Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different ...mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
It has recently been shown that mononuclear cells from murine skeletal muscle contain the potential to repopulate all major peripheral blood lineages in lethally irradiated mice, but the origin of ...this activity is unknown. We have fractionated muscle cells on the basis of hematopoietic markers to show that the active population exclusively expresses the hematopoietic stem cell antigens Sca-1 and CD45. Muscle cells obtained from 6- to 8-week-old C57BL/6-CD45.1 mice and enriched for cells expressing Sca-1 and CD45 were able to generate hematopoietic but not myogenic colonies in vitro and repopulated multiple hematopoietic lineages of lethally irradiated C57BL/6-CD45.2 mice. These data show that muscle-derived hematopoietic stem cells are likely derived from the hematopoietic system and are a result not of transdifferentiation of myogenic stem cells but instead of the presence of substantial numbers of hematopoietic stem cells in the muscle. Although CD45-negative cells were highly myogenic in vitro and in vivo, CD45-positive muscle-derived cells displayed only very limited myogenic activity and only in vivo.
Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian ...hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear.
We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene.
Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model.
These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.
Hematopoietic stem cells (HSC), normally resident in bone marrow, can be detected in the murine and human circulation. It is thought that HSC move in and out of bone marrow daily and that returning ...HSC are generally equivalent to their bone marrow counterparts in phenotype and function. However, large numbers of mononuclear blood cells are required to rescue animals from lethal irradiation, indicating either that the prevalence of circulating HSC is low, or they are inherently deficient in their repopulating ability. Accordingly, recent data suggest that circulating HSC may be unable to stably engraft WBM under homeostatic conditions. The purpose of this study was to explore these dynamics in detail using parabiosis and bone marrow transplantation.
The WBM and skeletal muscle HSC stem cell compartments of parabiosed CD45 congenic mice were analyzed functionally (via bone marrow transplantation) and phenotypically (via flow cytometry) for circulating stem cells at specific time points postparabiosis and after surgical separation.
Surprisingly, we find that stem cells trafficking out of bone marrow and into the circulation do not stably return to bone marrow, although long-lived lymphoid precursors do stably re-engraft. Circulating HSC do, however, take up residence in skeletal muscle, wherein they account for HSC activity.
Circulating HSC are not in flux with the bone marrow HSC and can persist in peripheral tissues.
Objective. Skeletal muscle–derived cells have the potential to repopulate the major peripheral blood lineages of lethally irradiated mice and thus behave like hematopoietic stem cells (HSC). We have ...recently shown that muscle cells with HSC activity (ms-HSC) express CD45 and Sca-1, suggesting a hematopoietic origin. Here we sought to clarify contradictions in the literature regarding the phenotype of ms-HSC and precisely define the hematopoietic origin of these cells.
Methods. Skeletal muscle–derived cells fractionated based on the expression of CD45 and c-kit and efflux of Hoechst 33342 and were examined for HSC activity in vivo. WBM HSC expressing β-galactosidase were transplanted into lethally irradiated recipients, whose ms-HSC compartment was later analyzed for β-galactosidase activity to determine if ms-HSC were derived from WBM HSC.
Results. Muscle-derived HSC fall exclusively in the c-kit
dimCD45
pos compartment of the muscle side population (msSP). Furthermore, the CD45
pos msSP compartment of skeletal muscle is derived from WBM HSC. CD45
posc-kit
dim msSP are about 22-fold less potent in HSC activity than WBM HSC cells in competitive repopulation assays and express low levels of c-kit relative to WBM HSC.
Conclusions. In our transplantation experiments, WBM HSC gave rise to ms-HSC, suggesting that WBM HSC and ms-HSC likely represent the same stem cell population in distinct environments. However, these two related populations are both functionally distinct in their ability to repopulate the peripheral blood of irradiated mice and phenotypically distinct.
Hematopoietic stem cells (HSCs) are the blood forming stem cells thought to be responsible for supporting the blood system throughout life. Transplantability has long been the flagship assay used to ...define and characterize HSCs throughout ontogeny. However, it has recently become clear that many cells emerge during ontogeny that lack transplantability, yet nevertheless are fated to ultimately contribute to the adult HSC pool. Here, we explore recent advances in understanding the numbers and kinetics of cells that emerge during development to support lifelong hematopoiesis—advances that are made possible by new technologies allowing interrogation of lifelong blood potential without embryo perturbation or transplantation. Illuminating the dynamics of these cells during normal development informs efforts to better understand the origins of hematologic disease and engineer HSCs from differentiating pluripotent stem cells.
In this paper we report on the timing resolution obtained in a beam test with pions of 180 GeV/c momentum at CERN for the first production of 45 μm thick Ultra-Fast Silicon Detectors (UFSD). UFSD are ...based on the Low- Gain Avalanche Detector (LGAD) design, employing n-on-p silicon sensors with internal charge multiplication due to the presence of a thin, low-resistivity diffusion layer below the junction. The UFSD used in this test had a pad area of 1.7 mm2. The gain was measured to vary between 5 and 70 depending on the sensor bias voltage. The experimental setup included three UFSD and a fast trigger consisting of a quartz bar readout by a SiPM. The timing resolution was determined by doing Gaussian fits to the time-of-flight of the particles between one or more UFSD and the trigger counter. For a single UFSD the resolution was measured to be 34 ps for a bias voltage of 200 V, and 27 ps for a bias voltage of 230 V. For the combination of 3 UFSD the timing resolution was 20 ps for a bias voltage of 200 V, and 16 ps for a bias voltage of 230 V.
This article shows an example of the peer review process for “Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis” (Ranzoni et al., 2021).
This article shows an ...example of the peer review process for “Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis” (Ranzoni et al., 2021).
Our work focuses on the clonal dynamics of hematopoietic stem and progenitor cells throughout embryogenesis and adult mammalian life. Via novel use of a multi-colored lineage trace reporter, we ...showed that hundreds of embryonic precursors contribute to life-long hematopoiesis during mammalian ontogeny. We recently used this approach to systematically and non-invasively analyze the number of clones supporting steady-state hematopoiesis throughout mammalian life. We found that the number of clones supporting the major blood and bone marrow (BM) hematopoietic compartments in mice declines with age by about 30% and 60%, respectively. As previously reported, aging dramatically reduced HSC in vivo repopulating activity, lymphoid potential and increased functional heterogeneity. Repeated challenge by serial transplantation provoked the clonal collapse of both young and aged hematopoietic systems. Whole exome sequencing of serially transplanted aged and young hematopoietic clones confirmed oligoclonal hematopoiesis and revealed mutations in at least 27 genes, including nonsense, missense and deletion mutations in Bcl11b, Hist1h2ac, Npy2r, Notch3, Ptprr and Top2b, implicating these genes in clonal selection during intense hematopoietic stress. Finally, to better understand the developmental bottlenecks that dictate which embryonic HSCs realize their potential to contribute to life-long hematopoiesis, we interrogated the phenotype, frequency and function of all major HSPC populations in the fetal and neonatal BM between E15.5 and early neonatal life. Here, we observed surprising shifts in the phenotype and frequency of specific HSPCs during this window of ontogeny. Cues present immediately following birth appear to play a major role in establishing the HSPC hierarchy of adult BM. We are currently working to identify these cues.
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