Nanoparticle-encapsulated siRNAs for gene silencing in the haematopoietic stem-cell niche.
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ABSTRACT:
SUBMITTER: Krohn-Grimberghe M
PROVIDER: S-EPMC7655681 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The haematopoietic stem cell (HSC) microenvironment in the bone marrow, termed the niche, ensures haematopoietic homeostasis by controlling the proliferation, self-renewal, differentiation and migration of HSCs and progenitor cells at steady state and in response to emergencies and injury. Improved methods for HSC isolation, driven by advances in single-cell and molecular technologies, have led to a better understanding of their behaviour, heterogeneity and lineage fate and of the niche cells and signals that regulate their function. Niche regulatory signals can be in the form of cell-bound or secreted factors and other local physical cues. A combination of technological advances in bone marrow imaging and genetic manipulation of crucial regulatory factors has enabled the identification of several candidate cell types regulating the niche, including both non-haematopoietic (for example, perivascular mesenchymal stem and endothelial cells) and HSC-derived (for example, megakaryocytes, macrophages and regulatory T cells), with better topographical understanding of HSC localization in the bone marrow. Here, we review advances in our understanding of HSC regulation by niches during homeostasis, ageing and cancer, and we discuss their implications for the development of therapies to rejuvenate aged HSCs or niches or to disrupt self-reinforcing malignant niches.
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Project description:Since we still know very little about stem cells in their natural environment, it is useful to explore their dynamics through modelling and simulation, as well as experimentally. Most models of stem cell systems are based on deterministic differential equations that ignore the natural heterogeneity of stem cell populations. This is not appropriate at the level of individual cells and niches, when randomness is more likely to affect dynamics. In this paper, we introduce a fast stochastic method for simulating a metapopulation of stem cell niche lineages, that is, many sub-populations that together form a heterogeneous metapopulation, over time. By selecting the common limiting timestep, our method ensures that the entire metapopulation is simulated synchronously. This is important, as it allows us to introduce interactions between separate niche lineages, which would otherwise be impossible. We expand our method to enable the coupling of many lineages into niche groups, where differentiated cells are pooled within each niche group. Using this method, we explore the dynamics of the haematopoietic system from a demand control system perspective. We find that coupling together niche lineages allows the organism to regulate blood cell numbers as closely as possible to the homeostatic optimum. Furthermore, coupled lineages respond better than uncoupled ones to random perturbations, here the loss of some myeloid cells. This could imply that it is advantageous for an organism to connect together its niche lineages into groups. Our results suggest that a potential fruitful empirical direction will be to understand how stem cell descendants communicate with the niche and how cancer may arise as a result of a failure of such communication.
| S-EPMC4154659 | biostudies-literature
Project description:Little is known about the formation of niches, local micro-environments required for stem-cell maintenance. Here we develop an in vivo assay for adult haematopoietic stem-cell (HSC) niche formation. With this assay, we identified a population of progenitor cells with surface markers CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1.1(-) (CD105(+)Thy1(-)) that, when sorted from 15.5 days post-coitum fetal bones and transplanted under the adult mouse kidney capsule, could recruit host-derived blood vessels, produce donor-derived ectopic bones through a cartilage intermediate and generate a marrow cavity populated by host-derived long-term reconstituting HSC (LT-HSC). In contrast, CD45(-)Tie2(-)alpha(V)(+)CD105(+)Thy1(+) (CD105(+)Thy1(+)) fetal bone progenitors form bone that does not contain a marrow cavity. Suppressing expression of factors involved in endochondral ossification, such as osterix and vascular endothelial growth factor (VEGF), inhibited niche generation. CD105(+)Thy1(-) progenitor populations derived from regions of the fetal mandible or calvaria that do not undergo endochondral ossification formed only bone without marrow in our assay. Collectively, our data implicate endochondral ossification, bone formation that proceeds through a cartilage intermediate, as a requirement for adult HSC niche formation.
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Project description:Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.
| S-EPMC6973286 | biostudies-literature
Project description:Haematopoietic stem cells (HSCs) are maintained by bone marrow niches in vivo1,2, but the ability of niche cells to maintain HSCs ex vivo is markedly diminished. Expression of niche factors by Nestin-GFP+ mesenchymal-derived stromal cells (MSCs) is downregulated upon culture, suggesting that transcriptional rewiring may contribute to this reduced HSC maintenance potential. Using an RNA sequencing screen, we identified five genes encoding transcription factors (Klf7, Ostf1, Xbp1, Irf3 and Irf7) that restored HSC niche function in cultured bone marrow-derived MSCs. These revitalized MSCs (rMSCs) exhibited enhanced synthesis of HSC niche factors while retaining their mesenchymal differentiation capacity. In contrast to HSCs co-cultured with control MSCs, HSCs expanded with rMSCs showed higher repopulation capacity and protected lethally irradiated recipient mice. Competitive reconstitution assays revealed an approximately sevenfold expansion of functional HSCs by rMSCs. rMSCs prevented the accumulation of DNA damage in cultured HSCs, a hallmark of ageing and replication stress. Analysis of the reprogramming mechanisms uncovered a role for myocyte enhancer factor 2c (Mef2c) in the revitalization of MSCs. These results provide insight into the transcriptional regulation of the niche with implications for stem cell-based therapies.
