow. Previous studies in our laboratory demonstrate that HSC reside within an endosteal stem cell niche, and identified several key molecules that play critical roles in their attraction to, and retention and regulation within this region. We have previously described Hyaluronic acid (HA) as one of these key molecules and shown that human and murine HSC synthesise and express HA. HSC express the 3 HAS genes and synthesis of this glycosaminoglycan by HSC as well as by cells within the hemopoietic microenvironment is critical for HSC engraftment and for regulating the HSC pool in vivo. In addition, analysis of HAS 1 and 3 knockout mice identified HA synthesised by HAS 3 to be responsible for these effects. In the absence of HAS-3 synthase the endosteal region is devoid of HA, and the hemopoietic microenvironment is significantly impaired in its ability to attract and support HSC post-transplant as well as regulate the HSC pool in vivo. Furthermore, transplanted HSC isolated from HAS-3−/− mice have a reduced ability to lodge within the endosteal region and reconstitute hemopoiesis. In vitro, binding of HA by a surrogate ligand, HABP, also has a negative regulatory effect of HSC proliferation and differentiation. Together these data suggest that HA on the surface of HSC is critical in both their lodgement and subsequent quiescence within the hemopoietic stem cell niche. Analysis of a cohort of childhood ALL samples revealed a significant correlation between the length of first complete remission and HA synthesis, cell surface HA expression, HAS-3 expression, the distribution of leukemic cells following transplantation and the development of disease in a murine model. In addition, anti-sense inhibition of HAS-3 expression in the Pre-B ALL cell line NALM6, resulted in a significantly reduced time to onset of leukaemia within a NOD/SCID murine model. Furthermore, retroviral mediated overexpression of HAS-3 in this cell line resulted in 2.5-fold increase in mRNA, a 20-fold increase in cell surface HA and a significant decrease in the proliferation potential of these cells in culture. Furthermore, following transplantation of HAS-3 overexpressing NALM6 cells into NOD/SCID recipients there was a significant delay in the onset of leukemia compared to that seen following the transplant of unmanipulated NALM6 cells. This demonstrates a primary role of HAS-3 in the onset and progression of common ALL and that HA expression levels may provide a novel prognostic indictor for this leukaemia. Furthermore, initial studies of HA expression in other leukemic bone marrow samples revealed that cells from AML, CML and CLL also exhibit increased levels of HA, with a correlation between disease progression and HA synthesis and expression; HA levels were elevated at diagnosis, decreasing in remission and increasing again at relapse. Overall, our data is strongly suggestive of a key role for this polysaccharide in both normal and aberrant hemopoietic stem cell biology.

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