| S-EPMC6499646 | biostudies-literature
Project description:BACKGROUND:Bone metastasis is one of the most common complications of advanced breast cancer. During dissemination to bone, breast cancer cells locate in a putative 'metastatic niche', a microenvironment that regulates the colonisation, maintenance of tumour cell dormancy and subsequent tumour growth. The precise location and composition of the bone metastatic niche is not clearly defined. We have used in vivo models of early breast cancer dissemination to provide novel evidence that demonstrates overlap between endosteal, perivascular, HSC and the metastatic niche in bone. METHODS:Estrogen Receptor (ER) +ve and -ve breast cancer cells were labelled with membrane dyes Vybrant-DiD and Vybrant-CM-DiI and injected via different routes in BALBc/nude mice of different ages. Two-photon microscopy was used to detect and quantitate tumour cells and map their location within the bone microenvironment as well as their distance to the nearest bone surface compared to the nearest other tumour cell. To investigate whether the metastatic niche overlapped with the HSC niche, animals were pre-treated with the CXCR4 antagonist AMD3100 to mobilise hematopoietic (HSCs) prior to injection of breast cancer cells. RESULTS:Breast cancer cells displayed a characteristic pattern of homing in the long bones, with the majority of tumour cells seeded in the trabecular regions, regardless of the route of injection, cell-line characteristics (ER status) or animal age. Breast cancer cells located in close proximity to the nearest bone surface and the average distance between individual tumour cells was higher than their distance to bone. Mobilisation of HSCs from the niche to the circulation prior to injection of cell lines resulted in increased numbers of tumour cells disseminated in trabecular regions. CONCLUSION:Our data provide evidence that homing of breast cancer cells is independent of their ER status and that the breast cancer bone metastasis niche is located within the trabecular region of bone, an area rich in osteoblasts and microvessels. The increased number of breast cancer cells homing to bone after mobilisation of HSCs suggests that the HSC and the bone metastasis niche overlap.
| S-EPMC6582079 | biostudies-other
Project description:Haematopoietic stem cells (HSCs) support maintenance of the haematopoietic and immune systems throughout the life of vertebrates, and are the therapeutic component of bone marrow transplants. Understanding native specification of HSCs, to uncover key signals that might help improve in vitro directed differentiation protocols, has been a long-standing biomedical goal. The current impossibility of specifying true HSCs in vitro suggests that key signals remain unknown. We speculated that such signals might be presented by surrounding 'niche' cells, but no such cells have been defined. Here we demonstrate in zebrafish, that trunk neural crest (NC) physically associate with HSC precursors in the dorsal aorta (DA) just prior to initiation of the definitive haematopoietic program. Preventing association of the NC with the DA leads to loss of HSCs. Our results define NC as key cellular components of the HSC specification niche that can be profiled to identify unknown HSC specification signals.
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Project description:Stem cells reside in a specialized regulatory microenvironment or niche, where they receive appropriate support for maintaining self-renewal and multi-lineage differentiation capacity. The niche may also protect stem cells from environmental insults including cytotoxic chemotherapy and perhaps pathogenic immunity. The testis, hair follicle and placenta are all sites of residence for stem cells and are immune-suppressive environments, called immune-privileged sites, where multiple mechanisms cooperate to prevent immune attack, even enabling prolonged survival of foreign allografts without immunosuppression. We sought to determine if somatic stem-cell niches more broadly are immune-privileged sites by examining the haematopoietic stem/progenitor cell (HSPC) niche in the bone marrow, a site where immune reactivity exists. We observed persistence of HSPCs from allogeneic donor mice (allo-HSPCs) in non-irradiated recipient mice for 30?days without immunosuppression with the same survival frequency compared to syngeneic HSPCs. These HSPCs were lost after the depletion of FoxP3 regulatory T (T(reg)) cells. High-resolution in vivo imaging over time demonstrated marked co-localization of HSPCs with T(reg) cells that accumulated on the endosteal surface in the calvarial and trabecular bone marrow. T(reg) cells seem to participate in creating a localized zone where HSPCs reside and where T(reg) cells are necessary for allo-HSPC persistence. In addition to processes supporting stem-cell function, the niche will provide a relative sanctuary from immune attack.
| S-EPMC3725645 | biostudies-literature
Project description:The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs), identified using nestin expression, constitute an essential HSC niche component. Nestin(+) MSCs contain all the bone-marrow colony-forming-unit fibroblastic activity and can be propagated as non-adherent 'mesenspheres' that can self-renew and expand in serial transplantations. Nestin(+) MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or beta3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin(+) cells and favours their osteoblastic differentiation, in vivo nestin(+) cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin(+) MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin(+) cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem-cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem-cell pairs.
| S-EPMC3146551 | biostudies-literature
Project description:In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units. Double-stranded RNA precursors of viral siRNAs can potentially be generated by host RDR-dependent RNA polymerase (RDR). However, genetic evidence revealed that CaLCuV siRNA biogenesis does not require RDR1, RDR2, or RDR6. By contrast, CaLCuV derivatives engineered to target 30 nt sequences of a GFP transgene by primary viral siRNAs trigger RDR6-dependent production of secondary siRNAs. Viral siRNAs targeting upstream of the GFP stop codon induce secondary siRNAs almost exclusively from sequences downstream of the target site. Conversely, viral siRNAs targeting the GFP 3'-untranslated region (UTR) induce secondary siRNAs mostly upstream of the target site. RDR6-dependent siRNA production is not necessary for robust GFP silencing, except when viral siRNAs targeted GFP 5'-UTR. Furthermore, viral siRNAs targeting the transgene enhancer region cause GFP silencing without secondary siRNA production. We conclude that the majority of viral siRNAs accumulating during geminiviral infection are RDR1/2/6-independent primary siRNAs. Double-stranded RNA precursors of these siRNAs are likely generated by bidirectional readthrough transcription of circular viral DNA by RNA polymerase II. Unlike transgenic mRNA, geminiviral mRNAs appear to be poor templates for RDR-dependent production of secondary siRNAs.
| S-EPMC3460622 | biostudies-literature